Interaction of leukemia blasts with the bone marrow extracellular matrix often results in protect... more Interaction of leukemia blasts with the bone marrow extracellular matrix often results in protection of leukemia cells from chemotherapy and in persistence of the residual disease which is on the basis of subsequent relapses. The adhesion signaling pathways have been extensively studied in adherent cells as well as in mature haematopoietic cells, but the adhesion structures and signaling in haematopoietic stem and progenitor cells, either normal or malignant, are much less explored. We analyzed the interaction of leukemia cells with fibronectin (FN) using interference reflection microscopy, immunofluorescence, measurement of adherent cell fraction, real-time microimpedance measurement and live cell imaging. We found that leukemia cells form very dynamic adhesion structures similar to early stages of focal adhesions. In contrast to adherent cells, where Src family kinases (SFK) belong to important regulators of focal adhesion dynamics, we observed only minor effects of SFK inhibitor dasatinib on leukemia cell binding to FN. The relatively weak involvement of SFK in adhesion structure regulation might be associated with the lack of cytoskeletal mechanical tension in leukemia cells. On the other hand, active Lyn kinase was found to specifically localize to leukemia cell adhesion structures and a less firm cell attachment to FN was often associated with higher Lyn activity (this unexpectedly occurred also after cell treatment with the inhibitor SKI-1). Lyn thus may be important for signaling from integrin-associated complexes to other processes in leukemia cells.
Mutations of the gene for nucleophosmin (NPM1) are the most frequent genetic aberration in patien... more Mutations of the gene for nucleophosmin (NPM1) are the most frequent genetic aberration in patients with acute myeloid leukemia (AML). The mechanism of leukemic transformation in this leukemia subtype is not fully understood, but aberrant cytoplasmic localization of mutated NPM (NPMmut) is widely considered as an important factor for leukemia manifestation. We analyzed the subcellular localization of three types of NPM with a C-terminal mutation (A, B and E). Genes for the individual NPM forms were fused with a gene for one of fluorescent protein variants in plasmids, which were transfected into three cell lines with different endogenous NPM expression. Subcellular localization of the fluorescent proteinlabeled NPM was further correlated with the relative expression of all NPM forms. We confirmed a high cytoplasmic expression of NPMmutA and NPMmutB whereas a substantial fraction of NPMmutE was found to be localized in nucleoli. Moreover, we revealed that the localization of fluorescently labeled NPM is affected by the interaction between various forms of the protein.
bioRxiv (Cold Spring Harbor Laboratory), Aug 25, 2020
P21-activated kinases (PAK) regulate many processes associated with cytoskeleton dynamics, includ... more P21-activated kinases (PAK) regulate many processes associated with cytoskeleton dynamics, including cell adhesion, migration, and apoptosis. PAK function is frequently altered in cancer, and PAK were proposed as therapy targets both in solid tumors and in hematological malignancies. However, current knowledge about PAK function in cell adhesion is mainly based on adherent cell models. Moreover, existing functional differences among the individual PAK family members are unsufficiently characterized. We measured expression of PAK group I members in leukemia cell lines and in primary leukemia cells, both on protein and mRNA levels. In functional assays, we analyzed the effect of two PAK inhibitors with different mechanisms of action, IPA-3 and FRAX597. Changes in cell interaction with fibronectin were monitored through impedance measurement and by interference reflection microscopy. Cytotoxic effects of inhibitors were assessed by Annexin V/propidium iodide test. PAK intracellular localization was analyzed by confocal microscopy. PAK2 transcript was dominant in cell lines, whereas primary leukemia cells also expressed comparable amount of PAK1, which was detected as two transcription isoforms: PAK1-full and PAK1Δ15. PAK1Δ15 and PAK2 transcript levels correlated with surface density of integrins β1 and αVβ3. PAK1-full, but not PAK2, was present in membrane protrusions. The inhibitors had partly opposed effects: IPA-3, which prevents PAK activation, induced cell contraction in semi-adherent HEL cells only. FRAX597, which inhibits PAK kinase activity, increased cell-surface contact area in all leukemia cells. Both inhibitors reduced the stability of cell attachment and induced cell death. Although many cells accumulated high FRAX597 amounts, low doses were sufficient to kill sensitive cells. FRAX597-induced cell death was fast in the MV4-11 cell line and in primary AML cells. Although PAK group I seem to be essential for leukemia cell adhesion and survival, and might thus serve as therapy targets, many PAK functions still remain to be attributed to individual isoforms and to their functional domains.
The rate and extent of uptake of the fluorescent probe diS-C3(3) reporting on membrane potential ... more The rate and extent of uptake of the fluorescent probe diS-C3(3) reporting on membrane potential in S. cerevisiae is affected by the strain under study, cell-growth phase, starvation and by the concentration of glucose both in the growth medium and in the monitored cell suspension under non-growth conditions. Killer toxin K1 brings about changes in membrane potential. In all types of cells tested, viz. in glucose-supplied stationary or exponential cells of the killer-sensitive strain $6/1 or a conventional strain RXII, or in glucose-free exponential cells of both strains, both active and heat-inactivated toxin slow down the potential-dependent uptake of diS-C3(3) into the cells. This may reflect "clogging" of pores in the cell wall that hinders, but does not prevent, probe passage to the plasma membrane and its equilibration. The clogging effect of heatinactivated toxin is stronger than that exerted by active toxin. In susceptible cells, Le. in exponential-phase glucose-supplied ceils of the sensitive strain $6/1, this phase of probe uptake retardation is followed by an irreversible red shift in probe fluorescence maximum)l.ma. x indicating damage to membrane integrity and cell permeabilization. A similar fast red shift in 2max signifying lethal cell damage was found in heat-killed or nystatin-treated cells.
