Onemol2024 Abstractbook Cshl2024 Single Biomolecules

Abstracts of papers presented
at the 2024 meeting on
SINGLE BIOMOLECULES
October 23–October 26, 2024
Abstracts of papers presented
at the 2024 meeting on
SINGLE BIOMOLECULES
October 23–October 26, 2024
Arranged by
Xavier Darzacq, University of California, Berkeley

Daniel Larson, National Cancer Institute
Jennifer Lippincott-Schwartz, HHMI Janelia Research Campus
Contributions from the following companies provide core support for the
Cold Spring Harbor meetings program.
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Corporate Sponsors
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Front Cover: Super resolution image of a single molecule of translating

HSPD1 mRNA at ~10 nm in fixed Human Bronchial Epithelial Cells detected
using 20 mer DNA oligonucleotide probes labeled with Quasor 670 dye and
imaged on Minflux 3D super resolution microscope.
Credit: M. Palangat, D. Ball.
SINGLE BIOMOLECULES
Wednesday, October 23 – Saturday, October 26, 2024
Wednesday 7:30 pm – 10:00 pm 1 Imaging Tools and Platforms
Thursday 9:00 am – 12:00 pm 2 Organelles
Thursday 2:00 pm – 5:00 pm 3 Cytoplasm
Thursday 5:00 pm Wine & Cheese Party
Thursday 7:30 pm – 10:30 pm Poster Session
Friday 9:00 am – 12:00 pm 4 Nucleus
Friday 2:00 pm – 5:00 pm 5 Membranes
Friday 6:00 pm Banquet
Saturday 9:00 am – 12:00 pm 6 Super Resolution Imaging
All times shown are US Eastern: Time Zone Converter
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Breakfast 7:30 am-9:00 am
Lunch 11:30 am-1:30 pm
Dinner 5:30 pm-7:00 pm
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Violations, please see the back of this book.
___________________________________________________________
Abstracts are the responsibility of the author(s) and publication of an

abstract does not imply endorsement by Cold Spring Harbor Laboratory of
the studies reported in the abstract.
These abstracts should not be cited in bibliographies. Material herein

should be treated as personal communications and should be cited as such
only with the consent of the author(s).
Please note that photography or video/audio recording of oral presentations

or individual posters is strictly prohibited except with the advance
permission of the author(s), the organizers, and Cold Spring Harbor
Laboratory.
Any discussion via social media platforms of material presented at this

meeting requires explicit permission from the presenting author(s).
Printed on 100% recycled paper.

PROGRAM
WEDNESDAY, October 23—7:30 PM
SESSION 1 IMAGING TOOLS AND PLATFORMS
Chairperson: Jennifer Lippincott-Schwartz, HHMI Janelia Research

Campus, Ashburn, Virginia
Designing brighter dyes for advanced fluorescence microscopy

and beyond
Luke D. Lavis.
Presenter affiliation: Janelia Research Campus, Ashburn, Virginia. 1
SPECTR - seeing conformational changes of single molecules in

live cells
Klaus M. Hahn.
Presenter affiliation: University of North Carolina - Chapel Hill, Chapel
Hill, North Carolina. 2
SPTNet—A deep learning based framework for end-to-end single

particle tracking and motion dynamics analysis
Cheng Bi, Kevin L. Scrudders, Yue Zheng, Hao-Cheng Gao, Li Fang,
Yilun Li, Shalini T. Low-Nam, Fang Huang.
Presenter affiliation: Purdue University, West Lafayette, Indiana. 3
Non-averaged single-molecule 3D structure revealed by

individual-particle cryo-electron tomography (IPET)
Gang Ren, Jianfang Liu, Meng Zhang.
Presenter affiliation: Lawrence Berkeley National Laboratory, Berkeley,
California. 4
Mapping the directionality of glucocorticoid receptor

nucleocytoplasmic transport in live cells using two-foci cross-
correlation in massively parallel fluorescence correlation
spectroscopy (mpFCS)
Sho Oasa, Stanko N. Nikolic, Aleksandar J. Krmpot, Lars Terenius,
Milivoj R. Belic, Vladana Vukojevic.
Presenter affiliation: Karolinska Institutet, Stockholm, Sweden. 5
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Live-cell single molecule imaging reveals the mechanism of
Polycomb-mediated transcription repression
Aleksander T. Szczurek, Rob J. Klose.
Presenter affiliation: University of Oxford, Oxford, United Kingdom. 6
Leveraging microfluidics for high-throughput single-molecule

force spectroscopy measurements
Matthew P. DeJong, Alexander R. Dunn, Polly M. Fordyce.
Presenter affiliation: Stanford University, Stanford, California; Chan
Zuckerberg Biohub, California. 7
in situ force mapping to quantify and visualize mechanical

communication in genomes of living cells
Maria Mukhina, Yulia Gromova, Auston Butterfield, Haley Young,
Connor McCormick, Raymond Schaak, Nancy Kleckner.
Presenter affiliation: University of Maryland, College Park, Maryland. 8
Systematic nascent RNA kinetics imaging unveils principles for

genetic information flow in real-time
Yihan Wan.
Presenter affiliation: Westlake University, Hangzhou, China. 9
THURSDAY, October 24—9:00 AM
SESSION 2 ORGANELLES
Chairperson: Dan Larson, National Cancer Institute, Bethesda,

Maryland
Illuminating organelle dynamics during differentiation and

neurodegeneration
Maria Clara Zanellati, Gregory Miner, Sarah Cohen.
Presenter affiliation: University of North Carolina, Chapel Hill, North
Carolina. 10
Vibrational photothermal fluorescence imaging of cellular

organelles—Bridging the best of two separate worlds
Ji-Xin Cheng, Meng wang, Jianpeng Ao, Jiaze Yin.
Presenter affiliation: Boston University, Boston, Massachusetts. 11
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Imaging the life cycle of a single mRNA molecule
Weihan Li, Clemence Saint-Donat, Louis Degroux, Young J. Yoon,
Robert H. Singer.
Presenter affiliation: Albert Einstein College of Medicine, Bronx, New
York; Albert Einstein College of Medicine, Bronx, New York. 12
Translation of mRNAs encoding membrane proteins regulated by

lysosomes and Lunapark
Heejun Choi, Ya-Cheng Liao, Young J. Yoon, Jonathan Grimm, Luke
D. Lavis, Robert H. Singer, Jennifer Lippincott-Schwartz.
Presenter affiliation: HHMI, Ashburn, Virginia. 13
Focusing on mitochondria
Stefan Jakobs.
Presenter affiliation: University Medical Center of Göttingen,
Göttingen, Germany; Max Planck Institute for Multidisciplinary
Sciences, Göttingen, Germany; Fraunhofer Institute for Translational
Medicine and Pharmacology ITMP, Göttingen, Germany. 14
The splicing factor U2AF regulates the translation and

localization of nuclear-encoded mitochondrial mRNAs
Gloria R. Garcia, Murali Palangat, Ben T. Donovan, Kathy McGraw,
Josquin Moraly, Naomi Taylor, Dan R. Larson.
Presenter affiliation: Center for Cancer Research, Bethesda, Maryland. 15
Direct visualization of retrotranslocation by the Hrd1 complex by

high speed single molecule tracking in living cells
Tim Abel, Sonya Neal, Thomas Sommer, Jennifer Lippincott-Schwartz,
Christopher Obara.
Presenter affiliation: University of California, San Diego, La Jolla,
California. 16
Exploring structure and function of non-canonical SMC protein

SMCHD1 with high-speed AFM and optical tweezers
Ting-Wei Liao, Fahad Rashid, Sushil Pangeni, James M. Berger,
Taekjip Ha.
Presenter affiliation: Johns Hopkins University, Baltimore, Maryland;
Boston Children’s Hospital, Boston, Massachusetts. 17
Charting live cell 4D chromatin landscapes—Unraveling neuronal

differentiation via chromatin imaging
Kirby Campbell, Marybeth Lupo, Pratik Kumar, Luke Lavis, Michael
Dyer, David Solecki.
Presenter affiliation: St. Jude Children’s Research Hospital, Memphis,
Tennessee. 18
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THURSDAY, October 24—2:00 PM
SESSION 3 CYTOPLASM
Chairperson: Luke Lavis, HHMI Janelia Farm Research Campus,

Ashburn, Virginia
Harnessing AI-driven protein design to rapidly generate

functional intrabodies for tracking gene regulation in living cells
Timothy J. Stasevich, Gabriel Galindo, Daiki Maejima, Jacob DeRoo,
Scott Burlingham, Gretchen Fixen, Ning Zhao, Christopher D. Snow,
Brian J. Geiss, Yuko Sato, Hiroshi Kimura.
Presenter affiliation: Colorado State University, Fort Collins, Colorado. 19
Rapid transcription repression induced by DNA double-strand

break
Shuaixin He, Bin Wu.
Presenter affiliation: Johns Hopkins University School of Medicine,
Baltimore, Maryland. 20
Kinesin-14 HSET and KlpA are non-processive microtubule

motors with load-dependent power strokes.
Xinglei Liu, Lu Rao, Florian Berger, Weihong Qiu, Arne Gennerich.
Presenter affiliation: Albert Einstein College of Medicine, New York,
New York. 21
Single-molecule imaging of the endogenous Camk2a mRNA

reveals activity-dependent local regulation of the mRNA at
dendritic spines
Dong-Woo Hwang, Sulagna Das, Robert H. Singer.
York. 22
YAP signal integration through charge-mediated transcriptional

co-condensates
Kirstin Meyer, Klaus Yserentant, Rasmi Cheloor Kovilakam, Kiersten
Ruff, Bo Huang, Orion Weiner.
Presenter affiliation: University of California San Francisco, San
Francisco, California; Yale University, New Haven, Connecticut. 23
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Single-molecule observation of Hedgehog diffusion, and a new
model for morphogen movement in crowded tissues
Gavin Schlissel, Pulin Li.
Presenter affiliation: Whitehead Institute, Cambridge, Massachusetts;
MIT, Cambridge, Massachusetts. 24
Capturing the transcriptional patterning of β-actin in the mouse

skin by live imaging
Han Yang, Zhongqi Lin, Valentina Greco.
Presenter affiliation: Yale University, New Haven, Connecticut. 25
Phosphorylation of FMRP granules alters their transport, protein

synthesis and phase separation
Young J. Yoon, Shivani C. Kharod, Dongwoo Hwang, Heejun Choi,
Pablo E. Castillo, Robert H. Singer.
York. 26
Nanoscale volumetric fluorescence imaging via photochemical

sectioning
Wei Wang, Xiongtao Ruan, Gaoxiang Liu, Daniel E. Milkie, Wenping
Li, Eric Betzig, Ruixuan Gao, Srigokul Upadhyayula.
Presenter affiliation: University of California, Berkeley, Berkeley,
California. 27
Wine and Cheese Party
POSTER SESSION
See. p. xiv for List of Posters
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FRIDAY, October 25—9:00 AM
SESSION 4 NUCLEUS
Chairperson: Melike Lakadamyali, Perelman School of Medicine,

Philadelphia, Pennsylvania
Transcription factor exchange enables prolonged transcriptional

bursts
Wim Pomp, Joseph V. Meeussen, Tineke L. Lenstra.
Presenter affiliation: The Netherlands Cancer Institute, Oncode
Institute, Amsterdam, the Netherlands. 28
Studying the dynamic regulation of transcription initiation and

elongation using live-cell single molecule imaging
Nayem Haque, Yessenia Cedeno, John Hobbs, Robert H. Singer,
Ulrich Steidl, Robert A. Coleman.
York. 29
Gene-specific hub kinetics regulate transcription factor

occupancy
Samantha Fallacaro, Apratim Mukherjee, Puttachai Ratchasanmuang,
Joseph Zinski, Kareena Shankta, Mustafa Mir.
Presenter affiliation: University of Pennsylvania, Philadelphia,
Pennsylvania; Children’s Hospital of Philadelphia, Philadelphia,
Pennsylvania. 30
Effective in vivo binding energy landscape illustrates kinetic

stability of TF-DNA binding
Duyen Huynh, Philipp Hoffmeister, Tobias Friedrich, Kefan Zhang,
Marek Bartkuhn, Francesca Ferrante, Benedetto Daniele Giaimo,
Rhett Kovall, Tilman Borggrefe, Franz Oswald, J. Christof M.
Gebhardt.
Presenter affiliation: Ulm University, Ulm, Germany. 31
Lattice light sheet microscopy—Innovations, applications and

future directions
Wesley R. Legant.
Presenter affiliation: University of North Carolina - Chapel Hill, Chapel
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Molecular kinetics of transcription regulation during embryonic
development
Mustafa Mir.
Pennsylvania. 33
Single molecule tracking in drug discovery and systems biology

Daniel J. Anderson.
Presenter affiliation: Eikon Therapeutics, Hayward, California. 34
Multimodal single-molecule imaging reveals the molecular

mechanism of class-switch recombination
Mariia Mikhova, Kefei Yu, Jens Schmidt.
Presenter affiliation: Michigan State University, East Lansing,
Michigan. 35
Uncovering the Complex mechanisms of antisense transcription

regulation at the Xist/Tsix locus through single-cell stochasticity
Benjamin K. Kesler, John E. Adams, Gregor Neuert.
Presenter affiliation: Vanderbilt University, Nashville, Tennessee. 36
FRIDAY, October 25—2:00 PM
SESSION 5 MEMBRANES
Chairperson: Robert Singer, Albert Einstein College of Medicine,

Bronx, New York
Single molecule microscopy of the assembly of giant pore

structures on membranes
Martin A. Do, James C. Walsh, Till Boecking, Conall Mc Guinness.
Presenter affiliation: University of New South Wales, Sydney,
Australia. 37
Activation of engineered T cells is shaped by single molecule,

single cell activities
Kevin L. Scrudders, Suriya Selvarajan, Philip S. Low, Shalini T. Low-
Nam.
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Direct observation of motor protein stepping in living cells using
MINFLUX
Jonas Ries.
Presenter affiliation: University of Vienna, Vienna, Austria. 39
Optical single-molecule recordings in membrane tethers

Madeleine R. Howell, Katherine M. Xiang, Adam E. Cohen.
Presenter affiliation: Harvard University, Cambridge, Massachusetts. 40
Biological tuning of the membrane phase transition facilitates

plasma membrane organization and function.
Sarah Veatch.
Presenter affiliation: University of Michigan, Ann Arbor, Michigan. 41
Cryogenic light microscopy with sub-nanometer resolution

resolves the structural conformations of PIEZO1
Hisham Mazal, Franz-Ferdinand Wieser, Daniel Bollschweiler,
Alexandra Schambony, Vahid Sandoghdar.
Presenter affiliation: Max Planck Institute for the Science of Light,
Erlangen, Germany; Max-Planck-Zentrum für Physik und Medizin,
Erlangen, Germany. 42
Mechanistic of force transmission across the nuclear envelope at

LINC complexes by single molecule imaging
Liying Wu, Remi Dore, Gaetan Bellot, Christine Doucet, Emmanuel
Margeat, Fabien Pinaud.
Presenter affiliation: University of Southern California, Los Angeles,
California. 43
Multimodal microscopy platform for 3D single-molecule super-

resolution imaging throughout mammalian cells
Sofía Vargas-Hernández, Tyler Nelson, Margareth Freire, Siyang
Cheng, Anna-Karin Gustsvsson.
Presenter affiliation: Rice University, Houston, Texas. 44
A strategy for quantifying the conformational changes of single

molecules in live cells
Pengning Xu, Nick Pinkin, Saygin Gulec, Bei Liu, Timothy Elston,
Klaus Hahn.
Presenter affiliation: UNC Chapel Hill, Chapel Hill, North Carolina. 45
FRIDAY, October 25—6:00 PM
COCKTAILS and BANQUET
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SATURDAY, October 26—9:00 AM
SESSION 6 SUPER RESOLUTION IMAGING
Chairperson: Xavier Darzacq, University of California, Berkeley
Visualization of translation dependent changes to shape of mRNA

in vivo at ~10 nm using Minflux
Murali Palangat, David Ball, Tatiana Karpova, Daniel R. Larson.
Presenter affiliation: National Institutes of Health, Bethesda, Maryland. 46
Single-molecule live imaging of endogenous protein interactions

using proximity-assisted photoactivation (PAPA)
Thomas G. Graham, Claire Dugast-Darzacq, Gina M. Dailey, Britney
Weng, Sathvik Anantakrishnan, Amir Hay, Gokul Upadhyayula, Eric
Betzig, Xavier Darzacq, Robert Tjian.
California; Johns Hopkins University, Baltimore, Maryland. 47
Using single molecule tracking to investigate DNA methylation in

live cells
Amir D. Hay, Thomas GW Graham, Kemal Achour, Xavier Darzacq,
Robert Tjian, Srigokul Upadhyayula, Eric Betzig.
Presenter affiliation: UC Berkeley, Berkeley, California. 48
Mechanisms of transcription control by distal enhancers from

high-resolution single-gene imaging
Alexandros Pertsinidis.
Presenter affiliation: MSKCC, New York, New York. 49
Super resolution imaging of chromatin in health and disease

Melike Lakadamyali.
Pennsylvania. 50
Single-molecule dynamics of pioneer transcription factors

interrogating chromatin in live cells
Zuhui Wang, Bo Wang, Di Niu, Claudia Cattoglio, Kyle Loh, Luke
Lavis, Hao Ge, Wulan Deng.
Presenter affiliation: Peking University, Beijing, China. 51
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Persistent and transient chromatin binding shapes single-
molecule dynamics of RNA polymerase I, II, and III machineries in
live cells
Yick Hin Ling, Chloe Liang, Sixiang Wang, Carl Wu.
Presenter affiliation: Johns Hopkins University, Baltimore, Maryland. 52
Single-molecule imaging of transcription regulation by DNA

topology
Nicholas Yahya, Sam Meyer, Jie Xiao.
Presenter affiliation: Johns Hopkins School of Medicine, Baltimore,
Maryland. 53
Ultra-resolution, multi-scale, live imaging of the 3D genome,

folded by motor proteins
Joo Lee, Liang-Fu Chen, Simon Gaudin, Alistair N. Boettiger.
Presenter affiliation: Stanford University, Stanford, California. 54
POSTER SESSION
The N- and C-terminal intrinsically disordered regions of TOX

regulate its chromatin binding and target search kinetics with
opposing effects
Santosh Adhikari, Matthew A. Sullivan, Aditi Chandra, Golnaz Vahedi,
E John Wherry, Mustafa Mir.
Presenter affiliation: Childrens Hospital of Philadelphia, Philadelphia,
Pennsylvania. 55
A methodology to reduce the localization error of multi-loci

microscopy data provides new insights into enhancer biology
Christopher H. Bohrer, Daniel R. Larson.
Presenter affiliation: National Cancer Institute, Bethesda, Maryland. 56
Dissecting NMNAT1’s role in dynamic regulation of transcription

Yessenia Cedeno, Samuel Taylor, Ulrich Steidl, Robert Coleman.
York. 57
GOLDFISH-X—Heat-free, exonuclease-based DNA FISH with

immunofluorescence compatibility
Po-Ta Chen, Tiantian Shang, Taekjip Ha.
Presenter affiliation: Boston Children’s Hospital, Boston,
Massachusetts. 58
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Live-cell single-molecule dynamics to unravel spatiotemporal
transcriptional modulation by the BAF chromatin remodeler
complex
Ziyuan Chen, Gina M. Dailey, Robert Tjian, Xavier Darzacq.
California. 59
ERα mediated gene state switching regulates the extent of the

single-cell estrogen response
Christopher R. Day, Pelin Yasar, Gloria Adedoyin, Brian D. Bennett,
Joseph Rodriguez.
Presenter affiliation: NIEHS, Durham, North Carolina. 60
Real-time visualization of spliceosome assembly reveals basic

principles of splice site selection
Benjamin T. Donovan, Bixuan Wang, Gloria R. Garcia, Stephen M.
Mount, Daniel R. Larson.
Presenter affiliation: NIH, Bethesda, Maryland. 61
Live-cell imaging uncovers dynamic coupling between enhancer

transcription and gene activation.
Nadezda A. Fursova, Christopher H. Bohrer, Simona Patange, Varun
Sood, Carson C. Chow, Daniel R. Larson.
Capturing mechanisms of transcription initiation using genome-

bound anisotropy of RNA Polymerase II
Nayem Haque, Shu-Hao Liou, Robert A. Coleman.
York. 63
Molecular kinetics of Polycomb body formation during

development
Gabriela Hayward-Lara, Apratim Mukherjee, Mustafa Mir.
Presenter affiliation: Perelman School of Medicine, University of
Pennsylvania, Philadelphia, Pennsylvania; Children’s Hospital of
Philadelphia, Philadelphia, Pennsylvania. 64
Utilizing single-molecule imaging to investigate hematopoietic

cell fate decision-making
John W. Hobbs IV, Lou Lou Dimple Maria Reenath Victor Jagaraja,
Samuel Taylor, Rajni Kumari, Robert A. Coleman, Ulrich Steidl.
York. 65
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A statistical resolution measure of fluorescence microscopy with
finite photons
Yilun Li, Fang Huang.
DNA supercoiling-mediated G4/R-loop formation tunes

transcription by controlling the access of RNA polymerase
Jihee Hwang, Chung-Ying Lee, Tapas Paul, Huijin Lee, Taekjip Ha,
Sua Myong.
Presenter affiliation: Boston Children's Hospital, Boston,
Massachusetts; Harvard Medical School, Boston, Massachusetts. 67
Visualization of multiple translation frames in Repeat Associated

Non-AUG (RAN) translation involved in neurodegenerative
diseases at single mRNA level in living cells
Hayato Ito, Timothy Stasevich, Yoshitaka Nagai, Hideki Taguchi.
Presenter affiliation: Tokyo Institute of Technology, Yokohama, Japan. 68
The biophysical properties of the ER lumen are affected by

mechanical perturbations and differentiation
Chao Jiang, Andrew Bazley, Liam Holt.
Presenter affiliation: Institute for Systems Genetics, New York, New
York. 69
Super-resolution, single-molecule imaging of transcriptional

bursts
Rebecca J. Kaddis Maldonado, Leslie J. Parent.
Presenter affiliation: Penn State College of Medicine, Hershey,
Pennsylvania. 70
Magnetic micromanipulation of loci on DNA

Veer Keizer, Yeonee Seol, Murali Palangat, Keir Neuman, Daniel
Larson.
Dynamics and function of long-range Polycomb interactions

Sumin Kim, Anders Sejr Hansen.
Presenter affiliation: Massachusetts Institute of Technology,
Cambridge, Massachusetts; Broad Institute of MIT and Harvard,
Cambridge, Massachusetts. 72
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Bursting translation on single mRNAs in live cells
Nathan M. Livingston, Jiwoong Kwon, Oliver Valera, James A. Saba,
Niladri K. Sinha, Pranav Reddy, Blake Nelson, Clara Wolfe, Taekjip
Ha, Rachel Green, Jian Liu, Bin Wu.
Presenter affiliation: Johns Hopkins University School of Medicine,
Baltimore, Maryland. 73
A close look at the nanocolumns of the calyx of Held synapses

under the MAGNIFY expansion microscopy
Xuelin Lou.
Presenter affiliation: Medical College of Wisconsin, Milwaukee,
Wisconsin. 74
Ribosomal DNA undergoes structural reorganization during

homologous recombination repair of double strand breaks
revealed by super-resolution fluorescence imaging
Ye Ma.
Presenter affiliation: Boston Children's Hospital, Harvard Medical
School, Boston, Massachusetts; Johns Hopkins University, Baltimore,
Maryland. 75
Single molecule kinetics of pathogenic gene activation by

transcriptional hubs formed by mutant ENL
Kaeli M. Mathias, Santosh Adhikari, Liling Wan, Mustafa Mir.
Presenter affiliation: Children's Hospital of Philadelphia, Philadelphia,
Pennsylvania; Perelman School of Medicine, University of
Pennsylvania, Philadelphia, Pennsylvania. 76
General principles to improve the measurement protein dynamics

at scale as a means of generating biological insight
David McSwiggen.
Presenter affiliation: Eikon Therapeutics, Hayward, California. 77
Molecular kinetics of transcription during embryonic

development
Apratim Mukherjee, Kareena Shankta, Manya Kapoor, Raymond D.
Carter, Yara Haloush, Mustafa Mir.
Pennsylvania. 78
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TrueSpot—A robust automated tool for quantifying signal puncta
in fluorescent imaging
Blythe G. Hospelhorn, Benjamin K. Kesler, Hossein Jashnsaz, Gregor
Neuert.
Presenter affiliation: Vanderbilt University, Nashville, Tennessee. 79
Single-molecule imaging of Mediator-RNA Polymerase II

interaction dynamics during early neurogenesis
Heta Patel, Gina Dailey, Xavier Darzacq, Robert Tjian.
California. 80
Exploring synaptic actin-myosin dynamics with super resolution

microscopy
Shayna J. Reed, Natasha Mayorga, Gavin Rumbaugh, Courtney A.
Miller.
Presenter affiliation: Scripps Research, La Jolla, California; The
Wertheim UF Scripps Institute for Biomedical Innovation &
Technology, Jupiter, Florida. 81
Single-molecule imaging defines distinct functional complexes of

telomere binding proteins
Tomas Janovic, Jens C. Schmidt.
Presenter affiliation: Michigan State University, East Lansing,
Michigan. 82
Chromatin and nuclear receptor single molecule dynamics and

gene regulation in vivo
Diana A. Stavreva, Le Hoang, Kaustubh Wagh, Hannah LaPoint,
Sohyoung Kim, Arpita Upadhyaya, Louis Schiltz, Raj Chari, Gordon L.
Hager.
Presenter affiliation: NCI, CCR, NIH, Bethesda, Maryland. 83
Chromosome contraction is not sufficient to repress transcription

revealed by CRISPR-CLIP
Yu-Chieh Chung, Dilshodbek Nishonov, Siou-Luan He, Nevin Wise, Li-
Chun Tu.
Presenter affiliation: The Ohio State University, Columbus, Ohio. 84
Compartmentalized diffusion between paired meiotic

chromosomes regulates crossovers in C. elegans
Lexy von Diezmann.
Presenter affiliation: University of Minnesota, Minneapolis, Minnesota. 85
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Transglutaminase 2-mediated post-translational modification of
vimentin in macrophage activation of sepsis mouse
Yali xu, Ting Su, Wenkui Yu, Yutaka Furutani, Harukazu Suzuki, Xian-
Yang Qin.
Presenter affiliation: RIKEN Center for Integrative Medical Sciences,
Yokohama, Japan; Nanjing University, Nanjing, China. 86
Using advanced microscopy techniques to study the effects of

condensates on the nuclear environment
Chelsea Q. Yang, Wesley Legant.
Presenter affiliation: University of North Carolina, Chapel Hill, Chapel
Proteolethargy is a pathogenic mechanism in chronic disease

Ming Zheng, Alessandra Dall’Agnese, Shannon Moreno, Jesse M.
Platt, Tong Ihn Lee, Richard A. Young.
Presenter affiliation: Whitehead Institute for Biomedical Research,
Cambridge, Massachusetts; Massachusetts Institute of Technology,
Cambridge, Massachusetts. 88
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AUTHOR INDEX
Abel, Tim, 16 Coleman, Robert A., 29, 63, 57,

