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Bershadsky, A. D.

Publications and source records attributed to Bershadsky, A. D..

8 recordsLinked to original sources

Fibrillar adhesions are the primary integrin complexes shaped by matrix topography

Mechanisms of matrix topography recognition are poorly understood. Here, we show that 5{beta}1-integrin mediated fibrillar adhesions serve this function. While on planar substrates, their formation requires fibronectin secretion, tensins, and actomyosin contractility, these requirements are bypassed on nanotopographical features. While focal adhesions avoid these features, fibrillar adhesions rapidly align along pre-existing fibrous cell-derived matrix or electrospun nanofibers where they can then template fibronectin fibrils. Topography-induced fibrillar adhesions depend primarily on 5{beta}1-integrin clustering and disassemble upon cortical stiffening driven by myosin-II overactivation or increased membrane tension. We propose a generic theoretical model where the adhesion receptor favours the membrane and substrate planes to be tilted relative to each other. This model matches experimental observations of preferential 5{beta}1-integrin clustering along nanofibers and concave edges of large negative curvature along micro-ridges. These findings establish fibrillar adhesions as primary adhesion complexes that form independently of focal adhesions in response to matrix topography.

cell biology↗

Crosstalk between myosin II and formin functions in the regulation of force generation and actomyosin dynamics in stress fibers

REF52 fibroblasts have a well-developed contractile machinery, the most prominent elements of which are actomyosin stress fibers with highly ordered organization of actin and myosin IIA filaments. The relationship between contractile activity and turnover dynamics of stress fibers is not sufficiently understood. Here, we simultaneously measured the forces exerted by stress fibers (using traction force microscopy or micropillar array sensors) and the dynamics of actin and myosin (using photoconversion-based monitoring of actin incorporation and high-resolution fluorescence microscopy of myosin II light chain). Our data revealed new features of the crosstalk between myosin II-driven contractility and stress fiber dynamics. During normal stress fiber turnover, actin incorporated all along the stress fibers and not only at focal adhesions. Incorporation of actin into stress fibers/focal adhesions, as well as actin and myosin II filaments flow along stress fibers, strongly depends on myosin II activity. Myosin II-dependent generation of traction forces does not depend on incorporation of actin into stress fibers per se, but still requires formin activity. This previously overlooked function of formins in maintenance of the actin cytoskeleton connectivity could be the main mechanism of formin involvement in traction force generation. Highlights* Cell traction forces are measured together with visualization of actomyosin flow * Actin turnover depends on formin and myosin II activities * Traction forces depend not only on myosin II, but also on formins * Traction force generation may require formin-dependent cytoskeleton connectivity

cell biology↗

Actin polymerization and crosslinking drive left-right asymmetry in single cell and cell collectives

Deviations from mirror symmetry in the development of bilateral organisms are highly stereotypic and genetically predetermined, but their mechanisms are not sufficiently understood. At the cellular level, self-organization of the actin cytoskeleton results in chiral actin swirling, and cells in groups confined to micropatterns demonstrate chiral cell alignment. The relationship between individual and collective cell chirality is unclear, and molecular players involved remain essentially unidentified. Here, by screening major actin-associated proteins and deep-learning-based morphometric analysis of actin patterns, we found that knockdowns of specific actin polymerization regulators, such as mDia1, ARPC2, and cofilins 1&2, abolished chiral actin swirling, while depletion of profilin 1 and CapZ{beta}, reversed its direction in an actin crosslinker -actinin1-dependent manner. Analysis of these and other knockdowns and pharmacological treatments revealed a robust correlation between their effects on the chirality of individual cells and confined cell groups. Thus, actin-driven cell chirality may underlie tissue and organ asymmetry. One Sentence SummaryCell chirality determined by specific regulators of actin polymerization drives left-right asymmetry emergence in cell groups

cell biology↗

Mechanosensitive calcium signaling in filopodia

Filopodia are ubiquitous membrane projections that play crucial role in guiding cell migration on rigid substrates and through extracellular matrix by utilizing yet unknown mechanosensing molecular pathways. As recent studies show that Ca2+ channels localized to filopodia play an important role in regulation of their formation and since some Ca2+ channels are known to possess mechanosensing properties, activity of filopodial Ca2+ channels might be tightly interlinked with the filopodia mechanosensing function. We tested this hypothesis by monitoring changes in the intra-filopodial Ca2+ level in response to application of stretching force to individual filopodia of several cell types. It has been found that stretching forces of tens of pN strongly promote Ca2+ influx into filopodia, causing persistent Ca2+ oscillations that last for minutes even after the force is released. Most of the known mechanosensitive Ca2+ channels, such as Piezo 1, Piezo 2 and TRPV4, were found to be dispensable for the observed force-dependent Ca2+ influx. In contrast, L-type Ca2+ channels appear to be a key component in the discovered phenomenon. Since previous studies have shown that intra-filopodial transient Ca2+ signals play an important role in guidance of cell migration, our results suggest that the force-dependent activation of L-type Ca2+ channels may contribute to this process. Overall, our study reveals an intricate interplay between mechanical forces and Ca2+ signaling in filopodia, providing novel mechanistic insights for the force-dependent filopodia functions in guidance of cell migration. Significance statementWe found that tensile forces of tens of pN applied to individual filopodia trigger Ca2+ influx through L-type Ca2+ channels, producing persistent Ca2+ oscillations inside mechanically stretched filopodia. Resulting elevation of the intra-filopodial Ca2+ level in turn leads to downstream activation of calpain protease, which is known to play a crucial role in regulation of the cell adhesion dynamics. Thus, our work suggests that L-type channel-dependent Ca2+ signaling and the mechanosensing function of filopodia are coupled to each other, synergistically governing cell adhesion and motion in a force-dependent manner. Since L-type Ca2+ channels have been previously found in many different cell types, such as neural or cancer cells, the above mechanism is likely to be widespread among various cell lines.

