Transiently crosslinked F-actin bundles

F-actin bundles are prominent cytoskeletal structures in eukaryotes. They provide mechanical stability in stereocilia, microvilli, filopodia, stress fibers and the sperm acrosome. Bundles are typically stabilized by a wide range of specific crosslinking proteins, most of which exhibit off-rates on the order of 1s⁻¹. Yet F-actin bundles exhibit structural and mechanical integrity on time scales that are orders of magnitude longer. By applying large deformations to reconstituted F-actin bundles using optical tweezers, we provide direct evidence of their differential mechanical response in vitro: bundles exhibit fully reversible, elastic response on short time scales and irreversible, elasto-plastic response on time scales that are long compared to the characteristic crosslink dissociation time. Our measurements show a broad range of characteristic relaxation times for reconstituted F-actin bundles. This can be reconciled by considering that bundle relaxation behavior is also modulated by the number of filaments, crosslinking type and occupation number as well as the consideration of defects due to filament ends.     

Cytoskeletal bundle mechanics

The mechanical properties of cytoskeletal actin bundles play an essential role in numerous physiological processes, including hearing, fertilization, cell migration, and growth. Cells employ a multitude of actin-binding proteins to actively regulate bundle dimensions and cross-linking properties to suit biological function. The mechanical properties of actin bundles vary by orders of magnitude depending on diameter and length, cross-linking protein type and concentration, and constituent filament properties. Despite their importance to cell function, the molecular design principles responsible for this mechanical behavior remain unknown. Here, we examine the mechanics of cytoskeletal bundles using a molecular-based model that accounts for the discrete nature of constituent actin filaments and their distinct cross-linking proteins. A generic competition between filament stretching and cross-link shearing determines three markedly different regimes of mechanical response that are delineated by the relative values of two simple design parameters, revealing the universal nature of bundle-bending mechanics. In each regime, bundle-bending stiffness displays distinct scaling behavior with respect to bundle dimensions and molecular composition, as observed in reconstituted actin bundles in vitro. This mechanical behavior has direct implications on the physiological bending, buckling, and entropic stretching behavior of cytoskeletal processes, as well as reconstituted actin systems. Results are used to predict the bending regimes of various in vivo cytoskeletal bundles that are not easily accessible to experiment and to generate hypotheses regarding implications of the isolated behavior on in vivo bundle function.     

Micromechanics and ultrastructure of actin filament networks crosslinked by human fascin: a comparison with alpha-actinin.

Fascin is an actin crosslinking protein that organizes actin filaments into tightly packed bundles believed to mediate the formation of cellular protrusions and to provide mechanical support to stress fibers. Using quantitative rheological methods, we studied the evolution of the mechanical behavior of filamentous actin (F-actin) networks assembled in the presence of human fascin. The mechanical properties of F-actin/fascin networks were directly compared with those formed by alpha-actinin, a prototypical actin filament crosslinking/bundling protein. Gelation of F-actin networks in the presence of fascin (fascin to actin molar ratio >1:50) exhibits a non-monotonic behavior characterized by a burst of elasticity followed by a slow decline over time. Moreover, the rate of gelation shows a non-monotonic dependence on fascin concentration. In contrast, alpha-actinin increased the F-actin network elasticity and the rate of gelation monotonically. Time-resolved multiple-angle light scattering and confocal and electron microscopies suggest that this unique behavior is due to competition between fascin-mediated crosslinking and side-branching of actin filaments and bundles, on the one hand, and delayed actin assembly and enhanced network micro-heterogeneity, on the other hand. The behavior of F-actin/fascin solutions under oscillatory shear of different frequencies, which mimics the cell's response to forces applied at different rates, supports a key role for fascin-mediated F-actin side-branching. F-actin side-branching promotes the formation of interconnected networks, which completely inhibits the motion of actin filaments and bundles. Our results therefore show that despite sharing seemingly similar F-actin crosslinking/bundling activity, alpha-actinin and fascin display completely different mechanical behavior. When viewed in the context of recent microrheological measurements in living cells, these results provide the basis for understanding the synergy between multiple crosslinking proteins, and in particular the complementary mechanical roles of fascin and alpha-actinin in vivo.     