Biochimica Et Biophysica Acta - General Subjects, 2019
BACKGROUND EGFP is a fluorescent tag extensively used in biological and biomedical research. Over... more BACKGROUND EGFP is a fluorescent tag extensively used in biological and biomedical research. Over the years many researches have gathered collections of cell lines bearing specific EGFP-tagged proteins. Despite its popularity some photochemical properties of EGFP remain undocumented and unused. We report on so far unexplored lifetime photoconversion of EGFP usable in FLIM. METHODS Fluorescence lifetime imaging and spectral FLIM has been used for characterization of the EGFP photoconversion and protein tracking. RESULT Our data suggest that EGFP can be permanently photoconverted to a short-fluorescencelifetime form (PC-EGFP) by intense blue irradiation. PC-EGFP cannot be reverted back by 405 nm light and exhibits the same spectral emission properties with blue-shifted absorption compared to the unconverted EGFP. Fluorescence of PC-EGFP is pH-independent and the photoconversion efficiency decreases with the solvent viscosity. Utilization of the EGFP photoconversion was demonstrated by tracking of a nucleophosmin mutant in live HEK 293T cells during its cytoplasm-nuclear relocalization induced by Leptomycin B. CONCLUSIONS Besides potential FLIM artifacts caused by an unintended EGFP photoconversion, the controlled photoconversion turns EGFP to an excellent tool for kinetic FLIM applications. Since the photoconversion occurs in the lifetime domain, PC-EGFP can be easily distinguished from the unconverted tag by time-resolved detection while all other spectral channels stay free for multicolor labeling.
The International Journal of Biochemistry & Cell Biology, Oct 1, 2018
Highlights Leukemia-related mutation of nucleophosmin disrupts its interaction with nucleolin ... more Highlights Leukemia-related mutation of nucleophosmin disrupts its interaction with nucleolin Stability of nucleophosmin oligomers is attenuated by C-terminal mutation All-trans retinoic acid stabilizes nucleophosmin oligomers Nucleolin/nucleophosmin ratio is enhanced in patients with nucleophosmin mutation Abstract C-terminal mutations of the nucleolar protein nucleophosmin (NPM) are the most frequent genetic aberration detected in acute myeloid leukemia (AML) with normal karyotype. The mutations cause aberrant cytoplasmic localization of NPM and lead to loss of functions associated with NPM nucleolar localization, e.g. in ribosome biogenesis or DNA-damage repair. NPM has many interaction partners and some of them were proved to interact also with the mutated form (NPMmut) and due to this interaction thereby to be withdrawn from their site of action. We analyzed the impact of the mutation on NPM interaction with nucleolin (NCL) which is also prevalently localized into the nucleolus and cooperates with wild-type NPM (NPMwt) in many cellular processes. We revealed that the NCL-NPM complex formation is completely abolished by the mutation and that the presence/absence of the interaction is not affected by drugs causing genotoxic stress or differentiation. Deregulation resulting from changes of NCL/NPMwt ratio may contribute to leukemogenesis.
Nucleophosmin (NPM) mutations causing its export from the nucleoli to the cytoplasm are frequent ... more Nucleophosmin (NPM) mutations causing its export from the nucleoli to the cytoplasm are frequent in acute myeloid leukemia (AML). Due to heterooligomerization of wild type NPM with the AML-related mutant, the wild-type becomes misplaced from the nucleoli and its functions are significantly altered. Dissociation of NPM heterooligomers may thus restore the proper localization and function of wildtype NPM. NSC348884 is supposed to act as a potent inhibitor of NPM oligomerization. The effect of NSC348884 on the NPM oligomerization was thoroughly examined by fluorescence lifetime imaging with utilization of FRET and by a set of immunoprecipitation and electrophoretic methods. Leukemiaderived cell lines and primary AML cells as well as cells transfected with fluorescently labeled NPM forms were investigated. Our results clearly demonstrate that NSC348884 does not inhibit formation of NPM oligomers neither in vivo nor in vitro. Instead, we document that NSC348884 cytotoxicity is rather associated with modified cell adhesion signaling. The cytotoxic mechanism of NSC348884 has therefore to be reconsidered. Abbreviations NPMwt Wild-type nucleophosmin NPMmut Nucleophosmin with AML-associated mutation type A C21 Cysteine 21 in nucleophosmin C21A Substitution of C21 to alanine C21F Substitution of C21 to phenylalanine Δ25 Nucleophosmin with deletion of the first 25 amino acids Δ100 Nucleophosmin with deletion of the first 100 amino acids Δ117 Nucleophosmin with deletion of the first 117 amino acids G_ EGFP-labeled protein at the N-terminus R_ MRFP1-labeled protein at the N-terminus The human NPM1 gene is located on the chromosome 5q35 and encodes a 32.6 kDa polypeptide. Nucleophosmin (NPM), encoded by the NPM1 gene, is a ubiquitously expressed phosphoprotein residing predominantly in the granular component of the nucleolus and shuttling dynamically among nucleoli, the nucleoplasm and the cytoplasm 1-4. It functions as a chaperone 5 and is engaged in regulation of various cellular processes including the ribosome biogenesis 6 , DNA-damage repair 7 , centrosome duplication 8 , and DNA replication 9. Furthermore, NPM is involved in apoptosis and can modulate p53 stability and activity 1,10. NPM overexpression, fusion, or mutation have oncogenic potential and are associated with cancer progression in many types of solid tumors 11 and in hematopoietic malignancies 12-17. Acute myeloid leukemia (AML) with mutated NPM1 accounts for about 1/3 of de novo adult AML, and NPM1 is the most frequently mutated gene in AML with normal karyotype (50-60% incidence) 12. To date, more than 100 NPM1 mutation types have been identified in AML, occurring almost exclusively in the last exon (exon 12) of the gene 18. A nucleotide insertion and/or deletion (indel mutations) lead to the frame shift in the region encoding the C-terminus of NPM. All these NPM1 mutations result in loss of tryptophan residues at positions 288 and/or 290, which form the main part of the nucleolar localization signal (NoLS) ensuring nucleolar localization of the wild-type NPM (NPMwt). Moreover, the indel mutations frequently generate an additional nuclear export sequence (NES), which labels the mutated protein for the nuclear exporter XPO1 and targets NPM into the cytoplasm 19. Both the loss of NoLS and