Achour, Kemal, 48 65
Adams, John E., 36
Adedoyin, Gloria, 60 Dailey, Gina M., 47, 59, 80
Adhikari, Santosh, 55, 76 Dall’Agnese, Alessandra, 88
Anantakrishnan, Sathvik, 47 Darzacq, Xavier, 47, 48, 59, 80
Anderson, Daniel J., 34 Das, Sulagna, 22
Ao, Jianpeng, 11 Day, Christopher R., 60
Degroux, Louis, 12
Ball, David, 46 DeJong, Matthew P., 7
Bartkuhn, Marek, 31 Deng, Wulan, 51
Bazley, Andrew, 69 DeRoo, Jacob, 19
Belic, Milivoj R., 5 Dimple Maria Reenath Victor
Bellot, Gaetan, 43 Jagaraja, Lou Lou, 65
Bennett, Brian D., 60 Do, Martin A., 37
Berger, Florian, 21 Donovan, Benjamin T., 15, 61
Berger, James M., 17 Dore, Remi, 43
Betzig, Eric, 27, 47, 48 Doucet, Christine, 43
Bi, Cheng, 3 Dugast-Darzacq, Claire, 47
Boecking, Till, 37 Dunn, Alexander R., 7
Boettiger, Alistair N., 54 Dyer, Michael, 18
Bohrer, Christopher H., 56, 62
Bollschweiler, Daniel, 42 Elston, Timothy, 45
Borggrefe, Tilman, 31
Burlingham, Scott, 19 Fallacaro, Samantha, 30
Butterfield, Auston, 8 Fang, Li, 3
Ferrante, Francesca, 31
Campbell, Kirby, 18 Fixen, Gretchen, 19
Carter, Raymond D., 78 Fordyce, Polly M., 7
Castillo, Pablo E., 26 Freire, Margareth, 44
Cattoglio, Claudia, 51 Friedrich, Tobias, 31
Cedeno, Yessenia, 29, 57 Fursova, Nadezda A., 62
Chandra, Aditi, 55 Furutani, Yutaka, 86
Chari, Raj, 83
Cheloor Kovilakam, Rasmi, 23 Galindo, Gabriel, 19
Chen, Liang-Fu, 54 Gao, Hao-Cheng, 3
Chen, Po-Ta, 58 Gao, Ruixuan, 27
Chen, Ziyuan, 59 Garcia, Gloria R., 15, 61
Cheng, Ji-Xin, 11 Gaudin, Simon, 54
Cheng, Siyang, 44 Ge, Hao, 51
Choi, Heejun, 13, 26 Gebhardt, J. Christof M., 31
Chow, Carson C., 62 Geiss, Brian J., 19
Chung, Yu-Chieh, 84 Gennerich, Arne, 21
Cohen, Adam E., 40 Giaimo, Benedetto Daniele, 31
Cohen, Sarah, 10 Graham, Thomas G., 47, 48
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Greco, Valentina, 25 Kumar, Pratik, 18
Green, Rachel, 73 Kumari, Rajni, 65
Grimm, Jonathan, 13 Kwon, Jiwoong, 73
Gromova, Yulia, 8
Gulec, Saygin, 45 Lakadamyali, Melike, 50
Gustsvsson, Anna-Karin, 44 LaPoint, Hannah, 83
Larson, Daniel R., 15, 46, 56, 61,
Ha, Taekjip, 17, 58, 67, 73 62, 71
Hager, Gordon L., 83 Lavis, Luke D., 1, 13, 18, 51
Hahn, Klaus, 2, 45 Lee, Chung-Ying, 67
Haloush, Yara, 78 Lee, Huijin, 67
Haque, Nayem, 29, 63 Lee, Joo, 54
Hay, Amir, 47, 48 Lee, Tong Ihn, 88
Hayward-Lara, Gabriela, 64 Legant, Wesley, 32, 87
He, Shuaixin, 20 Lenstra, Tineke L., 28
He, Siou-Luan, 84 Li, Pulin, 24
Hoang, Le, 83 Li, Weihan, 12
Hobbs IV, John W., 29, 65 Li, Wenping, 27
Hoffmeister, Philipp, 31 Li, Yilun, 3, 66
Holt, Liam, 69 Liang, Chloe, 52
Hospelhorn, Blythe G., 79 Liao, Ting-Wei, 17
Howell, Madeleine R., 40 Liao, Ya-Cheng, 13
Huang, Bo, 23 Lin, Zhongqi, 25
Huang, Fang, 3, 66 Ling, Yick Hin, 52
Huynh, Duyen, 31 Liou, Shu-Hao, 63
Hwang, Dong-Woo, 22, 26 Lippincott-Schwartz, Jennifer,
Hwang, Jihee, 67 13, 16
Ito, Hayato, 68 Liu, Bei, 45
Liu, Gaoxiang, 27
Jakobs, Stefan, 14 Liu, Jian, 73
Janovic, Tomas, 82 Liu, Jianfang, 4
Jashnsaz, Hossein, 79 Liu, Xinglei, 21
Jiang, Chao, 69 Livingston, Nathan M., 73
Loh, Kyle, 51
Kaddis Maldonado, Rebecca J., Lou, Xuelin, 74
70 Low, Philip S., 38
Kapoor, Manya, 78 Low-Nam, Shalini T., 3, 38
Karpova, Tatiana, 46 Lupo, Marybeth, 18
Keizer, Veer, 71
Kesler, Benjamin K., 36, 79 Ma, Ye, 75
Kharod, Shivani C., 26 Maejima, Daiki, 19
Kim, Sohyoung, 83 Margeat, Emmanuel, 43
Kim, Sumin, 72 Mathias, Kaeli M., 76
Kimura, Hiroshi, 19 Mayorga, Natasha, 81
Kleckner, Nancy, 8 Mazal, Hisham, 42
Klose, Rob J., 6 McGuinness, Conall, 37
Kovall, Rhett, 31 McCormick, Connor, 8
Krmpot, Aleksandar J., 5 McGraw, Kathy, 15
xxii
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McSwiggen, David, 77 Ratchasanmuang, Puttachai, 30
Meeussen, Joseph V., 28 Reddy, Pranav, 73
Meyer, Kirstin, 23 Reed, Shayna J., 81
Meyer, Sam, 53 Ren, Gang, 4
Mikhova, Mariia, 35 Ries, Jonas, 39
Milkie, Daniel E., 27 Rodriguez, Joseph, 60
Miller, Courtney A., 81 Ruan, Xiongtao, 27
Miner, Gregory, 10 Ruff, Kiersten, 23
Mir, Mustafa, 30, 33, 55, 64, 76, Rumbaugh, Gavin, 81
78
Moraly, Josquin, 15 Saba, James A., 73
Moreno, Shannon, 88 Saint-Donat, Clemence, 12
Mount, Stephen M., 61 Sandoghdar, Vahid, 42
Mukherjee, Apratim, 30, 64, 78 Sato, Yuko, 19
Mukhina, Maria, 8 Schaak, Raymond, 8
Myong, Sua, 67 Schambony, Alexandra, 42
Schiltz, Louis, 83
Nagai, Yoshitaka, 68 Schlissel, Gavin, 24
Neal, Sonya, 16 Schmidt, Jens, 35, 82
Nelson, Blake, 73 Scrudders, Kevin L., 3, 38
Nelson, Tyler, 44 Sejr Hansen, Anders, 72
Neuert, Gregor, 36, 79 Selvarajan, Suriya, 38
Neuman, Keir, 71 Seol, Yeonee, 71
Nikolic, Stanko N., 5 Shang, Tiantian, 58
Nishonov, Dilshodbek, 84 Shankta, Kareena, 30, 78
Niu, Di, 51 Singer, Robert H., 12, 13, 22, 26,
29
Oasa, Sho, 5 Sinha, Niladri K., 73
Obara, Christopher, 16 Snow, Christopher D., 19
Oswald, Franz, 31 Solecki, David, 18
Sommer, Thomas, 16
Palangat, Murali, 15, 46, 71 Sood, Varun, 62
Pangeni, Sushil, 17 Stasevich, Timothy, 19, 68
Parent, Leslie J., 70 Stavreva, Diana A., 83
Patange, Simona, 62 Steidl, Ulrich, 29, 57, 65
Patel, Heta, 80 Su, Ting, 86
Paul, Tapas, 67 Sullivan, Matthew A., 55
Pertsinidis, Alexandros, 49 Suzuki, Harukazu, 86
Pinaud, Fabien, 43 Szczurek, Aleksander T., 6
Pinkin, Nick, 45
Platt, Jesse M., 88 Taguchi, Hideki, 68
Pomp, Wim, 28 Taylor, Naomi, 15
Taylor, Samuel, 57, 65
Qin, Xian-Yang, 86 Terenius, Lars, 5
Qiu, Weihong, 21 Tjian, Robert, 47, 48, 59, 80
Tu, Li-Chun, 84
Rao, Lu, 21
Rashid, Fahad, 17 Upadhyaya, Arpita, 83
xxiii
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Upadhyayula, Srigokul, 27, 47, Zhang, Kefan, 31
48 Zhang, Meng, 4
Zhao, Ning, 19
Vahedi, Golnaz, 55 Zheng, Ming, 88
Valera, Oliver, 73 Zheng, Yue, 3
Vargas-Hernández, Sofía, 44 Zinski, Joseph, 30
Veatch, Sarah, 41
von Diezmann, Lexy, 85
Vukojevic, Vladana, 5
Wagh, Kaustubh, 83
Walsh, James C., 37
Wan, Liling, 76
Wan, Yihan, 9
Wang, Bixuan, 61
Wang, Bo, 51
Wang, Meng, 11
Wang, Sixiang, 52
Wang, Wei, 27
Wang, Zuhui, 51
Weiner, Orion, 23
Weng, Britney, 47
Wherry, E John, 55
Wieser, Franz-Ferdinand, 42
Wise, Nevin, 84
Wolfe, Clara, 73
Wu, Bin, 20, 73
Wu, Carl, 52
Wu, Liying, 43
Xiang, Katherine M., 40

Xiao, Jie, 53
Xu, Pengning, 45
Xu, Yali, 86
Yahya, Nicholas, 53
Yang, Chelsea Q., 87
Yang, Han, 25
Yasar, Pelin, 60
Yin, Jiaze, 11
Yoon, Young J., 12, 13, 26
Young, Haley, 8
Young, Richard A., 88
Yserentant, Klaus, 23
Yu, Kefei, 35
Yu, Wenkui, 86
Zanellati, Maria Clara, 10
xxiv
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DESIGNING BRIGHTER DYES FOR ADVANCED FLUORESCENCE
MICROSCOPY AND BEYOND
Luke D Lavis
Janelia Research Campus, HHMI, Ashburn, VA
Specific labeling of biomolecules with bright, photostable fluorophores is

the keystone of fluorescence microscopy. An established method to label
cellular components utilizes genetically encoded self labeling tags, which
enable the attachment of chemical fluorophores to specific proteins inside
living cells. This strategy combines the genetic specificity of fluorescent
proteins with the favorable photophysics of synthetic dyes. However,
intracellular labeling using these techniques requires small, cell-permeable
fluorophores, previously limiting utility to a small number of classic,
unoptimized dyes. We discovered a simple structural modification to
standard fluorophores that improves brightness and photostability while
preserving other spectral properties and cell permeability. Inspired by
computational experiments, we replaced the N,N-dimethylamino
substituents in tetramethylrhodamine with a four-membered azetidine ring.
This net addition of two carbon atoms doubles the quantum efficiency and
improves the photon yield in living cells. The novel substitution is
generalizable to fluorophores from different structural classes, yielding a
palette of synthetically tractable chemical dyes with improved quantum
efficiency and enabling multicolor single-molecule imaging experiments.
These brighter versions of classic fluorophores can be further modified to
fine-tune spectral and chemical properties for advanced imaging
experiments in increasingly complex biological samples.
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SPECTR - SEEING CONFORMATIONAL CHANGES OF SINGLE
MOLECULES IN LIVE CELLS
Klaus M Hahn
University of North Carolina - Chapel Hill, Pharmacology, Chapel Hill, NC
This talk will focus on SPECTR (small peptide exposure for conformation
tracking), a new approach to visualize the conformational changes of
individual molecules in living cells. A small peptide (7-12mer) is embedded
in the target protein where its interaction with a separate “binder protein”
occurs only in a given conformation. Simple colocalization of the target
protein and binder is sufficient to report the conformational state. Because
direct excitation of bright dyes can be used, single molecule detection is
readily achieved. The approach has been used to image movement of
autoinhibitory domains, stretch-induced exposure of binding sites in
mechanosensory proteins, and activation-induced changes at allosteric sites.
Using novel, environment-sensing dyes in biosensor designs reveals
changing conformational states along individual particle tracks.
Applications to clustering in membrane domains, adhesion biology and
feedback in GTPase signaling will be discussed.
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SPTNET: A DEEP LEARNING BASED FRAMEWORK FOR END-TO-
END SINGLE PARTICLE TRACKING AND MOTION DYNAMICS
ANALYSIS
Cheng Bi1, Kevin L Scrudders2, Yue Zheng2, Hao-Cheng Gao2, Li Fang2,

Yilun Li2, Shalini T Low-Nam3, Fang Huang2
1
Purdue University, Department of Biological Science, West Lafayette, IN,
2
Purdue University, Weldon School of Biomedical Engineering, West
Lafayette, IN, 3Purdue University, Tarpo Department of Chemistry, West
Lafayette, IN
Single particle tracking (SPT) provides high-resolution spatial-temporal

information on biomolecule dynamics. However, localization inaccuracies,
limited track lengths, along with heterogeneous fluorescence backgrounds
and potential molecular motion blur, pose significant challenges that hinder
the accurate extraction of movement trajectories and their underlying
motion behavior. Conventional SPT pipeline struggles to comprehensively
address detection, localization, linkage, and parameter inference
simultaneously, resulting in information loss during sequential processing.
To overcome these challenges, we propose SPTNet, an end-to-end deep
learning framework that leverages a Transformer-based architecture to
optimize trajectory and motion parameter estimations in parallel through a
global loss. SPTNet bypasses traditional SPT processes, directly inferring
molecular trajectories and motion parameters from fluorescence microscopy
videos with a precision approaching the statistical information limit. Our
results demonstrate that SPTNet outperforms conventional methods under
commonly encountered but challenging conditions such as short
trajectories, low SNR, structured background and motion blur cases and
especially when molecules exhibit non-Brownian behaviors.
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NON-AVERAGED SINGLE-MOLECULE 3D STRUCTURE
REVEALED BY INDIVIDUAL-PARTICLE CRYO-ELECTRON
TOMOGRAPHY (IPET)
Gang Ren, Jianfang Liu, Meng Zhang
Lawrence Berkeley National Laboratory, The Molecular Foundry, Berkeley,

CA
Although the atomic structure of biomolecules can be determined by current

techniques such as X-ray crystallography and cryo-electron microscopy
(cryo-EM), achieving a low-resolution 3D structure of some biomolecules
with flexible structures and continuously changing conformations—such as
lipoproteins, antibodies, and nucleotide nanostructures—remains
challenging. This difficulty arises because current techniques typically
require an averaging process, wherein a small population of homogeneous
particles must be pre-selected from a large pool and then averaged to
produce a static 3D structure. While averaging can improve the signal-to-
noise ratio and achieve high-resolution structures of rigid-body portions, it
can also result in the loss of density in flexible portions/domains and
produce an anisotropic resolution distribution on the averaged 3D map.
Notably, the structures of most un-selected particles remain elusive.
In recent years, we have enhanced the cryo-electron tomography (cryo-ET)

technique for determining the 3D structure of a single biomolecular particle
without averaging from different particles, called individual-particle
electron tomography (IPET, Zhang et al. in PLoS One, 2012;7(1)).
However, due to the extremely weak signal from a single biomolecule
caused by the electron dose limit necessary to prevent radiation damage,
only the low-resolution of a single biomolecule can be achieved (up to 2
nm), which is barely sufficient to reveal the tertiary structure of nucleotide
nanoparticles during their processes of self-folding and intramolecular
conformational changing.
In this study, we demonstrate an implementation of IPET technique in

investigation of the inner and intramolecular conformational changes of
tetra-nucleosome arrays during the processes of phase transition (Zhang,
M., et al., Nat Comm, 2024, 15, 4395 (2024); Mol Cell. 2022, 82(16):3000-
3014.e9). We found that the crucial parameter determining the structure
adopted by chromatin arrays is the angle between the entry and exit of the
DNA and the corresponding tangents to the nucleosomal disc. Additionally,
we discovered that the transition involves the exposure of nucleosome
hydrophobic surfaces, leading to modified inter-nucleosome interactions.
These results provide insights into the initial stages of intra-array
compaction and suggest a critical precursor to condensation in the
regulation of chromatin organization, and a physical mechanism by which
chromatin may transition from interphase to metaphase structures.
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MAPPING THE DIRECTIONALITY OF GLUCOCORTICOID
RECEPTOR NUCLEOCYTOPLASMIC TRANSPORT IN LIVE CELLS
USING TWO-FOCI CROSS-CORRELATION IN MASSIVELY
PARALLEL FLUORESCENCE CORRELATION SPECTROSCOPY
(mpFCS)
Sho Oasa*1, Stanko N Nikolić*1,2,3, Aleksandar J Krmpot*1,2,3, Lars

Terenius1, Milivoj R Belić3, Vladana Vukojević1
1
Karolinska Institutet, Clinical Neuroscience, Stockholm, Sweden,
2
University of Belgrade, Institute of Physics Belgrade, Belgrade, Serbia,
3
Texas A&M University at Qatar, Division of Arts and Sciences, Doha,
Qatar
The glucocorticoid receptor (GR) is a ligand-dependent transcription factor

that is conserved across species and plays a critical role in maintaining
homeostasis and responding to stress. It is also a significant therapeutic
target for treating inflammation and cancer. In the absence of stimulation,
the GR is located in the cytoplasm in a large multiprotein complex. Upon
stimulation by endogenous or synthetic ligands, the GR translocates to the
cell nucleus, where it regulates the transcription of numerous genes either
by directly binding to glucocorticoid response elements or by interacting
with other transcription factors. Therefore, the nucleocytoplasmic transport
of glucocorticoid receptor is crucial for its functions, and its disruption is a
significant contributor to various diseases. Although considerable
knowledge exists about the molecular mechanisms of GR function, the
directionality of its nucleocytoplasmic transport is less characterized and
not as much is known about how the bidirectional flow of GR between the
nucleus and cytoplasm is organized in stimulated cells. In this study, we
employed massively parallel fluorescence correlation spectroscopy
(mpFCS), a recently developed quantitative analytical method with single-
molecule sensitivity, and two-foci cross-correlation to map the
directionality of GR translocation at various positions along the nuclear
envelope in live cells. Our results suggest that during ligand-induced GR
translocation, the import and export of GR occur simultaneously but at
different locations within the cell nucleus. This suggests a complex
coordination of GR movement through the nuclear pore complex (NPC) to
prevent accumulation or depletion of transcription factors inside the
nucleus. The ability of mpFCS to detail the heterogeneity of directional
nucleocytoplasmic transport in live cells highlights its potential for studying
how bidirectional macromolecule flow through the NPC is managed in live
cells.
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LIVE-CELL SINGLE MOLECULE IMAGING REVEALS THE
MECHANISM OF POLYCOMB-MEDIATED TRANSCRIPTION
REPRESSION
Aleksander T Szczurek, Rob J Klose
University of Oxford, Biochemistry, Oxford, United Kingdom
The Polycomb Repressive Complex (PRC) plays fundamental role in

regulating gene expression of thousands genes during mammalian
development. However, how it controls transcription to enable gene
repression has remained enigmatic. Having previously characterized
components of PRC system responsible for transcription repression1,2 we
now set out to understand its mechanism via employing rapid degron-based
PRC depletion coupled with live-cell imaging of single nascent RNAs and
single-particle tracking. Curiously, when in transcribing state, transcription
of PRC-repressed genes remains similar to that from typical gene promoters
suggesting PRC acts upstream of but not directly on transcription bursts.
Further, we discover that PRC is not a constitutive block to transcription but
instead sustains a long-lived deep promoter OFF-state which limits the
frequency with which the promoter can enter into a transcribing state. Next,
using novel live-cell imaging approach we characterize the single-molecule
dynamics of the components of transcription machinery and demonstrate
that PRC sustains promoter OFF-state by counteracting the chromatin-
binding of factors that enable early transcription pre-initiation complex
formation leading to gene repression. Together these important discoveries
provide a new rationale for how the Polycomb system controls transcription
and suggests a universal mechanism that could enable the Polycomb system
to constrain transcription across diverse cellular contexts. In broad terms,
this work demonstrates how single molecule RNA and protein imaging
approaches will aid to discover the mechanisms of gene regulation in living
cells.
1. Blackledge, N. P. et al. PRC1 Catalytic Activity Is Central to Polycomb

System Function. Mol Cell 77, 857-874 e859 (2020).
https://doi.org:10.1016/j.molcel.2019.12.001
2. Dobrinic, P., Szczurek, A. T. & Klose, R. J. PRC1 drives Polycomb-
mediated gene repression by controlling transcription initiation and burst
frequency. Nat Struct Mol Biol 28, 811-824 (2021).
https://doi.org:10.1038/s41594-021-00661-y
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LEVERAGING MICROFLUIDICS FOR HIGH-THROUGHPUT
SINGLE-MOLECULE FORCE SPECTROSCOPY MEASUREMENTS
Matthew P DeJong1, Alexander R Dunn1, Polly M Fordyce2,3,4,5

1
Stanford University, Chemical Engineering, Stanford, CA, 2Stanford
University, Bioengineering, Stanford, CA, 3Stanford University, Genetics,
Stanford, CA, 4Stanford University, Sarafan ChEM-H, Stanford, CA, 5Chan
Zuckerberg Biohub, San Francisco, CA
Here, we report the development of a high-throughput microfluidic method

( [SM]3FS, for spatially multiplexed, single-molecule, simultaneous
microfluidic force spectroscopy) capable of quantifying force-dependent
bond lifetimes for many DNA sequence variants in parallel. Initial results
establish that [SM]3FS can directly quantify force-extension curves for
many molecules comprised of as many as 16 different sequence variants
with a spatial resolution of 10 nm. To demonstrate the power of the
technique, we applied it to systematically investigate the sequence-
dependence of catch vs. slip bond formation in > 50 designed double-
stranded DNA duplexes. These results reveal how simple molecular
architectures can encode complex mechanical responses to force. In future
work, we anticipate that the platform can be used to engineer novel DNA-
based molecular force sensors and elucidate catch-bond formation
mechanisms in more complex macromolecular systems.
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IN SITU FORCE MAPPING TO QUANTIFY AND VISUALIZE
MECHANICAL COMMUNICATION IN GENOMES OF LIVING CELLS
Maria Mukhina1, Yulia Gromova2, Auston Butterfield3, Haley Young3,

Connor McCormick3, Raymond Schaak3, Nancy Kleckner2
1
University of Maryland, Physics, College Park, MD, 2Harvard University,
Molecular and Cellular Biology, Cambridge, MA, 3The Pennsylvania State
University, Chemistry, University Park, PA
We are interested in how the production, sensing, and transduction of

mechanical forces at the molecular level relate to the global mechanics of
chromosomes. To answer this question, we are developing new approaches
to quantify and visualize intracellular forces with nanoscale precision, over
micron-scale distances, and in their physiological context. Our work aims to
apply nanotechnologies to measure forces acting within and among
chromosomes, with a focus on chromosome-wide mechanical patterning
and its contributions to genome function.
In this talk, I will discuss two nanosensors, which enable quantitative

luminescent imaging of mechanical effects in situ. The first sensor utilizes
nanoscale excitation of mechanoluminescence, i.e., emission of light
triggered by mechanical deformation, in faulted ZnMnS nanorods. This
approach allows for a direct optical readout, in which repetitive mechanical
stress applied to the sensor is converted to a series of photon flashes. The
lowest threshold for mechanoluminescence in ZnMnS detected in our
experiments is only 0.233 MPa or 0.233 pN/nm2 which is in the range of
intracellular forces. Since no photoexcitation is needed, the technique is free
of autofluorescent background and phototoxicity. The second sensor
represents a dynamic DNA nanostructure, which changes the color of
photoluminescence in response to the applied force. This probe can register
mechanically induced changes in its immediate environment on the scale
just above the thermal bath (0.23-0.36±0.01 pN applied over ~18 nm).
When attached to the chromosomes, these sensors report an (F,x,y,z,t) map
of intracellular forces. I will discuss the application of the sensors to
elucidate how cells synchronize and orchestrate global changes to ensure
accurate segregation of chromosome copies in preparation for cell division.
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SYSTEMATIC NASCENT RNA KINETICS IMAGING UNVEILS
PRINCIPLES FOR GENETIC INFORMATION FLOW IN REAL-TIME
Yihan Wan
Westlake University, School of Life Sciences, Hangzhou, China
Summary
Dynamic genetic information flow stands as the backbone of cellular

functionality. Real-time recording of transcription dynamics in live cells is
essential for understanding the principles of genetic information flow.
Studies using single-molecule live-cell imaging have revealed that
transcription is characterized by periods of bursting activity and sporadic
behavior. However, a comprehensive picture of real-time transcriptome
activity remains elusive. Here, we developed a novel technology for
Genome-wide Imaging of Nascent RNA Kinetics. This new approach
combines single-molecule nascent RNA imaging, gene barcoding, in situ
sequencing and computer vision to capture real-time transcription dynamics
in tens of thousands of cells. By measuring gene-expression dynamics of
127 genes, we were able to dissect the intricate properties and regulatory
mechanisms of transcription dynamics. Our findings underscore the crucial
roles of cis-regulatory elements, epigenetic modifications, and gene
neighborhood in orchestrating the temporal patterns of gene expression.
Notably, our approach achieves a combination of high temporal resolution
(100 seconds) and long-term recording (over 70 hours), enabling us to track
real-time transcription dynamics throughout the differentiation process of
human embryonic stem cells into neural lineages. This study sets the
stepping stone to reveal the genome-wide real-time information flow along
the central dogma within living cells.
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ILLUMINATING ORGANELLE DYNAMICS DURING
DIFFERENTIATION AND NEURODEGENERATION
Maria Clara Zanellati, Gregory Miner, Sarah Cohen
University of North Carolina, Cell Biology and Physiology, Chapel Hill,

NC
Emerging evidence indicates that organelle dynamics and interactions are

dysregulated in multiple types of neurodegenerative disease. For example,
many proteins implicated in Alzheimer’s disease, Parkinson’s disease, or
amyotrophic lateral sclerosis mediate or localize to membrane contact sites
at the interface between organelles. Organelle contacts implicated in these
neurodegenerative disorders include endoplasmic reticulum (ER)-
mitochondria, ER-endolysosome, and ER-lipid droplet (LD) contacts. I will
describe our work using live-cell multispectral imaging and dimerization-
dependent fluorescent probes to characterize the dynamics of organelle
contacts. We used multispectral imaging to visualize eight organelles –
plasma membrane, nuclei, ER, Golgi, lysosomes, mitochondria,
peroxisomes, and LDs – throughout differentiation of human induced
pluripotent stem cells (iPSCs) into cortical neurons. From these images, we
measured organelle size, shape, distribution, contacts with other organelles,
and speed (together termed morphodynamics). We found that organelle
morphology, contacts, and speed, all changed throughout differentiation.
Specifically, mitochondria-organelle contacts remodeled early during
neuronal differentiation, while peroxisome-organelle contacts remodeled
during synaptogenesis. These changes in organelle communication were
necessary for the synthesis of key lipids required for synapse formation. We
are currently testing the effects of mutations associated with Alzheimer’s
disease on organelle morphodynamics with the goal of identifying pathways
that are misregulated in neurodegenerative disease.
This work was supported by the National Institute of General Medical

Sciences of the National Institutes of Health under award number R35
GM133460, and by the Chan Zuckerberg Initiative under a
Neurodegeneration Challenge Network Collaborative Pairs Award.
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VIBRATIONAL PHOTOTHERMAL FLUORESCENCE IMAGING OF
CELLULAR ORGANELLES: BRIDGING THE BEST OF TWO
SEPARATE WORLDS.
Ji-Xin Cheng1, Meng wang2, Jianpeng Ao3, Jiaze Yin4

1
Boston University, BME, Boston, MA, 2Janelia Research Campus,
Ashburn, VA, 3Boston University, ECE, Boston, MA, 4Boston University,
ECE, Boston, MA
Cellular organelles are functional units of eukaryotic cells, each specialized

to perform specific tasks vital for cellular homeostasis. The metabolic
activities of these organelles are highly diverse, and their dysfunctions can
lead to tissue malfunction and diseases. Therefore, functional imaging of
cellular organelles is fundamental for molecule-based precision diagnosis
and treatment of diseases. While fluorescence microscopy enables
visualization of the spatial distribution and morphological dynamics of
specific organelles, it lacks chemical content information to elucidate their
functional heterogeneity. Advanced vibrational microscopy, including
coherent Raman scattering and mid-infrared photothermal techniques,
provides chemical content information but often lacks organelle specificity.
Here, we present a new platform called Vibrational Photothermal
Fluorescence (VIP-F) microscopy, designed to analyze the chemical content
of target organelles or subcellular structures in live cells. This technology
utilizes thermosensitive yet photostable fluorophores that label specific
subcellular structures, as well as act as reporters for generating vibrational
photothermal signals. We demonstrated that these signals can reveal the
chemical fingerprints of targeted organelles and their dynamic changes in
response to genetic perturbations in live organisms, as well as uncover the
structural properties of membraneless condensates in living cells.
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IMAGING THE LIFE CYCLE OF A SINGLE mRNA MOLECULE
Weihan Li1,2, Clemence Saint-Donat1,2,4, Louis Degroux4, Young J Yoon1,3,