cell biology↗

The Formin Inhibitor, SMIFH2, Inhibits Members of the Myosin Superfamily

The small molecular inhibitor of formin FH2 domains, SMIFH2, is widely used in cell biological studies. It was selected in a chemical screen as a compound inhibiting formin-driven actin polymerization in vitro, but not polymerization of pure actin, and found to be active against several types of formins from different species (Rizvi et al., 2009). Here, in experiments with cultured fibroblasts, we found that SMIFH2 inhibits retrograde flow of myosin 2 filaments and contraction of stress fibers. We further checked the effect of SMIFH2 on non-muscle myosin 2A and skeletal muscle myosin 2 in vitro and found that SMIFH2 inhibits myosin ATPase activity and ability to translocate actin filaments in the in vitro motility assay. While inhibition of myosin 2A in vitro required somewhat higher concentration of SMIFH2 than inhibition of retrograde flow and stress fiber contraction in cells, inhibition of several other non-muscle myosin types, e.g. mammalian myosin 10, Drosophila myosin 7a and Drosophila myosin 5 by SMIFH2, was equally or more efficient than inhibition of formins. Since actin polymerization and myosin contractility are linked in many cytoskeleton processes, additional careful analysis is needed in each case when function of formins was proposed solely on the basis of experiment with SMIFH2.

cell biology↗

Forces and constraints controlling podosome assembly and disassembly

Podosomes are a singular category of integrin-mediated adhesions important in the processes of cell migration, matrix degradation, and cancer cell invasion. Despite a wealth of biochemical studies, the effects of mechanical forces on podosome integrity and dynamics are poorly understood. Here, we show that podosomes are highly sensitive to two groups of physical factors. First, we describe the process of podosome disassembly induced by activation of myosin-IIA filament assembly. Next, we find that podosome integrity and dynamics depends upon membrane tension and can be experimentally perturbed by osmotic swelling and deoxycholate treatment. We have also found that podosomes can be disrupted in a reversible manner by single or cyclic radial stretching of the substratum. We show that disruption of podosomes induced by osmotic swelling is independent of myosin-II filaments. Inhibition of the membrane sculpting protein, dynamin-II, but not clathrin, resulted in activation of myosin-IIA filament formation and disruption of podosomes. The effect of dynamin-II inhibition on podosomes was however independent of myosin-II filaments. Moreover, formation of organized arrays of podosomes in response to microtopographic cues (the ridges with triangular profile) was not accompanied by reorganization of myosin-II filaments. Thus, mechanical elements such as myosin-II filaments and factors affecting membrane tension/sculpting independently modulate podosome formation and dynamics, underlying a versatile response of these adhesion structures to intracellular and extracellular cues.

cell biology↗

Force dependence of filopodia adhesion: involvement of myosin II and formins

Filopodia are dynamic membrane protrusions driven by polymerization of an actin filament core, mediated by formin molecules at the filopodia tips. Filopodia can adhere to the extracellular matrix and experience both external and cell generated pulling forces. The role of such forces in filopodia adhesion is however insufficiently understood. Here, we induced sustained growth of filopodia by applying pulling force to their tips via attached fibronectin-coated beads trapped by optical tweezers. Strikingly, pharmacological inhibition or knockdown of myosin IIA, which localized to the base of filopodia, resulted in weakening of filopodia adherence strength. Inhibition of formins, which caused detachment of actin filaments from formin molecules, produced similar effect. Thus, myosin IIA-generated centripetal force transmitted to the filopodia tips through interactions between formins and actin filaments are required for filopodia adhesion. Force-dependent adhesion led to preferential attachment of filopodia to rigid versus fluid substrates, which may underlie cell orientation and polarization.

cell biology↗

Microtubule regulation of integrin-based adhesions is mediated by myosin-IIA

The interrelationship between microtubules and the actin cytoskeleton in mechanoregulation of integrin-mediated adhesions is poorly understood. Here, we show that the effects of microtubules on two major types of cell-matrix adhesions, focal adhesions and podosomes, are mediated by KANK family proteins connecting the adhesion protein talin with microtubule tips. Both total microtubule disruption and microtubule uncoupling from adhesions by manipulations with KANKs trigger a massive assembly of myosin-IIA filaments. Myosin-IIA filaments, augmenting the focal adhesions and disrupting the podosomes, are indispensable effectors in the microtubule-dependent regulation of integrin-mediated adhesions. Myosin-IIA filament assembly depends on Rho activation by the RhoGEF, GEF-H1, which is trapped by microtubules when they are connected with integrin-mediated adhesions via KANK proteins but released after their disconnection. Thus, microtubule capturing by integrin-mediated adhesions modulates the GEF-H1-dependent effect of microtubules on the myosin-IIA filaments. Subsequent actomyosin reorganization then remodels the focal adhesions and podosomes, closing the regulatory loop.

cell biology↗