P21-activated kinases (PAK) are key effectors of the small GTPases Rac1 and Cdc42, as well as of ... more P21-activated kinases (PAK) are key effectors of the small GTPases Rac1 and Cdc42, as well as of Src family kinases. In particular, PAK1 has several well-documented roles, both kinase-dependent and kinase-independent, in cancer-related processes, such as cell proliferation, adhesion, and migration. However, PAK1 properties and functions have not been attributed to individual PAK1 isoforms: besides the full-length kinase (PAK1-full), a splicing variant lacking the exon 15 (PAK1Δ15) is annotated in protein databases. In addition, it is not clear if PAK1 and PAK2 are functionally overlapping. Using fluorescently tagged forms of human PAK1-full, PAK1Δ15, and PAK2, we analyzed their intracellular localization and mutual interactions. Effects of PAK inhibition (IPA-3, FRAX597) or depletion (siRNA) on cell-surface adhesion were monitored by real-time microimpedance measurement. Both PAK1Δ15 and PAK2, but not PAK1-full, were enriched in focal adhesions, indicating that the C-terminus might be important for PAK intracellular localization. Using coimmunoprecipitation, we documented direct interactions among the studied PAK group I members: PAK1 and PAK2 form homodimers, but all possible heterocomplexes were also detected. Interaction of PAK1Δ15 or PAK2 with PAK1-full was associated with extensive PAK1Δ15/PAK2 cleavage. The impedance measurements indicate, that PAK2 depletion slows down cell attachment to a surface, and that PAK1-full is involved in cell spreading. Altogether, our data suggest a complex interplay among different PAK group I members, which have non-redundant functions. PAK (p21-activated kinases) are a group of serine-threonine kinases originally identified as downstream effectors of p21 proteins, specifically of the Ras-related GTPases Rac1 and Cdc42 1,2. The initial phase of PAK research focused on their role as small GTPase effectors in the context of dynamic remodelling of the cytoskeleton and of cell adhesion structures 3. Later on, PAK were found to be involved in many cancer-related processes in different tumour types 4,5. In parallel, the discovery of PAK1 nuclear localization 6 prompted the analysis of PAK functions in the cell nucleus. The human PAK family is divided into the group I (PAK1 to PAK3) and group II (PAK4 to PAK6). In general, these kinases regulate the cytoskeleton dynamics, intracellular signaling, and gene expression 7. The current knowledge about PAK group I is derived mostly from adherent cell models, where PAK activity usually correlates with increased cell motility. In this area, the research was mainly focused to PAK1, although some important differences between PAK1 and PAK2 have been reported 8. In addition, PAK are known regulators of a wide range of cellular processes, including the dynamics of actin structures and microtubules, cell division, apoptosis, and adhesion to the extracellular matrix. Despite considerable sequence homology within PAK group I, the individual members appear to have distinct function in cell physiology. Whereas PAK2 is more or less ubiquitiously expressed, PAK1 expression is more restricted in adulthood, and PAK3 is expressed mainly in the brain 9. PAK2 and PAK4 are essential during embryonic development, since knockouts are embryonic lethal, at least in mice 9. Group I PAK share some domains that are not present in the group II members 10,11. In particular, the autoinhibitory domain (AID) is important for regulation of the kinase activity of the group I family members. The regulatory mechanism was described on the basis of PAK1 crystal structure 12 , and is likely valid also for PAK2 and PAK3. PAK1 forms homodimers in a trans arrangement, the AID of one molecule interacting with the kinase domain of its partner molecule 13,14. In this closed conformation, the kinase activity is very low. Binding of a
Enhanced green fluorescence protein (EGFP) is a fluorescent tag commonly used in cellular and bio... more Enhanced green fluorescence protein (EGFP) is a fluorescent tag commonly used in cellular and biomedical applications. Surprisingly, some interesting photochemical properties of EGFP have remained unexplored. Here we report on two-photon-induced photoconversion of EGFP, which can be permanently converted by intense IR irradiation to a form with a short fluorescence lifetime and spectrally conserved emission. Photoconverted EGFP thus can be distinguished from the unconverted tag by the time-resolved detection. Nonlinear dependence of the two-photon photoconversion efficiency on the light intensity allows for an accurate 3D localization of the photoconverted volume within cellular structures, which is especially useful for kinetic FLIM applications. For illustration, we used the two photon photoconversion of EGFP for measurements of redistribution kinetics of nucleophosmin and histone H2B in nuclei of live cells. Measurements revealed high mobility of fluorescently tagged histone H2B ...
Nucleophosmin (NPM) interaction with tumor suppressor p53 is a part of a complex interaction netw... more Nucleophosmin (NPM) interaction with tumor suppressor p53 is a part of a complex interaction network and considerably affects cellular stress response. The impact of NPM1 mutations on its interaction with p53 has not been investigated yet, although consequences of NPMmut-induced p53 export to the cytoplasm are important for understanding the oncogenic potential of these mutations. We investigated p53-NPM interaction in live HEK-293T cells by FLIM-FRET and in cell lysates by immunoprecipitation. eGFP lifetime-photoconversion was used to follow redistribution dynamics of NPMmut and p53 in Selinexor-treated cells. We confirmed the p53-NPMwt interaction in intact cells and newly documented that this interaction is not compromised by the NPM mutation causing displacement of p53 to the cytoplasm. Moreover, the interaction was not abolished for non-oligomerizing NPM variants with truncated oligomerization domain, suggesting that oligomerization is not essential for interaction of NPM forms...