Robert H Singer1,2
1
Albert Einstein College of Medicine, Department of Cell Biology, Bronx,
NY, 2Albert Einstein College of Medicine, Program in RNA Biology,
Bronx, NY, 3Albert Einstein College of Medicine, Dominick P. Purpura
Department of Neuroscience, Bronx, NY, 4Paris Cité University, Paris,
France
Imaging mRNA single molecules elucidates gene expression with high

spatial-temporal resolution. Tracking the mRNA’s trajectories reveals its
translational status and interaction partners. However, single-molecule
tracking has been limited to a few seconds due to the need for high-
frequency imaging, which causes photobleaching. This limitation has
hindered our ability to comprehensively capture longer events, such as the
complete life cycle of an mRNA.
To address this challenge, we developed a sparse labeling method that

controls the number of fluorescent mRNA molecules. In budding yeast, we
methodically optimized the system so that certain cells contained only one
fluorescent mRNA molecule, allowing for unambiguous tracking. This
approach extended the duration of single-molecule tracking from 4 seconds
to 20 minutes—a 300-fold increase. Using this method, we discovered
distinct dynamics in the localization of mRNA to mitochondria.
In conclusion, our advancement of long-term single-molecule tracking

allows us to capture cellular events across time scales ranging from
milliseconds to tens of minutes, offering new opportunities to uncover the
complex processes underlying gene regulation.
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TRANSLATION OF mRNAs ENCODING MEMBRANE PROTEINS
REGULATED BY LYSOSOMES AND LUNAPARK
Heejun Choi1, Ya-Cheng Liao2, Young J Yoon3, Jonathan Grimm1, Luke D

Lavis1, Robert H Singer3, Jennifer Lippincott-Schwartz1
1
HHMI, Janelia Research Campus, Ashburn, VA, 2Columbia University,
Biochemistry and Molecular Biophysics, New York, NY, 3Albert Einstein
College of Medicine, Neuroscience, Bronx, NY
The coordination between the translation of mRNAs encoding membrane

and lumenal proteins on the endoplasmic reticulum (ER) and the
positioning of intracellular organelles remains poorly understood, despite
the critical role of inter-organellar communication in proper protein folding
and transport. In this study, we employ live cell single-molecule imaging to
investigate mRNAs encoding lumenal and membrane proteins (referred to
as "secretome mRNAs"). Our findings reveal that the translation of these
mRNAs primarily occurs at ER junctions marked by the protein Lunapark
and in the vicinity of lysosomes. Depletion of Lunapark leads to a selective
reduction in the translation of secretome mRNAs, accompanied by a
simultaneous decrease in ribosome densities on secretome mRNAs located
near lysosomes. This effect depends on eIF2-dependent translation initiation
and can be reversed by the addition of the integrated stress response
inhibitor, ISRIB. Moreover, the translation of secretome mRNAs near
lysosomes is enhanced during external amino acid deprivation and inhibited
when lysosome pH is neutralized, suggesting a link between local protein
synthesis on the ER and local amino acid release by lysosomes. These
results support a novel regulatory mechanism in which lysosomes, along
with Lunapark, spatially pattern and regulate the translation of mRNAs
encoding secretory and membrane proteins.
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FOCUSING ON MITOCHONDRIA
Stefan Jakobs1,2,3
1
University Medical Center of Göttingen, High Resolution Microscopy,
Göttingen, Germany, 2Max Planck Institute for Multidisciplinary Sciences,
Mitochondrial Structure and Dynamics, Göttingen, Germany, 3Fraunhofer
Institute for Translational Medicine and Pharmacology ITMP, Translational
Neuroinflammation and Automated Microscopy, Göttingen, Germany
Mitochondria, known as the ‘powerhouses of the cell’, are double

membrane organelles that are essential for eukaryotic life. Their shapes
range from small oval fragments to interconnected networks of tubules that
are constantly moving, fusing and dividing. The innermost mitochondrial
compartment, the matrix, is enclosed by the highly convoluted inner
membrane, which in turn is surrounded by the outer membrane. The inner
membrane projects cristae into the matrix. A unique feature of mitochondria
is that they contain their own DNA (mtDNA), a gene-dense circular
molecule of about 16.6 kilobases. Each mtDNA molecule is compacted by
proteins into a nucleoid, with several hundred to a few thousand nucleoids
present per cell. Human mtDNA encodes 13 proteins, 2 ribosomal RNAs,
and 22 tRNAs, all of which are essential for respiration and eukaryotic life.
Due to their intracellular mobility, small size, and complex architecture,
mitochondria are notoriously challenging targets for for high-resolution
light microscopy. We utilize STED super-resolution microscopy, among
other techniques, to investigate the internal architecture of mitochondria.
We aim to understand how mitochondria develop and maintain the complex
folding of the inner membrane and how this relates to mitochondrial gene
expression. This talk will summarize our recent progress in using light
microscopy to study these intricate organelles.
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THE SPLICING FACTOR U2AF REGULATES THE TRANSLATION
AND LOCALIZATION OF NUCLEAR-ENCODED MITOCHONDRIAL
mRNAs
Gloria R Garcia, Murali Palangat, Ben T Donovan, Kathy McGraw, Josquin

Moraly, Naomi Taylor, Dan R Larson
Center for Cancer Research, National Cancer Institute, Bethesda, MD
Molecular targeting to mitochondria is crucial for understanding normal cellular

processes, yet it remains poorly characterized. In the cell, mitochondria are key
regulators of energy production, metabolic pathways, and cell fate decisions.
Over 95% of mitochondrial proteins are encoded by nuclear genes and
translated by cytosolic ribosomes, making molecular targeting critical for
mitochondrial function. The RNA binding proteins U2AF1/2 form a
heterodimer (U2AF) that shuttles between the nucleus and cytoplasm,
regulating splicing in the nucleus and translation in the cytoplasm. Our study
identifies an unexpected role for U2AF in mitochondrial function. Using a
combination of single-molecule imaging (TIRF microscopy, with DNA-paint
labeling) and IP-mass-spec, we demonstrate that U2AF localizes to the outer
mitochondrial membrane and interacts with nuclear-transcribed mitochondrial
proteins in the cytoplasm. PAR-CLIP analysis demonstrated that cytoplasmic
U2AF binds approximately 20% of all mitochondrial mRNAs, a proportion
higher than expected by chance. U2AF primarily binds to coding regions, with a
preference for the 5’ end of the message near the AUG start codon, a region
critical for targeting nascent proteins via the mitochondrial targeting sequence.
In vitro reporter assays using a mitochondrial model gene (MCL1) indicated
that U2AF acts as a translational repressor in a sequence- and position-
dependent manner. Additionally, polysome profiling followed by Western
blotting showed that U2AF1 is predominantly found in non-translating
fractions. To test if U2AF-induced changes in translation affect targeting to
mitochondria, we analyzed the spatial localization of nuclear-encoded
mitochondrial mRNAs relative to mitochondria using two-color smFISH. The
results show a decrease in mRNA targeting to mitochondria when U2AF-
mRNA interactions are perturbed, consistent with our model that U2AF1-
mediated translational regulation facilitates efficient mRNA targeting to
mitochondria. Moreover, an oncogenic point mutation in U2AF1 disrupts this
regulation, leading to altered mitochondrial structure, decreased mitochondrial
function, and increased cytoplasmic translation. This mutation also causes
OXPHOS-dependent metabolic remodeling. These disruptions recapitulate the
phenotypes observed in bone marrow stem and progenitor cells isolated from
patients with myelodysplastic syndromes (MDS). MDS is a clonal stem cell
disorder characterized by bone marrow failure, resulting in dysplasia and
peripheral cytopenias. Our findings reveal a novel cytoplasmic role for U2AF in
regulating nuclear-mitochondrial crosstalk, where U2AF regulates the
translation and targeting of nuclear-encoded mitochondrial mRNAs to
mitochondria and is required for proper mitochondrial physiology. Furthermore,
our results suggest that disruptions in U2AF-mediated translational regulation
and mitochondrial functions contribute to MDS pathogenesis, providing new
insights into the molecular mechanisms underlying this disease.
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DIRECT VISUALIZATION OF RETROTRANSLOCATION BY THE
HRD1 COMPLEX BY HIGH SPEED SINGLE MOLECULE TRACKING
IN LIVING CELLS:
Tim Abel1, Sonya Neal2, Thomas Sommer3, Jennifer Lippincott-Schwartz4,

Christopher Obara1
1
University of California, San Diego, Departments of Pharmacology,
Chemistry & Biochemistry, La Jolla, CA, 2University of California, San
Diego, Department of Cell and Developmental Biology, La Jolla, CA,
3
Max-Dellbrück-Center, Department for Cancer, Berlin, Germany, 4Janelia
Research Campus, Howard Hughes Medical Institute, Ashburn, VA
Terminally misfolded or otherwise damaged proteins of the Endoplasmic

Reticulum (ER) are degraded by the proteasome in a process termed
Endoplasmic Reticulum Associated Degradation (ERAD). The Hrd1
complex is a multicomponent transmembrane protein complex that
mediates ubiquitination and export of proteins from the ER to be degraded
in the cytosol. Despite substantial effort in the past two decades, the
biochemical characterization of its architecture and mechanism produced
inconsistent and even contradictory results, yielding no consensus on how it
mediates protein transport. Its elusive nature is representative of the
limitations of classical biochemical approaches, whose often harsh
experimental conditions directly interfere with the objects they study.
In this project I used fluorescence multi-color single molecule microscopy

to offer a new perspective on the architecture, formation and dynamics of
the Hrd1 complex. In this process I developed cell biological, experimental
and analytical tools to robustly quantify and characterize Hrd1
oligomerization in vivo. Combining live-cell dual-color single-particle
tracking with chemical inhibition, downregulation of complex components
and a novel, binding-competition based tracking assay, I demonstrated that
Hrd1 forms a stable homo-tetramer via its cytosolic domain Hrd1480-529.
By structural modeling via AlphaFold, results of which were validated with
both single-particle tracking and recombinant protein expression, I showed
that this domain assembles into a canonical coiled-coil domain
independently of other complex components or Hrd1's activity.
While yielding specific novel biological insight into Hrd1 complex

formation, it also serves as a general blueprint on how dual-color single
particle tracking can be used to address questions that bring classical
biochemistry to its limits.
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EXPLORING STRUCTURE AND FUNCTION OF NON-CANONICAL
SMC PROTEIN SMCHD1 WITH HIGH-SPEED AFM AND OPTICAL
TWEEZERS
Ting-Wei Liao1,2, Fahad Rashid3, Sushil Pangeni1,2, James M Berger3,

Taekjip Ha1,2,4,5
1
Johns Hopkins University, Department of Biophysics, Baltimore, MD,
2
Boston Children’s Hospital, Program in Cellular and Molecular Medicine,
Boston, MA, 3Johns Hopkins School of Medicine, Department of
Biophysics and Biophysical Chemistry, Baltimore, MD, 4Johns Hopkins
School of Medicine, Department of Biomedical Engineering, Baltimore,
MD, 5Howard Hughes Medical Institute, Baltimore, MD
Structural maintenance of chromosomes flexible hinge domain containing 1

(SMCHD1) is an SMC family protein involved in epigenetic repression and
X-chromosome inactivation. Genetic mutations in SMCHD1 are linked to
diseases such as facioscapulohumeral muscular dystrophy (FSHD) and
Bosma arhinia microphthalmia syndrome (BAMS). Much of our current
understanding of SMCHD1 function comes from genetic studies. However,
structural analysis indicates that SMCHD1 possesses non-canonical
features, differing from traditional SMCs. These differences include
forming a homodimer through hinge domain, having a flexible linker, and
containing GHKL ATPase domains, suggesting it likely operates through
distinct molecular mechanisms. Recent structural studies of truncated
SMCHD1, focusing on the hinge and ATPase domains, provide insights
into how SMC proteins may bind to DNA. However, despite these efforts,
the molecular mechanisms by which SMCHD1 is involved in chromatin
organization remain unknown, primarily because these snapshots only
capture the static states of the processes. A significant bottleneck in
understanding its functional modes has been the challenges in purifying the
full-length protein and characterizing its active functional dynamics with
DNA substrates. In this work, we combine biochemistry, high-speed atomic
force microscopy, and correlative optical trapping to study full-length
SMCHD1. We analyze the structural conformations of SMCHD1 and its
interactions with ssDNA and dsDNA substrates. SMCHD1 primarily
functions as a dimer and can diffuse on both ssDNA and dsDNA.
Furthermore, after binding to dsDNA, SMCHD1 can contact either cis or
trans DNA to initiate DNA bridging. These observations suggest how
SMCHD1 is involved in chromosome maintenance and provide insights
into how mutations affect these chromosomal functions.
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CHARTING LIVE CELL 4D CHROMATIN LANDSCAPES:
UNRAVELING NEURONAL DIFFERENTIATION VIA CHROMATIN
IMAGING
Kirby Campbell1, Marybeth Lupo2, Pratik Kumar3, Luke Lavis3, Michael

Dyer2, David Solecki1
St. Jude Children’s Research Hospital, Neuronal Cell Biology Division,

1
Department of Developmental Neurobiology, Memphis, TN, 2St. Jude

Children’s Research Hospital, Department of Developmental Neurobiology,
Memphis, TN, 3Howard Hughes Medical Institute Janelia Research
Campus, Ashburn, VA
Neuronal differentiation is characterized by nuclear condensation and

chromatin compaction, a process essential for newborn neurons to provision
when and where genes are expressed to support their maturation and
incorporation into developing neuronal circuits. However, the mechanisms
through which neuronal progenitor nuclei attain this unique chromatin state
and its implications for gene regulation remain inadequately explored.
Traditional transcription control methodologies, relying on reductionist
molecular biology, sequencing, and biochemistry, often fail to capture the
4D genome organization within live cells adequately. We have developed
innovative imaging probes based on cMAPs to address these limitations,
leveraging advanced microscopy and live-cell imaging techniques. These
probes utilize defined reader domains to intersectionally recognize specific
epigenetic marks and label histones within living cells, thereby enabling the
visualization of chromatin dynamics in real-time. Our application of these
innovative probes to neuronal differentiation has revealed novel insights.
We discovered that bivalent chromatin loss underlies a reduction of
euchromatin during granule neuron precursor (GNP) nuclear condensation,
a finding that surpasses traditional antibody-based methods by providing a
multifaceted view of histone modifications. We've expanded the range of
cMAP probes beyond bivalent chromatin by employing synthetic biology,
creating over 20 new variants targeting transcriptional start sites, enhancers,
gene bodies, and repressed chromatin. These probes illuminate novel
chromatin design principles like variegation of active chromatin marks,
lamination of repressed and active chromatin, and the dynamic movement
of chromatin domains, which significantly enhance our understanding of
chromatin organization in neuronal differentiation, inspiring new avenues of
research and discovery. We've also developed these innovative probes
beyond imaging, transforming them into drug delivery systems through
Halotag technology. This approach has enabled targeted manipulation of
chromatin remodelers, such as converting repressed chromatin to active
chromatin by adding a halo-tag ligand with an appended epigenetic drug.
Our work advances our understanding of neuronal differentiation at the
molecular level and opens new avenues for manipulating chromatin in
neural development, demonstrating the profound impact of integrating
synthetic biology with chromatin research
< Back 18
HARNESSING AI-DRIVEN PROTEIN DESIGN TO RAPIDLY
GENERATE FUNCTIONAL INTRABODIES FOR TRACKING GENE
REGULATION IN LIVING CELLS
Timothy J Stasevich1, Gabriel Galindo1, Daiki Maejima2, Jacob DeRoo3,

Scott Burlingham1, Gretchen Fixen1, Ning Zhao4, Christopher D Snow3,5,
Brian J Geiss6, Yuko Sato2, Hiroshi Kimura2
1
Colorado State University, Department of Biochemistry and Molecular
Biology, Fort Collins, CO, 2Tokyo Institute of Technology, Cell Biology
Center, Institute of Innovative Research, Yokohama, Japan, 3Colorado State
University, Department of Chemical and Biological Engineering, Fort
Collins, CO, 4University of Colorado, Anshchutz Medical Campus,
Department of Biochemistry and Molecular Biology, Aurora, CO,
5
Colorado State University, School of Biomedical Engineering, Fort
Collins, CO, 6Colorado State University, Department of Microbiology,
Immunology, and Pathology, Fort Collins, CO
My lab is developing technology to image gene regulation with single-

molecule precision in living cells. In this talk, I’ll discuss our ongoing
efforts to image the dynamics of post-translational modifications (PTMs) in
the context of single-gene activity and single-nucleosome movement [1,2].
Given the transient and chemical nature of PTMs, imaging them in living
cells has necessitated the use of PTM-specific intrabodies that dynamically
bind to and light them up. A long-term challenge has been that most natural
antibody sequences (>90%) are unsuitable for intrabody formats, often
encountering solubility issues and misfolding in the reducing environment
of living cells. To overcome this problem, we have combined AlphaFold
with Protein Message Passing Neural Network (ProteinMPNN) to redesign
antibody scaffold sequences for improved solubility when encoded in a
single chain variable fragment format. Testing in live cells reveals the
majority of designs work (>60%). Using this approach, we now have
functional intrabodies that can be encoded on plasmids and that bind and
light up a panel of histone modifications, including H3K4me2, H3K4me3,
H3S10ph, H3K27ac, H3K14ac, H3K36me3, H4K12ac, and H4K12ac. We
believe this approach will facilitate the rapid production and more
widespread use of intrabodies for diverse live-cell applications.
[1] Forero et al., Nature Communications, 12 (1), (2021).

[2] Saxton et al., Science Advances, in press (2023).
< Back 19
RAPID TRANSCRIPTION REPRESSION INDUCED BY DNA
DOUBLE-STRAND BREAK
Shuaixin He, Bin Wu
Johns Hopkins University School of Medicine, Department of Biophysics

and Biophysical Chemistry, Baltimore, MD
DNA double-strand break (DSB) jeopardizes genome integrity and

endangers cell viability. Actively transcribed genes are especially important
for cellular function and need to be reliably repaired if broken.
Transcription is repressed at the broken gene to prevent the accumulation of
truncated transcripts and prepare the chromatin for repair. However, it
remains elusive how fast the repression is initiated and how far it influences
the neighboring genes on the chromosome. We adopted a recently
developed very fast CRISPR (vfCRISPR) to generate DSB at a specific
genomic locus with precise timing. We employed MS2 technology to
monitor transcription in live cells and used chromatin immunoprecipitation
(ChIP) to measure the RNA polymerase II (RNAP2) occupancy on the cut-
and neighboring genes. We measured the dynamics of DSB-induced
transcription repression and investigated its molecular mechanism. We
observed that a single DSB repressed transcription of the damaged gene in
minutes, which coincided with the recruitment of damage repair protein
53BP1. Transcription repression propagated bi-directionally along the
chromosome from DSB for hundreds of kilo-bases in an attenuated manner.
Different from what the literature suggested, transcription repression
induced by a single DSB did not depend on PRC1-mediated H2A K119
mono ubiquitination (H2AK119ub). Instead, proteasome was evoked to
remove elongating RNAP2. Repression of neighboring genes was
influenced by the propagation of repair signals and DNA looping complex
cohesin. Taken together, our kinetic analysis revealed the rapid transcription
repression around a single DSB and uncovered the repression creation and
propagation mechanism, which is essential for repairing broken DNAs and
maintaining genome integrity.
< Back 20
KINESIN-14 HSET AND KlpA ARE NON-PROCESSIVE
MICROTUBULE MOTORS WITH LOAD-DEPENDENT POWER
STROKES.
Xinglei Liu1, Lu Rao1, Florian Berger2, Weihong Qiu3, Arne Gennerich1

1
Albert Einstein College of Medicine, Biochemistry, New York, NY,
2
Utrecht University, Biology, Utrecht, Netherlands, 3Oregon State
University, Biochemistry & Biophysics, and Physics, Corvallis, OR
Accurate chromosome segregation during cell division relies on coordinated

actions of microtubule (MT)-based motor proteins in the mitotic spindle.
Kinesin-14 motors play vital roles in spindle assembly and maintenance by
crosslinking antiparallel MTs at the spindle midzone and anchoring spindle
MTs’ minus ends at the poles. We investigate the force generation and
motility of the Kinesin-14 motors HSET and KlpA, revealing that both
motors function as non-processive motors under load, producing single ~25
nm power strokes per MT encounter. Each homodimeric motor generates
forces of ~0.5 pN, but when assembled in teams, they cooperate to generate
forces of 1 pN or more. Importantly, cooperative activity among multiple
motors leads to increased MT-sliding velocities. Our findings quantitatively
elucidate the structure-function relationship of Kinesin-14 motors and
underscore the significance of cooperative behavior in their cellular
functions.
< Back 21
SINGLE-MOLECULE IMAGING OF THE ENDOGENOUS CAMK2A
mRNA REVEALS ACTIVITY-DEPENDENT LOCAL REGULATION
OF THE mRNA AT DENDRITIC SPINES
Dong-Woo Hwang1, Sulagna Das2, Robert H Singer1

1
Albert Einstein College of Medicine, Cell Biology, Bronx, NY, 2Emory
University School of Medicine, Cell Biology and Human Genetics, Atlanta,
GA
Background: Calcium/calmodulin-dependent protein kinase type II

(CaMKII) integrates a transient Ca2+ signal into long-term synaptic
plasticity and structural changes at the excitatory postsynaptic
compartments – dendritic spines. Among the subunits that make up the
CaMKII holoenzyme, CaMKIIα has been shown to be critical for the
maintenance of long-lasting synaptic changes at dendritic spines. Notably,
CaMKIIα’s persistent abundance in an individual spine during the
maintenance period renders it a key molecule that facilitates perpetuation of
synaptic transmission, triggered by an otherwise fleeting Ca 2+ signal.
However, it has yet to be understood how CaMKIIα becomes persistently
available even if proteins in spines are subject to dynamic exchange through
diffusion and protein turnover.
Aims: We hypothesized that the major CaMKIIα subunits become locally

supplied to individual dendritic spines via activity-dependent on-site protein
synthesis. We, therefore, set out to investigate whether the Camk2a mRNA,
encoding the CaMKIIα subunit, localize to the stimulated spines and
undergo rounds of on-site translation during synaptic changes.
Methods: We implemented single-molecule mRNA imaging approaches to

examine spatiotemporal regulation of the endogenous Camk2a mRNAs at
individual dendritic spines in dissociated mouse hippocampal neurons.
Results: By single tracking a fluorescently tagged single mRNA, we found

a rapid activity-dependent localization of the endogenous Camk2a mRNAs
to stimulated dendritic spines (~1 minute). The spine-localized mRNAs
underwent on-site protein synthesis, which persisted over extended periods
(~30 minutes). The interplay between the cis regulatory elements in the 3’
UTR of the Camk2a mRNA and the cognate RNA-binding Cpeb proteins
(1,2,3 or 4) was required for the spine localization.
Conclusion: These findings suggest that activity-dependent spine

localization and local protein synthesis of Camk2a mRNA may serve to tag
an individual dendritic spine by supplying a pool of CaMKIIα in situ during
the maintenance of long-lasting synaptic changes.
< Back 22
YAP SIGNAL INTEGRATION THROUGH CHARGE-MEDIATED
TRANSCRIPTIONAL CO-CONDENSATES
Kirstin Meyer1,2, Klaus Yserentant1, Rasmi Cheloor Kovilakam1, Kiersten

Ruff3, Bo Huang1, Orion Weiner1
1
University of California San Francisco, San Francisco, Department of
Biochemistry and Biophysics, San Francisco, CA, 2Yale University,
Department of Molecular, Cellular, and Developmental Biology, New
Haven, CT, 3Washington University in St. Louis, James McKelvey School
of Engineering, St. Louis, MO
YAP is a transcriptional regulator that forms condensates, an attractive

signaling module for emergent gene regulatory behavior. Yet, how YAP
signals are integrated through condensates to control transcriptional
dynamics has remained unclear. Leveraging light sheet single-molecule
imaging, synthetic condensates, and IDR sequence analysis we probed the
molecular basis of YAP condensate formation and function. We
demonstrate that YAP condensate formation is mediated through
electrostatics interactions with the oppositely charged transcriptional
regulator Mediator. However, protein engineering demonstrates that,
instead of the net charge, the interaction is mediated through YAP’s charge
pattern that specifically interacts with the opposing charge pattern of
Mediator. The co-condensation of both proteins drives the transcriptional
activation but is counteracted by delayed negative feedback from the
transcriptional output, generating an adaptive transcriptional response. Our
work unravels a molecular mechanism for YAP condensate formation and
provides a new framework for their role in emergent gene regulatory
behavior.
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SINGLE-MOLECULE OBSERVATION OF HEDGEHOG DIFFUSION,
AND A NEW MODEL FOR MORPHOGEN MOVEMENT IN
CROWDED TISSUES
Gavin Schlissel1,2, Pulin Li1,2

1
Whitehead Institute, Cambridge, MA, 2MIT, Biology, Cambridge, MA
Developing animals use a small number of evolutionarily conserved

morphogens to pattern tissues of varying sizes, however it is not clear how
evolution can modulate the effective signaling range of a morphogen. This
problem is particularly acute in Hedgehog signaling, where there is deep
disagreement about whether the poorly soluble Hedgehog morphogen is
capable of extracellular diffusion. Using single-molecule microscopy in live
cells, we found that Hedgehog family morphogens diffused extracellularly
in four distinct states with diffusion rates ranging from near 0µm2/s to
~20µm2/s, corresponding to distinct biochemical complexes. Individual
Hedgehog molecules dynamically transitioned between states, and the
evolutionarily novel protein SCUBE accelerated Hedgehog diffusion by
catalytically accelerating the transition rate between each state. Based on
our single-molecule observations of Hedgehog dynamics, we propose a new
model for morphogen diffusion, in which a morphogen molecule either
diffuses laterally in association with a cell, or transiently becomes free to
“jump” between adjacent cells. Under this model, regulating a morphogen’s
“jump” probability is sufficient to regulate its effective diffusion rate, and a
tissue’s topological organization is a critical determinant of signaling
outcomes. Our work spans a wide range of spatial and temporal scales,
connecting single-molecule biophysical measurements to long-range pattern
formation over hours or days. Through this work, we resolved a decades-old
dispute about the mechanism of Hedgehog movement through tissues, and
revealed unexpected knobs that nature can use to tune morphogen gradient
sizes across tissues or organisms. I will discuss our multiscale
understanding of Hedgehog diffusion, and our efforts to extend this
framework to understand unrelated signaling proteins, including
developmental morphogens, immunological cytokines, and fully synthetic
signaling agonists.
< Back 24
CAPTURING THE TRANSCRIPTIONAL PATTERNING OF β-ACTIN
IN THE MOUSE SKIN BY LIVE IMAGING
Han Yang, Zhongqi Lin, Valentina Greco
Yale University, Department of Genetics, New Haven, CT
β-actin is an essential component of the cytoskeleton and involved in

cellular processes including migration, proliferation, chromatin remodeling
and gene regulation, where its mRNA localization and local translation has
been shown to be important for cell mobility and polarity. While most
studies on the function and transcriptional regulation of β-actin have been
performed at the single cell level, our understanding of how its expression is
regulated and locally tuned to fuel tissue building in a living organism is
still limited. Leveraging the power of tracking mRNAs in vivo, this study is
one of its first to investigate the spatiotemporal organization and regulation
of β-actin transcription in the skin of a live mouse. Specifically, we
examine the transcriptional patterning of β-actin in the developmental tissue
context, where different cell types exhibit diverse cycling and mobility
characteristics. Ultimately, this study aims to dissect the regulatory
principles underlying the establishment and maintenance of tissue
architecture fueled by β-actin. Our initial characterization has found distinct
transcriptional activity of β-actin across different cell types (epithelial cells,
fibroblasts and endothelial cells), spatial and temporal axes, and tissue
states. Our preliminary results showed that this housekeeping gene was not
transcribed by all cells at all times but could be induced to an almost
ubiquitous coverage under perturbation. Efforts are now underway to
understand the underpinning mechanisms and functional significance of this
dynamic transcriptional patterning. These findings shed light on how
transcription is controlled and coordinated in the tissue and how different
cell types integrate the cellular and molecular cues from other cells
surrounding them to execute a proper regulatory program.
< Back 25
PHOSPHORYLATION OF FMRP GRANULES ALTERS THEIR
TRANSPORT, PROTEIN SYNTHESIS AND PHASE SEPARATION
Young J Yoon1,2, Shivani C Kharod1, Dongwoo Hwang1,2, Heejun Choi3,

Pablo E Castillo1, Robert H Singer1,2
1
Albert Einstein College of Medicine, Neuroscience, Bronx, NY, 2Albert
Einstein College of Medicine, Cell Biology, Bronx, NY, 3Janelia Research
Campus, Ashburn, VA
Fragile X messenger ribonucleoprotein (FMRP) is an RNA-binding protein

involved in mRNA transport and protein synthesis with links to autism.
Expression of fluorescently-tagged FMRP leads to formation of punctate
granular structures that coincide with ribosomes and move bidirectionally
along dendrites and axons. Here, we show that phosphorylation on serine
499 (S499) can lead to differences in FMRP puncta size, intensity, contrast,
and transport between phospho-deficient (S499A) and phospho-mimic
(S499D) mutant FMRP granules. Next, to determine whether FMRP
granules are liquid-liquid phase separated (LLPS) structures, we performed
fluorescence recovery after photobleaching to determine exchange of FMRP
within granules. We found that recovery was relatively slow and required
translation for FMRP exchange on granules. Intriguingly, the constitutively
phosphorylated S499D mutant displayed rapid recovery in the presence of a
translation inhibitor, cycloheximide, suggesting that phospho-FMRP
exchange is increased when ribosomes are completely stalled. To better
understand FMRP granules, we utilized a patient-derived FMRP I304N
mutant that was incapable of binding ribosomes. When we fused a CRY2
light-inducible clustering domain to FMRP I304N, we observed that it
associated with preexisting FMRP granules rather than form independent
granules or condensates. These results suggest that FMRP granules may
behave like liquid droplets in dendrites and we are investigating how phase
separation may regulate local protein synthesis in neurons. Thus, the
phospho-state of FMRP can alter the structure or phase of individual
granules leading to changes in transport and translation as a means to
regulate local protein synthesis.
< Back 26
NANOSCALE VOLUMETRIC FLUORESCENCE IMAGING VIA
PHOTOCHEMICAL SECTIONING
Wei Wang, Xiongtao Ruan, Gaoxiang Liu, Daniel E Milkie, Wenping Li,
Eric Betzig, Ruixuan Gao*, Srigokul Upadhyayula*
University of California, Berkeley, Berkeley, CA
†These authors contributed equally to this work.

