Vimentin filaments integrate low-complexity domains in a complex helical structure.

Intermediate filaments (IFs) are integral components of the cytoskeleton. They provide cells with tissue-specific mechanical properties and are involved in numerous cellular processes. Due to their intricate architecture, a 3D structure of IFs has remained elusive. Here we use cryo-focused ion-beam milling, cryo-electron microscopy and tomography to obtain a 3D structure of vimentin IFs (VIFs). VIFs assemble into a modular, intertwined and flexible helical structure of 40 α-helices in cross-section, organized into five protofibrils. Surprisingly, the intrinsically disordered head domains form a fiber in the lumen of VIFs, while the intrinsically disordered tails form lateral connections between the protofibrils. Our findings demonstrate how protein domains of low sequence complexity can complement well-folded protein domains to construct a biopolymer with striking mechanical strength and stretchability.

Allosteric activation of vinculin by talin.

The talin-vinculin axis is a key mechanosensing component of cellular focal adhesions. How talin and vinculin respond to forces and regulate one another remains unclear. By combining single-molecule magnetic tweezers experiments, Molecular Dynamics simulations, actin-bundling assays, and adhesion assembly experiments in live cells, we here describe a two-ways allosteric network within vinculin as a regulator of the talin-vinculin interaction. We directly observe a maturation process of vinculin upon talin binding, which reinforces the binding to talin at a rate of 0.03 s-1. This allosteric transition can compete with force-induced dissociation of vinculin from talin only at forces up to 10 pN. Mimicking the allosteric activation by mutation yields a vinculin molecule that bundles actin and localizes to focal adhesions in a force-independent manner. Hence, the allosteric switch confines talin-vinculin interactions and focal adhesion build-up to intermediate force levels. The 'allosteric vinculin mutant' is a valuable molecular tool to further dissect the mechanical and biochemical signalling circuits at focal adhesions and elsewhere.

The Actin Network Interfacing Diverse Integrin-Mediated Adhesions.

The interface between the cellular actin network and diverse forms of integrin-mediated cell adhesions displays a unique capacity to serve as accurate chemical and mechanical sensors of the cell's microenvironment. Focal adhesion-like structures of diverse cell types, podosomes in osteoclasts, and invadopodia of invading cancer cells display distinct morphologies and apparent functions. Yet, all three share a similar composition and mode of coupling between a protrusive structure (the lamellipodium, the core actin bundle of the podosome, and the invadopodia protrusion, respectively), and a nearby adhesion site. Cytoskeletal or external forces, applied to the adhesion sites, trigger a cascade of unfolding and activation of key adhesome components (e.g., talin, vinculin, integrin), which in turn, trigger the assembly of adhesion sites and generation of adhesion-mediated signals that affect cell behavior and fate. The structural and molecular mechanisms underlying the dynamic crosstalk between the actin cytoskeleton and the adhesome network are discussed.

Enhanced statistical sampling reveals microscopic complexity in the talin mechanosensor folding energy landscape.

Statistical mechanics can describe the major conformational ensembles determining the equilibrium free-energy landscape of a folding protein. The challenge is to capture the full repertoire of low-occurrence conformations separated by high kinetic barriers that define complex landscapes. Computationally, enhanced sampling methods accelerate the exploration of molecular rare events. However, accessing the entire protein's conformational space in equilibrium experiments requires technological developments to enable extended observation times. We developed single-molecule magnetic tweezers to capture over a million individual transitions as a single talin protein unfolds and refolds under force in equilibrium. When observed at classically-probed timescales, talin folds in an apparently uncomplicated two-state manner. As the sampling time extends from minutes to days, the underlying energy landscape exhibits gradually larger signatures of complexity, involving a finite number of well-defined rare conformations. A fluctuation analysis allows us to propose plausible structures of each low-probability conformational state. The physiological relevance of each distinct conformation can be connected to the binding of the cytoskeletal protein vinculin, suggesting an extra layer of complexity in talin-mediated mechanotransduction. More generally, our experiments directly test the fundamental notion that equilibrium dynamics depend on the observation timescale.

A network of mixed actin polarity in the leading edge of spreading cells.