One way how to study individual processes that underlie the complex behaviour of living systems i... more One way how to study individual processes that underlie the complex behaviour of living systems is to construct artificial structures resembling those that compose the living systems. By using such a structure, the process of interest can be studied and characterised separately, e.g. with regard to its physical basis, and the basic principles applicable to living systems can be postulated. Of course, such approach provides only partial information the importance of which has to be considered with respect to the complexity of living systems. In this study, we want to show that liposomes or proteoliposomes offer a useful tool for testing fluorescent probes used for studying living cells, chapter 2, for studying membrane transport systems, chapter 3, or for modelling transport processes across the membrane, chapter 4.Available from STL Prague, CZ / NTK - National Technical LibrarySIGLECZCzech Republi
P21-activated kinases (PAK) regulate processes associated with cytoskeleton dynamics. PAK express... more P21-activated kinases (PAK) regulate processes associated with cytoskeleton dynamics. PAK expression in leukemia cells was measured on protein and mRNA levels. In functional assays, we analyzed the effect of PAK inhibitors IPA-3 and FRAX597 on cell adhesivity and viability. PAK2 was dominant in cell lines, whereas primary cells also expressed comparable amount of PAK1 transcription isoforms: PAK1-full and PAK1Δ15. PAK1Δ15 and PAK2 levels correlated with surface density of integrins β1 and αVβ3. PAK1-full, but not PAK2, was present in membrane protrusions. IPA-3, which prevents PAK activation, induced cell contraction in semi-adherent HEL cells only. FRAX597, which inhibits PAK kinase activity, increased cell-surface contact area in all leukemia cells. Both inhibitors reduced the stability of cell attachment and induced cell death.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2003
Potential-sensitive fluorescent probes oxonol V and oxonol VI were employed for monitoring membra... more Potential-sensitive fluorescent probes oxonol V and oxonol VI were employed for monitoring membrane potential (Dw) generated by the Schizosaccharomyces pombe plasma membrane H +-ATPase reconstituted into vesicles. Oxonol VI was used for quantitative measurements of the Dw because its response to membrane potential changes can be easily calibrated, which is not possible with oxonol V. However, oxonol V has a superior sensitivity to Dw at very low concentration of reconstituted vesicles, and thus it is useful for testing quality of the reconstitution. Oxonol VI was found to be a good emission-ratiometric probe. We have shown that the reconstituted H +-ATPase generates Dw of about 160 mV on the vesicle membrane. The generated Dw was stable at least over tens of minutes. An influence of the H + membrane permeability on the Dw buildup was demonstrated by manipulating the H + permeability with the protonophore CCCP. Ratiometric measurements with oxonol VI thus offer a promising tool for studying processes accompanying the yeast plasma membrane H +-ATPasemediated Dw buildup.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1997
A novel fluorescent technique for direct assessment of membrane potential was tested on suspensio... more A novel fluorescent technique for direct assessment of membrane potential was tested on suspensions of large Ž. unilamellar vesicles LUV. The method is based on monitoring shifts in the fluorescence maximum, l , of the max Ž. redistribution dye diSC 3 caused by dye binding to the LUV membrane. A theory describing the behavior of this dye in 3 LUV suspensions was elaborated and tested. The diffusion potentials across the LUV membrane were adjusted by ion gradients in the absence of valinomycin. When using KCl and choline chloride without valinomycin the potential can be set as high as y70 mV. These potentials exhibited long-term stability and the theory allowed to determine the upper limits of membrane permeabilities for Cl y , choline cations, protons and hydroxyls relative to the K q permeability. The absolute values of membrane potential set by ion gradients were calibrated using valinomycin. The monitoring of the l shift max permitted us to show real-time changes in membrane potential, since addition of valinomycin to the LUV was followed by an immediate depolarization. The setting of the potential and the dye re-equilibration after valinomycin addition took place within a second.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2012
FLIM-photobleaching technique Homo/hetero di(oligo)mers Plasma membrane protein interaction Donor... more FLIM-photobleaching technique Homo/hetero di(oligo)mers Plasma membrane protein interaction Donor lifetime Each of the three plasma membrane Ato proteins is involved in ammonium signalling and the development of yeast colonies. This suggests that although these proteins are homologous, they do not functionally substitute for each other, but may form a functional complex. Here, we present a detailed combined FRET, FLIM and photobleaching study, which enabled us to detect interactions between Ato proteins found in distinct compartments of yeast cells. We thus show that the proteins Ato1p and Ato2p interact and can form complexes when present in the plasma membrane. No interaction was detected between Ato1p and Ato3p or Ato2p and Ato3p. In addition, using specially prepared strains, we were able to detect an interaction between molecules of the same Ato protein, namely Ato1p-Ato1p and Ato3p-Ato3p, but not Ato2p-Ato2p.
The effect of actinomycin D on HeLa cells was studied by live fluorescence and transmission-throu... more The effect of actinomycin D on HeLa cells was studied by live fluorescence and transmission-through-dye microscopy-a recently developed technique that permits volume measurements in live cells. In particular, it is well suited for the observation and quantification of the apoptotic volume decrease (AVD), which is widely viewed as an essential feature of apoptosis. The main results from our study are as follows. (1) Apoptosis caused in HeLa cells by actinomycin D proceeds in two morphologically distinct stages: the early stage is characterized by extensive blebbing, and the late stage by a more compact shape. The loss of mitochondrial membrane potential occurs at about the same time as blebbing, and chromatin condensation follows 30-90 min later. Caspase-3 and 7 become activated during the late stage. (2) Because blebbing occurs before activation of caspase-3, it has to be initiated by a different mechanism. Although blebbing is one of the earliest observable changes, it can be selectively inhibited without affecting other apoptotic reactions. (3) The majority of cells experience a temporary volume increase after the appearance of blebs. Eventually, AVD takes over and the cells shrink by approximately 40 % of their initial volume; the volume loss becomes noticeable at the end of the blebbing phase and continues through the late stage. Sometimes, at the end of long incubations, shrinkage gives way to swelling, possibly indicating secondary necrosis. (4) Both early and late apoptosis are accompanied by intracellular accumulation of Na(+), while low-sodium medium prevents apoptosis. Except for a partial protective effect of quinine, all of the tested blockers of Na(+), K(+) and Cl(-) channels failed to prevent apoptosis or AVD.
