Cytoskeletal dynamics

The ability of a eukaryotic cell to resist deformation, to transport intracellular cargo and to change shape during movement depends on the cytoskeleton, an interconnected network of filamentous polymers and regulatory proteins. Recent work has demonstrated that both internal and external physical forces can act through the cytoskeleton to affect local mechanical properties and cellular behaviour. Attention is now focused on how cytoskeletal networks generate, transmit and respond to mechanical signals over both short and long timescales. An important insight emerging from this work is that long-lived cytoskeletal structures may act as epigenetic determinants of cell shape, function and fate. In a 1960 lecture, cell and developmental biologist Paul A. Weiss encouraged his audience to think of the cell as an integrated whole " lest our necessary and highly successful preoccupation with cell fragments and fractions obscure the fact that the cell is not just an inert playground for a few almighty masterminding molecules, but is a system, a hierarchically ordered system, of mutually interdependent species of molecules, molecular groupings, and supramolecular entities; and that life, through cell life, depends on the order of their interactions " 1. This statement may be more relevant today than it was 50 years ago. Despite tremendous progress, fundamental gaps remain between our understanding of individual molecules and our understanding of how these molecules function collectively to form living cells. The sequencing of genomes outpaces characterization of the cellular components they encode and far exceeds our ability to reassemble these components into the types of complex system that can provide mecha-nistic insight into cellular behaviour. An even more difficult task is to connect the behaviour of cells in culture with that of more complex living tissues and organisms. Ever since muscle fibres were first examined under rudimentary microscopes in the seventeenth century, researchers have been motivated to understand how the process of self-organization generates dynamic, robust and elaborate structures that organize and 'animate' cells. The biological importance of establishing order over diverse length scales and timescales, as well as the challenges of understanding how systems of self-organizing molecules carry out cellular functions, is perhaps best illustrated by studies of the cytoskeleton. The cytoskeleton carries out three broad functions: it spatially organizes the contents of the cell; it connects the cell physically and biochemically to the external environment; and it generates coordinated forces that enable the cell to move and change shape. To achieve these functions, the cytoskeleton integrates the activity of a multitude of cytoplasmic proteins and organelles. Despite the connotations of the word 'skeleton' , the cytoskeleton is not a fixed structure whose function can be understood in isolation. Rather, it is a dynamic and adaptive structure whose component polymers and regulatory proteins are in constant flux. Many basic building blocks of the cytoskeleton have been identified and characterized extensively in vitro, and researchers are now using advanced light microscopy to determine, with great spatial and temporal precision, the locations and dynamics of these cytoskeletal proteins during processes such as cell division and motility. For example, more than 150 proteins have so far been found to contain binding domains for the protein actin, which polymerizes to form one of the key cytoskeletal filaments in cells 2. One set of actin regulators forms a macromolecular ensemble called the WAVE complex that promotes assembly of actin-filament networks at the leading edge of motile cells 3. High-resolution light microscopy of rapidly crawling leukocytes revealed that the WAVE complex forms highly coherent travelling waves whose movement correlates with cell protrusion 4. Such observations in living cells can stimulate the formation of detailed hypotheses for how molecules collaborate to form functional cytoskeletal structures, but to test these hypotheses definitively, the components must be isolated from cells and purified. Remarkably, experiments that combine a small number of purified proteins have demonstrated that many complex cytoskeletal structures observed in cells can be reconstituted in vitro from purified components. For example, only three proteins are required to actively track and transport cargo on the growing end of microtubules, which are formed by the polymerization of subunits consisting of αβ-tubulin heterodimers and are another key cytoskeletal filament in cells 5. Although the list of proteins associated with the cytoskeleton continues to grow, the ultimate goal remains — understanding how the interactions of the individual molecules of the cytoskeleton give rise to the large-scale cellular behaviours that depend on them. In this Review, we discuss recent progress towards an integrated understanding of the cytoskeleton. In particular, we focus on the mechanics of cytoskeletal networks and the roles that mechanics have in many cell biological processes. Rather than focusing on one cellular process or cytoskel-etal filament, we identify a set of basic concepts and link them to work in several cytoskeleton-related fields. We begin with a brief introduction to the major polymers that constitute the cytoskeleton and then shift focus from molecules to more complex structures, emphasizing three concepts that echo Weiss's 1960 challenge to view cells as an integrated whole. The first concept is that long-range order arises from the regulated self-assembly of components guided by spatial cues and physical constraints. The second is that beyond simply composition, it is the architecture of the cytoskeleton that controls the physical properties of the cell. And the third is that cytoskeletal links to the external microenvironment can mediate both short and long timescale changes in cellular behaviour. We finish by discussing the intriguing and under-appreciated question of whether long-lived cytoskeletal structures can function as a cellular 'memory' that integrates past interactions with the mechanical microenvironment and influences future cellular behaviour.