Physical interactions of cells with the underlying extracellular matrix (ECM) play key roles in multiple cellular processes. The actin cytoskeleton is a central driver and regulator of cellular dynamics, that produces membrane-protrusions such as lamellipodia and filopodia. Here, we examined actin organization in expanding lamellipodia during early stages of cell spreading. To gain insight into the 3D actin organization, we plated fibroblasts on galectin-8 coated EM grids, an ECM protein presents in disease states. We then combined cryo-electron tomography with advanced image processing tools for reconstructing the structure of F-actin in the lamellipodia. This approach enabled us to resolve the polarity and orientation of filaments, and the structure of the Arp2/3 complexes associated with F-actin branches. We show that F-actin in lamellipodial protrusions forms a dense network with three distinct sub-domains. One consists primarily of radial filaments, with their barbed ends pointing towards the membrane, the other is enriched with parallel filaments that run between the radial fibers, in addition to an intermediate sub-domain. Surprisingly, a minor, yet significant (~10%) population of actin filaments, are oriented with their barbed-ends towards the cell center. Our results provide structural insights into F-actin assembly and dynamic reorganization in the leading edge of spreading cells.

The Multicellular Effects of VDAC1 N-Terminal-Derived Peptide.

The mitochondrial voltage-dependent anion channel-1 (VDAC1) protein functions in a variety of mitochondria-linked physiological and pathological processes, including metabolism and cell signaling, as well as in mitochondria-mediated apoptosis. VDAC1 interacts with about 150 proteins to regulate the integration of mitochondrial functions with other cellular activities. Recently, we developed VDAC1-based peptides that have multiple effects on cancer cells and tumors including apoptosis induction. Here, we designed several cell-penetrating VDAC1 N-terminal-derived peptides with the goal of identifying the shortest peptide with improved cellular stability and activity. We identified the D-Δ(1-18)N-Ter-Antp comprising the VDAC1 N-terminal region (19-26 amino acids) fused to the Antp, a cell-penetrating peptide. We demonstrated that this peptide induced apoptosis, autophagy, senescence, cell volume enlargement, and the refusion of divided daughter cells into a single cell, it was responsible for reorganization of actin and tubulin filaments, and increased cell adhesion. In addition, the peptide induced alterations in the expression of proteins associated with cell metabolism, signaling, and division, such as enhancing the expression of nuclear factor kappa B and decreasing the expression of the nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha. These cellular effects may result from the peptide interfering with VDAC1 interaction with its interacting proteins, thereby blocking multiple mitochondrial/VDAC1 pathways associated with cell functions. The results of this study further support the role of VDAC1 as a mitochondrial gatekeeper protein in controlling a variety of cell functions via interaction with associated proteins.

Talin-activated vinculin interacts with branched actin networks to initiate bundles.

Vinculin plays a fundamental role in integrin-mediated cell adhesion. Activated by talin, it interacts with diverse adhesome components, enabling mechanical coupling between the actin cytoskeleton and the extracellular matrix. Here we studied the interactions of activated full-length vinculin with actin and the way it regulates the organization and dynamics of the Arp2/3 complex-mediated branched actin network. Through a combination of surface patterning and light microscopy experiments we show that vinculin can bundle dendritic actin networks through rapid binding and filament crosslinking. We show that vinculin promotes stable but flexible actin bundles having a mixed-polarity organization, as confirmed by cryo-electron tomography. Adhesion-like synthetic design of vinculin activation by surface-bound talin revealed that clustered vinculin can initiate and immobilize bundles from mobile Arp2/3-branched networks. Our results provide a molecular basis for coordinate actin bundle formation at nascent adhesions.

Force Production by a Bundle of Growing Actin Filaments Is Limited by Its Mechanical Properties.

Bundles of actin filaments are central to a large variety of cellular structures such as filopodia, stress fibers, cytokinetic rings, and focal adhesions. The mechanical properties of these bundles are critical for proper force transmission and force bearing. Previous mathematical modeling efforts have focused on bundles' rigidity and shape. However, it remains unknown how bundle length and buckling are controlled by external physical factors. In this work, we present a biophysical model for dynamic bundles of actin filaments submitted to an external load. In combination with in vitro motility assays of beads coated with formins, our model allowed us to characterize conditions for bead movement and bundle buckling. From the deformation profiles, we determined key biophysical properties of tethered actin bundles such as their rigidity and filament density.