Interaction of leukemia blasts with the bone marrow extracellular matrix often results in protect... more Interaction of leukemia blasts with the bone marrow extracellular matrix often results in protection of leukemia cells from chemotherapy and in persistence of the residual disease which is on the basis of subsequent relapses. The adhesion signaling pathways have been extensively studied in adherent cells as well as in mature haematopoietic cells, but the adhesion structures and signaling in haematopoietic stem and progenitor cells, either normal or malignant, are much less explored. We analyzed the interaction of leukemia cells with fibronectin (FN) using interference reflection microscopy, immunofluorescence, measurement of adherent cell fraction, real-time microimpedance measurement and live cell imaging. We found that leukemia cells form very dynamic adhesion structures similar to early stages of focal adhesions. In contrast to adherent cells, where Src family kinases (SFK) belong to important regulators of focal adhesion dynamics, we observed only minor effects of SFK inhibitor dasatinib on leukemia cell binding to FN. The relatively weak involvement of SFK in adhesion structure regulation might be associated with the lack of cytoskeletal mechanical tension in leukemia cells. On the other hand, active Lyn kinase was found to specifically localize to leukemia cell adhesion structures and a less firm cell attachment to FN was often associated with higher Lyn activity (this unexpectedly occurred also after cell treatment with the inhibitor SKI-1). Lyn thus may be important for signaling from integrin-associated complexes to other processes in leukemia cells.
Mutations of the gene for nucleophosmin (NPM1) are the most frequent genetic aberration in patien... more Mutations of the gene for nucleophosmin (NPM1) are the most frequent genetic aberration in patients with acute myeloid leukemia (AML). The mechanism of leukemic transformation in this leukemia subtype is not fully understood, but aberrant cytoplasmic localization of mutated NPM (NPMmut) is widely considered as an important factor for leukemia manifestation. We analyzed the subcellular localization of three types of NPM with a C-terminal mutation (A, B and E). Genes for the individual NPM forms were fused with a gene for one of fluorescent protein variants in plasmids, which were transfected into three cell lines with different endogenous NPM expression. Subcellular localization of the fluorescent proteinlabeled NPM was further correlated with the relative expression of all NPM forms. We confirmed a high cytoplasmic expression of NPMmutA and NPMmutB whereas a substantial fraction of NPMmutE was found to be localized in nucleoli. Moreover, we revealed that the localization of fluorescently labeled NPM is affected by the interaction between various forms of the protein.
bioRxiv (Cold Spring Harbor Laboratory), Aug 25, 2020
P21-activated kinases (PAK) regulate many processes associated with cytoskeleton dynamics, includ... more P21-activated kinases (PAK) regulate many processes associated with cytoskeleton dynamics, including cell adhesion, migration, and apoptosis. PAK function is frequently altered in cancer, and PAK were proposed as therapy targets both in solid tumors and in hematological malignancies. However, current knowledge about PAK function in cell adhesion is mainly based on adherent cell models. Moreover, existing functional differences among the individual PAK family members are unsufficiently characterized. We measured expression of PAK group I members in leukemia cell lines and in primary leukemia cells, both on protein and mRNA levels. In functional assays, we analyzed the effect of two PAK inhibitors with different mechanisms of action, IPA-3 and FRAX597. Changes in cell interaction with fibronectin were monitored through impedance measurement and by interference reflection microscopy. Cytotoxic effects of inhibitors were assessed by Annexin V/propidium iodide test. PAK intracellular localization was analyzed by confocal microscopy. PAK2 transcript was dominant in cell lines, whereas primary leukemia cells also expressed comparable amount of PAK1, which was detected as two transcription isoforms: PAK1-full and PAK1Δ15. PAK1Δ15 and PAK2 transcript levels correlated with surface density of integrins β1 and αVβ3. PAK1-full, but not PAK2, was present in membrane protrusions. The inhibitors had partly opposed effects: IPA-3, which prevents PAK activation, induced cell contraction in semi-adherent HEL cells only. FRAX597, which inhibits PAK kinase activity, increased cell-surface contact area in all leukemia cells. Both inhibitors reduced the stability of cell attachment and induced cell death. Although many cells accumulated high FRAX597 amounts, low doses were sufficient to kill sensitive cells. FRAX597-induced cell death was fast in the MV4-11 cell line and in primary AML cells. Although PAK group I seem to be essential for leukemia cell adhesion and survival, and might thus serve as therapy targets, many PAK functions still remain to be attributed to individual isoforms and to their functional domains.
The rate and extent of uptake of the fluorescent probe diS-C3(3) reporting on membrane potential ... more The rate and extent of uptake of the fluorescent probe diS-C3(3) reporting on membrane potential in S. cerevisiae is affected by the strain under study, cell-growth phase, starvation and by the concentration of glucose both in the growth medium and in the monitored cell suspension under non-growth conditions. Killer toxin K1 brings about changes in membrane potential. In all types of cells tested, viz. in glucose-supplied stationary or exponential cells of the killer-sensitive strain $6/1 or a conventional strain RXII, or in glucose-free exponential cells of both strains, both active and heat-inactivated toxin slow down the potential-dependent uptake of diS-C3(3) into the cells. This may reflect "clogging" of pores in the cell wall that hinders, but does not prevent, probe passage to the plasma membrane and its equilibration. The clogging effect of heatinactivated toxin is stronger than that exerted by active toxin. In susceptible cells, Le. in exponential-phase glucose-supplied ceils of the sensitive strain $6/1, this phase of probe uptake retardation is followed by an irreversible red shift in probe fluorescence maximum)l.ma. x indicating damage to membrane integrity and cell permeabilization. A similar fast red shift in 2max signifying lethal cell damage was found in heat-killed or nystatin-treated cells.