Out of the three main cytoskeletal fibers—actin filaments, intermediate filaments, and microtubules (MTs)—MTs have the largest diameter (outer diameter is ~25 nm). These microscopic hollow tubes are composed of the proteins α- and β-tubulin (γ-tubulin is localized to the centrosome and sometimes form caps on the minus ends of MTs) and are involved in a multitude of cellular activities and functions, including maintaining/carrying out/helping cell structural integrity, motility, division, organization, mechanotransduction, and intracellular transport. Other proteins that associate with MTs (MT-associated proteins (MAPs)) such as the motor proteins and MT-severing proteins regulate MTs and greatly increase the functionality of MTs.

This project is interested in the musica humana, the complex dynamics of biochemistry and how this gives rise to life. It investigates how acoustic waves may affect the physical chemistry of bio-molecules and therefore how this may also affect the biological mechanisms in which they participate.

Phenotypic characterization of non-haernopoietic small cell tumours of childhood with monocloaal antibodies to leucocytes, epithelial cells and cytoskeletal proteins Recently, great interest has been shown in the histological identification of small cell tumours or childhood-nephroblastoma (Wilms' tumour), neuroblastoma. rhabdomyosarcorna and Ewing's sarcoma-using immunohistochemical methods. However, several antigens operationally specific for leucocyte typing in blood and marrow are also expressed on cells of epithelial and neural origin. We undertook phenotypic characteri:ration of 17 non-haemopoietic small cell tumours of childhood using a panel of 30 monoclonal antibodies to leucocyte, epithelial and cytoskeletal antigens using a sensitive alkaline phosphatase-anti-alkaline phosphatase technique on cryostat scctions of fresh tumour. Our results demonstrated frequcnt expression of the leucocyte-associated antigens CDlO (CALLA), CD9 (p24) and CDw32 (FcRII) in these small cell turnours and occasional expression of MHC class I1 (HLA-DR) and HNK-I antigens. However, the leococyte-associated antigens CD4S (leucocyte common), CD22 (pan B-cell), CDI Ib (C3bi receptor), CD15 (Lewisa) or CDw42 (platelet g,p Ib) were not detected on any tumour. Aberrant expression of desmin, neurofilarnent and UJ13A antigen was found in nephroblastoma and of epithelialassociated markers (CIBrl7 and 43-9F) in neuroblastoma. Our results also demonstrated broad reactivity in frozen section with two monoclonal antibodies specific for melanoma (NKI/C-3) or epithelial cells (OM-I) in paraffin sections. Hence, it is necessary to include monoclonal antibodies to CD4S and pan-epithelial antigens, e.g. LP34 (cytokeratin) or HEAI2S for the precise immunohistochemical identifica:ion of small round cell malignancies of childhood.

LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation, and remodeling of the cell cytoskeleton. Human Four-and-a-half LIM-only protein 2 (FHL2) is expressed predominantly in human heart and is only slightly expressed in skeletal muscle. Since FHL2 is an abundant protein in human heart, it may play an important role in the regulation of cell differentiation and myofibrillogenesis of heart at defined subcellular compartment. Therefore, we hypothesized that FHL2 act as a multi-functional protein by the specific arrangement of the LIM domains of FHL2 and that one of the LIM domains of FHL2 can function as an anchor and localizes it into a specific subcellular compartment in a cell type specific manner to regulate myofibrillogenesis. From our reuslts, we observed that FHL2 is localized at the focal adhesions of the C2C12, H9C2 myoblast as well as a nonmyogenic cell line, HepG2 cells. Colocalization of vinculin-CFP and FHL2-GFP at focal adhesions was also observed in cell lines. Site-directed mutagen-esis, in turn, suggested that the second LIM domain-LIM2 is essential for its specific localization to focal adhesions. Moreover, FHL2 was observed along with F-actin and focal adhesion of C2C12 and H9C2 myotubes. Finally, we believe that FHL2 moves from focal adhesions and then stays at the Z-discs of terminally differentiated heart muscle. Cell Motil. Cytoskeleton 48:11–23, 2001.

During mitosis in budding yeast the nucleus first moves to the mother-bud neck and then into the neck. Both movements depend on interactions of cytoplasmic microtubules with the cortex. We investigated the mechanism of these movements in living cells using video analysis of GFP-labeled microtubules in wild-type cells and in EB1 and Arp1 mutants, which are defective in the first and second steps, respectively. We found that nuclear movement to the neck is largely mediated by the capture of microtubule ends at one cortical region at the incipient bud site or bud tip, followed by microtubule depolymerization. Efficient microtubule interactions with the capture site require that microtubules be sufficiently long and dynamic to probe the cortex. In contrast, spindle movement into the neck is mediated by microtubule sliding along the bud cortex, which requires dynein and dynactin. Free microtubules can also slide along the cortex of both bud and mother. Capture/shrinkage of microtubule ends also contributes to nuclear movement into the neck and can serve as a backup mechanism to move the nucleus into the neck when microtubule sliding is impaired. Conversely, microtubule sliding can move the nucleus into the neck even when capture/shrinkage is impaired.