Quantitative regulation of the dynamic steady state of actin networks.

Principles of regulation of actin network dimensions are fundamentally important for cell functions, yet remain unclear. Using both in vitro and in silico approaches, we studied the effect of key parameters, such as actin density, ADF/Cofilin concentration and network width on the network length. In the presence of ADF/Cofilin, networks reached equilibrium and became treadmilling. At the trailing edge, the network disintegrated into large fragments. A mathematical model predicts the network length as a function of width, actin and ADF/Cofilin concentrations. Local depletion of ADF/Cofilin by binding to actin is significant, leading to wider networks growing longer. A single rate of breaking network nodes, proportional to ADF/Cofilin density and inversely proportional to the square of the actin density, can account for the disassembly dynamics. Selective disassembly of heterogeneous networks by ADF/Cofilin controls steering during motility. Our results establish general principles on how the dynamic steady state of actin network emerges from biochemical and structural feedbacks.

Tiopronin-Protected Gold Nanoparticles as a Potential Marker for Cryo-EM and Tomography.

Gold nanoparticles (AuNPs) and their conjugation to biological samples have numerous potential applications. When combined with cryo-electron microscopy and tomography analysis, AuNPs may provide a versatile and powerful tool to identify and precisely localize proteins even when attached to cellular components. Here, we describe a general and facile approach for the synthesis of homogeneous and stable AuNPs, which can readily be conjugated to a molecule of interest and imaged by cryo-electron tomography (cryo-ET). We demonstrate the synthesis of 2.2 ± 0.45-nm tiopronin-protected AuNPs, followed by their conjugation with recombinant proteins and peptides. Visualization of the ∼2.2-nm gold-tagged peptides by cryo-ET reveals the potential use of this strategy to label and localize accessible proteins in a cellular environment with nanometric resolution.

Network heterogeneity regulates steering in actin-based motility.

The growth of branched actin networks powers cell-edge protrusions and motility. A heterogeneous density of actin, which yields to a tunable cellular response, characterizes these dynamic structures. We study how actin organization controls both the rate and the steering during lamellipodium growth. We use a high-resolution surface structuration assay combined with mathematical modeling to describe the growth of a reconstituted lamellipodium. We demonstrate that local monomer depletion at the site of assembly negatively impacts the network growth rate. At the same time, network architecture tunes the protrusion efficiency, and regulates the rate of growth. One consequence of this interdependence between monomer depletion and network architecture effects is the ability of heterogeneous network to impose steering during motility. Therefore, we have established that the general principle, by which the cell can modulate the rate and the direction of a protrusion, is by varying both density and architecture of its actin network.Protrusive cellular structures contain a heterogeneous density of actin, but whether this influences motility is not known. Using an in vitro system and modelling, here the authors show that local actin monomer depletion and network architecture can tune the rate of network growth to impose steering during motility.

Directed actin assembly and motility.

The actin cytoskeleton is a key component of the cellular architecture. However, understanding actin organization and dynamics in vivo is a complex challenge. Reconstitution of actin structures in vitro, in simplified media, allows one to pinpoint the cellular biochemical components and their molecular interactions underlying the architecture and dynamics of the actin network. Previously, little was known about the extent to which geometrical constraints influence the dynamic ultrastructure of these networks. Therefore, in order to study the balance between biochemical and geometrical control of complex actin organization, we used the innovative methodologies of UV and laser patterning to design a wide repertoire of nucleation geometries from which we assembled branched actin networks. Using these methods, we were able to reconstitute complex actin network organizations, closely related to cellular architecture, to precisely direct and control their 3D connections. This methodology mimics the actin networks encountered in cells and can serve in the fabrication of innovative bioinspired systems.

Autonomous and in trans functions for the two halves of Srv2/CAP in promoting actin turnover.