*Corresponding authors. Email: gaor@uic.edu (R.G.); sup@berkeley.edu
(S.U.)
Building on our previous work on expansion lattice light-sheet microscopy

(Gao et al., Science, 2019; DOI: 10.1126/science.aau8302), we address a
key limitation of current optical microscopy methods that drastically
restricts the size and shape of intact specimens that can be imaged at
nanoscale resolution. We developed “photochemical sectioning”, a spatially
precise, light-based sample sectioning capability. By combining
photochemical sectioning with serial on-block volumetric fluorescence
imaging, our approach enables super-resolution imaging of whole-mount
specimens at petabyte scale and beyond. We demonstrated this technology
by imaging and reconstructing axons and myelin sheaths across two
complete adult mouse olfactory bulbs at sub-diffraction-limited resolution
in all three dimensions via sequential volumetric lattice light-sheet imaging
and photochemical sectioning. This work revealed unique axonal
degeneration and de-/dysmyelination patterns across the neurodegenerative
mouse olfactory bulb at single axon resolution. Our method, when
combined with large expansion factors and pan-membrane labeling, may
provide a viable alternative to existing volume imaging methods like
ssTEM and FIB-SEM, while offering the added advantage of multispectral
molecular contrast for specific biomolecules. Additionally, our approach
can be valuable for large-scale connectomics, as well as stereotypy and
disease perturbation studies at the whole mammalian brain level.
Reference:
https://www.biorxiv.org/content/10.1101/2024.08.01.605857v1
< Back 27
TRANSCRIPTION FACTOR EXCHANGE ENABLES PROLONGED
TRANSCRIPTIONAL BURSTS
Wim Pomp, Joseph V Meeussen, Tineke L Lenstra
The Netherlands Cancer Institute, Oncode Institute, Division of Gene

Regulation, Amsterdam, Netherlands
Single-molecule imaging inside living cells has revealed that transcription

factors (TFs) bind to DNA transiently, but a long-standing question is how
this transient binding is related to transcription activation. Here, we devised
a microscopy method to simultaneously measure transient TF binding at a
single locus and the effect of these binding events on transcription. We
show that DNA binding of the yeast TF Gal4 activates transcription of a
target gene within a few seconds, with at least ~20% efficiency and with a
high initiation rate of ~1 RNA/s. Gal4 DNA dissociation decreases
transcription rapidly. Moreover, at a gene with multiple binding sites,
individual Gal4 molecules only rarely stay bound throughout the entire
burst, but instead frequently exchange during a burst to increase the
transcriptional burst duration. Our results suggest a mechanism for enhancer
regulation in more complex eukaryotes, where TF cooperativity and
exchange enable robust and responsive transcription regulation.
< Back 28
STUDYING THE DYNAMIC REGULATION OF TRANSCRIPTION
INITIATION AND ELONGATION USING LIVE-CELL SINGLE
MOLECULE IMAGING
Nayem Haque1, Yessenia Cedeno1, John Hobbs1, Robert H Singer1,2, Ulrich

Steidl1, Robert A Coleman1
1
Albert Einstein College of Medicine, Department of Cell Biology, Bronx,
NY, 2Howard Hughes Medical Institute, Janelia Research Campus,
Ashburn, VA
Transcription initiation and early elongation events are intensely regulated

to primarily keep genes in a default off state. This is evidenced by live-cell
imaging studies showing that approximately 1% of Pol II that is loaded onto
the genome ever escapes into gene bodies. Global levels of transcription
become further restricted upon DNA damage or the stress-response. We
previously found that p53 could bind and initiate structural changes in Pol II
in a manner similar to factors that regulate transcription elongation. In vitro
biochemical experiments with highly purified factors revealed that p53
could directly regulate Pol II transcription elongation. To better determine
the significance of p53’s impact on Pol II’s function in cells, we have now
developed a live-cell single molecule method called GEANIS that monitors
changes in Pol II’s movement on the genome.
Using GEANIS, we have found that Pol II’s movement on the genome is
highly dependent on its residence time. These results suggest that we have
defined a new metric to monitor the presence of different functional states
of Pol II or interactions with co-regulatory complexes linked to the stability
of Pol II on a promoter. Furthermore, the expression of p53 alters Pol II’s
movement in a manner analogous to stalled or paused Pol II suggesting that
p53 may have the ability to regulate Pol II elongation in vivo. We also find
that Pol II’s movement temporally fluctuates during a binding trajectory
which is altered in the presence of inhibitors of initiation and elongation.
We will discuss these and additional findings indicating that GEANIS can
be generally applied to discern different time-dependent functional states of
many nuclear factors that engage chromatin in live cells.
< Back 29
GENE-SPECIFIC HUB KINETICS REGULATE TRANSCRIPTION
FACTOR OCCUPANCY
Samantha Fallacaro1,2, Apratim Mukherjee1,2, Puttachai Ratchasanmuang2,3,

Joseph Zinski1,2, Kareena Shankta1,2, Mustafa Mir1,2,3
1
University of Pennsylvania, Cell and Developmental Biology,
Philadelphia, PA, 2Children’s Hospital of Philadelphia, Center for
Computational and Genomic Medicine, Philadelphia, PA, 3Howard Hughes
Medical Institute, Children's Hospital of Pennsylvania, Philadelphia, PA
Transcription factors (TFs) robustly occupy their target genes to regulate

gene expression, despite having very short residence times on chromatin (1-
10s of seconds). This high occupancy is driven by an increase in the
frequency of binding events that counteracts the short individual residence
times. This increased binding frequency is driven by the formation of high-
local concentration clusters, or hubs, that concentrate factors around
genomic targets. How distinct types of TF hubs are directed to form at
genes and how their biophysical properties vary between target sites to
drive different levels of TF occupancy is poorly understood. Here, we use
sub-second resolution volumetric lattice light-sheet microscopy and single-
molecule tracking to quantify the molecular kinetics underlying gene-
specific hub formation of the key developmental TFs, Zelda and Dorsal, in
Drosophila embryos. To address how hubs are localized, we utilize a Zelda
mutant with perturbed recognition of its canonical motif. We find that
despite a decreased residence time, mutant Zelda still forms hubs, but they
are relocalized to different genomic sites that are enriched with motifs of a
co-binding partner. Our data suggest that hub localization is driven by a
combination of DNA motif recognition and a fine-tuned kinetic balance of
interactions between TFs and their co-binding partners. To address how
biophysical hub properties vary between target sites to generate differential
TF occupancy, we simultaneously visualized Dorsal hubs along with
nascent transcription of single target genes. We find that the stability,
frequency, and lifetime of TF hubs are finely tuned at different target genes.
Our data show that TF occupancy and transcriptional activity are
determined by the biophysical properties of hubs in a gene-specific manner.
Collectively, this work suggests that the physical mechanisms of hub
formation and their biophysical properties are dictated by local determinants
rather than global nucleus-wide phenomena.
< Back 30
EFFECTIVE IN VIVO BINDING ENERGY LANDSCAPE
ILLUSTRATES KINETIC STABILITY OF TF-DNA BINDING
Duyen Huynh1, Philipp Hoffmeister2, Tobias Friedrich3,4, Kefan Zhang1,

Marek Bartkuhn4, Francesca Ferrante3, Benedetto Daniele Giaimo3, Rhett
Kovall5, Tilman Borggrefe3, Franz Oswald2, J. Christof M Gebhardt1
1
Ulm University, Institute of Biophysics, Ulm, Germany, 2University
Medical Center Ulm, Clinic of Internal Medicine I, Ulm, Germany, 3Justus-
Liebig-Universitaet Giessen, Institute of Biochemistry, Giessen, Germany,
4
Justus-Liebig-Universitaet Giessen, Biomedical Informatics and Systems
Medicine, Giessen, Germany, 5University of Cincinnati College of
Medicine, Department of Molecular Genetics, Biochemistry and
Microbiology, Cincinnati, OH
Transcription factors (TFs) such as the central DNA-binding hub in Notch

signal transduction, RBPJ, bind to specific DNA sequences to regulate gene
transcription. How the efficiency of gene regulation depends on the TF-
DNA binding kinetics and cofactor interactions is mostly unknown. We
determined the DNA binding kinetics and the transcriptional activity of
RBPJ and several mutant variants by live-cell single-molecule tracking and
reporter assays, and measured their genome-wide chromatin occupation by
ChIP-Seq. We observed that cofactor binding, in addition to DNA binding,
was required for target site specificity. Importantly, the target site search
time of RBPJ was longer than its residence time, indicating kinetic rather
than thermodynamic binding stability. Impaired DNA binding, e.g. by
mutation K195E related to Adams-Oliver-Syndrome, modulated not only
dissociation, but also association to target sites. Impaired cofactor binding
mainly altered the rates of unspecific binding and target site association. For
other TFs, we also observed longer search than residence times, indicating
that kinetic rather than thermodynamic stability of DNA binding might be a
general feature of TFs in vivo. We propose that an effective in vivo binding
energy landscape of TF-DNA interactions constitutes an instructive
visualization of TF-DNA binding kinetics and the changes upon mutations.
< Back 31
LATTICE LIGHT SHEET MICROSCOPY – INNOVATIONS,
APPLICATIONS AND FUTURE DIRECTIONS
Wesley R Legant1,2
1
University of North Carolina - Chapel Hill, Biomedical Engineering,
Chapel Hill, NC, 2University of North Carolina - Chapel Hill,
Pharmacology, Chapel Hill, NC
Living specimens are both animate and three-dimensional. Lattice Light

Sheet Microscopy (LLSM) utilizes optically structured beams to perform
fast 3D imaging of dynamic processes in vivo with improved resolution and
beam uniformity. I will provide updates on our work to apply light sheet
microscopes together with single-molecule imaging to understand the
relationship between chromatin architecture and nuclear functions.
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MOLECULAR KINETICS OF TRANSCRIPTION REGULATION
DURING EMBRYONIC DEVELOPMENT
Mustafa Mir1,2,3
1
University of Pennsylvania, Department of Cell and Developmental
Biology, Philadelphia, PA, 2Children’s Hospital of Philadelphia, Center for
Computational and Genomic Medicine, Philadelphia, PA, 3Children’s
Hospital of Philadelphia, Howard Hughes Medical Institute, Philadelphia,
PA
During embryonic development gene expression patterns progressively

emerge as cell fates are determined and the embryo takes form. I will
discuss the application of high-resolution light-sheet microscopy, single-
molecule tracking, and new analysis approaches to quantify the molecular
scale interactions that orchestrate this patterning. I will show how the
spatial and temporal distributions of transcriptional regulators and
transcriptional machinery shape the nucleoplasm to regulate gene
expression during zygotic genomic activation. I will illustrate how
combining live imaging, genomics, and small molecule perturbations allows
us to elucidate how transcriptional regulators efficiently find and bind their
genomic targets. Finally, I will discuss how applying these integrated
approaches has changed our understanding of how the nucleoplasm is
functionally organized to achieve the robust patterns of transcription
necessary for proper embryonic development.
< Back 33
SINGLE MOLECULE TRACKING IN DRUG DISCOVERY AND
SYSTEMS BIOLOGY
Daniel J Anderson
Eikon Therapeutics, Research, Hayward, CA
At Eikon Therapeutics, we have industrialized a single molecule tracking

(SMT) platform to enable measurement of protein motions in up to 15
million cells per day. We have leveraged this platform to screen for drug
candidates against targets where cellular context matters and where there is
clear unmet need. SMT is a central driver of understanding molecule
potency and structure-activity relationships. Additionally, SMT is being
leveraged to better understand the cellular mechanisms and how small
molecule inhibitors affect targets.
WRN is a DNA helicase that is a target of interest in microsatellite instable

(MSI) tumors. Small molecule inhibitors of WRN are being developed,
however, the molecular consequence of WRN inhibition is poorly
understood. We have leveraged SMT and other molecular biology analyses
to uncover an MSI-specific mechanism of DNA trapping and protein
degradation.
More recently, we have begun conducting large-scale genetic SMT screens

to map protein interactions and signaling pathways. These screens provide
high-dimensional data sets that describe the interactions and functions of
target proteins within the cellular context. We are using this approach to
study key targets in cancer such as WRN to refine our understanding of key
oncogenic pathways.
< Back 34
MULTIMODAL SINGLE-MOLECULE IMAGING REVEALS THE
MOLECULAR MECHANISM OF CLASS-SWITCH RECOMBINATION
Mariia Mikhova1, Kefei Yu*2, Jens Schmidt*3

1
Michigan State University, Department of Biochemistry and Molecular
Biology, East Lansing, MI, 2Michigan State University, Department of
Microbiology, Genetics, and Immunology, East Lansing, MI, 3Michigan
State University, Department of Obstetrics, Gynecology, and Reproductive
Biology, East Lansing, MI
The adaptive immune system is a critical defense mechanism against a large

variety of pathogens, which requires diversification of both antigen
recognition and effector function of the antibodies produced by B cells.
Antibodies are composed of the immunoglobulin heavy chain (IgH) and
light chain proteins, and the diversification of their antigen binding variable
regions occurs via V(D)J recombination. Antibody effector function is
specified by the constant region of the immunoglobulin heavy chain. In the
IgH locus, the coding sequences for the distinct constant regions are
separated by switch regions. During class switch recombination (CSR),
DNA double strand breaks are induced in two switch regions, and DNA
repair results in the deletion of the intervening chromosomal sequence,
leading to a rearranged IgH locus.
Two key requirements for CSR are the active transcription of the switch
region to be targeted and the expression of the activation-induced
deaminase (AID). AID deaminates cytosines in the exposed single stranded
DNA of the switch regions, which generates uracils that trigger DNA repair
and ultimately lead to formation of DNA double strand breaks. Therefore,
AID is highly mutagenic, and its enzymatic activity must be tightly
controlled and restricted to the IgH locus. Importantly, the molecular
mechanisms underlying the recruitment of AID to the IgH locus are poorly
understood.
To analyze how AID localizes to the actively transcribed switch regions of
the IgH locus, we integrated a HaloTag in the endogenous AID locus and a
PP7 hairpin in a switch region of the IgH locus in the CH12 B-cell line.
This approach allows us to simultaneously detect single-molecules of AID
and mark the actively transcribed switch region in the nuclei of living B-
cells. Using this powerful approach, we have defined the recruitment
mechanism and binding kinetics of the AID to the IgH locus. Our results are
consistent with the model that AID is recruited to the IgH locus via binding
to the switch region RNA that is tethered to the IgH locus during the
transcription process. In addition, we demonstrate that RNA transcripts
containing the switch region accumulate in the nucleus of B-cells over time,
potentially serving as a sponge for AID to prevent non-specific targeting of
other chromosomal loci by AID.
In total, this work represents both a technological achievement and a
significant advancement in our mechanistic understanding of CSR, which is
critical for the adaptive immune response in mammals.
< Back 35
UNCOVERING THE COMPLEX MECHANISMS OF ANTISENSE
TRANSCRIPTION REGULATION AT THE XIST/TSIX LOCUS
THROUGH SINGLE-CELL STOCHASTICITY.
Benjamin K Kesler, John E Adams, Gregor Neuert
Vanderbilt University, Department of Molecular Physiology and

Biophysics, Nashville, TN
The process of X-chromosome inactivation (XCI) and its regulation by long

noncoding RNAs (lncRNAs) such as XIST and TSIX in mouse embryonic
stem cells provide a useful model for studying lncRNA regulation and gene
silencing. While various models have been proposed for how lncRNAs can
regulate their targets, they have not been directly compared to one another.
This study proposes a quantitative single-cell approach to investigate the
regulation of the Xist/Tsix locus using transcription and histone
modification analysis. By analyzing the natural stochastic transcription
variability of Xist and Tsix expression in over 14,000 individual embryonic
stem cells before and after two days of differentiation, the study
distinguishes between different mechanisms of regulation. The results show
that at high-levels of nascent transcription, transcriptional interference is
prevalent. At medium levels of transcription, polymerases transcribing Xist
and Tsix do not interfere with each other. Instead, individual polymerases
transcribe either Xist or Tsix, which coincides with enriched histone
methylation. This study shows that stochastic gene expression can lead to
diverse mechanisms of transcription regulation in a single cell population.
The methodology can be used to investigate other mRNAs and lncRNAs
and their effects on gene regulation and silencing.
< Back 36
SINGLE MOLECULE MICROSCOPY OF THE ASSEMBLY OF GIANT
PORE STRUCTURES ON MEMBRANES
Martin A Do, James C Walsh, Till Boecking, Conall Mc Guinness
University of New South Wales, Department of Molecular Medicine,

Sydney, Australia
Cholesterol dependent cytolysins (CDCs) are toxins released by gram-

positive bacteria that create giant aqueous pores in cell membranes. Pore
formation causes leakage of proteins and metabolites and ultimately cell
death. CDCs are major virulence factors in clinically relevant bacterial
infections such as gas gangrene and necrotizing fasciitis.
The pore formation pathway is an ancient, conserved and complex

mechanism. Soluble CDC monomers bind transiently to cholesterol present
in the cell membrane. Membrane- bound monomers can interact to
dimerize, irreversibly nucleating a stably growing arc-shaped oligomer that
ultimately closes to form a ring-shaped pre-pore complex consisting of 25-
50 subunits. The pre-pore complex undergoes conformational changes to
insert a giant beta barrel pore (25-50 nm diameter) into the membrane.
Recent studies suggest a different assembly pathway whereby pre-pore arcs
(<25 subunits) insert into the membrane to form a toroidal pore, which can
continue to add subunits and ultimately form a full ring. We hypothesize
that pore assembly and formation pathways are kinetically controlled.
However, most studies into the kinetics of CDC pore formation rely on bulk
ensemble signals that lack the ability to temporally resolve individual pore
formation steps and events.
To overcome limitations of other assays, we developed a total internal

reflection fluorescence (TIRF) microscopy assay to visualise assembly of
fluorescently labelled CDCs on virus-like particles (VLPs) immobilised on
glass coverslips. The VLPs are derived from the plasma membrane of
producer cells and encapsulate soluble GFP as a fluid phase marker. Our
TIRF assay visualises CDC oligomer nucleation and growth as the increase
of the CDC signal on individual VLPs. Pore opening leads to the release of
GFP from VLPs, which is detected by signal loss in the GFP channel. Using
our single molecule analysis pipeline, we can determine the number of CDC
subunits present on a single VLP throughout the assembly pathway and
measure the kinetics of monomer binding, nucleation, oligomerisation and
pore opening as a function of CDC concentration. Using this approach, we
compare the pore formation kinetics of the CDCs Streptolysin O (SLO) and
Perfringolysin O (PFO) to identify common pathways and specialised
mechanisms as well as characterise CDC behaviour on different model
membranes. Finally, by adding fluorescently labelled anti-SLO antibody to
our model system, we can identify which intermediates in the pore
assembly pathway are inhibited.
< Back 37
ACTIVATION OF ENGINEERED T CELLS IS SHAPED BY SINGLE
MOLECULE, SINGLE CELL ACTIVITIES
Kevin L Scrudders1, Suriya Selvarajan2, Philip S Low1, Shalini T Low-

Nam1,2
1
Purdue University, Chemistry, West Lafayette, IN, 2Purdue University,
Physics, West Lafayette, IN
T cells expressing engineered chimeric antigen receptors (CARs)

consolidate key components of T cell triggering machinery into a single
transmembrane receptor capable of scanning for specific pathogenic
markers. These programmable therapeutics are capable of identifying and
destroying target cells but have had limited success and often exhibit
toxicity from hyperactivation of the immune response. Normal T cells
generate global responses from the integration of signals from small
numbers of binding events, collected stochastically. Responses to weak,
physiological ligands are the result of spatiotemporal coordination of T cell
receptor (TCR) triggering events. By comparison, the mechanistic
determinants for threshold setting in CAR T cells are still poorly
understood. Signaling onset, at the cell surface, may be tuned by
spatiotemporal, topographic, mechanical, and chemical parameters. We map
CAR T cell inputs to cellular activation and cytotoxic responses using
single molecule, single cell in vitro reconstitution assays. We monitor the
collection of binding interactions that culminate in polarization of lytic
granules that are trafficked to the intercellular junction and may undergo
exocytosis to release cytolytic factors. We find that, surprisingly, CAR T
cells can mobilize cytotoxic responses to a small number of antigenic
binding events, suggesting a different molecular activation threshold than
previously appreciated. An in vitro cell:cell killing assay using low CAR T
cell:target cell ratios showed that small numbers of binding interactions by
CAR T cells are sufficient to induce target cell death. Additionally, CAR T
cells experiencing high environmental stiffness are most efficient in
generating killing outcomes. Taken together, our data argue that CAR T cell
activation is tuned by mechanochemical features of antigen binding at the
single molecule level and may suggest actionable insights to optimize these
therapies.
< Back 38
DIRECT OBSERVATION OF MOTOR PROTEIN STEPPING IN
LIVING CELLS USING MINFLUX
Jonas Ries
University of Vienna, Max Perutz Labs, Vienna, Austria
Super-resolution fluorescence microscopy is a powerful tool for structural

cell biology to reveal detailed spatial arrangements of biomolecules.
However, it has been a challenge to study dynamic structural changes of
protein machines in living cells due to their small size and fast dynamics.
Among all super-resolution techniques, MINFLUX utilizes the limited
photon budget of fluorescent markers most efficiently. It can achieve
nanometer resolution in living cells and was shown to reach a sub-
millisecond temporal resolution for tracking single fluorophores in vitro.
In this study, we focus on the motor protein kinesin-1, which is an
intracellular cargo transporter and exhibits processive motility along
microtubules. Driven by ATP hydrolysis, each motor domain moves with
16 nm steps in a hand-over-hand manner. Due to the small step size and fast
stepping rate, previous studies of kinesin stepping behavior have been
limited to in vitro work with purified proteins.
We optimized MINFLUX to track fluorescently labeled kinesin as it walks
on cellular microtubules. Using motor-PAINT in fixed cells, we could
precisely track the path of microtubules in 2D and 3D and reveal kinesin
side-stepping between neighboring protofilaments. In living cells, we were
able to clearly resolve the 16 nm steps of the kinesin motor domain at ~2
nm localization precision with sub-millisecond temporal resolution. This
allowed us to determine the precise step size and step time distributions
from thousands of steps.
I will conclude by discussing how dual-color MINFLUX co-tracking will
enable us to study conformational changes of proteins during their function
in the living cell and I will share new ideas on how to improve resolution,
speed, and robustness of future MINFLUX instruments.
Our results will facilitate further in vivo studies of precise motor protein
kinetics, enable the super-resolution mapping of complex microtubule
arrays, and pave the way towards monitoring functional conformational
changes of protein machines at high spatiotemporal resolution in living
systems.
< Back 39
OPTICAL SINGLE-MOLECULE RECORDINGS IN MEMBRANE
TETHERS
Madeleine R Howell1, Katherine M Xiang2, Adam E Cohen1,2

1
Harvard University, Department of Chemistry and Chemical Biology,
Cambridge, MA, 2Harvard University, Department of Physics, Cambridge,
MA
Single-molecule recording techniques have revolutionized our

understanding of processes such as transcription, translation, transport, and
signaling. Single-channel electrophysiology probes ion channel gating, but
there remains a need for analogous tools to record gating of channels or
transporters where the single-unit current is undetectably small or zero. We
used membrane tethers – thin tubes of membrane with length ~10 µm,
diameter ~100 nm, surface area ~1 µm2, and volume ~0.1 fL contiguous
with the parent cell – to isolate individual transmembrane proteins and to
restrict the diffusion of their small molecule transport products. Fluctuation
in the concentration of these products within the tether were detected via a
fluorescent sensor. This approach probed activity of single membrane
proteins in living cells. We demonstrated our approach by imaging Ca 2+ in
membrane tethers pulled from HEK293 cells expressing a T-type Ca2+
channel, CaV3.2. Depolarizing electrical stimuli elicited isolated Ca2+
transients, consistent with single-channel gating events. Ongoing work aims
to use these tools to study CaV3.2 gating in detail and to extend this method
to other transporter/fluorescent sensor pairs. We believe that the approach
may also generalize to studying turnover of single enzymes, provided that
the product can be monitored via a fluorescent sensor.
< Back 40
BIOLOGICAL TUNING OF THE MEMBRANE PHASE TRANSITION
FACILITATES PLASMA MEMBRANE ORGANIZATION AND
FUNCTION.
Sarah Veatch
University of Michigan, Biophysics, Ann Arbor, MI
Isolated cell plasma membranes are biologically tuned to be in a single

phase at growth temperature but close to a critical point of the membrane
phase transition. This talk will explore several consequences of this
biological tuning through experiments in model and intact cell membranes.
For example, near-critical membranes have a high compositional
susceptibility, meaning that stable membrane domains can assemble in
response to membrane proximal forces. This is demonstrated in live B cells
through quantitative super-resolution nanoscopy measurements that detect
the emergence of functional domains upon B cell receptor clustering in live
cells. Near-critical tuning of the membrane phase transition can also
enhance the stability of proteins condensed at membranes, and this is
demonstrated through simulation and experiments in model and cellular
systems.
< Back 41
CRYOGENIC LIGHT MICROSCOPY WITH SUB-NANOMETER
RESOLUTION RESOLVES THE STRUCTURAL CONFORMATIONS
OF PIEZO1
Hisham Mazal1,2, Franz-Ferdinand Wieser1,2,3, Daniel Bollschweiler4,

Alexandra Schambony1,2,3, Vahid Sandoghdar1,2,3
1
Max Planck Institute for the Science of Light, Nano-Optics, Erlangen,
Germany, 2Max-Planck-Zentrum für Physik und Medizin, Nano-Optics,
Erlangen, Germany, 3Friedrich-Alexander University of Erlangen-
Nürnberg, Erlangen, Germany, 4Max Planck Institute of Biochemistry,
Cryo-EM Facility, Planegg, Germany
PIEZO proteins are large transmembrane mechanosensitive ion channels

that mediate the translation of mechanical force into biological signals,
aiding in regulation of various physiological processes. The structure of the
protein, partially resolved by cryo-electron microscopy (cryo-EM), forms a
symmetric trimer. The core of the channel is formed by the C-terminal
domain, while the N-terminal side of the protein forms long transmembrane
alpha-helices, creating a blade-like structure. However, little is known about
the large-scale conformational changes of the blade domain and their
functional significance in the near-native cell membrane. To address these
challenges, we implemented single-particle cryogenic light microscopy
(spCryo-LM) to resolve the conformational states of mouse PIEZO1
(mPIEZO1) protein with high optical resolution. To avoid chemical fixation
protocols and to persevere the sample in its near-native state, we developed
a dedicated vitrification and transfer system that allows shock-frozen
biological samples to be transferred in and out of our cryogenic microscope
(operation at 4K) without devitrification. We, thus, obtain sub-nanometer
localization precision, yielding super-resolution images of vitrified COS7
cell membranes expressing mPIEZO1 protein. By implementing a 2D
single-particle image classification scheme, together with structural
modeling analysis, we were able to resolve three distinct conformations of
the mPIEZO1 blade domain. While one of the conformations agree with the
structures resolved by cryo-EM and AlphaFold predictions, our data reveal
two novel structural conformations, suggesting a strongly curved dome-
shaped structure, and a fully flat unbent blade conformation. We believe
spCryo-LM introduces a powerful method for structural studies of
membrane protein conformations without chemical fixation or artificial
membrane reconstitution. Additionally, our experimental pipeline opens the
door to direct correlative measurements with cryo-EM techniques,
facilitating high-resolution structural validation within the cellular
ultrastructure context.
< Back 42
MECHANISTIC OF FORCE TRANSMISSION ACROSS THE
NUCLEAR ENVELOPE AT LINC COMPLEXES BY SINGLE
MOLECULE IMAGING
Liying Wu1, Remi Dore2, Gaetan Bellot2, Christine Doucet2, Emmanuel