Functional synergy of actin filament cross-linking proteins

The organization of filamentous actin (F-actin) in resilient networks is coordinated by various F-actin cross-linking proteins. The relative tolerance of cells to null mutations of genes that code for a single actin cross-linking protein suggests that the functions of those proteins are highly redundant. This apparent functional redundancy may, however, reflect the limited resolution of available assays in assessing the mechanical role of F-actin cross-linking/bundling proteins. Using reconstituted F-actin networks and rheological methods, we demonstrate how alpha-actinin and fascin, two F-actin cross-linking/bundling proteins that co-localize along stress fibers and in lamellipodia, could synergistically enhance the resilience of F-actin networks in vitro. These two proteins can generate microfilament arrays that "yield" at a strain amplitude that is much larger than each one of the proteins separately. F-actin/alpha-actinin/fascin networks display strain-induced hardening, whereby the network "stiffens" under shear deformations, a phenomenon that is non-existent in F-actin/fascin networks and much weaker in F-actin/alpha-actinin networks. Strain-hardening is further enhanced at high rates of deformation and high concentrations of actin cross-linking proteins. A simplified model suggests that the optimum results of the competition between the increased stiffness of bundles and their decreased density of cross-links. Our studies support a re-evaluation of the notion of functional redundancy among cytoskeletal regulatory proteins.     

F-actin aggregates in transformed cells contain alpha-actinin and fimbrin but apparently lack tropomyosin

Transformation-specific F-actin structures are examined in tumor cells after in vitro tumor cell growth alone or on an untransformed cell monolayer. In transformed cells F-actin aggregates near the ventral plasma membrane in close substrate adhesion areas contain the cytoskeletal proteins alpha-actinin and fimbrin but, unlike microfilament bundles, are not labeled with antibody against tropomyosin. By electron microscopy the dense ventral aggregates in transformed cells resemble stress fiber termini found at the membrane in normal cells. These transformed-cell cytoskeletal structures are not limited solely to substrate adhesion areas; they are also expressed at cell-cell contacts about 48 h after transformed cells are plated on untransformed cells. These specialized F-actin aggregates appear to be implicated in the processes of penetration of these transformed cells between adjoining untransformed cells in vitro.     

A mechanochemical model of actin filaments

In eukaryotic cells, actin filaments are involved in important processes such as motility, division, cell shape regulation, contractility, and mechanosensation. Actin filaments are polymerized chains of monomers, which themselves undergo a range of chemical events such as ATP hydrolysis, polymerization, and depolymerization. When forces are applied to F-actin, in addition to filament mechanical deformations, the applied force must also influence chemical events in the filament. We develop an intermediate-scale model of actin filaments that combines actin chemistry with filament-level deformations. The model is able to compute mechanical responses of F-actin during bending and stretching. The model also describes the interplay between ATP hydrolysis and filament deformations, including possible force-induced chemical state changes of actin monomers in the filament. The model can also be used to model the action of several actin-associated proteins, and for large-scale simulation of F-actin networks. All together, our model shows that mechanics and chemistry must be considered together to understand cytoskeletal dynamics in living cells.     

Modelling the dynamics of F-actin in the cell

The regulation of the interactions between the actin binding proteins and the actin filaments are known to affect the cytoskeletal structure of F-actin. We develop a model depicting the formation of actin cytoskeleton, bundles and orthogonal networks, via activation or inactivation of different types of actin binding proteins. It is found that as the actin filament density increases in the cell, a spontaneous tendency to organize into bundles or networks occurs depending on the active actin binding protein concentration. Also, a minute change in the relative binding affinity of the actin binding proteins in the cell may lead to a major change in the actin cytoskeleton. Both the linear stability analysis and the numerical results indicate that the structures formed are highly sensitive to changes in the parameters, in particular to changes in the parameter ϕ, denoting the relative binding affinity and concentration of the actin binding proteins.     

