Focal adhesions are controlled by microtubules through local contractility regulation.

Microtubules regulate cell polarity and migration via local activation of focal adhesion turnover, but the mechanism of this process is insufficiently understood. Molecular complexes containing KANK family proteins connect microtubules with talin, the major component of focal adhesions. Here, local optogenetic activation of KANK1-mediated microtubule/talin linkage promoted microtubule targeting to an individual focal adhesion and subsequent withdrawal, resulting in focal adhesion centripetal sliding and rapid disassembly. This sliding is preceded by a local increase of traction force due to accumulation of myosin-II and actin in the proximity of the focal adhesion. Knockdown of the Rho activator GEF-H1 prevented development of traction force and abolished sliding and disassembly of focal adhesions upon KANK1 activation. Other players participating in microtubule-driven, KANK-dependent focal adhesion disassembly include kinases ROCK, PAK, and FAK, as well as microtubules/focal adhesion-associated proteins kinesin-1, APC, and αTAT. Based on these data, we develop a mathematical model for a microtubule-driven focal adhesion disruption involving local GEF-H1/RhoA/ROCK-dependent activation of contractility, which is consistent with experimental data.

Membrane Tilt Drives Phase Separation of Adhesion Receptors.

Cell adhesion receptors are transmembrane proteins that bind cells to their environment. These proteins typically cluster into disk-shaped or linear structures. Here, we show that such clustering patterns spontaneously emerge when the receptor senses the membrane deformation gradient, for example, by reaching a lower-energy conformation when the membrane is tilted relative to the underlying binding substrate. Increasing the strength of the membrane gradient-sensing mechanism first yields isolated disk-shaped clusters and then long linear structures. Our theory is coherent with experimental estimates of the parameters, suggesting that a tilt-induced clustering mechanism is relevant in the context of cell adhesion.

Chiral growth of adherent filopodia.

Adherent filopodia are elongated finger-like membrane protrusions, extending from the edges of diverse cell types and participating in cell adhesion, spreading, migration, and environmental sensing. The formation and elongation of filopodia are driven by the polymerization of parallel actin filaments, comprising the filopodia cytoskeletal core. Here, we report that adherent filopodia, formed during the spreading of cultured cells on galectin-8-coated substrates, tend to change the direction of their extension in a chiral fashion, acquiring a left-bent shape. Cryoelectron tomography examination indicated that turning of the filopodia tip to the left is accompanied by the displacement of the actin core bundle to the right of the filopodia midline. Reduction of the adhesion to galectin-8 by treatment with thiodigalactoside abolished this filopodia chirality. By modulating the expression of a variety of actin-associated filopodia proteins, we identified myosin-X and formin DAAM1 as major filopodia chirality promoting factors. Formin mDia1, actin filament elongation factor VASP, and actin filament cross-linker fascin were also shown to be involved. Thus, the simple actin cytoskeleton of filopodia, together with a small number of associated proteins are sufficient to drive a complex navigation process, manifested by the development of left-right asymmetry in these cellular protrusions.

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 inhibits formin-driven actin polymerization in vitro, but not polymerization of pure actin. It is active against several types of formin from different species. Here, 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 activity of myosin ATPase and the ability to translocate actin filaments in the gliding actin in vitro motility assay. Inhibition of non-muscle myosin 2A in vitro required a higher concentration of SMIFH2 compared with that needed to inhibit retrograde flow and stress fiber contraction in cells. We also found that SMIFH2 inhibits several other non-muscle myosin types, including bovine myosin 10, Drosophila myosin 7a and Drosophila myosin 5, more efficiently than it inhibits formins. These off-target inhibitions demand additional careful analysis in each case when solely SMIFH2 is used to probe formin functions. This article has an associated First Person interview with Yukako Nishimura, joint first author of the paper.

Differential cellular responses to adhesive interactions with galectin-8- and fibronectin-coated substrates.