Margeat2, Fabien Pinaud1,3
1
University of Southern California, Biological Sciences, Los Angeles, CA,
2
University of Montpellier, CNRS, INSERM, Center for Structural Biology,
Montpellier, France, 3University of Southern California, Physics and
Astronomy, Los Angeles, CA
The nucleus is an important coordinator of cell adaption to environmental

forces and its defective response to mechanical cues can lead to severe
diseases, including premature aging and muscular dystrophies. At the
nuclear membrane, LINC complexes (SUN1, SUN2, emerin, nesprin) form
mechanotransducing hubs to transmit bidirectional forces between the
cytoskeleton and the nucleoskeleton. Within LINC, SUN proteins bridge the
space across the outer nuclear envelope (ONE) and the inner nuclear
envelope (INE). How LINC complexes integrate and convey forces across
the two membranes of the NE remains unclear.
Here we combined single molecule tracking, super-resolution microscopy
and modulation of nuclear mechanics to study the spatial distribution and
the mobility of SUN1 and elucidate the mechanisms underlying force
transmission at LINC complexes embedded in the NE. We found that SUN1
protomers rapidly assemble as trimers after ER membrane insertion and
accumulate as slow diffusing homo-trimers (33%) and near-immobile
nanoclusters (61%) at the INE. Mechanical challenges to the nucleus induce
an increased mobility of SUN1 and a local de-clustering of SUN1
homotrimers in a SUN1/nesprin interaction-dependent manner. We also
identified amino acid residues in SUN1 that participate in two distinct force
transmission steps across the NE: (i) force transfer initiation at the ONE via
SUN1/nesprin contacts and (ii) force transfer regulation across the NE by
the SUN1 coiled-coil backbone, via (de)clustering of SUN1. Those residues
and the clustering state of SUN1 homotrimers impact the stability of the
NE.
To assess the piconewton forces transmitted by LINC complexes across the
NE, we are also developing fluorescent optical force sensors (OFS) that are
inserted into SUN proteins. We calibrate those OFS with a novel approach
involving the use of DNA origami nanoactuators and single molecule FRET
detection.
Together, our research provides an initial physical model of how LINC
complexes function as mechanotransducing hubs across the NE to modulate
nuclear mechanics in cells.
< Back 43
MULTIMODAL MICROSCOPY PLATFORM FOR 3D SINGLE-
MOLECULE SUPER-RESOLUTION IMAGING THROUGHOUT
MAMMALIAN CELLS
Sofía Vargas-Hernández1,2, Tyler Nelson1,3,4, Margareth Freire1, Siyang

Cheng1,3,4, Anna-Karin Gustsvsson1,2,3,4
1
Rice University, Chemistry, Houston, TX, 2Rice University, Systems,
Synthetic, and Physical Biology Program, Houston, TX, 3Rice University,
Applied Physics Program, Houston, TX, 4Rice University, Smalley-Curl
Institute, Houston, TX, 5Rice University, Department of Biosciences,
Houston, TX, 6Rice University, Department of Electrical and Computer
Engineering, Houston, TX, 7The University of Texas MD Anderson Cancer
Center, Department of Cancer Biology, Houston, TX, 8Rice University,
Center for Nanoscale Imaging Sciences, Houston, TX
Single-molecule super-resolution imaging is instrumental for investigating

cellular architecture and organization at the nanoscale. However, obtaining
3D information with nanometric precision requires careful selection of the
illumination scheme, especially when imaging subcellular structures
throughout samples several microns thick, such as mammalian cells. Here
we present a multimodal microscopy platform for 3D single-molecule
super-resolution imaging that integrates and allows for easy switching
between light sheet illumination and flat-field epi- and TIRF illumination.
The uniform intensity profile of our flat-field modality combined with TIRF
illumination offers reduced photodamage and excellent optical sectioning
close to the coverslip while homogenizing fluorophore blinking kinetics,
fluorescence signal, and localization precision over the entire field of view,
when compared with a conventional Gaussian intensity profile. To enable
optical sectioning away from the coverslip our setup integrates light sheet
illumination. Light sheet illumination reduces photobleaching,
photodamages, and fluorescence background, making it an excellent
alternative for imaging throughout mammalian cells. In addition, our setup
combines the fast switching between illumination modalities with point
spread function engineering to enable 3D single-molecule super-resolution
imaging.
We validated our platform by 3D super-resolution imaging of actin and

paxillin, a protein located in the focal adhesion complex, in human
osteosarcoma cells. In close agreement with the literature-reported values,
the separation between actin filaments and paxillin in the focal adhesion
complexes was measured to be 59.5 ± 6.9 nm, which demonstrates the
quantitative performance of this setup for 3D imaging at the nanoscale.
< Back 44
A STRATEGY FOR QUANTIFYING THE CONFORMATIONAL
CHANGES OF SINGLE MOLECULES IN LIVE CELLS
Pengning Xu1, Nick Pinkin1, Saygin Gulec1, Bei Liu2, Timothy Elston1,
Klaus Hahn1
1
UNC Chapel Hill, Pharmacology, Chapel Hill, NC, 2Peking University,
Beijing, China
Traditional techniques that probe individual protein interactions such as

smFRET can be challenging for live cell single molecule experiments
because of FRET’s challenging signal/noise ratio, and the relatively low
photon budget of most fluorescent proteins. Emergent live cell labeling
systems such as SNAP and Halo tag provide a practical means to covalently
attach dyes to proteins in living cells and could enable the use of
environment-sensing dyes for single molecule sensing of conformational
changes. Here we explore this strategy to study single-molecule GTPase
conformational changes. The solvent-sensitive fluorophore Nile Red, and
other novel dyes, were attached to Cdc42 using a SNAP tag. The SNAP was
in a linker connecting Cdc42 to a WASP-derived peptide that binds only to
the active conformation of Cdc42. Upon Cdc42 activation, the Nile Red
emission maximum shifted from 622 nm to 597 nm and fluorescence
lifetime increased from 3.6 to 4.0 ns. The probe was used to quantify local
activation in membrane nanoclusters for polarized cell motility, and to
quantify the rates of membrane translocation and GTPase
activation/inactivation.
< Back 45
VISUALIZATION OF TRANSLATION DEPENDENT CHANGES TO
SHAPE OF mRNA IN VIVO AT ~10 nm USING MINFLUX.
Murali Palangat, David Ball, Tatiana Karpova, Daniel R Larson
National Institutes of Health, Laboratory of Receptor Biology & Gene

Expression, Bethesda, MD
Macromolecular motor machines likely alter the structure and shape of the
nucleic acids they interact with. How the interaction of eukaryotic
translational machinery with mRNA alters the shape of mRNA is not
known. Although fluorescence microscopy has provided many insights into
translating ribosomes in vivo, the spatial resolution necessary to visualize
changes to the mRNA being translated is still lacking. We have taken a
single molecule RNA-FISH (smRNA-FISH) approach that takes advantage
of the blinking properties of fluorophores to image single molecules of
fluorescently labeled probes tiled along an mRNA molecule using the
Minflux microscope at a resolution of ~10 nm. To study translation
dependent changes to mRNA, we targeted the gene FTL, that codes for
ferritin, and its translation is regulated by the levels of iron in the cell.
Translation of FTL mRNA can be readily down regulated by decreasing the
level of iron in cells using the iron chelator Desferoxamine (DFO). We
detected single molecules of FTL mRNA in cells either treated or untreated
with DFO, by smRNA-FISH using fluorescently labeled (Quasar 670)
probes directed to FTL mRNA. Imaging individual fluorophores at super-
resolution in 3D using the Minflux revealed a substantially significant
decrease in the volume of individual mRNAs in cells treated with DFO,
suggesting that downregulation of translation of this mRNA resulted in the
compaction of the RNA in treated cells relative to actively translated
mRNA in untreated cells. On the contrary, we did not observe any DFO
dependent change in volume of the control mRNA, HSPD1, that is not
regulated by iron, and codes for a nuclear encoded mitochondrial protein.
These results suggest that mRNA is in a compact shape when they are not
being translated and assume a more relaxed state with increased volume
when being actively translated resulting in a measurable change in mRNA
shape. We have now applied this change in mRNA shape as a metric to
assess where translation occurs of mRNA of nuclear encoded mitochondrial
protein. We assessed the translation status of HSPD1 mRNA, a nuclear
encoded mitochondrial protein, translated both in the cytoplasm and co-
localized on the mitochondria. While almost all the mRNA that co-localized
on the mitochondria appear to be translationally active with an increased
volume and shape, only ~63% of HSPD1 mRNA in the cytoplasm and not
co-localized on mitochondria were actively translated. Collectively, these
results suggest that actively translating mRNA assume a more relaxed shape
occupying a larger volume, and this metric can be used to assess the
translation status of an mRNA in cells at super resolution. This powerful
tool now allows us to resolve changes at tens of nanometers previously not
observed.
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SINGLE-MOLECULE LIVE IMAGING OF ENDOGENOUS PROTEIN
INTERACTIONS USING PROXIMITY-ASSISTED
PHOTOACTIVATION (PAPA)
Thomas G Graham1,2, Claire Dugast-Darzacq1,3, Gina M Dailey1, Britney

Weng1, Sathvik Anantakrishnan1, Amir Hay1, Gokul Upadhyayula1, Eric
Betzig1,5, Xavier Darzacq1,5, Robert Tjian1,5
1
University of California, Berkeley, Molecular and Cell Biology, Berkeley,
CA, 2Johns Hopkins University, Biophysics, Baltimore, MD, 3Université
Paris Cité, CNRS, Institut Jacques Monod, Paris, France, 4UC Berkeley,
Physics, Berkeley, CA, 5UC Berkeley, HHMI, Berkeley, CA
Fast single-molecule tracking (fSMT) exploits changes in the mobility of

molecules to probe their interactions in live cells, yet it does not tell us with
which partner a protein interacts. Proximity-assisted photoactivation
(PAPA) can supply this crucial missing information. PAPA uses excitation
of a “sender” fluorophore to reactivate a nearby “receiver” fluorophore from
a photochemical dark state, permitting detection of protein interactions with
single-molecule sensitivity, a broad distance range, and no spectral
crosstalk.1 Using a newly developed automated fSMT system, we have
applied PAPA to endogenous human proteins for the first time, focusing on
ribonucleoprotein complexes that regulate the master transcription
elongation factor P-TEFb.2 Contrary to existing models, PAPA-fSMT
measurements reveal that the P-TEFb:7SK complex is predominantly
unbound to chromatin in live cell nuclei. Upon treatment with P-TEFb
kinase inhibitors, P-TEFb dissociates rapidly from the 7SK complex and is
replaced by heterogeneous nuclear ribonucleoproteins (hnRNPs). Our
results show that PAPA powerfully augments the fSMT toolkit, making it
possible to monitor not just specific proteins but specific complexes at
single-molecule resolution in live cells. Finally, I will discuss the ongoing
development of new kinetic PAPA (kPAPA) methods, empowered by high-
throughput oblique line-scan (OLS) microscopy, to measure previously
inaccessible molecular interaction rates in live cells.
References:
1) Graham TGW, Ferrie JJ, Dailey GM, Tjian R, Darzacq X. Detecting
molecular interactions in live-cell single-molecule imaging with proximity-
assisted photoactivation (PAPA). Elife. 2022 Aug 17;11:e76870. doi:
10.7554/eLife.76870. PMID: 35976226
2) Thomas G.W. Graham, Claire Dugast-Darzacq, Gina M. Dailey, Britney
Weng, Xavier Darzacq, Robert Tjian. Single-molecule live imaging of
subunit interactions and exchange within cellular regulatory complexes.
bioRxiv 2024.06.25.600644; doi:
https://doi.org/10.1101/2024.06.25.600644
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USING SINGLE MOLECULE TRACKING TO INVESTIGATE DNA
METHYLATION IN LIVE CELLS
Amir D Hay, Thomas GW Graham, Kemal Achour, Xavier Darzacq, Robert

Tjian, Srigokul Upadhyayula, Eric Betzig
UC Berkeley, Department of Molecular and Cell Biology, Berkeley, CA
DNA methylation is an essential modification of mammalian genomes, yet

its function and regulation in vivo are unclear. To study the protein kinetics
that underlie the process of DNA methylation deposition in live cells, we
built an Oblique Line Scan (OLS) microscope for single molecule tracking
(SMT) that can generate quality data at a higher throughput compared to
any other similar and available technology. The OLS improves upon highly
inclined and laminated sheet (HILO) microscopy by homogenizing
illumination across the sample and minimizing out-of-focus fluorescence,
thereby enabling fast frame rate imaging with a large field of view (e.g.
5ms, 250um by 100um) to capture single molecule dynamics in 30-40 cells,
as opposed to just one or two. This is achieved by shaping a collimated laser
beam into a very thin (~2um) light sheet that is swept, obliquely, across the
sample by a galvanometer whose motion is synced with the rolling shutter
of an sCMOS camera. Using this tool on multiple gene-edited human cell
lines, we can observe biophysical properties, such as the diffusion and
chromatin residence time of single proteins across thousands of cells,
providing a powerful new platform to examine how replication,
transcription, and differentiation influence methyltransferase activity.
Describing and analyzing such kinetic processes will answer fundamental
open questions in the DNA methylation field.
< Back 48
MECHANISMS OF TRANSCRIPTION CONTROL BY DISTAL
ENHANCERS FROM HIGH-RESOLUTION SINGLE-GENE IMAGING
Alexandros Pertsinidis
MSKCC, Structural Biology, New York, NY
Distal enhancers can activate target genes over large genomic distances, but
physically how this communication is achieved in space and time, and the
nature of the signals transmitted, is poorly understood. On a fundamental
level, enhancers could control transcription by regulating the function of the
RNA Polymerase II machinery at target promoters. Consistent with this
notion, enhancers control the frequency of transcriptional bursts, regulating
how often a promoter is active. However, the precise molecular transactions
and/or biochemical activities facilitated by enhancer-promoter
communication to activate remain obscure. Moreover, the molecular and
biophysical means of achieving such facilitation are presently not well
defined.
Using real-time single-gene super-resolution imaging and acute targeted
perturbations, we define physical parameters of enhancer-promoter
communication and elucidate processes that underlie target gene activation.
Our experiments reveal that productive enhancer-promoter encounters are
dynamic, happen at distances ≲100-150 nm, and are temporally correlated
with transcriptional bursts.
Our study further reveals that activities of certain general transcription
factor (GTF) Pol II machinery components are on-pathway for
transcriptional burst initiation. Enhancer-controlled bursting thus involves a
multi-step process, that, unexpectedly, comprises several steps of the early
phases of the Pol II transcription cycle.
Intriguingly, we discover that some GTFs form nano-scale clusters at distal
enhancers, akin to enhancer-associated regulatory factor clusters we had
described previously. GTF clustering at enhancers is important for
transcriptional bursting. As the enhancer and promoter come in proximity to
commence a transcriptional burst, enhancer-associated GTF clusters also
move closer to the promoter. More frequent embedding of a promoter into
such environments of high local GTF concentration increases bursting
frequency, providing a mechanism for how enhancer-promoter interactions
facilitate GTF activities taking place at the promoter during the initiation of
transcription bursts.
Finally, our study also uncovers new relations between long-lived
transcriptionally refractory off states, antagonistic action of transcription
activators and repressors, and cohesin-dependent long-range genomic
interactions. Overall, our findings provide key insights into molecular and
biophysical mechanisms of long-range gene regulation by distal enhancers
and help understand how promoter-enhancer spatio-temporal relations
translate into transcriptional outputs.
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SUPER RESOLUTION IMAGING OF CHROMATIN IN HEALTH AND
DISEASE
Melike Lakadamyali
University of Pennsylvania, Physiology, Philadelphia, PA
Super-resolution imaging has revolutionized our understanding of the

folding and spatial organization of the genome. I will talk about how this
organization is cell type specific and becomes remodeled in disease states
and how we can leverage machine learning approaches to classify cells
based on chromatin states captured using super-resolution microscopy.
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SINGLE-MOLECULE DYNAMICS OF PIONEER TRANSCRIPTION
FACTORS INTERROGATING CHROMATIN IN LIVE CELLS
Zuhui Wang1, Bo Wang1, Di Niu1, Claudia Cattoglio2, Kyle Loh3, Luke

Lavis4, Hao Ge1, Wulan Deng1
1
Peking University, Biomedical Pioneering Innovation Center, Academy for
Advanced Interdisciplinary Studies, Peking-Tsinghua Center for Life,
Beijing, China, 2University of California, Department of Molecular and Cell
Biology, Berkeley, CA, 3Stanford University, School of Medicine, Stanford,
CA, 4Howard Hughes Medical Institute, Janelia Research Campus,
Ashburn, VA
Pioneer transcription factors (PTFs) possess the unique capability to access

closed chromatin regions and initiate cell fate changes, yet the underlying
mechanisms remain elusive. Here, we characterized the single-molecule
dynamics of PTFs targeting chromatin in living cells, revealing a notable
“confined target search” mechanism. PTFs like FOXA1, FOXA2, SOX2,
OCT4 and KLF4 sampled chromatin more frequently than non-pioneer
factor MYC, alternating between fast free diffusion in the nucleus and
slower confined diffusion within mesoscale zones. Super-resolved
microscopy showed closed chromatin organized as mesoscale nucleosome-
dense domains, confining FOXA2 diffusion locally and enriching its
binding. We pinpointed specific histone-interacting disordered regions,
distinct from DNA-binding domain, crucial for confined target search
kinetics and pioneer activity within closed chromatin. Fusion to other
factors enhanced pioneer activity. Kinetic simulations suggested transient
confinement could increase target association rate by shortening search time
and binding repeatedly. Our findings illuminate how PTFs recognize and
exploit closed chromatin organization to access targets, revealing a pivotal
aspect of gene regulation.
< Back 51
PERSISTENT AND TRANSIENT CHROMATIN BINDING SHAPES
SINGLE-MOLECULE DYNAMICS OF RNA POLYMERASE I, II, AND
III MACHINERIES IN LIVE CELLS
Yick Hin Ling1, Chloe Liang1, Sixiang Wang1, Carl Wu1,2

1
Johns Hopkins University, Department of Biology, Baltimore, MD, 2Johns
Hopkins University, Department of Molecular Biology and Genetics,
Baltimore, MD
Eukaryotic gene expression in the nucleus is orchestrated by three RNA

polymerases (RNAP-I, -II, and -III) and associated factors. Despite
extensive biochemical, genomic, structural, and imaging studies, the real-
time dynamics of these transcription complexes remain obscure. Here, we
employ single-molecule tracking in living yeast to assess the physiological
kinetics of over 50 representative proteins encompassing all three RNAP
machineries. Components of RNAPI and RNAPIII pre-initiation complexes
(PICs) engage in long-lived interactions on chromatin, reflecting their roles
for constitutive rRNA and tRNA synthesis, in contrast to the transient
RNAPII PIC. We further report the dynamics of key components across the
RNAPII transcription cycle—factors for upstream regulation, elongation,
histone modification, RNAPII C-terminal domain (CTD) modification,
RNA processing, and termination—revealing unprecedented insights into
the temporal landscape of RNAPII transcription. Strikingly, many
elongation factors, previously thought to travel processively with RNAPII,
display transient residence times, suggesting highly dynamic interactions
rather than constant association. Systematic screening of RNAPII-
associated factors shows that truncation of RNAPII-CTD substantially
reduces U1 snRNP residence time and decreases intron retention in
ribosomal protein genes, providing insights into how CTD length influences
co-transcriptional splicing. Our findings establish a framework for dynamic
chromatin interactions of RNA polymerase machineries in living cells.
< Back 52
SINGLE-MOLECULE IMAGING OF TRANSCRIPTION REGULATION
BY DNA TOPOLOGY
Nicholas Yahya1, Sam Meyer2, Jie Xiao1

1
Johns Hopkins School of Medicine, Biophysics and Biophysical
Chemistry, Baltimore, MD, 2INSA Lyon, Unite of Microbiology, Lyon,
France
The classic bacterial gene regulation dogma depicts that a gene’s

transcription activity is regulated through the recognition of specific DNA
and/or RNA sequences by proteins such as RNA polymerase and
transcription factors. In recent years an increasing number of studies have
shown that the supercoiling state of chromosomal DNA is a fundamental
factor impacting chromosome organization and transcription regulation in
bacteria. The topological organization of chromosomal DNA into individual
domains, between which the diffusion of supercoiling is prohibited, thus
plays an important role in gene regulation. In this talk I will discuss our
single-molecule approaches in vivo and in vitro to investigate how DNA
topology impacts chromosome organization and transcription regulation.
< Back 53
ULTRA-RESOLUTION, MULTI-SCALE, LIVE IMAGING OF THE 3D
GENOME, FOLDED BY MOTOR PROTEINS
Joo Lee1, Liang-Fu Chen1, Simon Gaudin1,2, Alistair N Boettiger1

1
Stanford University, Developmental Biology, Stanford, CA, 2Stanford
University, Genetics, Stanford, CA
In order to function, the mammalian genome must constantly refold itself.

In the last decade, sequencing methods like Hi-C have made it clear the
genome is intricately folded (e.g. in compartments, TADs, stripes and dots),
and that this organization contributes significantly to the control of gene
expression and thence cell fate and behavior. Single cell genome tracing
methods such as Optical Reconstruction of Chromatin Architecture and
polymer physics-based simulations of genome folding have proposed these
patterns reflect heterogeneous dynamic behaviors, rather than stable 3D
structures, indicating that motion, rather than structure, is key to
understanding genome function. However tools to directly observe this
motion have been severely limited in their genomic coverage, spatial
resolution, and temporal resolution, such that previously observed genome
motion is indistinguishable from the minimal Rouse polymer model -
lacking evidence for the predicted complex, motor-driven organization.
I will describe TRansposon Assisted Chromatin Kinetic Imaging
Technology, which by addressing all three of these limitations, enables
observation of the motor-assisted search process. With brighter, smaller,
and more stable fluorescent labels, we increase the spatial resolution five-
fold in comparison to current state-of-the-art work and the number of
frames per movie one-hundred fold. With improvements in optical setup,
we increase the temporal resolution forty-fold. We refer to this as ‘ultra-
resolution’ as the live cell data achieves the ~30 nm spatial resolution of our
previous fixed-cell super-resolution imaging methods while also
significantly improving the temporal resolution of the state-of-the-art.
Through transposon mediated tiling of self-mapping labels and automated
live imaging, we increase the genomic coverage to span length scales from
3 to 73,000 kilobases in a single experiment. With this deep, ultra-
resolution dataset, we find that genomic motion at all length scales is faster
than previously anticipated: Elements at submegabase scales exhibit search
times of a few to a few tens of seconds. Remarkably, search-times within
TADs increase much slower with genomic distance than expected by
polymer theory. We show that both the rapid search times and the
unexpected scaling are dependent on the loop-extruding motor protein,
cohesin, providing first direct in vivo evidence of its motor activity.
< Back 54
THE N- AND C-TERMINAL INTRINSICALLY DISORDERED
REGIONS OF TOX REGULATE ITS CHROMATIN BINDING AND
TARGET SEARCH KINETICS WITH OPPOSING EFFECTS
Santosh Adhikari1, Matthew A Sullivan2, Aditi Chandra2, Golnaz Vahedi2, E

John Wherry2, Mustafa Mir1,2,3
1
Childrens Hospital of Philadelphia, Pathology, Philadelphia, PA, 2University of
Pennsylvania, Perelman School of Medicine, Philadelphia, PA, 3Howard
Hughes Medical Institute, College of Developmental Biology, Philadelphia, PA
CD8 T cell exhaustion (Tex) is a coordinated dysfunctional differentiation state

of T cells driven by persistent antigen stimulation during chronic viral
infections and cancer. Tex are key targets of revolutionary immune checkpoint
blockade therapies such as anti-PD-1 that, at least temporarily, reverse Tex
dysfunction with clinical benefit for many patients. However, Tex
reinvigoration is often temporary and not all patients respond. Tex undergo
extensive transcriptional and epigenetic changes during their development that
collectively limit effector and memory function, reduce cytokine production,
and maintain sustained inhibitory receptor expression. This epigenetic
landscape likely underlies the incomplete and only temporary benefit of Tex-
targeted immunotherapies. The high mobility group (HMG)-box transcription
factor TOX is a critical driver of the Tex epigenetic landscape and is required
for Tex development. TOX binds to chromatin without strong sequence
specificity and its DNA target selection mechanisms are unknown. The N- and
C-terminal domains (“NTD,” “CTD”) of TOX are highly disordered, with large
net negative N-terminal charge, and were previously not functionally
characterized. Thus, it is unclear how TOX programs Tex-specific epigenetics.
To improve our mechanistic understanding of how Tex identity is established
and how TOX activity may be modulated, we used single molecule tracking
(SMT) in mouse fibroblasts to test the roles of these intrinsically disordered
regions (IDRs) in regulating TOX chromatin binding and search kinetics. We
find a 150% increase in the chromatin-bound fraction of full-length (FL) TOX
versus the HMG alone, suggesting that the TOX IDRs play a critical role in
target search and binding. Further analysis of diffusion kinetics shows that the
TOX IDRs mediate compact nuclear exploration. Individual deletion of either
the NTD or CTD shows that both tune chromatin-binding behavior via opposing
kinetic effects, with NTD-driven control of off-rate and CTD-driven control of
on-rate. Within the NTD, negatively charged residues are required for TOX-
dependent PD-1 expression. We further show that increasing NTD net negative
charge or decreasing serine content can have opposing kinetic effects on
chromatin binding, suggesting that NTD-focused perturbations could be used to
tune TOX activity. We propose a two-state dynamic conformational switching
model in which the negatively charged NTD screens low affinity protein-
protein and protein-DNA interactions to limit promiscuous binding and
concomitantly accelerate target search. Collectively, our SMT data can inform
strategies to engineer the molecular kinetics of synthetic TOX variants to
selectively modulate functional activity for therapeutic applications.
< Back 55
A METHODOLOGY TO REDUCE THE LOCALIZATION ERROR OF
MULTI-LOCI MICROSCOPY DATA PROVIDES NEW INSIGHTS
INTO ENHANCER BIOLOGY
Christopher H Bohrer, Daniel R Larson
National Cancer Institute, Laboratory of Receptor Biology and Gene

Expression, Bethesda, MD
Numerous functions hinge on the spatial dynamics of different genomic

loci; hence, microscopy techniques, such as chromatin tracing, have been
developed to localize multiple loci in living and fixed cells. Depending on
the throughput and specifics of the experiment, localization errors can still
obscure the true spatial locations. We have developed a post-processing
methodology to address this challenge without the need for additional
experimentation. By leveraging the fact that localization errors increase the
variability of the displacements between loci, and given a rough
approximation of the localization error, we can determine the "true" degree
of variation for each pair of loci to guide the error correction process. After
validating our approach with simulation and experiment, we used this new
methodology to improve the resolution of existing live-cell microscopy data
and showed that enhancer-promoter spatial proximity correlates with
transcriptional burst size within individual transcriptional bursts, and that
"communication" between the enhancer and the promoter happens quickly,
with a timescale less than 30 seconds. We then applied our approach to
existing chromatin tracing data that probed the linkage between chromatin
organization and Sox2 regulation, where previous work found no linkage
between enhancer-promoter proximity and transcription activity in
individual cells. We found a linkage previously obscured due to localization
error, demonstrating the need for the methodology and providing an
important result for enhancer biology.
< Back 56
DISSECTING NMNAT1’s ROLE IN DYNAMIC REGULATION OF
TRANSCRIPTION
Yessenia Cedeno1,2, Samuel Taylor2, Ulrich Steidl2, Robert Coleman1,2