The intermediate filament network in cultured human keratinocytes is remarkably extensible and resilient

The prevailing model of the mechanical function of intermediate filaments in cells assumes that these 10 nm diameter filaments make up networks that behave as entropic gels, with individual intermediate filaments never experiencing direct loading in tension. However, recent work has shown that single intermediate filaments and bundles are remarkably extensible and elastic in vitro, and therefore well-suited to bearing tensional loads. Here we tested the hypothesis that the intermediate filament network in keratinocytes is extensible and elastic as predicted by the available in vitro data. To do this, we monitored the morphology of fluorescently-tagged intermediate filament networks in cultured human keratinocytes as they were subjected to uniaxial cell strains as high as 133%. We found that keratinocytes not only survived these high strains, but their intermediate filament networks sustained only minor damage at cell strains as high as 100%. Electron microscopy of stretched cells suggests that intermediate filaments are straightened at high cell strains, and therefore likely to be loaded in tension. Furthermore, the buckling behavior of intermediate filament bundles in cells after stretching is consistent with the emerging view that intermediate filaments are far less stiff than the two other major cytoskeletal components F-actin and microtubules. These insights into the mechanical behavior of keratinocytes and the cytokeratin network provide important baseline information for current attempts to understand the biophysical basis of genetic diseases caused by mutations in intermediate filament genes.     

The Switch-associated Protein 70 (SWAP-70) Bundles Actin Filaments and Contributes to the Regulation of F-actin Dynamics

Coordinated assembly and disassembly of actin into filaments and higher order structures such as stress fibers and lamellipodia are fundamental for cell migration and adhesion. However, the precise spatiotemporal regulation of F-actin structures is not completely understood. SWAP-70, a phosphatidylinositol 3,4,5-trisphosphate-interacting, F-actin-binding protein, participates in actin rearrangements through yet unknown mechanisms. Here, we show that SWAP-70 is an F-actin-bundling protein that oligomerizes through a Gln/Glu-rich stretch within a coiled-coil region. SWAP-70 bundles filaments in parallel and anti-parallel fashion through its C-terminal F-actin binding domain and delays dilution-induced F-actin depolymerization. We further demonstrate that SWAP-70 co-localizes and directly interacts with cofilin, an F-actin severing and depolymerization factor, and contributes to the regulation of cofilin activity in vivo. In line with these activities, upon stem cell factor stimulation, murine bone marrow-derived mast cells lacking SWAP-70 display aberrant regulation of F-actin and actin free barbed ends dynamics. Moreover, proper stem cell factor-dependent cofilin activation via dephosphorylation and subcellular redistribution into a detergent-resistant cytoskeletal compartment also require SWAP-70. Together, these findings reveal an important role of SWAP-70 in the dynamic spatiotemporal regulation of F-actin networks.     

Actin and microtubules in neurite initiation: are MAPs the missing link?

During neurite initiation microtubules align to form a tight bundle and actin filaments reorganize to produce a growth cone. The mechanisms that underlie these highly coordinated cytoskeletal rearrangements are not yet fully understood. Recently, various levels of coordination between the actin- and microtubule-based cytoskeletons have been observed during cellular migration and morphogenesis, processes that share some similarities to neurite initiation. Direct, physical association between both cytoskeletons has been suggested, because microtubules often preferentially grow along actin bundles and transiently target actin-rich adhesion complexes. We propose that such physical association might be involved in force-based interactions and spatial organization of the two networks during neurite initiation as well. In addition, many signaling cascades that affect actin filaments are also involved in the regulation of microtubule dynamics, and vice versa. Although several candidates for mediating these effects have been identified in non-neuronal cells, the general mechanism is still poorly understood. In neurons certain plakins and neuron-specific microtubule associated proteins (MAPs), like MAP1B and MAP2, which can bind to both microtubules and F-actin, are promising candidates to play key roles in the specific cytoskeletal rearrangements controlling the transition from an undifferentiated state to neurite-bearing morphology. Here we review the effects of MAPs on microtubules and actin, as well as the coordination of both cytoskeletons during neurite initiation.     