The mechanisms underlying the cellular response to extracellular matrices (ECMs) that consist of multiple adhesive ligands are still poorly understood. Here, we address this topic by monitoring specific cellular responses to two different extracellular adhesion molecules - the main integrin ligand fibronectin and galectin-8, a lectin that binds ß-galactoside residues  - as well as to mixtures of the two proteins. Compared with cell spreading on fibronectin, cell spreading on galectin-8-coated substrates resulted in increased projected cell area, more-pronounced extension of filopodia and, yet, the inability to form focal adhesions and stress fibers. These differences can be partially reversed by experimental manipulations of small G-proteins of the Rho family and their downstream targets, such as formins, the Arp2/3 complex and Rho kinase. We also show that the physical adhesion of cells to galectin-8 was stronger than adhesion to fibronectin. Notably, galectin-8 and fibronectin differently regulate cell spreading and focal adhesion formation, yet act synergistically to upregulate the number and length of filopodia. The physiological significance of the coherent cellular response to a molecularly complex matrix is discussed. This article has an associated First Person interview with the first author of the paper.

Actin cytoskeleton self-organization in single epithelial cells and fibroblasts under isotropic confinement.

Actin cytoskeleton self-organization in two cell types, fibroblasts and epitheliocytes, was studied in cells confined to isotropic adhesive islands. In fibroblasts plated onto islands of optimal size, an initially circular actin pattern evolves into a radial pattern of actin bundles that undergo asymmetric chiral swirling before finally producing parallel linear stress fibers. Epitheliocytes, however, did not exhibit succession through all the actin patterns described above. Upon confinement, the actin cytoskeleton in non-keratinocyte epitheliocytes was arrested at the circular stage, while in keratinocytes it progressed as far as the radial pattern but still could not break symmetry. Epithelial-mesenchymal transition pushed actin cytoskeleton development from circular towards radial patterns but remained insufficient to cause chirality. Knockout of cytokeratins also did not promote actin chirality development in keratinocytes. Left-right asymmetric cytoskeleton swirling could, however, be induced in keratinocytes by treatment with small doses of the G-actin sequestering drug, latrunculin A in a transcription-independent manner. Both the nucleus and the cytokeratin network followed the induced chiral swirling. Development of chirality in keratinocytes was controlled by DIAPH1 (mDia1) and VASP, proteins involved in regulation of actin polymerization.This article has an associated First Person interview with the first author of the paper.

Environmental sensing through focal adhesions.

Recent progress in the design and application of artificial cellular microenvironments and nanoenvironments has revealed the extraordinary ability of cells to adjust their cytoskeletal organization, and hence their shape and motility, to minute changes in their immediate surroundings. Integrin-based adhesion complexes, which are tightly associated with the actin cytoskeleton, comprise the cellular machinery that recognizes not only the biochemical diversity of the extracellular neighbourhood, but also its physical and topographical characteristics, such as pliability, dimensionality and ligand spacing. Here, we discuss the mechanisms of such environmental sensing, based on the finely tuned crosstalk between the assembly of one type of integrin-based adhesion complex, namely focal adhesions, and the forces that are at work in the associated cytoskeletal network owing to actin polymerization and actomyosin contraction.

Publisher Correction: A mechano-signalling network linking microtubules, myosin IIA filaments and integrin-based adhesions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A mechano-signalling network linking microtubules, myosin IIA filaments and integrin-based adhesions.

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 adhesion, 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, augmenting focal adhesions and disrupting podosomes. Myosin IIA filaments are indispensable effectors in the microtubule-driven 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 capture by integrin-mediated adhesions modulates the GEF-H1-dependent effect of microtubules on the assembly of myosin IIA filaments. Subsequent actomyosin reorganization then remodels the focal adhesions and podosomes, closing the regulatory loop.

Registry Kinetics of Myosin Motor Stacks Driven by Mechanical Force-Induced Actin Turnover.

Actin filaments associated with myosin motors constitute the cytoskeletal force-generating machinery for many types of adherent cells. These actomyosin units are structurally ordered in muscle cells and, in particular, may be spatially registered across neighboring actin bundles. Such registry or stacking of myosin filaments have been recently observed in ordered actin bundles of even fibroblasts with super-resolution microscopy techniques. We introduce here a model for the dynamics of stacking arising from long-range mechanical interactions between actomyosin units through mutual contractile deformations of the intervening cytoskeletal network. The dynamics of registry involve two key processes: 1) polymerization and depolymerization of actin filaments and 2) remodeling of cross-linker-rich actin adhesion zones, both of which are, in principle, mechanosensitive. By calculating the elastic forces that drive registry and their effect on actin polymerization rates, we estimate a characteristic timescale of tens of minutes for registry to be established, in agreement with experimentally observed timescales for individual kinetic processes involved in myosin stack formation, which we track and quantify. This model elucidates the role of actin turnover dynamics in myosin stacking and explains the loss of stacks seen when actin assembly or disassembly and cross-linking is experimentally disrupted in fibroblasts.