Recent evidence has suggested that Srv2/CAP (cyclase-associated protein) has two distinct functional roles in regulating actin turnover, with its N-terminus enhancing cofilin-mediated severing of actin filaments and its C-terminus catalyzing actin monomer recycling. However, it has remained unclear to what degree these two activities are coordinated by being linked in one molecule, or whether they can function autonomously. To address this, we physically divided the protein into two separate halves, N-Srv2 and C-Srv2, and asked whether they are able to function in trans both in living cells and in reconstituted assays for F-actin turnover and actin-based motility. Remarkably, in F-actin turnover assays the stimulatory effects of N-Srv2 and C-Srv2 functioning in trans were quantitatively similar to those of intact full-length Srv2. Further, in bead motility assays and in vivo, the fragments again functioned in trans, although not with the full effectiveness of intact Srv2. From these data, we conclude that the functions of the two halves of Srv2/CAP are largely autonomous, although their linkage improves coordination of the two functions in specific settings, possibly explaining why the linkage is conserved across distant plant, animal, and fungal species.

Geometrical control of actin assembly and contractility.

The actin cytoskeleton is a fundamental player in many cellular processes. Ultrastructural studies have revealed its extremely complex organization, where actin filaments self-organize into defined and specialized structures of distinct functions and, yet, are able to selectively recruit biochemical regulators that are available in the entire cell volume. To overcome this extraordinary complexity, simplified reconstituted systems significantly improve our understanding of actin dynamics and self-organization. However, little is known regarding physical rules governing actin networks organization and to which extent network structure may direct and regulate selective interactions with specific regulators. Here, we describe the first method to direct actin filament assembly to specific 2D motifs with a finely tuned geometry and relative distribution. This method enables the study of how geometrical confinement governs actin network structural organization and how, in return, structural cues can control selective contraction by myosin motor. The protocol relies on the use of surface micropatterning and functionalization procedures in order to selectively direct actin filament assembly to specific sites of nucleation.

Actin dynamics, architecture, and mechanics in cell motility.

Tight coupling between biochemical and mechanical properties of the actin cytoskeleton drives a large range of cellular processes including polarity establishment, morphogenesis, and motility. This is possible because actin filaments are semi-flexible polymers that, in conjunction with the molecular motor myosin, can act as biological active springs or "dashpots" (in laymen's terms, shock absorbers or fluidizers) able to exert or resist against force in a cellular environment. To modulate their mechanical properties, actin filaments can organize into a variety of architectures generating a diversity of cellular organizations including branched or crosslinked networks in the lamellipodium, parallel bundles in filopodia, and antiparallel structures in contractile fibers. In this review we describe the feedback loop between biochemical and mechanical properties of actin organization at the molecular level in vitro, then we integrate this knowledge into our current understanding of cellular actin organization and its physiological roles.

How actin network dynamics control the onset of actin-based motility.

Cells use their dynamic actin network to control their mechanics and motility. These networks are made of branched actin filaments generated by the Arp2/3 complex. Here we study under which conditions the microscopic organization of branched actin networks builds up a sufficient stress to trigger sustained motility. In our experimental setup, dynamic actin networks or "gels" are grown on a hard bead in a controlled minimal protein system containing actin monomers, profilin, the Arp2/3 complex and capping protein. We vary protein concentrations and follow experimentally and through simulations the shape and mechanical properties of the actin gel growing around beads. Actin gel morphology is controlled by elementary steps including "primer" contact, growth of the network, entanglement, mechanical interaction and force production. We show that varying the biochemical orchestration of these steps can lead to the loss of network cohesion and the lack of effective force production. We propose a predictive phase diagram of actin gel fate as a function of protein concentrations. This work unveils how, in growing actin networks, a tight biochemical and physical coupling smoothens initial primer-caused heterogeneities and governs force buildup and cell motility.

Actin network architecture can determine myosin motor activity.