Biochimica Et Biophysica Acta - General Subjects, 2019
BACKGROUND EGFP is a fluorescent tag extensively used in biological and biomedical research. Over... more BACKGROUND EGFP is a fluorescent tag extensively used in biological and biomedical research. Over the years many researches have gathered collections of cell lines bearing specific EGFP-tagged proteins. Despite its popularity some photochemical properties of EGFP remain undocumented and unused. We report on so far unexplored lifetime photoconversion of EGFP usable in FLIM. METHODS Fluorescence lifetime imaging and spectral FLIM has been used for characterization of the EGFP photoconversion and protein tracking. RESULT Our data suggest that EGFP can be permanently photoconverted to a short-fluorescencelifetime form (PC-EGFP) by intense blue irradiation. PC-EGFP cannot be reverted back by 405 nm light and exhibits the same spectral emission properties with blue-shifted absorption compared to the unconverted EGFP. Fluorescence of PC-EGFP is pH-independent and the photoconversion efficiency decreases with the solvent viscosity. Utilization of the EGFP photoconversion was demonstrated by tracking of a nucleophosmin mutant in live HEK 293T cells during its cytoplasm-nuclear relocalization induced by Leptomycin B. CONCLUSIONS Besides potential FLIM artifacts caused by an unintended EGFP photoconversion, the controlled photoconversion turns EGFP to an excellent tool for kinetic FLIM applications. Since the photoconversion occurs in the lifetime domain, PC-EGFP can be easily distinguished from the unconverted tag by time-resolved detection while all other spectral channels stay free for multicolor labeling.
The International Journal of Biochemistry & Cell Biology, Oct 1, 2018
Highlights Leukemia-related mutation of nucleophosmin disrupts its interaction with nucleolin ... more Highlights Leukemia-related mutation of nucleophosmin disrupts its interaction with nucleolin Stability of nucleophosmin oligomers is attenuated by C-terminal mutation All-trans retinoic acid stabilizes nucleophosmin oligomers Nucleolin/nucleophosmin ratio is enhanced in patients with nucleophosmin mutation Abstract C-terminal mutations of the nucleolar protein nucleophosmin (NPM) are the most frequent genetic aberration detected in acute myeloid leukemia (AML) with normal karyotype. The mutations cause aberrant cytoplasmic localization of NPM and lead to loss of functions associated with NPM nucleolar localization, e.g. in ribosome biogenesis or DNA-damage repair. NPM has many interaction partners and some of them were proved to interact also with the mutated form (NPMmut) and due to this interaction thereby to be withdrawn from their site of action. We analyzed the impact of the mutation on NPM interaction with nucleolin (NCL) which is also prevalently localized into the nucleolus and cooperates with wild-type NPM (NPMwt) in many cellular processes. We revealed that the NCL-NPM complex formation is completely abolished by the mutation and that the presence/absence of the interaction is not affected by drugs causing genotoxic stress or differentiation. Deregulation resulting from changes of NCL/NPMwt ratio may contribute to leukemogenesis.
Nucleophosmin (NPM) mutations causing its export from the nucleoli to the cytoplasm are frequent ... more Nucleophosmin (NPM) mutations causing its export from the nucleoli to the cytoplasm are frequent in acute myeloid leukemia (AML). Due to heterooligomerization of wild type NPM with the AML-related mutant, the wild-type becomes misplaced from the nucleoli and its functions are significantly altered. Dissociation of NPM heterooligomers may thus restore the proper localization and function of wildtype NPM. NSC348884 is supposed to act as a potent inhibitor of NPM oligomerization. The effect of NSC348884 on the NPM oligomerization was thoroughly examined by fluorescence lifetime imaging with utilization of FRET and by a set of immunoprecipitation and electrophoretic methods. Leukemiaderived cell lines and primary AML cells as well as cells transfected with fluorescently labeled NPM forms were investigated. Our results clearly demonstrate that NSC348884 does not inhibit formation of NPM oligomers neither in vivo nor in vitro. Instead, we document that NSC348884 cytotoxicity is rather associated with modified cell adhesion signaling. The cytotoxic mechanism of NSC348884 has therefore to be reconsidered. Abbreviations NPMwt Wild-type nucleophosmin NPMmut Nucleophosmin with AML-associated mutation type A C21 Cysteine 21 in nucleophosmin C21A Substitution of C21 to alanine C21F Substitution of C21 to phenylalanine Δ25 Nucleophosmin with deletion of the first 25 amino acids Δ100 Nucleophosmin with deletion of the first 100 amino acids Δ117 Nucleophosmin with deletion of the first 117 amino acids G_ EGFP-labeled protein at the N-terminus R_ MRFP1-labeled protein at the N-terminus The human NPM1 gene is located on the chromosome 5q35 and encodes a 32.6 kDa polypeptide. Nucleophosmin (NPM), encoded by the NPM1 gene, is a ubiquitously expressed phosphoprotein residing predominantly in the granular component of the nucleolus and shuttling dynamically among nucleoli, the nucleoplasm and the cytoplasm 1-4. It functions as a chaperone 5 and is engaged in regulation of various cellular processes including the ribosome biogenesis 6 , DNA-damage repair 7 , centrosome duplication 8 , and DNA replication 9. Furthermore, NPM is involved in apoptosis and can modulate p53 stability and activity 1,10. NPM overexpression, fusion, or mutation have oncogenic potential and are associated with cancer progression in many types of solid tumors 11 and in hematopoietic malignancies 12-17. Acute myeloid leukemia (AML) with mutated NPM1 accounts for about 1/3 of de novo adult AML, and NPM1 is the most frequently mutated gene in AML with normal karyotype (50-60% incidence) 12. To date, more than 100 NPM1 mutation types have been identified in AML, occurring almost exclusively in the last exon (exon 12) of the gene 18. A nucleotide insertion and/or deletion (indel mutations) lead to the frame shift in the region encoding the C-terminus of NPM. All these NPM1 mutations result in loss of tryptophan residues at positions 288 and/or 290, which form the main part of the nucleolar localization signal (NoLS) ensuring nucleolar localization of the wild-type NPM (NPMwt). Moreover, the indel mutations frequently generate an additional nuclear export sequence (NES), which labels the mutated protein for the nuclear exporter XPO1 and targets NPM into the cytoplasm 19. Both the loss of NoLS and