Mechanical stimulation induces formin-dependent assembly of a perinuclear actin rim.

Cells constantly sense and respond to mechanical signals by reorganizing their actin cytoskeleton. Although a number of studies have explored the effects of mechanical stimuli on actin dynamics, the immediate response of actin after force application has not been studied. We designed a method to monitor the spatiotemporal reorganization of actin after cell stimulation by local force application. We found that force could induce transient actin accumulation in the perinuclear region within ∼ 2 min. This actin reorganization was triggered by an intracellular Ca(2+) burst induced by force application. Treatment with the calcium ionophore A23187 recapitulated the force-induced perinuclear actin remodeling. Blocking of actin polymerization abolished this process. Overexpression of Klarsicht, ANC-1, Syne Homology (KASH) domain to displace nesprins from the nuclear envelope did not abolish Ca(2+)-dependent perinuclear actin assembly. However, the endoplasmic reticulum- and nuclear membrane-associated inverted formin-2 (INF2), a potent actin polymerization activator (mutations of which are associated with several genetic diseases), was found to be important for perinuclear actin assembly. The perinuclear actin rim structure colocalized with INF2 on stimulation, and INF2 depletion resulted in attenuation of the rim formation. Our study suggests that cells can respond rapidly to external force by remodeling perinuclear actin in a unique Ca(2+)- and INF2-dependent manner.

Mechanosensing Controlled Directly by Tyrosine Kinases.

To understand how cells form tissues, we need to understand how the tyrosine kinases are involved in controlling cell mechanics, whether they act directly as parts of mechanosensing machines or indirectly. Cells test the critical parameter of matrix rigidity by locally contracting ("pinching") matrices and measuring forces, and the depletion of contractile units causes transformation. We report here that knocking down the receptor tyrosine kinases (RTKs), AXL, and ROR2, alters rigidity sensing and increases the magnitude or duration of local contraction events, respectively. Phospho-AXL and ROR2 localize to contraction units and bind major contractile components, tropomyosin 2.1 (AXL), myosin IIA (AXL), and filamin A (ROR2). At a molecular level, phosphorylated AXL localizes to active myosin filaments and phosphorylates tropomyosin at a tyrosine critical for adhesion formation. ROR2 binding of ligand is unnecessary, but binding filamin A helps function. Thus, AXL and ROR2 alter rigidity sensing and consequently morphogenic processes by directly controlling local mechanosensory contractions without ligands.

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

Deviations from mirror symmetry in the development of bilateral organisms are common but the mechanisms of initial symmetry breaking are insufficiently understood. The actin cytoskeleton of individual cells self-organises in a chiral manner, but the molecular players involved remain essentially unidentified and the relationship between chirality of an individual cell and cell collectives is unclear. Here, we analysed self-organisation of the chiral actin cytoskeleton in individual cells on circular or elliptical patterns, and collective cell alignment in confined microcultures. Screening based on deep-learning analysis of actin patterns identified actin polymerisation regulators, depletion of which suppresses chirality (mDia1) or reverses chirality direction (profilin1 and CapZß). The reversed chirality  is mDia1-independent but requires the function of actin-crosslinker α-actinin1. A robust correlation between the effects of a variety of actin assembly regulators on chirality of individual cells and cell collectives is revealed. Thus, actin-driven cell chirality may underlie tissue and organ asymmetry.

Physical model for self-organization of actin cytoskeleton and adhesion complexes at the cell front.

Cell motion is driven by interplay between the actin cytoskeleton and the cell adhesions in the front part of the cell. The actin network segregates into lamellipodium and lamellum, whereas the adhesion complexes are characteristically distributed underneath the actin system. Here, we suggest a computational model for this characteristic organization of the actin-adhesion system. The model is based on the ability of the adhesion complexes to sense mechanical forces, the stick-slip character of the interaction between the adhesions and the moving actin network, and a hypothetical propensity of the actin network to disintegrate upon sufficiently strong stretching stresses. We identify numerically three possible types of system organization, all observed in living cells: two states in which the actin network exhibits segregation into lamellipodium and lamellum, whereas the cell edge either remains stationary or moves, and a state where the actin network does not undergo segregation. The model recovers the asynchronous fluctuations and outward bulging of the cell edge, and the dependence of the edge protrusion velocity on the rate of the nascent adhesion generation, the membrane tension, and the substrate rigidity.