The bimodal role of filamin in controlling the architecture and mechanics of F-actin networks

Reconstituted actin filament networks have been used extensively to understand the mechanics of the actin cortex and decipher the role of actin cross-linking proteins in the maintenance and deformation of cell shape. However, studies of the mechanical role of the F-actin cross-linking protein filamin have led to seemingly contradictory conclusions, in part due to the use of ill-defined mechanical assays. Using quantitative rheological methods that avoid the pitfalls of previous studies, we systematically tested the complex mechanical response of reconstituted actin filament networks containing a wide range of filamin concentrations and compared the mechanical function of filamin with that of the cross-linking/bundling proteins alpha-actinin and fascin. At steady state and within a well defined linear regime of small non-destructive deformations, F-actin solutions behave as highly dynamic networks (actin polymers are still sufficiently mobile to relax the stress) below the cross-linking-to-bundling threshold filamin concentration, and they behave as covalently cross-linked gels above that threshold. Under large deformations, F-actin networks soften at low filamin concentrations and strain-harden at high filamin concentrations. Filamin cross-links F-actin into networks that are more resilient, stiffer, more solid-like, and less dynamic than alpha-actinin and fascin. These results resolve the controversy by showing that F-actin/filamin networks can adopt diametrically opposed rheological behaviors depending on the concentration in cross-linking proteins.     

Dynamic changes of microtubule and actin structures in CV-1 cells during electrofusion

To study the involvement of the cytoskeletal system in the fusion of animal cells, we examined the dynamic changes of cytoskeletal proteins during the various stages of cell fusion. CV-1 cells were fused by applying a radio-frequency electrical pulse. Structural changes of microtubules (MTs) and F-actin were monitored simultaneously by double-label fluorescence microscopy. It was observed that in a few minutes after the initiation of cell fusion, MT bundles began to extend into the cytoplasmic bridges which were formed by fusing the membranes of neighboring cells. Later, a network of parallel MT bundles appeared between the adjacent nuclei of the fusing cells; such MT bundles may provide the mechanical links that are responsible for nuclear aggregation. The structural changes of F-actin during cell fusion were more complicated. We observed many different patterns of actin distribution in the fusing cells, including some giant, ring-shaped structures. Reorganization of actin is unlikely to be involved in the nuclear aggregation process. Instead, actin bundles condensed at the cell edges may help to widen the cytoplasmic bridges to allow merging of cellular contents between the fusing cells.     

Dynamics and regulation of contractile actin-myosin networks in morphogenesis

Contractile actin-myosin networks generate forces that drive cell shape changes and tissue remodeling during development. These forces can also actively regulate cell signaling and behavior. Novel features of actin-myosin network dynamics, such as pulsed contractile behaviors and the regulation of myosin localization by tension, have been uncovered in recent studies of Drosophila. In vitro studies of single molecules and reconstituted protein networks reveal intrinsic properties of motor proteins and actin-myosin networks, while in vivo studies have provided insight into the regulation of their dynamics and organization. Analysis of the complex behaviors of actin-myosin networks will be crucial for understanding force generation in actively remodeling cells and the coordination of cell shape and movement at the tissue level.     