1
Albert Einstein College of Medicine, Gruss-Lipper Biophotonics Center,
Bronx, NY, 2Albert Einstein College of Medicine, Department of Cell
Biology, Bronx, NY
Gene expression is achieved through a series of transcriptional bursts,

which are regulated by the cycling of chromatin-associated factors on and
off the genome. NMNAT1 is a metabolic enzyme responsible for local
NAD+ production in the nucleus, which is used by the PARP1 and SIRT1
chromatin-modifying enzymes. Our goal is to define the role of NMNAT1
in regulating chromatin-modifying enzyme activity, chromatin structure,
and gene expression. We hypothesize that the cycling of NMNAT1 on the
genome produces local NAD+ gradients that regulate PARP1 and SIRT1
activity, influencing rapid changes in chromatin structure and gene
expression of target genes. To address this hypothesis, we have developed
an NMNAT1 knockout system in U-2OS cells and integrated powerful
genomic approaches alongside live-cell single-molecule imaging. In this
system, we found that NMNAT1 is responsible for producing up to 70% of
total cellular NAD+ levels. We observed that NMNAT1, PARP1, and
SIRT1 display similar binding kinetics on the genome. These results
suggest that NMNAT1 potentially serves as a local “power plant” to fuel
and regulate the action of NAD+-dependent enzymes such as PARP1 and
SIRT1. Genomic analysis revealed that NMNAT1 is primarily located at the
promoter-TSS region where PARP1 and SIRT1 are known to function. To
gain a deeper understanding of how NMNAT1 regulates the transcriptional
dynamics of target genes, we have imaged changes in transcriptional burst
dynamics at the p21 gene in a NMNAT1 knockout cell line. We will discuss
potential mechanisms of how NMNAT1 is directly regulating expression of
its target genes. Together, these results enhance our understanding of the
intersection of metabolism and transcription, as well as define new
paradigms regarding regulation of gene expression.
< Back 57
GOLDFISH-X: HEAT-FREE, EXONUCLEASE-BASED DNA FISH
WITH IMMUNOFLUORESCENCE COMPATIBILITY
Po-Ta Chen1, Tiantian Shang2, Taekjip Ha1,3,4

1
Program in Cellular and Molecular Medicine, Boston Children’s Hospital,
Boston, MA, 2Department of Biophysics, Krieger School of Arts and
Sciences, Johns Hopkins University, Baltimore, MD, 3Department of
Pediatrics, Harvard Medical School, Boston, MA, 4Howard Hughes Medical
Institute, Harvard Medical School, Boston, MA
DNA fluorescence in situ hybridization (FISH) is a powerful technique for

localizing specific genomic loci within cells and tissues. Traditional DNA
FISH methods necessitate a sample heating step to denature double-
stranded DNA for oligo probe hybridization. However, this high
temperature treatment disrupts protein molecules, posing challenges for
quantitative imaging of proteins along with chromatin. Additionally, global
denature of the entire genome increases background signals due to non-
specific binding of oligo probes. To address these issues, we developed an
exonuclease-based genome oligopaint via local denature (GOLD) FISH
method, termed GOLDFISH-X. This technique uses Cas9 nickase for
precise target sequence identification, followed by exonuclease treatment to
create local single-stranded DNA regions for probe hybridization.
Consequently, GOLDFISH-X eliminates the need for heat denaturation of
chromatin, making it readily compatible with immunofluorescence imaging.
We demonstrate that GOLDFISH-X achieves a high signal-to-background
ratio for non-repetitive genomic targets and can detect single point mutation
when incorporated with a specificity-enhanced Cas9 variant. In summary,
GOLDFISH-X represents a versatile DNA FISH method, featuring local
denature and single nucleotide detection sensitivity, thereby advancing the
field of genomic imaging.
< Back 58
LIVE-CELL SINGLE-MOLECULE DYNAMICS TO UNRAVEL
SPATIOTEMPORAL TRANSCRIPTIONAL MODULATION BY THE
BAF CHROMATIN REMODELER COMPLEX
Ziyuan Chen1, Gina M Dailey1, Robert Tjian1,2, Xavier Darzacq1

1
University of California, Berkeley, Department of Molecular and Cell
Biology, Berkeley, CA, 2University of California, Berkeley, 2Howard
Hughes Medical Institute, Berkeley, CA
The BRG1/BRM-associated factor (BAF) complex is a critical regulator of

gene expression and chromatin remodeling, functioning as part of the
SWI/SNF remodeler family. The ATPase module of BAF consumes ATP
energy to alter chromatin structure, thereby facilitating or repressing gene
expression. Chromatin remodelers cooperate with transcription factors to
regulate chromatin accessibility. Sox2 and Oct4, two pluripotency-related
pioneer factors, open chromatin at motif-specific sites with the assistance of
the BAF ATPase Brg1. However, the live-cell dynamics of this cooperation
between transcription factors and the BAF complex remain inadequately
explored. Here, we employ single-molecule tracking (SMT) to investigate
the dynamics of BAF complex components and their interactions with
transcription factors in live mouse embryonic stem cells. Using the
proximity-assisted photoactivation (PAPA) imaging technique, we observe
that Brg1 exhibits a higher chromatin-bound fraction when in close spatial
proximity to Sox2. This finding supports the model in which pioneer factors
and chromatin remodelers cooperate to change chromatin accessibility.
< Back 59
ERα MEDIATED GENE STATE SWITCHING REGULATES THE
EXTENT OF THE SINGLE-CELL ESTROGEN RESPONSE
Christopher R Day1, Pelin Yasar1, Gloria Adedoyin1, Brian D Bennett2,

Joseph Rodriguez1
1
NIEHS, Epigenetics and Stem Cell Biology Laboratory, Durham, NC,
2
NIEHS, Biostatistics and Computational Biology Branch, Durham, NC
Gene regulation is a complex process and involves the coordination of

hundreds of proteins to initiate transcription. At individual loci,
transcriptional initiation is stochastic and results in short periods of nascent
RNA synthesis known as transcriptional bursts. In the hormone response,
the ligand Estradiol (E2) binds its canonical receptor Estrogen Receptor α
(ERα). The receptor then binds DNA regulatory elements and initiates
transcriptional bursts. We utilized single molecule imaging of estrogen
responsive genes to understand how transcriptional activation by ERα is
altered when bound by non-canonical ligands such as Bisphenol A (BPA).
We observe that cells treated with BPA exhibited TFF1 burst initiation rates
and sizes which were indistinguishable from cells treated with E2.
However, we observed a 50% reduction in the number of active alleles in
BPA treated cells. This effect is gene specific, as bursting from the estrogen
responsive GREB1 gene was unperturbed. Using genomics, we observed no
difference in chromatin accessibility. However, the TFF1 promoter
exhibited altered nucleosome positioning which coincided with reduced
ERα and cofactor binding. Lastly, BPA treatment prevented enhancer
mediated TFF1 bursting by inhibiting enhancer function. Our results
demonstrate gene specific effects of altered ERα recruitment and function
which lead to a reduction of transcriptionally permissive states. Our work
supports the model that the initial estrogen response occurs from alleles in
transcriptionally permissive states with additional inactive alleles
contributing to the cellular response over time.
< Back 60
REAL-TIME VISUALIZATION OF SPLICEOSOME ASSEMBLY
REVEALS BASIC PRINCIPLES OF SPLICE SITE SELECTION
Benjamin T Donovan1, Bixuan Wang1,2, Gloria R Garcia1, Stephen M

Mount2, Daniel R Larson1
1
NIH, NCI, Bethesda, MD, 2University of Maryland, Department of Cell
Biology and Molecular Genetics, College Park, MD
Among the first steps of spliceosome assembly is recognition of the 3’

splice site (3’SS) by the U2AF heterodimer. However, U2AF occupies
many more non-functional than functional sites throughout a pre-mRNA.
Therefore, how the spliceosome selects the correct 3’SS among this vast
pool of highly similar non-functional sites is not known. To address this
question, we developed a kinetic model of splice site selection with
parameters that can be measured with single-molecule imaging and high-
throughput binding assays performed both with purified proteins and in live
cells. This kinetic model, which successfully predicts alternative splicing
patterns, indicates that 3’SS selection occurs while U2AF is in complex
with the spliceosome, not during initial binding. Therefore, we sought to
identify proteins that interact with U2AF during spliceosome assembly by
immunoprecipitation mass spectrometry and to determine how these
proteins influence U2AF occupancy and subsequent splice site selection
with single-molecule tracking and RNA sequencing. Together, these assays
establish that the RNA helicase DDX42 regulates splice site selection by
accelerating U2AF dissociation while in complex with the spliceosome. In
addition to measuring the time scales of the earliest steps of spliceosome
assembly, this work establishes that the spliceosome operates in a ‘partial’
proofreading regime which achieves high sensitivity to the underlying
U2AF binding site while still allowing for efficient spliceosome forward
progression.
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LIVE-CELL IMAGING UNCOVERS DYNAMIC COUPLING
BETWEEN ENHANCER TRANSCRIPTION AND GENE
ACTIVATION.
Nadezda A Fursova1, Christopher H Bohrer1, Simona Patange2, Varun

Sood1, Carson C Chow3, Daniel R Larson1
1
Expression, Bethesda, MD, 2National Institute of Standards and
Technology, Material Measurement Laboratory, Gaithersburg, MD,
3
National Institute of Diabetes and Digestive and Kidney Diseases,
Laboratory of Biological Modeling, Bethesda, MD
Despite 40 years of investigation, how enhancers find and activate their

target genes remains a central unanswered question in the field. Recent
studies have identified enhancer transcription as a robust signature of
enhancer activity, yet its regulation and potential role in gene activation are
still poorly understood. To address these fundamental questions, we
characterized the transcriptional dynamics of the endogenous MYC gene
and its upstream super-enhancer in a non-transformed human cell line,
focusing on the coordination between enhancer and promoter activities.
Using dual-color live-cell imaging, we discovered that both MYC gene and
enhancer transcription occur in bursts, but with markedly different
dynamics, pointing to distinct regulatory mechanisms. Intriguingly, we also
found that the MYC gene and its enhancer are transcribed in alternating
phases, suggesting a functional link between these processes. Consistently,
perturbation of the MYC enhancer or its RNA results in a significant
reduction in MYC gene transcription. Taken together, these results indicate
that enhancer transcription is temporally coordinated with gene
transcription, facilitating gene activation. In summary, our work uncovers
dynamic coupling between enhancer and gene transcription, previously
obscured by the lack of temporal information, offering new insights into the
mechanisms of enhancer function.
< Back 62
CAPTURING MECHANISMS OF TRANSCRIPTION INITIATION
USING GENOME-BOUND ANISOTROPY OF RNA POLYMERASE II
Nayem Haque, Shu-Hao Liou, Robert A Coleman
Albert Einstein College of Medicine, Cell Biology, Bronx, NY
Approximately 1 in every 90 RNA Polymerase II (Pol II) molecules that

engage the genome successfully initiate and continue transcribing into the
gene body. To successfully proceed with transcription into the gene body,
Pol II must pass through multiple transition states during initiation. These
transitions depend on the coordinate interactions with various factors that
occur on the order of seconds, thus requiring a robust and powerful assay to
capture and characterize these complex dynamic events. In this study, we
employ single-molecule imaging techniques to characterize the behavior of
Pol II and associated factors within live cells. Our major question revolves
around how the early steps of transcription initiation can be dissected based
upon the dynamic behavior of genome-bound factors.
To address this question, we developed a novel single-particle tracking
algorithm called GEANIS (for GEnome-bound ANISotropy) which
measures the directional bias of proteins bound to the genome. Using
GEANIS, we tracked the dynamic movement of Pol II, which allowed us to
gain insights into its kinetics and function. Our preliminary results reveal
distinct GEANIS fingerprints associated with different modes of Pol II
activity. More specifically, we were able to identify time-dependent
changes in Pol II anisotropy during promoter escape and pause-release by
using inhibitors targeting these two processes. In addition, we analyzed how
the GEANIS fingerprint of Pol II compares to components of the pre-
initiation complex. Strikingly, we found that Pol II and TAF1, a component
of the TFIID complex, display overlapping GEANIS signatures seconds
after Pol II engages the genome. We will further discuss how these results
relate to known structural changes in the PIC upon transcription initiation
and elongation. Overall, these results demonstrate the power of GEANIS to
capture changes in dynamic protein interactions at an unprecedented
temporal resolution in live cells.
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MOLECULAR KINETICS OF POLYCOMB BODY FORMATION
DURING DEVELOPMENT
Gabriela Hayward-Lara1,2,3, Apratim Mukherjee2,3, Mustafa Mir2,3,4

1
Perelman School of Medicine, University of Pennsylvania, Developmental,
Stem Cell, and Regenerative Biology Graduate Group, Philadelphia, PA,
2
Perelman School of Medicine, University of Pennsylvania, Department of
Cell and Developmental Biology, Philadelphia, PA, 3Children’s Hospital of
Philadelphia, Center for Computational and Genomic Medicine,
Philadelphia, PA, 4Howard Hughes Medical Institute, Children’s Hospital
of Philadelphia, Philadelphia, PA
Cell fates are determined by the progressive activation and repression of

developmental genes. This process is essential to correctly pattern the
embryo. Recent work using live single-molecule imaging has shown that
gene activation is a highly dynamic process: short-lived multi-protein
microenvironments called hubs increase the frequency of transcription
factor interactions at target genes, where they bind for a few seconds and
initiate short (~minutes) bursts of gene expression. Repressive Polycomb
group proteins bind chromatin for similar seconds-scale durations and form
multi-protein microenvironments known as Polycomb bodies, but they
mediate continuous and near-absolute repression of genes. These bodies are
proposed to mediate repression by compacting chromatin, enriching
Polycomb group proteins, and/or excluding transcriptional machinery, but it
is not well understood which, if any, of these mechanisms are responsible
for repression. Here, we use lattice light-sheet microscopy and single-
molecule tracking in live Drosophila embryos to study the kinetics of
Polycomb body formation during early embryonic development. We
observe that Polycomb bodies form progressively and stabilize as cell fates
become specified. Once formed, they persist over the course of a cell cycle
and become progressively enriched. Single-molecule diffusion analysis
shows that over the same time period, the fraction of Polycomb proteins
bound to chromatin increases. Our data suggest that Polycomb bodies
mediate repression by maintaining a persistent local enrichment of proteins
at target sites. We propose that this increases the likelihood of protein-
chromatin interactions to generate continuous occupancy of Polycomb
group proteins at gene targets over long periods of time.
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UTILIZING SINGLE-MOLECULE IMAGING TO INVESTIGATE
HEMATOPOIETIC CELL FATE DECISION-MAKING
John W Hobbs IV, Lou Lou Dimple Maria Reenath Victor Jagaraja, Samuel
Taylor, Rajni Kumari, Robert A Coleman, Ulrich Steidl
Albert Einstein College of Medicine, Cell Biology, Bronx, NY
Hematopoiesis is a tightly regulated process that involves the self-renewal

of hematopoietic stem cells and the production of hematopoietic stem
progenitor cells (HSPCs) which develop into specialized blood cells.
Current models of hematopoiesis suggest that HSPCs have a distinct
transcriptional state, characterized by the expression or absence of specific
antagonistic TFs at a given lineage state. For example, GATA2 and PU.1
are critical TFs that enforce cell lineage progression from HSPCs towards
either terminally differentiated erythroid or myeloid cells respectively.
GATA2 and PU.1 function in a mutually antagonistic manner. Thus, HSC
lineage-specific gene expression is generally thought to be determined by
the relative expression of GATA2 and PU.1 at a given cell state. However,
our lab has previously adapted the single-molecule mRNA fluorescent in
situ hybridization (smFISH) to detect TF RNA expression throughout HSC
development and found PU.1 transcripts in erythroid progenitors (thought to
be GATA2 exclusive) and GATA2 transcripts in myeloid progenitors
(thought to be PU.1 exclusive). We hypothesize that hematopoietic
differentiation is driven by not only lineage regulated TF expression but
also the TF genomic binding dynamics within a particular cell. To test this
hypothesis, we utilize live-cell single-molecule microscopy to study how
TF activity and gene expression regulate lineage fate decision making. To
visualize single-molecule dynamics for in live cells, we fused GATA2 and
PU.1 to a HaloTag or SNAP-tag. Tagged TF constructs were virally
transduced into cell culture models with either single lineage or
multilineage commitment capacity. Live-cell single-molecule tracking was
performed at different stages of differentiation. To determine how TFs
direct hematopoiesis in vivo, we are currently generating transgenic mouse
models for fluorescent labeling of key hematopoietic TFs at both the mRNA
and protein levels. This will allow us to quantify endogenously TF activity
and gene expression in a live cell setting. With this live-cell single-molecule
technique we can understand TF behaviors in differentiating HSPCs at
unprecedented sensitivity and spatiotemporal resolution. This information
will provide important insight as to how TF genomic binding changes are
linked to normal blood development and hematopoietic diseases.
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A STATISTICAL RESOLUTION MEASURE OF FLUORESCENCE
MICROSCOPY WITH FINITE PHOTONS
Yilun Li, Fang Huang
Purdue University, Weldon School of Biomedical Engineering, West

Lafayette, IN
First discovered by Ernest Abbe in 1873, the resolution limit of a far-field

microscope is considered determined by the numerical aperture and
wavelength of light, approximately λ/2NA. With the advent of modern
fluorescence microscopy and nanoscopy methods over the last century, this
definition is insufficient to fully describe a microscope’s resolving power.
To determine the practical resolution limit of a fluorescence microscope,
photon noise remains one essential factor yet to be incorporated in a
statistics-based theoretical framework. We proposed an information density
measure quantifying the theoretical resolving power of a fluorescence
microscope in the condition of finite photons. The developed approach not
only allows us to quantify the practical resolution limit of various
fluorescence and super-resolution microscopy modalities but also offers the
potential to predict the achievable resolution of a microscopy design under
different photon levels.
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DNA SUPERCOILING-MEDIATED G4/R-LOOP FORMATION TUNES
TRANSCRIPTION BY CONTROLLING THE ACCESS OF RNA
POLYMERASE
Jihee Hwang1,2, Chung-Ying Lee1,2, Tapas Paul1,2, Huijin Lee1,2, Taekjip Ha

1,2
, Sua Myong 1,2
1
Boston Children's Hospital, Program in Cellular and Molecular Medicine,
Boston, MA, 2Harvard Medical School, Pediatrics, Boston, MA
RNA polymerase (RNAP) is a processive motor that modulates DNA

supercoiling and reshapes DNA structures. The feedback loop between the
DNA topology and transcription remains elusive. Here, we investigate the
impact of potential G-quadruplex forming sequences (PQS) on transcription
in response to DNA supercoiling. We find that supercoiled DNA increases
transcription frequency 10-fold higher than relaxed DNA, which leads to an
abrupt formation of G-quadruplex (G4) and R-loop structures. Moreover,
the stable R-loop relieves topological strain, facilitated by G4 formation.
The cooperative formation of G4/R-loop effectively alters the DNA
topology around the promoter and suppresses transcriptional activity by
impeding RNAP loading. These findings highlight negative supercoiling as
a built-in spring that triggers a transcriptional burst followed by a rapid
suppression upon G4/R-loop formation. This study sheds light on the
intricate interplay between DNA topology and structural change in
transcriptional regulation, with implications for understanding gene
expression dynamics.
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VISUALIZATION OF MULTIPLE TRANSLATION FRAMES IN
REPEAT ASSOCIATED NON-AUG (RAN) TRANSLATION
INVOLVED IN NEURODEGENERATIVE DISEASES AT SINGLE
mRNA LEVEL IN LIVING CELLS
Hayato Ito1, Timothy Stasevich2, Yoshitaka Nagai3, Hideki Taguchi1,4

1
Tokyo Institute of Technology, School of Life Science and Technology,
Yokohama, Japan, 2Colorado State University, Biochemistry & Molecular
Biology, Fort Collins, CO, 3Kindai University, Faculty of Medicine, Osaka-
Sayama, Japan, 4Tokyo Institute of Technology, Institute of Innovative
Research, Yokohama, Japan
Nucleotide repeat expansion of GGGGCC (G4C2) in the non-coding region

of C9orf72 is the most common genetic cause underlying amyotrophic
lateral sclerosis (ALS) and frontotemporal dementia (FTD). Transcripts
harboring this repeat expansion undergo the translation of dipeptide repeats
via a non-canonical process termed repeat-associated non-AUG (RAN)
translation. One of the key features of RAN translation is translation of all
potential reading frames within the mRNA independent of the AUG codon.
A contributing factor to this phenomenon is the occurrence of ribosomal
frameshifting during translation elongation. However, this process is
typically inefficient, and the presence of specific translation initiation sites
corresponding to each reading frame is believed to play a significant role in
multiple translation frames in RAN translation. It remains unclear whether
these multiple frames are homogeneous, with all mRNAs translating
equally, or heterogeneous, with mRNAs adopting specific frames. To
clalify this issue, we employed a recently developed Multi-Frame (MF) tag
system to visualize multiple frame translation in live cells (Lyon K et al.,
Mol. Cell 2019).
First, we improved the MF tag to detect RAN translations with low
translation efficiency with high sensitivity. We constructed new MF tag
system encoded Sun/HA tag array that replaced the previously used FLAG
epitope tag array with an HA epitope tag array. This Sun/HA MF tag was
placed downstream of the GGGGCC repeat sequence that induces RAN
translation, and nascent chain tracking was applied to detect nascent
polypeptide chains being translated from the ribosome on mRNA in the
living cell. We observed active translation sites that reflect nascent chains
on the single mRNA. The GA frame (0 Frame; GGG-GCC), which is
considered to have the highest amount of translation, was translated most
frequently on the mRNA. Interestingly, in addition to the GA frame, the GP
frame (+1 Frame; GGG-CCG) was also present on the mRNA, and some
mRNAs were found to have only the GP frame translated, suggesting that
the mRNAs occurring RAN translation are heterogeneous.
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THE BIOPHYSICAL PROPERTIES OF THE ER LUMEN ARE
AFFECTED BY MECHANICAL PERTURBATIONS AND
DIFFERENTIATION
Chao Jiang, Andrew Bazley, Liam Holt
Institute for Systems Genetics, New York University School of Medicine,

New York, NY,
The extracellular mechanical environment regulates cell behavior during

tissue development and homeostasis. Recent studies have illuminated the
influence of mechanical forces on the interactions between the endoplasmic
reticulum (ER) and various cellular constituents. However, whether and
how the ER lumen senses and reacts to mechanical stimulation remains
elusive. Here, we utilized ER-targeted Genetically Encoded Multimeric
nanoparticles (ER-GEMs) to probe ER luminal diffusivity by single particle
tracking. Our findings reveal that mechanical compression notably enhances
the diffusivity of ER-GEMs, marking a distinct response when compared to
the mechanical behavior of the cytosol. Subsequently, we demonstrated that
this increase in diffusivity is not attributable to osmotic compression or the
unfolded protein response. Further investigation indicated that the
augmented luminal diffusivity could be rescued by depleting calcium or
ATP levels, and by reducing the contractility of myosin II. Finally, we have
shown that the nucleus, which has been identified as a mechanical sensor
under confinement, can transduce compression to ER and accelerate ER
luminal diffusivity. Future studies will aim to elucidate the role of luminal
diffusivity in modulating secretion and facilitating cellular adaptation to
mechanical stimuli.
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SUPER-RESOLUTION, SINGLE-MOLECULE IMAGING OF
TRANSCRIPTIONAL BURSTS
Rebecca J Kaddis Maldonado1,2, Leslie J Parent1,2

1
Penn State College of Medicine, Medicine, Hershey, PA, 2Penn State
College of Medicine, Microbiology & Immunology, Hershey, PA
Eukaryotic transcription by RNA polymerase II (RNAPII) occurs in a

stochastic fashion. When transcription is active, high quantities of RNA are
synthesized, forming transcriptional bursts which can be visualized as large
and discrete nuclear foci via microscopy. To image transcriptional burst
sites, we are using Rous sarcoma virus (RSV), an avian retrovirus that co-
opts host RNAPII machinery to express high levels of viral transcripts from
its chromosomally-integrated proviral DNA. We labeled RSV transcription
sites with single molecule FISH probes specific for unspliced viral RNA
and compared confocal microscopy versus STED super-resolution
microscopy. There were dramatic differences observed, in which
amorphous, large, single foci imaged with traditional confocal microscopy
were revealed to be made up of several clusters of RNA likely representing
individual foci of nascent viral RNAs that were presumably emanating from
a single proviral integration site, when imaged using STED. We speculate
that these clusters may represent RNAs moving from initiation, to
elongation, and finally termination, within a transcriptional condensate.
Packaging the unspliced viral genome into virions is essential for spreading
infection from cell to cell. The process of assembling virions is driven by
the retroviral Gag structural protein. Using single molecule labeling of Gag
with a SNAPTag, we have evidence that binding to the unspliced viral RNA
genome occurs co-transcriptionally, as the unspliced RNA is being made.
We have interrogated this model using various imaging approaches such as
FRET, BiFC, live cell imaging with particle tracking, and labeling of
nascent RNA using click chemistry. Exciting results using live cell time-
resolved, confocal imaging revealed a “kissing” interaction between Gag
and nascent unspliced viral RNA foci, similar to the dynamic interaction of
enhancer regions (marked by Mediator protein) with actively transcribing
RNA (Science, 361:412). We found that many Gag foci are within ~1 µm of
the RSV transcriptional burst, suggesting that Gag utilizes either chromatin-
bound proteins or other transcription machinery, such as Mediator proteins,
to localize in close proximity to transcriptional bursts. With live-cell STED
microscopy, we visualized Gag-viral RNA complexes trafficking through
the nuclear pore into the cytoplasm, suggesting that the ribonucleoprotein
complexes formed in the nucleus are transported to the site of virion
assembly and release.
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MAGNETIC MICROMANIPULATION OF LOCI ON DNA
Veer Keizer1, Yeonee Seol2, Murali Palangat1, Keir Neuman2, Daniel

Larson1
1
Expression, Bethesda, MD, 2National Heart, Lung and Blood Institute,
Biochemistry and Biophysics Center, Bethesda, MD
To gain a better understanding of the material properties of chromatin and

successfully link them to chromatin organization and functions such as
transcription, we need to develop tools to actively and directly interrogate
these links. To address this gap, we develop novel methods to actively
manipulate DNA both in cells and in vitro. Previously, we have developed a
method to manipulate a single genomic locus within the nucleus of a living
cell through magnetic micromanipulation1. However, to understand how
two genomically distant loci, such as an enhancer and a promoter, exchange
information and regulate gene expression we need to manipulate these two
loci with respect to each other.
Building on earlier work we aim to exploit magnetic repulsion and

attraction to separate or bring together two genomic loci, respectively 2. To
fully characterize the magnetic repulsion of two genomic loci, we have
developed an in vitro-reconstituted system. Briefly, we use a linker protein
to attach iron-containing ferritin cages to a plasmid, where they form
ferritin clusters. Upon approximation of a large magnet the dipoles within
the ferritin cluster align with the field of the external magnet forming a
single magnetic moment. Due to their alignment, the ferritin cluster on one
plasmid will repel the cluster on another plasmid in the plane orthogonal to
the external magnet, whereas two clusters that are in line with the direction
of the external magnetic field will be attracted to each other. This system
allows us to manipulate two plasmids with respect to each other with
incredible precision. Moreover, this system can be applied broadly to
manipulate single biomolecules with respect to each other in vitro. The in
vitro-reconstituted model forms a strong foundation for the manipulation of
these biomolecules in live cells, allowing us to tackle fundamental questions
in the field of gene regulation.
1
Keizer VIP, Grosse-Holz S, Woringer M, Zambon L, Aizel K, Bongaerts
M, Delille F, Kolar-Znika L, Scolari VF, Hoffmann S, Banigan EJ, Mirny
LA, Dahan M, Fachinetti D, Coulon A. Live-cell micromanipulation of a
genomic locus reveals interphase chromatin mechanics. Science. 2022 Jul
29;377(6605):489-495.
2
Vecchiarelli AG, Neuman KC, Mizuuchi K. A propagating ATPase
gradient drives transport of surface-confined cellular cargo. Proc Natl Acad
Sci U S A. 2014 Apr 1;111(13):4880-5.
< Back 71
DYNAMICS AND FUNCTION OF LONG-RANGE POLYCOMB
INTERACTIONS
Sumin Kim1,2,3, Anders Sejr Hansen1,2,3

1
Massachusetts Institute of Technology, Department of Biological
Engineering, Cambridge, MA, 2Broad Institute of MIT and Harvard, Gene
Regulation Observatory, Cambridge, MA, 3Massachusetts Institute of
Technology, Koch Institute for Integrative Cancer Research, Cambridge,
MA
The Polycomb repressive system is a critical regulator of gene expression

during development. Polycomb proteins are conserved across multicellular
eukaryotes and are essential in mammals, in which their deletion causes
embryonic lethality or developmental defects. However, how Polycomb
establishes repression of target genes and how these genes are derepressed
in the correct spatiotemporal context during differentiation remain
unknown. Polycomb proteins form complexes that modify chromatin and
mediate 3D interactions between target genomic regions. In particular,
distal Polycomb regions form long-range loops that span tens of megabases.
Long-range loops are conserved across species and appear to be a key
feature of Polycomb function. Yet, little is known about the dynamics of
these long-range loops in living cells, including whether these interactions
are stable or form frequently enough to mediate a functional role and their
relationship to transcription. Despite the importance of understanding the
dynamics of Polycomb interactions, methods applied thus far capture only
snapshots in fixed cells.
Here, we aim to elucidate the dynamics of long-range Polycomb
interactions using 3D Super-Resolution Live-Cell Imaging (SRLCI). We
generated mouse embryonic stem cells (mESCs) in which interacting
Polycomb loci can be imaged and tracked in real-time. We use these cell
lines to conduct live-cell imaging and analyze the frequency and lifetime of
long-range Polycomb loops. We additionally integrated neurogenin 2 in
these cells to enable neural differentiation, allowing us to address whether
changes in Polycomb looping dynamics correspond to gene activation. If
long-range Polycomb interactions are indeed critical for gene repression, we
expect them to be frequent and stable in mESCs, and become more transient
during differentiation. Together, this study will provide key insights into
how these 3D chromatin interactions contribute to transcriptional
regulation.
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BURSTING TRANSLATION ON SINGLE mRNAs IN LIVE CELLS
Nathan M Livingston*1,4, Jiwoong Kwon*1,4,7, Oliver Valera3,4, James A