Application of piconewton forces to individual filopodia reveals mechanosensory role of L-type Ca2+ channels.

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 be mechanosensitive, force-dependent activity of filopodial Ca2+ channels might be linked to filopodia's 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 using optical tweezers. Results show 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. Several known mechanosensitive Ca2+ channels, such as Piezo 1, Piezo 2 and TRPV4, were found to be dispensable for the observed force-dependent Ca2+ influx, while L-type Ca2+ channels appear to be a key player in the discovered phenomenon. As 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.

Dual control of formin-nucleated actin assembly by the chromatin and ER in mouse oocytes.

The first asymmetric meiotic cell divisions in mouse oocytes are driven by formin 2 (FMN2)-nucleated actin polymerization around the spindle. In this study, we investigated how FMN2 is recruited to the spindle peripheral ER and how its activity is regulated in mouse meiosis I (MI) oocytes. We show that this process is regulated by the Ran GTPase, a conserved mediator of chromatin signal, and the ER-associated protein VAPA. FMN2 contains a nuclear localization sequence (NLS) within a domain (SLD) previously shown to be required for FMN2 localization to the spindle periphery. FMN2 NLS is bound to the importin α1/ß complex, and the disruption of this interaction by RanGTP is required for FMN2 accumulation in the area proximal to the chromatin and the MI spindle. The importin-free FMN2 is then recruited to the surface of ER around the spindle through the binding of the SLD with the ER-membrane protein VAPA. We further show that FMN2 is autoinhibited through an intramolecular interaction between the SLD with the C-terminal formin homology 2 (FH2) domain that nucleates actin filaments. VAPA binding to SLD relieves the autoinhibition of FMN2, leading to localized actin polymerization. This dual control of formin-mediated actin assembly allows actin polymerization to initiate the movement of the meiotic spindle toward the cortex, an essential step in the maturation of the mammalian female gamete.

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.

A novel mechanism of actin filament processive capping by formin: solution of the rotation paradox.

The FH2 domains of formin family proteins act as processive cappers of actin filaments. Previously suggested stair-stepping mechanisms of processive capping imply that a formin cap rotates persistently in one direction with respect to the filament. This challenges the formin-mediated mechanism of intracellular cable formation. We suggest a novel scenario of processive capping that is driven by developing and relaxing torsion elastic stresses. Based on the recently discovered crystal structure of an FH2-actin complex, we propose a second mode of processive capping-the screw mode. Within the screw mode, the formin dimer rotates with respect to the actin filament in the direction opposite to that generated by the stair-stepping mode so that a combination of the two modes prevents persistent torsion strain accumulation. We determine an optimal regime of processive capping, whose essence is a periodic switch between the stair-stepping and screw modes. In this regime, elastic energy does not exceed feasible values, and supercoiling of actin filaments is prevented.

The heel and toe of the cell's foot: a multifaceted approach for understanding the structure and dynamics of focal adhesions.

Focal adhesions (FAs) are large clusters of transmembrane receptors of the integrin family and a multitude of associated cytoplasmic "plaque" proteins, which connect the extracellular matrix-bound receptors with the actin cytoskeleton. The formation of nearly stationary FAs defines a boundary between the dense and highly dynamic actin network in lamellipodium and the sparser and more diverse cytoskeletal organization in the lamella proper, creating a template for the organization of the entire actin network. The major "mechanical" and "sensory" functions of FAs; namely, the nucleation and regulation of the contractile, myosin-II-containing stress fibers and the mechanosensing of external surfaces depend, to a major extent, on the dynamics of molecular components within FAs. A central element in FA regulation concerns the positive feedback loop, based on the most intriguing feature of FAs; that is, their dependence on mechanical tension developing by the growing stress fibers. FAs grow in response to such tension, and rapidly disassemble upon its relaxation. In this article, we address the mechanistic relationships between the process of FA development, maturation and dissociation and the dynamic molecular events, which take place in different regions of the FA, primarily in the distal end of this structure (the "toe") and the proximal "heel," and discuss the central role of local mechanical forces in orchestrating the complex interplay between FAs and the actin system.