Multifunctional roles of tropomodulin-3 in regulating actin dynamics

Tropomodulins (Tmods) are proteins that cap the slow-growing (pointed) ends of actin filaments (F-actin). The basis for our current understanding of Tmod function comes from studies in cells with relatively stable and highly organized F-actin networks, leading to the view that Tmod capping functions principally to preserve F-actin stability. However, not only is Tmod capping dynamic, but it also can play major roles in regulating diverse cellular processes involving F-actin remodeling. Here, we highlight the multifunctional roles of Tmod with a focus on Tmod3. Like other Tmods, Tmod3 binds tropomyosin (Tpm) and actin, capping pure F-actin at submicromolar and Tpm-coated F-actin at nanomolar concentrations. Unlike other Tmods, Tmod3 can also bind actin monomers and its ability to bind actin is inhibited by phosphorylation of Tmod3 by Akt2. Tmod3 is ubiquitously expressed and is present in a diverse array of cytoskeletal structures, including contractile structures such as sarcomere-like units of actomyosin stress fibers and in the F-actin network encompassing adherens junctions. Tmod3 participates in F-actin network remodeling in lamellipodia during cell migration and in the assembly of specialized F-actin networks during exocytosis. Furthermore, Tmod3 is required for development, regulating F-actin mesh formation during meiosis I of mouse oocytes, erythroblast enucleation in definitive erythropoiesis, and megakaryocyte morphogenesis in the mouse fetal liver. Thus, Tmod3 plays vital roles in dynamic and stable F-actin networks in cell physiology and development, with further research required to delineate the mechanistic details of Tmod3 regulation in the aforementioned processes, or in other yet to be discovered processes.     

Actin-Myosin Spatial Patterns from a Simplified Isotropic Viscoelastic Model

F-actin networks are involved in cell mechanical processes ranging from motility to endocytosis. The mesoscale architecture of assemblies of individual F-actin polymers that gives rise to micrometer-scale rheological properties is poorly understood, despite numerous in vivo and vitro studies. In vitro networks have been shown to organize into spatial patterns when spatially confined, including dense spherical shells inside spherical emulsion droplets. Here we develop a simplified model of an isotropic, compressible, viscoelastic material continually assembling and disassembling. We demonstrate that spherical shells emerge naturally when the strain relaxation rate (corresponding to internal network reorganization) is slower than the disassembly rate (corresponding to F-actin depolymerization). These patterns are consistent with recent experiments, including a collapse of shells to a central high-density focus of F-actin when either assembly or disassembly is reduced with drugs. Our results demonstrate how complex spatio-temporal patterns can emerge without spatially distributed force generation, polar alignment of F-actin polymers, or spatially nonuniform regulation of F-actin by upstream biochemical networks.     

Actin-Myosin Spatial Patterns from a Simplified Isotropic Viscoelastic Model

F-actin networks are involved in cell mechanical processes ranging from motility to endocytosis. The mesoscale architecture of assemblies of individual F-actin polymers that gives rise to micrometer-scale rheological properties is poorly understood, despite numerous in vivo and vitro studies. In vitro networks have been shown to organize into spatial patterns when spatially confined, including dense spherical shells inside spherical emulsion droplets. Here we develop a simplified model of an isotropic, compressible, viscoelastic material continually assembling and disassembling. We demonstrate that spherical shells emerge naturally when the strain relaxation rate (corresponding to internal network reorganization) is slower than the disassembly rate (corresponding to F-actin depolymerization). These patterns are consistent with recent experiments, including a collapse of shells to a central high-density focus of F-actin when either assembly or disassembly is reduced with drugs. Our results demonstrate how complex spatio-temporal patterns can emerge without spatially distributed force generation, polar alignment of F-actin polymers, or spatially nonuniform regulation of F-actin by upstream biochemical networks.     