Saba2, Niladri K Sinha2, Pranav Reddy1,4, Blake Nelson1,4, Clara Wolfe1,
Taekjip Ha1,4,6,7, Rachel Green2,6, Jian Liu3,4, Bin Wu1,4,5
1
Johns Hopkins University School of Medicine, Department of Biophysics
and Biophysical Chemistry, Baltimore, MD, 2Johns Hopkins University
School of Medicine, Department of Molecular Biology and Genetics,
Baltimore, MD, 3Johns Hopkins University School of Medicine,
Department of Cell Biology, Baltimore, MD, 4Johns Hopkins University
School of Medicine, Center for Cell Dynamics, Baltimore, MD, 5Johns
Hopkins University School of Medicine, Solomon H Snyder Department of
Neuroscience, Baltimore, MD, 6Howard Huges Medical Institute, Chevy
Chase, MD, 7Boston Children's Hospital, Program in Cellular and
Molecular Medicine, Boston, MA
Stochasticity has emerged as a mechanism of gene regulation. Much of this

so-called ‘‘noise’’ has been attributed to bursting transcription. Although
bursting transcription has been studied extensively, the role of stochasticity
in translation has not been fully investigated due to the lack of enabling
imaging technology. In this study, we developed techniques to track single
mRNAs and their translation in live cells for hours, allowing the
measurement of previously uncharacterized translation dynamics. We
applied genetic and pharmacological perturbations to control translation
kinetics and found that, like transcription, translation is not a constitutive
process but instead cycles between inactive and active states, or ‘‘bursts.’’
However, unlike transcription, which is largely frequency-modulated,
complex structures in the 50-untranslated region alter burst amplitudes.
Bursting frequency can be controlled through cap-proximal sequences and
trans-acting factors such as eIF4F. We coupled single-molecule imaging
with stochastic modeling to quantitatively determine the kinetic parameters
of translational bursting.
* These authors contributed equally.
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A CLOSE LOOK AT THE NANOCOLUMNS OF THE CALYX OF
HELD SYNAPSES UNDER THE MAGNIFY EXPANSION
MICROSCOPY
Xuelin Lou
Medical College of Wisconsin, Cell Biology and Neurobiology, Milwaukee,

WI
The calyx of Held is the inhibitory center of sound processing in the

brainstem, it plays an essential role in sound localization by its remarkably
high-frequency transmission at up to 800 Hz. This unique feature requires
the synapses to operate with a large readily-releasable vesicle pool, tight
channel-vesicle coupling, fast kinetics of postsynaptic receptors, and
possible well-organized trans-synaptic alignments between presynaptic
active zoon and postsynapses density at ultrastructure levels, named trans-
synaptic nanocolumns. Here we applied MAGNIFY expansion microscopy
in combination with structured-illumination microscopy to visualize the
ultrastructure of nanocolumns. The nanocolumns are immunostained with
key active zone proteins and postsynaptic density proteins at the calyx of
Held at different developmental stages from the postnatal day-8, day-15 to
6-month-old mice. We find diverse spatial arrangements of nanocolumns
against the primary MNTB neuron surfaces, and there is a trend of tightened
nanocolumns during brain maturation. When comparing nanocolumns from
healthy adult mice and 5xFAD mice, a subset of abnormally aligned
nanocolumns was identified in 6-month-old 5xFAD mice, indicating the
misaligned nanocolumns may contribute to impairment of synaptic
transmission. This work suggests that MAGNIFY expansion microscopy
enables reliably visualizing synaptic ultrastructure under the optical
microscope. The tightened alignment of nanocolumns at the calyx of Held
contributes to the reliability of sensory input at high frequency. The
disrupted nanocolumn alignment observed in 5xFAD calyx may impair the
fidelity and accuracy of sound localization and hearing in Alzheimer’s
disease.
< Back 74
RIBOSOMAL DNA UNDERGOES STRUCTURAL REORGANIZATION
DURING HOMOLOGOUS RECOMBINATION REPAIR OF DOUBLE
STRAND BREAKS REVEALED BY SUPER-RESOLUTION
FLUORESCENCE IMAGING
Ye Ma1,2
1
Boston Children's Hospital, Harvard Medical School, PCMM, Boston,
MA, 2Johns Hopkins University, Biomedical Engineering, Baltimore, MD
Double strand breaks (DSBs) within the ribosomal DNA (rDNA) region
trigger a structural rearrangement termed nucleolar segregation, during
which a multitude of proteins associated with transcription and DSB repair
gather at the periphery of the nucleolus, forming what is known as the
nucleolar cap region. Currently, however, there is no available information
on whether sub-structures form within the nucleolar cap region. Using
super-resolution stimulated emission depletion (STED) microscopy and
lattice light sheet microscopy (LLSM), we revealed that the rDNA arrays
undergo significant structural re-organization during homologous
recombination (HR). We established that the reorganization depends on
cohesin, SMC5/6 and LINC complex. End-resection mediated by MRE11 is
required as well. Reorganization occurs in a subset of rDNA arrays in cells
during the G2/S phase, but not in the G1 phase. Our findings suggest that
chromatin organization plays a crucial role in DNA double-strand break
(DSB) repair processes. This could help explain the challenge of
maintaining rDNA integrity against mutagenic DNA recombination in this
repetitive tandem region through spatial rearrangement.
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SINGLE MOLECULE KINETICS OF PATHOGENIC GENE
ACTIVATION BY TRANSCRIPTIONAL HUBS FORMED BY
MUTANT ENL
Kaeli M Mathias1,2, Santosh Adhikari1, Liling Wan2, Mustafa Mir1,3

1
Children's Hospital of Philadelphia, Center for Computational and
Genomic Medicine, Philadelphia, PA, 2Perelman School of Medicine,
University of Pennsylvania, Cancer Biology, Philadelphia, PA, 3Howard
Hughes Medical Institute, Children's Hospital of Philadelphia, Philadelphia,
PA
Proper regulation of gene expression is essential to the health of all

organisms and disruptions to this process often leads to cancer and other
diseases. Recent in vivo single-molecule tracking has revealed that most
transcriptional regulators associate only transiently with chromatin and that
their occupancy at target genes may instead be driven through the formation
of high local-concentration assemblies, called condensates or hubs. We
recently discovered a series of oncogenic mutations in the chromatin
regulator, Eleven-nineteen-leukemia (ENL) protein, which can drive
aberrant formation of transcriptional hubs at target genes. ENL mutant hub
formation drives the hyper-activation of target genes, but the biophysical
properties and molecular kinetics that drive this hyperactivation are unclear.
Here, we combine fluorescence recovery after photobleaching (FRAP) and
single molecule tracking (SMT) assays to explore the effect of oncogenic
mutation on ENL kinetics. We observe through FRAP that ENL mutant
hubs have limited recovery over several minutes, when ENL is expressed at
close to endogenous levels. This contrasts with both ENL mutant hubs
when the protein is overexpressed and many transcriptional regulatory
proteins, both of which often have full recovery on the order of seconds.
Using SMT, we have found that the ENL mutant is more stabilized on
chromatin compared to wildtype and that molecules incorporated into a hub
have longer residence times on chromatin then non-hub molecules.
Together, these results suggest that ENL molecules are stably incorporated
into hubs and that ENL mutant hubs stabilize DNA-protein interactions to
alter transcription at target sites. Overall, our study supports a model by
which pathogenic mutations can drive aberrant hub formation and stabilize
interactions to increase transcriptional activity, driving disease outcomes.
< Back 76
GENERAL PRINCIPLES TO IMPROVE THE MEASUREMENT
PROTEIN DYNAMICS AT SCALE AS A MEANS OF GENERATING
BIOLOGICAL INSIGHT
David McSwiggen
Eikon Therapeutics, Eikon Systems, Hayward, CA
Live cell single molecule tracking has offered scientists a powerful tool to
understand how biomolecules navigate a complex cellular environment. The
power in these approaches is that they are often applicable across diverse
areas of study and have thus revealed new insights in various cellular
compartments and across time scales. Still, the challenge remains that a
high degree of experimental resources and technical sophistication are
required to collect and analyze single molecule tracking data, often with
bespoke solutions for each research study. It is therefore hard to distill
learnings from targeted studies on a specific protein, typically performed on
a specific microscope with a limited sample size, to more universal
principles applied to expand SMT to broad scientific questions across fields
of study.
In an effort to use single molecule tracking as a modality for the

identification of new therapeutic compounds, we have screened hundreds of
tagged proteins with distinct biological functions. From this effort we have
identified important principles that have guided the design of our oblique
line scanning (OLS) microscope and particle tracking analysis suite,
extending both the size of the field of acquisition and the range of
resolvable protein states by more an order of magnitude. Using both theory-
guided results as well as empirically validated findings, we’ll highlight the
variables we consider most important for producing robust and reliable
single molecule tracking results. By implementing these findings, we were
able to identify distinct mechanisms of inhibition of the Proliferating Cell
Nuclear Antigen (PCNA) as a function of cell cycle state. Our results
underscore the importance in capturing cell state heterogeneity as a means
of understanding underlying biological mechanisms. We hope these results
will serve as a resource for the community to improve the quality and
interpretability of future studies.
< Back 77
MOLECULAR KINETICS OF TRANSCRIPTION DURING
EMBRYONIC DEVELOPMENT
Apratim Mukherjee1,2, Kareena Shankta2, Manya Kapoor2,3, Raymond D

Carter2,5, Yara Haloush2,4, Mustafa Mir1,2,4,5
1
Department of Cell and Developmental Biology, University of
Pennsylvania, Philadelphia, PA, 2Center for Computational and Genomic
Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA,
3
Department of Bioengineering, University of Pennsylvania, Philadelphia,
PA, 4Howard Hughes Medical Institute, Children’s Hospital of
Philadelphia, Philadelphia, PA, 5Biochemistry Biophysics and Chemical
Biology Group, University of Pennsylvania, Philadelphia, PA
RNA Polymerase II (RNAPII) is the enzyme that transcribes all protein-

coding genes. Live imaging of RNAPII has shown that it organizes into
transient clusters within nuclei that last on the order of tens of seconds or
less. These clusters are thought to boost the efficiency of the transcription
reaction by coordinating the transcription of multiple, functionally related
genes. However, the conflicting evidence regarding the stability of RNAPII
clusters upon transcription disruption raises critical questions about their
functionality and the specific stages of the transcription cycle that govern
their formation.
Here we combined single-molecule tracking (SMT) and rapid volumetric

imaging of RNAPII in live Drosophila embryos during zygotic genome
activation (ZGA), a period marked by a nearly four-fold increase in
transcriptional activity. SMT revealed that the chromatin-bound fraction of
RNAPII increases by only 1.4-fold during ZGA, which is insufficient to
account for the 4-fold increase in transcription. Sub-second volumetric
imaging showed that RNAPII forms transient clusters during ZGA. SMT
further revealed that these clusters increase in number and are enriched with
chromatin-bound RNAPII molecules during ZGA. Drug perturbation
experiments targeting different stages of the transcription cycle (initiation
and elongation) indicate that cluster formation and RNAPII enrichment
within clusters are driven by initiation, while the kinetic composition within
clusters is influenced by both initiation and elongation. Overall, this study
demonstrates that during ZGA in developing embryos, the increase in
transcriptional activity is driven by increased RNAPII clustering that
primarily enhances the transcriptional initiation rate.
< Back 78
TRUESPOT: A ROBUST AUTOMATED TOOL FOR QUANTIFYING
SIGNAL PUNCTA IN FLUORESCENT IMAGING.
Blythe G Hospelhorn, Benjamin K Kesler, Hossein Jashnsaz, Gregor Neuert
Vanderbilt University, Department of Molecular Physiology and

Biophysics, Nashville, TN
Characterizing the movement and behavior of biomolecules in single cells

quantitatively is essential to understanding fundamental biological
mechanisms. One approach to studying such dynamics is tagging and
imaging molecules of interest. RNA fluorescent in situ hybridization (RNA-
FISH) is a frequently used technique for visualizing RNA in fixed cells
using fluorescent probes. Automated processing of the resulting images is
essential for large datasets that may include many different targets and time
points. Here we demonstrate using our data set of 1597 simulated and 869
experimentally obtained images that our MATLAB based RNA-FISH
image processing pipeline, TrueSpot, is a useful tool for automatically
detecting the 3D locations of cell boundaries and RNA transcripts at single
molecule resolution in an RNA-FISH image stack. In particular, this tool is
effective for facilitating quantitative analyses of FISH data such as
determining the colocalization of multiple transcripts or the relative amount
of RNA in various subcellular compartments. TrueSpot also performs well
on images with immunofluorescent (IF) and GFP tagged protein targets that
form clusters. Overall, TrueSpot is capable of performing analysis for a
variety of imaging-based approaches to studying biomolecular dynamics,
which can aid in endeavors such as advancing understanding of eukaryotic
transcription regulation mechanisms by studying the quantities and
behaviors of transcripts, regulatory RNA, and other targets at discrete time
points.
< Back 79
SINGLE-MOLECULE IMAGING OF MEDIATOR-RNA POLYMERASE
II INTERACTION DYNAMICS DURING EARLY NEUROGENESIS
Heta Patel1, Gina Dailey1, Xavier Darzacq1, Robert Tjian1,2

1
University of California, Berkeley, Molecular Cell Biology, Berkeley, CA,
2
Howard Hughes Medical Institute, Chevy Chase, MD
Cellular differentiation programs are controlled by transcription, which

involves extensive protein-protein interactions between master transcription
factors, Mediator complex, and RNA polymerase II (Pol II). The Mediator
complex aids transcription factors to recruit Pol II to target promoters and is
crucial for activating all protein-coding genes. In yeast and metazoans, the
most well-established Mediator-Pol II interaction occurs between Med17,
an essential Mediator architectural protein, and Rpb1, a subunit of Pol II.
However, the real-time diffusivity, stability, and kinetics of Mediator and
Mediator-Pol II interactions have not been characterized in metazoans,
especially during differentiation when subunits can transition from
functionally essential to dispensable. We utilize single-molecule imaging to
investigate Med17-Rpb1 interaction dynamics in living mouse embryonic
stem cells (mESCs) during early neurogenesis. Our results yield novel
mechanistic insights about how Mediator binds to and recruits Pol II to
facilitate transcription and how these dynamics may evolve during early
development.
< Back 80
EXPLORING SYNAPTIC ACTIN-MYOSIN DYNAMICS WITH SUPER
RESOLUTION MICROSCOPY
Shayna J Reed1,2,3, Natasha Mayorga2, Gavin Rumbaugh1,3, Courtney A

Miller1,2,3
1
Scripps Research, Skaggs Graduate School of Chemical and Biological
Sciences, La Jolla, CA, 2The Wertheim UF Scripps Institute for Biomedical
Innovation & Technology, Molecular Medicine, Jupiter, FL, 3The Wertheim UF
Scripps Institute for Biomedical Innovation & Technology, Neuroscience,
Jupiter, FL
Nonmuscle Myosin IIB (NMIIB) is an ATPase motor complex that generates

force on actin filaments. This force generation is an essential driver of the actin
re-organization that occurs in dendritic spines, actin dense post-synaptic
structures, that allows spines to enlarge when stimulated. When a neuron is
stimulated, actin mobilization in spines, spinal enlargement, and then actin
stabilization of the enlarged structures occurs, and this dynamic process results
in plasticity. Spine plasticity in regions of the brain such as the hippocampus
(HPC) and basolateral amygdala (BLA) contributes to the molecular basis of
learning and memory storage. While NMIIB is known to be a critical
contributor to the structural plasticity underlying learning and memory,
surprisingly little is known about its action and regulation in mature excitatory
neurons. Our group established that NMIIB is a driver of actin polymerization
in rodent hippocampal neurons and that it is regulated as a part of the NMDA
receptor pathway upon synaptic stimulation. Inhibiting NMIIB in the HPC
results in disruption of memory. With advances in molecular level imaging, a
comprehensive cellular biological study of NMIIB is now possible to elucidate
its regulation of synaptic actin dynamics. To support this, we have generated
and validated a novel endogenously tagged NMIIB knock-in mouse line
containing both 3x FLAG and Halo tags as a tool compatible with super
resolution imaging and biochemical analysis. Super resolution imaging is
necessary to address our questions about NMIIB localization and dynamics
because at 300nm, myosin filaments are just above diffraction limit and any
non-filamentous myosin structures will be even smaller. Our data shows NMIIB
interacts with proteins in the shaft and at tips of spines, suggesting that dynamic
changes in subcellular localization are occurring on a scale < 1 micron and
therefore super resolution, and even more specifically, single molecule
localization microscopy (SMLM) is most suitable to investigate. In this study
we show the validation of our mouse line generation and tagging strategy, the
subcellular distribution of NMIIB in dendritic spines, and the compatibility of
this mouse line with stochastic optical reconstruction microscopy (STORM).
Current analysis is focused on super-resolution evaluation of NMIIB
distribution in spines under basal conditions and synaptic plasticity stimulation.
Our findings are expected to be of broad value to the field’s understanding of
actin mobilization related to synapse plasticity and future studies will
interrogate NMII’s dynamics and function in maintaining stable synaptic
structure.
< Back 81
SINGLE-MOLECULE IMAGING DEFINES DISTINCT FUNCTIONAL
COMPLEXES OF TELOMERE BINDING PROTEINS
Tomas Janovic2, Jens C Schmidt1,2

1
Michigan State University, OBGYN, East Lansing, MI, 2Michigan State
University, Institute for Quantitative Health Science and Engineering, East
Lansing, MI
Chromosome ends resemble DNA double strand breaks and therefore must
be protected from the DNA repair machinery to prevent chromosome
fusions. The six protein shelterin complex composed of TRF1, TRF2,
RAP1, TIN2, POT1, and TPP1 carries out this critical end protection
function. In addition, the shelterin complex regulates telomerase
recruitment to telomeres and access of telomerase to the 3’-overhang of the
chromosome end to compensate for its incomplete DNA replication. How
the shelterin complex can simultaneously prevent the DNA repair
machinery from recognizing the chromosome end and allow telomerase to
elongate the telomere is one of the key unaddressed questions in telomere
biology. While the shelterin complex can assemble into a large complex
containing all of its 6 subunits in vitro, it is unclear whether it can form sub-
complexes that carry out its distinct function in cells. To study the shelterin
complex components at the single-molecule level, we have introduced the
HaloTag at the endogenous loci of all six shelterin components in
telomerase positive HeLa cells and U2OS cells, which use a cancer specific
alternative lengthening of telomeres mechanism to maintain their telomeres.
Using these 12 cell lines we systematically defined the total protein
abundance, and telomeric copy number of the shelterin proteins.
Furthermore, we used single-molecule live cell imaging to define the
biophysical properties of the shelterin proteins in the nucleoplasm and
bound to telomeres. Surprisingly, we identified two distinct complexes
composed of TRF1-TIN2-TPP1-POT1 and TRF2-RAP1, respectively.
While TRF1-TIN2-TPP1-POT1 is tightly bound to telomeres, TRF2-RAP1
is highly dynamic. These observations, suggest that the shelterin complex
exist in multiple sub-complexes that carry out distinct functions. TRF1-
TIN2-TPP1-POT1 controls telomeric DNA replication and telomerase
recruitment, while the dynamic binding of TRF2-RAP1 likely facilitates the
recruitment of a variety of effector proteins that mediate protection of the
chromosome end from the DNA repair machinery. Our work highlights the
tremendous power single-molecule imaging to define biochemical and
biophysics properties of biomolecules in living cells.
< Back 82
CHROMATIN AND NUCLEAR RECEPTOR SINGLE MOLECULE
DYNAMICS AND GENE REGULATION IN VIVO
Diana A Stavreva1, Le Hoang1, Kaustubh Wagh1, Hannah LaPoint1,

Sohyoung Kim1, Arpita Upadhyaya2, Louis Schiltz1, Raj Chari3, Gordon L
Hager1
1
NCI, CCR, NIH, Laboratory of Receptor Biology and Gene Expression,
Bethesda, MD, 2University of Maryland, Department of Physics, College
Park, MD, 3Frederick National Laboratory for Cancer Research, Genome
Modification Core, Frederick, MD
To study the role of chromatin and transcription factor (TF) dynamics for
gene regulation in vivo, we tagged the endogenous H2B as well as the
glucocorticoid and estrogen receptor (GR and ER, respectively) with
HaloTag in MCF7 cells. We utilized single molecule tracking (SMT) and
analysis methods in combination with other microscopy and biochemical
approaches to i) map the dynamics of these TFs in response to constant and
ultradian (pulsatile) hormone stimulations, and ii) determine the effects of
hormone fluctuations on transcription regulation. We found that nuclear
receptor (NR)/hormone dissociation time plays a critical role in the ability
of the NR to detect hormone level fluctuations. In contrast to GR, the strong
ER complex with estradiol (E2) results in continuous gene response of the
ER-regulated genes, even under discontinuous (ultradian) E2 stimulation.
The less stable interaction of ER and estriol (E3) or BPA allow the ER to
detect E3 and BPA fluctuations, albeit with a delay. The changes in gene
responses under these different treatments is accompanied with
corresponding changes in the ER bound fraction, as measured by SMT.
Even though most studies on hormonal signaling are performed under
continuous stimulation, secretion of many hormones follows a circadian
pattern, comprised of periodic (ultradian) pulses with short duration.
Understanding how the pattern of hormone stimulation impact gene
transcription could have important implications for human health.
< Back 83
CHROMOSOME CONTRACTION IS NOT SUFFICIENT TO REPRESS
TRANSCRIPTION REVEALED BY CRISPR-CLIP
Yu-Chieh Chung1, Dilshodbek Nishonov1, Siou-Luan He1, Nevin Wise1, Li-

Chun Tu1,2,3,4
1
The Ohio State University College of Medicine, Biological Chemistry and
Pharmacology, Columbus, OH, 2The Ohio State University, Center for
RNA Biology, Columbus, OH, 3The Ohio State University, Comprehensive
Cancer Center, Columbus, OH, 4The Ohio State University,
Interdisciplinary Biophysics Graduate Program, Columbus, OH
Nanotools that can manipulate genomic DNA folding inside living cells are
crucial for studying chromatin function and have the potential to be used as
novel therapeutic tools to treat genetic diseases. Chromosome organization
has been correlated with its function, such as gene activities. However, the
cause and effect of chromatin folding and transcription control remain
unclear. In this study, we developed a live-cell nanotool called CRISPR-
CLIP, which uses fused CRISPR-Sirius guide RNAs (gRNAs) to bring two
distant genomic regions into close proximity, generating artificial chromatin
loops. CRISPR-CLIP gRNAs can be fluorescently labeled to allow real-
time visualization of targeted genomic loci as loop anchors. We used
CRISPR-CLIP to manipulate chromatin folding at different genomic scales,
including creating individual stable chromatin loops and collapsing large-
scale chromosomal regions along single chromosomes. We found that
collapsing chromosomes did not affect the gene expression of non-targeted
genes but could repress genes targeted by CRISPR-CLIP which creates
cross-links on chromosomal DNA, a function similar to HP1
(heterochromatin protein 1). Importantly, our findings suggest that
CRISPR-CLIP could be a potential tool for gene therapy for gene
repression. Additionally, our results indicate that chromatin density alone is
not a sufficient criterion to characterize chromatin function.
< Back 84
COMPARTMENTALIZED DIFFUSION BETWEEN PAIRED MEIOTIC
CHROMOSOMES REGULATES CROSSOVERS IN C. ELEGANS
Lexy von Diezmann
University of Minnesota, Genetics, Cell Biology, and Development,

Minneapolis, MN
Sexually reproducing organisms use meiosis to partition homologous

chromosomes into haploid gametes. A critical step is the formation of physical
linkages known as crossovers between each pair of homologous chromosomes,
and errors in this process are a leading cause of age-related infertility and
conditions such as Down syndrome in humans. Accordingly, meiotic nuclei
ensure at least one crossover forms per pair of chromosomes, and that when
multiple crossovers form, they are well-separated. Termed “crossover
interference,” this phenomenon was first observed genetically in 1916, but the
mechanism by which interference acts remains hotly debated.
The synaptonemal complex is a known regulator of crossover interference. This

liquid-like protein assembly forms a 100-nm thick interface between aligned
chromosome pairs and recruits the signaling proteins that localize to and
determine crossover sites. This has inspired a “coarsening” model of
interference, wherein signaling proteins diffuse along the synaptonemal
complex and are sequestered by emerging crossovers, preventing further
crossovers from forming nearby. However, such dynamics are difficult or
impossible to measure using conventional techniques such as FRAP.
To understand how information flows in living meiotic nuclei, my lab develops

methods to image within the reproductive tract of the model nematode worm C.
elegans at single-molecule resolution. Using biplane detection of
photoconvertible dyes, we achieve single-molecule trajectories with tens to
hundreds of localizations (up to 10s of seconds) at < 25 nm lateral resolution
and a 1 µm depth of field. Tracking pro-crossover signaling proteins and
synaptonemal complex subunits relative to the GFP-labeled chromosome axis,
we discovered that both types of proteins exhibited one-dimensional Brownian
motion along the chromosomes. Simulations predicted the signaling protein can
explore the full length of the chromosomes within the timescale of crossover
designation, confirming a core assumption of the coarsening model.
This model further predicts a strong dependence on the rate of turnover into the
nucleoplasm and into/from recombination sites. We have directly observed
these transitions, and I will discuss our use of stroboscopic imaging to
accurately quantitate their rates despite order of magnitude differences in the
diffusion coefficients in different regions. I will also describe our use of dual-
labeling using the HaloTag system to create “guide stars” at crossover
intermediates, allowing us to accurately describe motion at these nanoscale
protein assemblies despite the inherently dynamic environment of live nuclei.
< Back 85
TRANSGLUTAMINASE 2-MEDIATED POST-TRANSLATIONAL
MODIFICATION OF VIMENTIN IN MACROPHAGE ACTIVATION OF
SEPSIS MOUSE
Yali Xu1,2, Ting Su2, Wenkui Yu2, Yutaka Furutani3, Harukazu Suzuki1,
Xian-Yang Qin1
1
RIKEN Center for Integrative Medical Sciences, Laboratory for Cellular
Function Conversion Technology, Yokohama, Japan, 2Nanjing University,
School of Medicine, Nanjing, China, 3The Jikei University School of
Medicine, Department of Laboratory Medicine, Tokyo, Japan
Sepsis is a major cause of death in hospitals and involves an uncontrolled

inflammatory response to infection, leading to multiple organ dysfunction,
including acute liver failure. Transglutaminase 2 (TG2) is a crosslinking
enzyme involved in diverse cellular processes, such as cytoskeletal
function, signal transduction, and cell survival. Methods: Sepsis mice were
established by intraperitoneal injection of lipopolysaccharide (LPS) or cecal
ligation and puncture. In vitro and ex vivo transamidase activity of TG2 was
measured based on the incorporation of 5-biotinamidopentylamine, a
biotinylated substrate for TG2. Loss-of-function analysis was performed
with TG2 inhibitor cystamine (CTM), clodronate liposome for macrophage
depletion, and siRNA against vimentin. LC-MS/MS-based proteome
analysis and RNA-seq transcriptome analysis was applied to explore the
underlying mechanism. Streptavidin-conjugated magnetic bead was used to
isolate biotinylated substrate of TG2. RAW264 cell was introduced for in
vitro validation. Results: Pharmacological inhibition of TG2 activity with
CTM improved the survival of sepsis mice and ameliorated LPS-induced
liver injury. Omics analysis showed that CTM inhibited LPS-induced
inflammation especially in the livers of sepsis mice. Increased TG2 activity
was mainly observed in TG2-expressing and F4/80-positive midzonal M1
macrophages in the livers of sepsis mice. Macrophage depletion
ameliorated LPS-induced liver inflammation. Vimentin was identified as a
substrate crosslinked by TG2 and knockdown of vimentin inhibited LPS-
induced cytokine expression in RAW264 cells. Conclusion: This study
elucidated the role of TG2 in mediating sepsis-induced inflammation by
facilitating the crosslinking of vimentin in macrophage and the therapeutic
potential of TG2 as a molecular target for sepsis-associated liver injury.
< Back 86
USING ADVANCED MICROSCOPY TECHNIQUES TO STUDY THE
EFFECTS OF CONDENSATES ON THE NUCLEAR ENVIRONMENT
Chelsea Q Yang1, Wesley Legant2