The organization of actin filaments into higher-ordered structures governs eukaryotic cell shape and movement. Global actin network size and architecture are maintained in a dynamic steady state through regulated assembly and disassembly. Here, we used experimentally defined actin structures in vitro to investigate how the activity of myosin motors depends on network architecture. Direct visualization of filaments revealed myosin-induced actin network deformation. During this reorganization, myosins selectively contracted and disassembled antiparallel actin structures, while parallel actin bundles remained unaffected. The local distribution of nucleation sites and the resulting orientation of actin filaments appeared to regulate the scalability of the contraction process. This "orientation selection" mechanism for selective contraction and disassembly suggests how the dynamics of the cellular actin cytoskeleton can be spatially controlled by actomyosin contractility.

Turnover of branched actin filament networks by stochastic fragmentation with ADF/cofilin.

Cell motility depends on the rapid assembly, aging, severing, and disassembly of actin filaments in spatially distinct zones. How a set of actin regulatory proteins that sustains actin-based force generation during motility work together in space and time remains poorly understood. We present our study of the distribution and dynamics of Arp2/3 complex, capping protein (CP), and actin-depolymerizing factor (ADF)/cofilin in actin "comet tails," using a minimal reconstituted system with nucleation-promoting factor (NPF)-coated beads. The Arp2/3 complex concentrates at nucleation sites near the beads as well as in the first actin shell. CP colocalizes with actin and is homogeneously distributed throughout the comet tail; it serves to constrain the spatial distribution of ATP/ADP-P(i) filament zones to areas near the bead. The association of ADF/cofilin with the actin network is therefore governed by kinetics of actin assembly, actin nucleotide state, and CP binding. A kinetic simulation accurately validates these observations. Following its binding to the actin networks, ADF/cofilin is able to break up the dense actin filament array of a comet tail. Stochastic severing by ADF/cofilin loosens the tight entanglement of actin filaments inside the comet tail and facilitates turnover through the macroscopic release of large portions of the aged actin network.

Cofilin tunes the nucleotide state of actin filaments and severs at bare and decorated segment boundaries.

Actin-based motility demands the spatial and temporal coordination of numerous regulatory actin-binding proteins (ABPs), many of which bind with affinities that depend on the nucleotide state of actin filament. Cofilin, one of three ABPs that precisely choreograph actin assembly and organization into comet tails that drive motility in vitro, binds and stochastically severs aged ADP actin filament segments of de novo growing actin filaments. Deficiencies in methodologies to track in real time the nucleotide state of actin filaments, as well as cofilin severing, limit the molecular understanding of coupling between actin filament chemical and mechanical states and severing. We engineered a fluorescently labeled cofilin that retains actin filament binding and severing activities. Because cofilin binding depends strongly on the actin-bound nucleotide, direct visualization of fluorescent cofilin binding serves as a marker of the actin filament nucleotide state during assembly. Bound cofilin allosterically accelerates P(i) release from unoccupied filament subunits, which shortens the filament ATP/ADP-P(i) cap length by nearly an order of magnitude. Real-time visualization of filament severing indicates that fragmentation scales with and occurs preferentially at boundaries between bare and cofilin-decorated filament segments, thereby controlling the overall filament length, depending on cofilin binding density.

Actin dynamics in plant cells: a team effort from multiple proteins orchestrates this very fast-paced game.

Gazing at a giant redwood tree in the Pacific Northwest, that has grown to enormous heights over centuries, does little to convince one that plants are built for speed and versatility. Even at the cellular level, a system of polymers-the cell skeleton or cytoskeleton-integrates signals and generates subcellular structures spanning scales of a few nanometers to hundreds of micrometers that coordinate cell growth. The term cytoskeleton itself connotes a stable structure. Clearly, this is not the case. Recent studies using advanced imaging modalities reveal the plant actin cytoskeleton to be a highly dynamic, ever changing assemblage of polymers. These insights along with growing evidence about the biochemical/biophysical properties of plant cytoskeletal polymers, especially those obtained by single filament imaging and reconstituted systems of purified proteins analyzed by total internal reflection fluorescence microscopy, allow the generation of a unique model for the dynamic turnover of actin filaments, termed stochastic dynamics. Here, we review several significant advances and highlight opportunities that will position plants at the vanguard of research on actin organization and turnover. A challenge for the future will be to apply the power of reverse-genetics in several model organisms to test the molecular details of this new model.