Mechanical response of a living human epidermal keratinocyte sheet as measured in a composite diaphragm inflation experiment

Sheets of normal human epidermal keratinocytes (NHEKs) were reconstituted in vitro on tensed but highly compliant, freestanding polydimethylsiloxane (PDMS) membranes, 5.0 mm in diameter and 10 mum thick. NHEK-PDMS composite diaphragm (CD) specimens were then subjected to cyclical axisymmetric inflation tests to investigate epithelial sheet rheology under conditions of physiologically severe deformations (~50% nominal polar biaxial strains). Because the compliance of the specially formulated PDMS membrane was greater than that of the attached cell layer, the finite load-deformation behavior (mechanical response) of the living NHEK sheet was inferred from differences between the mechanical behavior of the CD specimen and the response of the underlying PDMS membrane measured prior to cell culture. In these composite diaphragm inflation (CDI) experiments, interconnected NHEKs exhibited rheological behaviors that were suggestive of a viscoelastic-plastic stress response. Remarkably, specimens returned to quiescent culture following a sequence of inflation tests recovered at least 80% of their original ability to store elastic strain energy, evidence of biological adaptation and recovery or restitutio ad integrum. Unlike methodologies that assay the morphological or biochemical response of cultured cells to an applied mechanostimulus, CDI experiments can be used to probe the load-bearing functions of desmosomes and adherens junctions within a living epithelial sheet, as well as to assess the rheological behaviors of the intermediate filament and microfilament networks that these cell-cell junctions serve to interconnect.     

Many ways to build an actin filament

Cells rely on extensive networks of protein fibres to help maintain their proper form and function. For species ranging from bacteria to humans, this 'cytoskeleton' is integrally involved in diverse processes including movement, DNA segregation, cell division and transport of molecular cargoes. The most abundant cytoskeletal filament-forming protein, F-actin, is remarkably well conserved across eukaryotic species. From yeast to human - an evolutionary distance of over one billion years - only about 10% of residues in actin have changed and the filament structure has been highly conserved. Surprisingly, recent structural data show this to be not the case for filamentous bacterial actins, which exhibit highly divergent helical symmetries in conjunction with structural plasticity or polymorphism, and dynamic properties that may make them uniquely suited for the specific cellular processes in which they participate. Bacterial actin filaments often organize themselves into complex structures within the prokaryotic cell, driven by molecular crowding and cation association, to form bundles (ParM) or interwoven sheets (MreB). The formation of supramolecular structures is essential for bacterial cytoskeleton function. We discuss the underlying physical principles that lead to complex structure formation and the implications these have on the physiological functions of cytoskeletal proteins.     

Contraction mechanisms in composite active actin networks

Simplified in vitro systems are ideally suited for studying the principle mechanisms of the contraction of cytoskeletal actin systems. To shed light on the dependence of the contraction mechanism on the nature of the crosslinking proteins, we study reconstituted in vitro active actin networks on different length scales ranging from the molecular organization to the macroscopic contraction. Distinct contraction mechanisms are observed in polar and apolar crosslinked active gels whereas composite active gels crosslinked in a polar and apolar fashion at the same time exhibit both mechanisms simultaneously. In polar active actin/fascin networks initially bundles are formed which are then rearranged. In contrast, apolar cortexillin-I crosslinked active gels are bundled only after reorganization of actin filaments by myosin-II motor filaments.     

Brushes,cables, and anchors: Recent insights into multiscale assembly and mechanics of cellular structural networks

The remarkable ability of living cells to sense, process, and respond to mechanical stimuli in their environment depends on the rapid and efficient interconversion of mechanical and chemical energy at specific times and places within the cell. For example, application of force to cells leads to conformational changes in specific mechanosensitive molecules which then trigger cellular signaling cascades that may alter cellular structure, mechanics, and migration and profoundly influence gene expression. Similarly, the sensitivity of cells to mechanical stresses is governed by the composition, architecture, and mechanics of the cellular cytoskeleton and extracellular matrix (ECM), which are in turn driven by molecular-scale forces between the constituent biopolymers. Understanding how these mechanochemical systems coordinate over multiple length and time scales to produce orchestrated cell behaviors represents a fundamental challenge in cell biology. Here, we review recent advances in our understanding of these complex processes in three experimental systems: the assembly of axonal neurofilaments, generation of tensile forces by actomyosin stress fiber bundles, and mechanical control of adhesion assembly.