1
University of North Carolina, Chapel Hill, Biochemistry and Biophysics,
Chapel Hill, NC, 2University of North Carolina, Chapel Hill, Pharmacology,
Chapel Hill, NC
Biomolecular condensates have been implicated in numerous cellular

processes, from stress granule formation to super-enhancer activity. This is
due to the functional implication that intracellular phase transitions lead to
the formation of membrane-less, chemically distinct microenvironments,
which play a role in spatiotemporal regulation. Nuclear condensates, in
particular, have been suggested to play a large role in processes such as
transcription and DNA repair. However, though there is evidence that
condensates play a role in these processes, we often lack a mechanistic
understanding of the biophysical principles by which this occurs. One well-
known nuclear condensate system is that of Acute Myeloid Leukemia, a
cancer in which aberrant chromosomal recombination results in the
formation of phase-separating chimera proteins. Nup98-HoxA9 is one of
the most common of these chimeras and contains the DNA-binding region
of transcription factor, HoxA9, and the intrinsically disordered region of
nucleoporin protein Nup98, resulting in a protein that both binds DNA and
phase separates. These condensates have been shown to drive aberrant gene
expression and promote cancer progression. Using this biological system,
we combine high resolution highly inclined and laminated optical sheet
(HiLo) microscopy and protein tracking to study how these condensates
change the physical environment of the nucleus, both in terms of changes to
chromatin structure and movement as well as protein localization and
dynamics. We observe that the condensates do not change local chromatin
organization but do slow chromatin movement within. We also find that the
condensates appear to change the organization of chromatin marks and
concentrate leukemia associated methyltransferase, MLL1. These data show
the utility of high-resolution imaging to study how condensates alter the
local nuclear environment and suggest potential mechanisms by which this
might regulate gene expression.
< Back 87
PROTEOLETHARGY IS A PATHOGENIC MECHANISM IN CHRONIC
DISEASE
Ming Zheng1,2, Alessandra Dall’Agnese1, Shannon Moreno1,3, Jesse M

Platt1,4, Tong Ihn Lee1, Richard A Young1,3
1
Whitehead Institute for Biomedical Research, Massachusetts Institute of
Technology, Cambridge, MA, 2Department of Physics, Massachusetts
Institute of Technology, Cambridge, MA, 3Department of Biology,
Massachusetts Institute of Technology, Cambridge, MA, 4Division of
Gastroenterology, Department of Medicine, Massachusetts General
Hospital, Boston, MA
The pathogenic mechanisms of many diseases are well understood at the

molecular level, but there are prevalent syndromes associated with
pathogenic signaling, such as diabetes and chronic inflammation, where our
understanding is more limited. Here we leveraged single-particle tracking
and fluorescence recovery after photobleaching to study physical motion of
protein molecules in cell models of chronic disease. We found pathogenic
signaling suppresses the mobility of a spectrum of proteins that play
essential roles in cellular functions known to be dysregulated in these
chronic diseases. The reduced protein mobility, which we call
proteolethargy, was linked to cysteine residues in the affected proteins and
signaling-related increases in excess reactive oxygen species. Diverse
pathogenic stimuli, including hyperglycemia, dyslipidemia and
inflammation, produce similar reduced protein mobility phenotypes. We
propose that proteolethargy is an overlooked biophysical cause of disease
that may account for various pathogenic features of diverse chronic
syndromes.
< Back 88
NOTES
NOTES
NOTES
NOTES
NOTES
NOTES
Participant List
Dr. Tim Abel Dr. Siu Chiu Chan
UC San Diego Stony Brook University
timabel@web.de siuchiu.chan@stonybrookmedicine.edu
Dr. Santosh Adhikari Dr. Ziyuan Chen

Childrens Hospital of Philadelphia UC Berkeley
adhikaris2@chop.edu ziyuanc@berkeley.edu
Dr. Daniel Anderson Mr. Po-Ta Chen

Eikon Therapeutics Boston Children's Hospital, Harvard
andersond@eikontx.com Medical School
po-ta.chen@childrens.harvard.edu
Prof. Nurit Ballas
Stony Brook University Prof. Ji-Xin Cheng
nurit.ballas@stonybrook.edu Boston University
jxcheng@bu.edu
Mr. Cheng Bi
Purdue University Dr. Heejun Choi
bi19@purdue.edu Janelia Research Campus
choih@janelia.hhmi.org
Dr. Alistair Boettiger
Stanford University Dr. Sarah Cohen
boettiger.alistair@gmail.com University of North Carolina at Chapel Hill
sarahcoh@med.unc.edu
Dr. Christopher Bohrer
NIH/NCI Dr. Robert Coleman
bohrerch@nih.gov Albert Einstein College of Medicine
robert.coleman2@einsteinmed.edu
Andrea Callegari
University of Ulm Ms. Xu Cong
andrea.callegari@uni-ulm.de Icahn School of Medicine at Mount Sinai
xu.cong@mssm.edu
Yessenia Cedeno
Albert Einstein College of Medicine Dr. Robert Coukos
yessenia.cedeno@einsteinmed.edu Northwestern University
RWCOUKOS@GMAIL.COM
Mr. Raafat Chala
SUNY Stony Brook University Prof. Xavier Darzacq
raafat.chalar@stonybrookmedicine.edu University of California, Berkeley
darzacq@berkeley.edu
Mr. Christopher Day Dr. Thomas Graham
NIH/NIEHS University of California, Berkeley
christopher.day@nih.gov tgwgraham@gmail.com
Dr. Wulan Deng Dr. Klaus Hahn

Peking University University of North Carolina Chapel Hill
wdeng@pku.edu.cn khahn@med.unc.edu
Mr. Martin Do Mr. Nayem Haque

University of New South Wales Albert Einstein College of Medicine
z5113049@ad.unsw.edu.au nayem.haque@einsteinmed.edu
Ben Donovan Dr. Amir Hay

NIH UC Berkeley
ben.donovan@nih.gov amirdhay@berkeley.edu
Ms. Samantha Fallacaro Gabriela Hayward-Lara

University of Pennsylvania University of Pennsylvania
samantha@fallacaro.com gabriela.hayward-
lara@pennmedicine.upenn.edu
Dr. Matt Ferguson
Boise State University Mr. John Hobbs IV
mattferguson@boisestate.edu Albert Einstein College of Medicine
john.hobbs@einsteinmed.edu
Dr. Polly Fordyce
Stanford University Madeleine Howell
pfordyce@stanford.edu Harvard University
madeleinehowell@fas.harvard.edu
Dr. Nadezda Fursova
National Cancer Institute Dr. Huaiyu Hu
nadya.fursova@nih.gov Upstate Medical University
huh@upstate.edu
Dr. Gloria Garcia
NIH Dr. Wan-Chen Huang
gloria.garcia@nih.gov Academia Sinica
wchuang1@gate.sinica.edu.tw
Prof. J. Christof Gebhardt
Ulm University Fang Huang
christof.gebhardt@uni-ulm.de Purdue University
huang892@purdue.edu
Prof. Arne Gennerich
Albert Einstein College of Medicine Dr. Dong-Woo Hwang
arne.gennerich@einsteinmed.edu Albert Einstein College of Medicine
dongwoo.hwang@einsteinmed.edu
Dr. Jihee Hwang Dr. Jiwoong Kwon
Boston Children's Hospital Boston Children's Hospital
jihee.hwang@childrens.harvard.edu jiwoong.kwon@childrens.harvard.edu
Mr. Hayato Ito Dr. Melike Lakadamyali

Tokyo Institute of Technology University of Pennsylvania
ito.h.au@m.titech.ac.jp melikel@pennmedicine.upenn.edu
Dr. Stefan Jakobs Dr. Daniel Larson

Max Planck Institute, University of NIH
Gottingen dan.larson@nih.gov
jakobs-lab@gwdg.de
Dr. Luke Lavis
Dr. Chao Jiang Janelia Farm Research Campus
New York University lavisl@janelia.hhmi.org
jiangchao1901@gmail.com
Dr. Wesley Legant
Dr. Rebecca Kaddis Maldonado University of North Carolina at Chapel Hill
Penn State College of Medicine legantw@email.unc.edu
rjk297@psu.edu
Dr. Tineke Lenstra
Dr. Veer Keizer Netherlands Cancer Institute
National Cancer Institute, NIH t.lenstra@nki.nl
veer.keizer@nih.gov
Dr. Weihan Li
Chris Kim Albert Einstein College of Medicine
University of Washington Weihan.Li@einsteinmed.edu
hwanhee@uw.edu
Mr. Ting-Wei Liao
Dr. Sumin Kim Boston Children's Hospital, Harvard
Massachusetts Institute of Technology Medical School
suminkim@mit.edu Ting-Wei.liao@childrens.harvard.edu
Dr. Benjamin Kroeger Dr. Yick Hin Ling

Monash University Johns Hopkins Univeristy
Benjamin.Kroeger@monash.edu yling7@jhu.edu
Dr. Anjani Kumari Dr. Jennifer Lippincott-Schwartz

National Institutes of Health Howard Hughes Medical Inst. Janelia
anjani.kumari@nih.gov Research Campu
lippincottschwartzJ@janelia.hhmi.org
Nemanja Kutlesic
Cold Spring Harbor Laboratory
kutlesic@cshl.edu
Prof. Xuelin Lou Dr. Maria Mukhina
Medical College of Wisconsin University of Maryland, College Park
xulou@mcw.edu mukhina@umd.edu
Ms. Shalini Low-Nam Dr. RK Narayanan

Purdue University Cold Spring Harbor Laboratory
slownam@purdue.edu narayan@cshl.edu
Dr. Ye Ma Prof. Gregor Neuert

Boston Children's Hospital Vanderbilt University
Ye.Ma@childrens.harvard.edu gregor.neuert@vanderbilt.edu
Mr. Maximilian Madern Dr. Attila Oravecz

Hubrecht Insitute Institut de Génétique et de Biologie
m.madern@hubrecht.eu Moléculaire et Cellulaire (IGBMC)
oravecz@igbmc.fr
Kaeli Mathias
Children's Hospital of Philadelphia Dr. Meng Ouyang
kaelim@pennmedicine.upenn.edu Cold Spring Harbor Laboratory
ouyang@cshl.edu
Dr. Hisham Mazal
Max Planck Institute for the Science of Dr. Murali Palangat
Light NIH
hisham.mazal@mpl.mpg.de murali.palangat@nih.gov
Dr. David McSwiggen Dr. Leslie Parent

Eikon Therapeutics Penn State College of Medicine
mcswiggend@eikontx.com lparent@psu.edu
Kirstin Meyer Dr. Heta Patel

Yale University UC Berkeley
kirstin.meyer@yale.edu hetapatel@berkeley.edu
Ms. Mariia Mikhova Sebastian Peck

Michigan State University Eikon Therapeutics
mikhovam@msu.edu pecks@eikontx.com
Prof. Mustafa Mir Dr. Alexandros Pertsinidis

University of Pennsylvania Memorial Sloan Kettering Cancer Center
mirm@chop.edu pertsina@mskcc.org
Dr. Apratim Mukherjee Dr. Fabien Pinaud

Children's Hospital of Philadelphia University of Southern California
mukherjea1@chop.edu pinaud@usc.edu
Ms. Shayna Reed Dr. Diana Stavreva
The Scripps Research Institute/UF Scripps NCI, CCR, NIH
Biomedic stavrevd@mail.nih.gov
shayna.reed@scripps.edu
Dr. Hillary Sussman
Dr. Gang (Gary) Ren Genome Research, Executive Editor
Lawrence Berkeley National Laboratory hsussman@cshl.edu
gren@lbl.gov
Dr. Aleksander Szczurek
Dr. Jonas Ries University of Oxford
European Molecular Biology Laboratory aleksander.szczurek@bioch.ox.ac.uk
(EMBL) - Heidelberg
ries@embl.de Dr. Li-Chun Tu
Ohio State University
Dr. Joseph Rodriguez
tu.277@osu.edu
National Institute of Environmental Health
Science
Dr. Srigokul Upadhyayula
Joseph.Rodriguez@nih.gov
University of California, Berkeley
sup@berkeley.edu
Dr. Gavin Schlissel
Whitehead Institute
Sofia Vargas
gschliss@gmail.com
Rice University
xjv1@rice.edu
Dr. Jens Schmidt
Michigan State University
Dr. Sarah Veatch
schmi706@msu.edu
University of Michigan
sveatch@umich.edu
Dr. Robert Singer
Albert Einstein College of Medicine
Lexy von Diezmann
robert.singer@einsteinmed.edu
University of Minnesota
lvondiez@umn.edu
Dr. David Solecki
St. Jude Children's Research Hospital
Dr. Vladana Vukojevic
david.solecki@stjude.org
Karolinska Institutet
vladana.vukojevic@ki.se
Dr. Linda Song
Eikon Therapeutics, Inc.
Dr. Yihan Wan
songl@eikontx.com
Westlake University
wanyihan@westlake.edu.cn
Prof. Timothy Stasevich
Colorado State University
Dr. Mei Wang
tim.stasevich@colostate.edu
Cold Spring Harbor Laboratory
meiwang@cshl.edu
Mr. Paul Welzl Ms. Angela Yung
Research Institute of Molecular Pathology Eikon Therapeutics
(IMP) yunga@eikontx.com
paul.welzl@imp.ac.at
Mr. Ming Zheng
Prof. Jiahui (Chris) Wu Whitehead Institute
University Of Massachusetts ming@wi.mit.edu
jiahuiwu@umass.edu
David Zimmerman
Dr. Bin Wu Cold Spring Harbor Laboratory
Johns Hopkins University School of zimmerman@cshl.edu
Medicine
bwu20@jhmi.edu
Dr. Jie Xiao

John Hopkins University
xiao@jhmi.edu
Ms. Yali Xu
RIKEN
yali.xu@riken.jp
Dr. Pengning Xu
UNC Chapel Hill
pengning@email.unc.edu
Han Yang
Yale University
han.yang@yale.edu
Ms. Chelsea Yang

University of North Carolina, Chapel Hill
cheyang@ad.unc.edu
Dr. Johannes Yeh

CSHL
jyeh@cshl.edu
Prof. Young Yoon

Albert Einstein College of Medicine
young.yoon@einsteinmed.edu
CODE OF CONDUCT FOR ALL PARTICIPANTS IN CSHL MEETINGS
Cold Spring Harbor Laboratory (CSHL or the Laboratory) is dedicated to
pursuing its twin missions of research and education in the biological
sciences. The Laboratory is committed to fostering a working environment
that encourages and supports unfettered scientific inquiry and the free and
open exchange of ideas that are the hallmarks of academic freedom. To this
end, the Laboratory aims to maintain a safe and respectful environment that
is free from harassment and discrimination for all attendees of our meetings
and courses as well as associated support staff, in accordance with federal,
state and local laws.
Consistent with the Laboratory's missions, commitments and policies, the
purpose of this Code is to set forth expectations for the professional conduct
of all individuals participating in the Laboratory's meetings program, both in
person and virtually, including organizers, session chairs, invited speakers,
presenters, attendees and sponsors. This Code’s prohibition against
discrimination and harassment is consistent with the Laboratory’s internal
policies governing conduct by its own faculty, trainees, students and
employees.
By registering for and attending a CSHL meeting, either in person or
virtually, participants agree to:
1. Treat fellow meeting participants and CSHL staff with respect, civility
and fairness, without bias based on sex, gender, gender identity or
expression, sexual orientation, race, ethnicity, color, religion, nationality
or national origin, citizenship status, disability status, veteran status,
marital or partnership status, age, genetic information, or any other
criteria prohibited under applicable federal, state or local law.
2. Use all CSHL facilities, equipment, computers, supplies and resources
responsibly and appropriately if attending in person, as you would at
your home institution.
3. Abide by the CSHL Meeting Alcohol Policy (see below).
Similarly, meeting participants agree to refrain from:

1. Harassment and discrimination, either in person or online, in violation
of Laboratory policy based on actual or perceived sex, pregnancy
status, gender, gender identity or expression, sexual orientation, race,
ethnicity, color, religion, creed, nationality or national origin,
immigration or citizenship status, mental or physical disability status,
veteran status, military status, marital or partnership status, marital or
partnership status, familial status, caregiver status, age, genetic
information, status as a victim of domestic violence, sexual violence, or
stalking, sexual reproductive health decisions, or any other criteria
prohibited under applicable federal, state or local law.
2. Sexual harassment or misconduct.
3. Disrespectful, uncivil and/or unprofessional interpersonal behavior,
either in person or online, that interferes with the working and learning
environment.
4. Misappropriation of Laboratory property or excessive personal use of
resources, if attending in person.
BREACHES OR VIOLATIONS OF THE CODE OF CONDUCT
Cold Spring Harbor Laboratory aims to maintain in-person and virtual
conference environments that accord with the principles and expectations
outlined in this Code of Conduct. Meeting organizers are tasked with
providing leadership during each meeting, and may be approached
informally about any breach or violation. Breaches or violations should also
be reported to program leadership in person or by email:
• Dr. David Stewart, Grace Auditorium Room 204, 516-367-8801 or

x8801 from a campus phone, stewart@cshl.edu
• Dr. Charla Lambert, Hershey Laboratory Room 214, 516-367-5058

or x5058 from a campus phone, clambert@cshl.edu
Reports may be submitted by those who experience
harassment or discrimination as well as by those who
witness violations of the behavior laid out in this Code.
The Laboratory will act as needed to resolve the
matter, up to and including immediate expulsion of the offending
participant(s) from the meeting, dismissal from the Laboratory, and exclusion
from future academic events offered by CSHL.
If you have questions or concerns, you can contact the meeting organizers,
CSHL staff.
For meetings and courses funded by NIH awards:
Participants may contact the Health & Human Services Office for Civil
Rights (OCR). See this page for information on filing a civil rights complaint
with the OCR; filing a complaint with CSHL is not required before filing a
complaint with OCR, and seeking assistance from CSHL in no way prohibits
filing complaints with OCR. You may also notify NIH directly about sexual
harassment, discrimination, and other forms of inappropriate conduct at NIH-
supported events.
For meetings and courses funded by NSF awards:
Participants may file a complaint with the NSF. See this page for information
on how to file a complaint with the NSF.
Law Enforcement Reporting:
• For on-campus incidents, reports to law enforcement can be made to

the Security Department at 516-367-5555 or x5555 from a campus
phone.
• For off-campus incidents, report to the local department where the

incident occurred.
In an emergency, dial 911.
DEFINITIONS AND EXAMPLES
Uncivil/disrespectful behavior is not limited to but may take the following
forms:
• Shouting, personal attacks or insults, throwing objects, and/or

sustained disruption of talks or other meeting-related events
Harassment is any unwelcome verbal, visual, written, or physical conduct
that occurs with the purpose or effect of creating an intimidating, hostile,
degrading, humiliating, or offensive environment or unreasonably interferes
with an individual’s work performance. Harassment is not limited to but may
take the following forms:
• Threatening, stalking, bullying, demeaning, coercive, or hostile acts

that may have real or implied threats of physical, professional, or
financial harm
• Signs, graphics, photographs, videos, gestures, jokes, pranks,
epithets, slurs, or stereotypes that comment on a person’s sex,
gender, gender identity or expression, sexual orientation, race,
ethnicity, color, religion, nationality or national origin, citizenship
status, disability status, veteran status, marital or partnership status,
age, genetic information, or physical appearance
Sexual Harassment includes harassment on the basis of sex, sexual
orientation, self-identified or perceived sex, gender expression, gender
identity, and the status of being transgender. Sexual harassment is not
limited to sexual contact, touching, or expressions of a sexually suggestive
nature. Sexual harassment includes all forms of gender discrimination
including gender role stereotyping and treating employees differently
because of their gender. Sexual misconduct is not limited to but may take
the following forms:
• Unwelcome and uninvited attention, physical contact, or inappropriate

touching
• Groping or sexual assault
• Use of sexual imagery, objects, gestures, or jokes in public spaces or
presentations
• Any other verbal or physical contact of a sexual nature when such
conduct creates a hostile environment, prevents an individual from
fulfilling their professional responsibilities at the meeting, or is made a
condition of employment or compensation either implicitly or explicitly
MEETING ALCOHOL POLICY
Consumption of alcoholic beverages is not permitted in CSHL's public areas
other than at designated social events (wine and cheese reception, picnic,
banquet, etc.), in the Blackford Bar, or under the supervision of a licensed
CSHL bartender.
No provision of alcohol by meeting sponsors is permitted unless arranged
through CSHL.
Meeting participants consuming alcohol are expected to drink only in
moderation at all times during the meeting.
Excessive promotion of a drinking culture at any meeting is not acceptable
or tolerated by the Laboratory. No meeting participant should feel pressured
or obliged to consume alcohol at any meeting-related event or activity.
VISITOR INFORMATION
EMERGENCY (to dial outside line, press 3+1+number)

CSHL Security 516-367-8870 (x8870 from house phone)
CSHL Emergency 516-367-5555 (x5555 from house phone)
Local Police / Fire 911
Poison Control (3) 911
CSHL SightMD Center for Health and 516-422-4422

Wellness (call for appointment)
Dolan Hall, East Wing, Room 111 x4422 from house
phone
cshlwellness@northwell.edu
Emergency Room
Huntington Hospital 631-351-2000
270 Park Avenue, Huntington
Dentists
Dr. William Berg 631-271-2310
Dr. Robert Zeman 631-271-8090
Drugs - 24 hours, 7 days
Rite-Aid 631-549-9400
391 W. Main Street, Huntington
GENERAL INFORMATION
Meetings & Courses Main Office

Hours during meetings: M-F 9am – 9pm, Sat 8:30am – 1pm
After hours – See information on front desk counter
For assistance, call Security at 516-367-8870
(x8870 from house phone)
Dining, Bar
Blackford Dining Hall (main level):
Breakfast 7:30–9:00, Lunch 11:30–1:30, Dinner 5:30–7:00
Blackford Bar (lower level): 5:00 p.m. until late
House Phones
Grace Auditorium, upper / lower level; Cabin Complex; Blackford
Hall; Dolan Hall, foyer
Books, Gifts, Snacks, Clothing

CSHL Bookstore and Gift Shop
516-367-8837 (hours posted on door)
Grace Auditorium, lower level.
Computers, E-mail, Internet access

Grace Auditorium
Upper level: E-mail and printing in the business center area
WiFi Access: GUEST (no password)
Announcements, Message Board Mail, ATM, Travel info

Grace Auditorium, lower level
Russell Fitness Center
Dolan Hall, east wing, lower level
PIN#: (On your registration envelope)
Laundry Machines
Dolan Hall, lower level
Photocopiers, Journals, Periodicals, Books

CSHL Main Library
Open 24 hours (with PIN# or CSHL ID)
Staff Hours: 9:00 am – 9:00 pm
Use PIN# (On your registration envelope) to enter Library
See Library staff for photocopier code.
Library room reservations (hourly) available on request between
9:00 am – 9:00 pm
Swimming, Tennis, Jogging, Hiking

June–Sept. Lifeguard on duty at the beach. 12:00 noon–6:00 p.m.
Two tennis courts open daily.
Local Interest
Fish Hatchery 631-692-6758
Sagamore Hill 516-922-4788
Whaling Museum 631-367-3418
Heckscher Museum 631-351-3250
CSHL DNA Learning x 5170
Center
New York City

Helpful tip -
Take CSHL Shuttle OR Uber/Lyft/Taxi to Syosset Train Station
Long Island Railroad to Penn Station
Train ride about one hour.
TRANSPORTATION
Limo, Taxi
Syosset Limousine 516-364-9681
Executive Limo Service 516-826-8172
Limos Long Island 516-400-3364
Syosset Taxi 516-921-2141

Orange & White Taxi 631-271-3600
Uber / Lyft
Trains
Long Island Rail Road 718-217-LIRR (5477)
Amtrak 800-872-7245
MetroNorth 877-690-5114
New Jersey Transit 973-275-5555
CSHL Campus Map
9
-- Meetings and Courses Locations --
Cold
Spring 1. Grace Auditorium 39. Cole Cottage
Harbor 3. Nicholls Biondi Haff 41. Glass Cabin
Laboratory
5. Bush Lecture Hall 42. Eagle Cabin
6. Blackford Dining Hall 43. Stahl Cabin
6. Blackford Residence 44. Luria Cabin
8. Hooper House 45. Stent Cabin
10. Delbruck 46. Boyer Cabin
47. Maniatis Cabin
48. Alumni Cabin
49. Zinder Cabin
50. Wendt Cabin
38. Williams House 51. Pall Cabin
0 e Laboratories
e Facilities Support eoffice
e Housing e Public Assembly
b. Meetings & Courses b. Clarkson
Bookstore & Gift Shop Racker
Lockers Mayr
ATM Bar
b. Poster Pavillions b, Fitness Center
Health Center
Laundry
Parking Hiking Walking

Trail Path
I
I
/fl
Main campus
1 Bungtown Road.
Cold Spring Harbor, NY 11724 1,,Sr4L raph Arts :Jl,t,024

Onemol2024 Abstractbook Cshl2024 Single Biomolecules

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Abstracts of papers presented

at the 2024 meeting on

Xavier Darzacq, University of California, Berkeley

Estée Lauder Companies

Front Cover: Super resolution image of a single molecule of translating

Wednesday 7:30 pm – 10:00 pm 1 Imaging Tools and Platforms

Thursday 9:00 am – 12:00 pm 2 Organelles

Thursday 2:00 pm – 5:00 pm 3 Cytoplasm

Thursday 5:00 pm Wine & Cheese Party

Thursday 7:30 pm – 10:30 pm Poster Session

Friday 9:00 am – 12:00 pm 4 Nucleus

Friday 2:00 pm – 5:00 pm 5 Membranes

Friday 6:00 pm Banquet

Saturday 9:00 am – 12:00 pm 6 Super Resolution Imaging

All times shown are US Eastern: Time Zone Converter

Mealtimes at Blackford Hall are as follows:

Bar is open from 5:00 pm until late

Abstracts are the responsibility of the author(s) and publication of an

These abstracts should not be cited in bibliographies. Material herein

Please note that photography or video/audio recording of oral presentations

Any discussion via social media platforms of material presented at this

Printed on 100% recycled paper.

WEDNESDAY, October 23—7:30 PM

SESSION 1 IMAGING TOOLS AND PLATFORMS

Chairperson: Jennifer Lippincott-Schwartz, HHMI Janelia Research

Designing brighter dyes for advanced fluorescence microscopy

SPECTR - seeing conformational changes of single molecules in

SPTNet—A deep learning based framework for end-to-end single

Non-averaged single-molecule 3D structure revealed by

Mapping the directionality of glucocorticoid receptor

Leveraging microfluidics for high-throughput single-molecule

in situ force mapping to quantify and visualize mechanical

Systematic nascent RNA kinetics imaging unveils principles for

THURSDAY, October 24—9:00 AM

Chairperson: Dan Larson, National Cancer Institute, Bethesda,

Illuminating organelle dynamics during differentiation and

Vibrational photothermal fluorescence imaging of cellular

Translation of mRNAs encoding membrane proteins regulated by

The splicing factor U2AF regulates the translation and

Direct visualization of retrotranslocation by the Hrd1 complex by

Exploring structure and function of non-canonical SMC protein

Charting live cell 4D chromatin landscapes—Unraveling neuronal

Chairperson: Luke Lavis, HHMI Janelia Farm Research Campus,

Harnessing AI-driven protein design to rapidly generate

Rapid transcription repression induced by DNA double-strand

Kinesin-14 HSET and KlpA are non-processive microtubule

Single-molecule imaging of the endogenous Camk2a mRNA

YAP signal integration through charge-mediated transcriptional

Capturing the transcriptional patterning of β-actin in the mouse

Phosphorylation of FMRP granules alters their transport, protein

Nanoscale volumetric fluorescence imaging via photochemical

THURSDAY, October 24—5:00 PM

Wine and Cheese Party

THURSDAY, October 24—7:30 PM

See. p. xiv for List of Posters

Chairperson: Melike Lakadamyali, Perelman School of Medicine,

Transcription factor exchange enables prolonged transcriptional

Studying the dynamic regulation of transcription initiation and

Gene-specific hub kinetics regulate transcription factor

Effective in vivo binding energy landscape illustrates kinetic

Sho Oasa1, Stanko N Nikolić1,2,3, Aleksandar J Krmpot*1,2,3, Lars