P21-activated kinases (PAK) are key effectors of the small GTPases Rac1 and Cdc42, as well as of ... more P21-activated kinases (PAK) are key effectors of the small GTPases Rac1 and Cdc42, as well as of Src family kinases. In particular, PAK1 has several well-documented roles, both kinase-dependent and kinase-independent, in cancer-related processes, such as cell proliferation, adhesion, and migration. However, PAK1 properties and functions have not been attributed to individual PAK1 isoforms: besides the full-length kinase (PAK1-full), a splicing variant lacking the exon 15 (PAK1Δ15) is annotated in protein databases. In addition, it is not clear if PAK1 and PAK2 are functionally overlapping. Using fluorescently tagged forms of human PAK1-full, PAK1Δ15, and PAK2, we analyzed their intracellular localization and mutual interactions. Effects of PAK inhibition (IPA-3, FRAX597) or depletion (siRNA) on cell-surface adhesion were monitored by real-time microimpedance measurement. Both PAK1Δ15 and PAK2, but not PAK1-full, were enriched in focal adhesions, indicating that the C-terminus might be important for PAK intracellular localization. Using coimmunoprecipitation, we documented direct interactions among the studied PAK group I members: PAK1 and PAK2 form homodimers, but all possible heterocomplexes were also detected. Interaction of PAK1Δ15 or PAK2 with PAK1-full was associated with extensive PAK1Δ15/PAK2 cleavage. The impedance measurements indicate, that PAK2 depletion slows down cell attachment to a surface, and that PAK1-full is involved in cell spreading. Altogether, our data suggest a complex interplay among different PAK group I members, which have non-redundant functions. PAK (p21-activated kinases) are a group of serine-threonine kinases originally identified as downstream effectors of p21 proteins, specifically of the Ras-related GTPases Rac1 and Cdc42 1,2. The initial phase of PAK research focused on their role as small GTPase effectors in the context of dynamic remodelling of the cytoskeleton and of cell adhesion structures 3. Later on, PAK were found to be involved in many cancer-related processes in different tumour types 4,5. In parallel, the discovery of PAK1 nuclear localization 6 prompted the analysis of PAK functions in the cell nucleus. The human PAK family is divided into the group I (PAK1 to PAK3) and group II (PAK4 to PAK6). In general, these kinases regulate the cytoskeleton dynamics, intracellular signaling, and gene expression 7. The current knowledge about PAK group I is derived mostly from adherent cell models, where PAK activity usually correlates with increased cell motility. In this area, the research was mainly focused to PAK1, although some important differences between PAK1 and PAK2 have been reported 8. In addition, PAK are known regulators of a wide range of cellular processes, including the dynamics of actin structures and microtubules, cell division, apoptosis, and adhesion to the extracellular matrix. Despite considerable sequence homology within PAK group I, the individual members appear to have distinct function in cell physiology. Whereas PAK2 is more or less ubiquitiously expressed, PAK1 expression is more restricted in adulthood, and PAK3 is expressed mainly in the brain 9. PAK2 and PAK4 are essential during embryonic development, since knockouts are embryonic lethal, at least in mice 9. Group I PAK share some domains that are not present in the group II members 10,11. In particular, the autoinhibitory domain (AID) is important for regulation of the kinase activity of the group I family members. The regulatory mechanism was described on the basis of PAK1 crystal structure 12 , and is likely valid also for PAK2 and PAK3. PAK1 forms homodimers in a trans arrangement, the AID of one molecule interacting with the kinase domain of its partner molecule 13,14. In this closed conformation, the kinase activity is very low. Binding of a
Enhanced green fluorescence protein (EGFP) is a fluorescent tag commonly used in cellular and bio... more Enhanced green fluorescence protein (EGFP) is a fluorescent tag commonly used in cellular and biomedical applications. Surprisingly, some interesting photochemical properties of EGFP have remained unexplored. Here we report on two-photon-induced photoconversion of EGFP, which can be permanently converted by intense IR irradiation to a form with a short fluorescence lifetime and spectrally conserved emission. Photoconverted EGFP thus can be distinguished from the unconverted tag by the time-resolved detection. Nonlinear dependence of the two-photon photoconversion efficiency on the light intensity allows for an accurate 3D localization of the photoconverted volume within cellular structures, which is especially useful for kinetic FLIM applications. For illustration, we used the two photon photoconversion of EGFP for measurements of redistribution kinetics of nucleophosmin and histone H2B in nuclei of live cells. Measurements revealed high mobility of fluorescently tagged histone H2B ...
Nucleophosmin (NPM) interaction with tumor suppressor p53 is a part of a complex interaction netw... more Nucleophosmin (NPM) interaction with tumor suppressor p53 is a part of a complex interaction network and considerably affects cellular stress response. The impact of NPM1 mutations on its interaction with p53 has not been investigated yet, although consequences of NPMmut-induced p53 export to the cytoplasm are important for understanding the oncogenic potential of these mutations. We investigated p53-NPM interaction in live HEK-293T cells by FLIM-FRET and in cell lysates by immunoprecipitation. eGFP lifetime-photoconversion was used to follow redistribution dynamics of NPMmut and p53 in Selinexor-treated cells. We confirmed the p53-NPMwt interaction in intact cells and newly documented that this interaction is not compromised by the NPM mutation causing displacement of p53 to the cytoplasm. Moreover, the interaction was not abolished for non-oligomerizing NPM variants with truncated oligomerization domain, suggesting that oligomerization is not essential for interaction of NPM forms...
One way how to study individual processes that underlie the complex behaviour of living systems i... more One way how to study individual processes that underlie the complex behaviour of living systems is to construct artificial structures resembling those that compose the living systems. By using such a structure, the process of interest can be studied and characterised separately, e.g. with regard to its physical basis, and the basic principles applicable to living systems can be postulated. Of course, such approach provides only partial information the importance of which has to be considered with respect to the complexity of living systems. In this study, we want to show that liposomes or proteoliposomes offer a useful tool for testing fluorescent probes used for studying living cells, chapter 2, for studying membrane transport systems, chapter 3, or for modelling transport processes across the membrane, chapter 4.Available from STL Prague, CZ / NTK - National Technical LibrarySIGLECZCzech Republi
P21-activated kinases (PAK) regulate processes associated with cytoskeleton dynamics. PAK express... more P21-activated kinases (PAK) regulate processes associated with cytoskeleton dynamics. PAK expression in leukemia cells was measured on protein and mRNA levels. In functional assays, we analyzed the effect of PAK inhibitors IPA-3 and FRAX597 on cell adhesivity and viability. PAK2 was dominant in cell lines, whereas primary cells also expressed comparable amount of PAK1 transcription isoforms: PAK1-full and PAK1Δ15. PAK1Δ15 and PAK2 levels correlated with surface density of integrins β1 and αVβ3. PAK1-full, but not PAK2, was present in membrane protrusions. IPA-3, which prevents PAK activation, induced cell contraction in semi-adherent HEL cells only. FRAX597, which inhibits PAK kinase activity, increased cell-surface contact area in all leukemia cells. Both inhibitors reduced the stability of cell attachment and induced cell death.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2003
Potential-sensitive fluorescent probes oxonol V and oxonol VI were employed for monitoring membra... more Potential-sensitive fluorescent probes oxonol V and oxonol VI were employed for monitoring membrane potential (Dw) generated by the Schizosaccharomyces pombe plasma membrane H +-ATPase reconstituted into vesicles. Oxonol VI was used for quantitative measurements of the Dw because its response to membrane potential changes can be easily calibrated, which is not possible with oxonol V. However, oxonol V has a superior sensitivity to Dw at very low concentration of reconstituted vesicles, and thus it is useful for testing quality of the reconstitution. Oxonol VI was found to be a good emission-ratiometric probe. We have shown that the reconstituted H +-ATPase generates Dw of about 160 mV on the vesicle membrane. The generated Dw was stable at least over tens of minutes. An influence of the H + membrane permeability on the Dw buildup was demonstrated by manipulating the H + permeability with the protonophore CCCP. Ratiometric measurements with oxonol VI thus offer a promising tool for studying processes accompanying the yeast plasma membrane H +-ATPasemediated Dw buildup.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1997
A novel fluorescent technique for direct assessment of membrane potential was tested on suspensio... more A novel fluorescent technique for direct assessment of membrane potential was tested on suspensions of large Ž. unilamellar vesicles LUV. The method is based on monitoring shifts in the fluorescence maximum, l , of the max Ž. redistribution dye diSC 3 caused by dye binding to the LUV membrane. A theory describing the behavior of this dye in 3 LUV suspensions was elaborated and tested. The diffusion potentials across the LUV membrane were adjusted by ion gradients in the absence of valinomycin. When using KCl and choline chloride without valinomycin the potential can be set as high as y70 mV. These potentials exhibited long-term stability and the theory allowed to determine the upper limits of membrane permeabilities for Cl y , choline cations, protons and hydroxyls relative to the K q permeability. The absolute values of membrane potential set by ion gradients were calibrated using valinomycin. The monitoring of the l shift max permitted us to show real-time changes in membrane potential, since addition of valinomycin to the LUV was followed by an immediate depolarization. The setting of the potential and the dye re-equilibration after valinomycin addition took place within a second.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2012
FLIM-photobleaching technique Homo/hetero di(oligo)mers Plasma membrane protein interaction Donor... more FLIM-photobleaching technique Homo/hetero di(oligo)mers Plasma membrane protein interaction Donor lifetime Each of the three plasma membrane Ato proteins is involved in ammonium signalling and the development of yeast colonies. This suggests that although these proteins are homologous, they do not functionally substitute for each other, but may form a functional complex. Here, we present a detailed combined FRET, FLIM and photobleaching study, which enabled us to detect interactions between Ato proteins found in distinct compartments of yeast cells. We thus show that the proteins Ato1p and Ato2p interact and can form complexes when present in the plasma membrane. No interaction was detected between Ato1p and Ato3p or Ato2p and Ato3p. In addition, using specially prepared strains, we were able to detect an interaction between molecules of the same Ato protein, namely Ato1p-Ato1p and Ato3p-Ato3p, but not Ato2p-Ato2p.
The effect of actinomycin D on HeLa cells was studied by live fluorescence and transmission-throu... more The effect of actinomycin D on HeLa cells was studied by live fluorescence and transmission-through-dye microscopy-a recently developed technique that permits volume measurements in live cells. In particular, it is well suited for the observation and quantification of the apoptotic volume decrease (AVD), which is widely viewed as an essential feature of apoptosis. The main results from our study are as follows. (1) Apoptosis caused in HeLa cells by actinomycin D proceeds in two morphologically distinct stages: the early stage is characterized by extensive blebbing, and the late stage by a more compact shape. The loss of mitochondrial membrane potential occurs at about the same time as blebbing, and chromatin condensation follows 30-90 min later. Caspase-3 and 7 become activated during the late stage. (2) Because blebbing occurs before activation of caspase-3, it has to be initiated by a different mechanism. Although blebbing is one of the earliest observable changes, it can be selectively inhibited without affecting other apoptotic reactions. (3) The majority of cells experience a temporary volume increase after the appearance of blebs. Eventually, AVD takes over and the cells shrink by approximately 40 % of their initial volume; the volume loss becomes noticeable at the end of the blebbing phase and continues through the late stage. Sometimes, at the end of long incubations, shrinkage gives way to swelling, possibly indicating secondary necrosis. (4) Both early and late apoptosis are accompanied by intracellular accumulation of Na(+), while low-sodium medium prevents apoptosis. Except for a partial protective effect of quinine, all of the tested blockers of Na(+), K(+) and Cl(-) channels failed to prevent apoptosis or AVD.
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Papers by Ales Holoubek