the-mechanics-poro-elastic-contractile-actomyosin-networks-as-model

The Mechanics of (Poro-)Elastic Contractile Actomyosin Networks As a Model System of the Cell Cytoskeleton

Cells can actively change their shapes and become motile, a property that depends on their ability to actively reorganize their internal structure. This ...

Ben Gurion University of the Negev

The cellular cytoskeleton is a complex dynamical network that constantly remodels as cells divide and move. This reorganization process occurs not only at the cell membrane, but also in the cell interior (bulk). During locomotion, regulated actin assembly near the plasma membrane produces lamellipodia and filopodia. Therefore, most in vitro experiments explore phenomena taking place in the vicinity of a surface. To understand how the molecular machinery of a cell self-organizes in a more general way, we studied bulk polymerization of actin in the presence of actin-related protein 2/3 complex and a nucleation promoting factor as a model for actin assembly in the cell interior separate from membranes. Bulk polymerization of actin in the presence of the verprolin homology, cofilin homology, and acidic region, domain of Wiskott-Aldrich syndrome protein, and actin-related protein 2/3 complex results in spontaneous formation of diffuse aster-like structures. In the presence of fascin these asters transition into stars with bundles of actin filaments growing from the surface, similar to star-like structures recently observed in vivo. The transition from asters to stars depends on the ratio [fascin]/[G actin]. The polarity of the actin filaments during the transition is preserved, as in the transition from lamellipodia to filopodia. Capping protein inhibits star formation. Based on these experiments and kinetic Monte Carlo simulations, we propose a model for the spontaneous self-assembly of asters and their transition into stars. This mechanism may apply to the transition from lamellipodia to filopodia in vivo.

Reconstitution of the transition from lamellipodium to filopodium in a membrane-free system.

Bundles of filamentous actin form the primary building blocks of a broad range of cytoskeletal structures, including filopodia, stereocilia and microvilli. In each case, the cell uses specific associated proteins to tailor the dynamics, dimensions and mechanical properties of the bundles to suit a specific cellular function. While the length distribution of actin bundles was extensively studied, almost nothing is known about the thickness distribution. Here, we use high-resolution cryo-TEM to measure the thickness distribution of actin/fascin bundles, in vitro. We find that the thickness distribution has a prominent peak, with an exponential tail, supporting a scenario of an initial fast formation of a disc-like nucleus of short actin filaments, which only later elongates. The bundle thicknesses at steady state are found to follow the distribution of the initial nuclei indicating that no lateral coalescence occurs. Our results show that the distribution of bundles thicknesses can be controlled by monitoring the initial nucleation process. In vivo, this is done by using specific regulatory proteins complexes.

Thickness distribution of actin bundles in vitro.

Myosin II motors play several important roles in a variety of cellular processes, some of which involve active assembly/disassembly of cytoskeletal substructures. Myosin II motors have been shown to function in actin bundle turnover in neuronal growth cones and in the recycling of actin filaments during cytokinesis. Close examination had shown an intimate relationship between myosin II motor adenosine triphosphatase activity and actin turnover rate. However, the direct implication of myosin II in actin turnover is still not understood. Herein, we show, using high-resolution cryo-transmission electron microscopy, that myosin II motors control the turnover of actin bundles in a concentration-dependent manner in vitro. We demonstrate that disassembly of actin bundles occurs through two main stages: the first stage involves unbundling into individual filaments, and the second involves their subsequent depolymerization. These evidence suggest that, in addition to their "classical" contractile abilities, myosin II motors may be directly implicated in active actin depolymerization. We believe that myosin II motors may function similarly in vivo (e.g., in the disassembly of the contractile ring by fine tuning the local concentration/activity of myosin II motors).

A cytoskeletal demolition worker: myosin II acts as an actin depolymerization agent.

During cellular migration, regulated actin assembly takes place at the cell leading edge, with continuous disassembly deeper in the cell interior. Actin polymerization at the plasma membrane results in the extension of cellular protrusions in the form of lamellipodia and filopodia. To understand how cells regulate the transformation of lamellipodia into filopodia, and to determine the major factors that control their transition, we studied actin self-assembly in the presence of Arp2/3 complex, WASp-VCA and fascin, the major proteins participating in the assembly of lamellipodia and filopodia. We show that in the early stages of actin polymerization fascin is passive while Arp2/3 mediates the formation of dense and highly branched aster-like networks of actin. Once filaments in the periphery of an aster get long enough, fascin becomes active, linking the filaments into bundles which emanate radially from the aster's surface, resulting in the formation of star-like structures. We show that the number of bundles nucleated per star, as well as their thickness and length, is controlled by the initial concentration of Arp2/3 complex ([Arp2/3]). Specifically, we tested several values of [Arp2/3] and found that for given initial concentrations of actin and fascin, the number of bundles per star, as well as their length and thickness are larger when [Arp2/3] is lower. Our experimental findings can be interpreted and explained using a theoretical scheme which combines Kinetic Monte Carlo simulations for aster growth, with a simple mechanistic model for bundles' formation and growth. According to this model, bundles emerge from the aster's (sparsely branched) surface layer. Bundles begin to form when the bending energy associated with bringing two filaments into contact is compensated by the energetic gain resulting from their fascin linking energy. As time evolves the initially thin and short bundles elongate, thus reducing their bending energy and allowing them to further associate and create thicker bundles, until all actin monomers are consumed. This process is essentially irreversible on the time scale of actin polymerization. Two structural parameters, L, which is proportional to the length of filament tips at the aster periphery and b, the spacing between their origins, dictate the onset of bundling; both depending on [Arp2/3]. Cells may use a similar mechanism to regulate filopodia formation along the cell leading edge. Such a mechanism may allow cells to have control over the localization of filopodia by recruiting specific proteins that regulate filaments length (e.g., Dia2) to specific sites along lamellipodia.

Arp2/3 branched actin network mediates filopodia-like bundles formation in vitro.

The cooperative action of many molecular motors is essential for dynamic processes such as cell motility and mitosis. This action can be studied by using motility assays which track the motion of cytoskeletal filaments over a surface coated with motor proteins. Here, we propose to use a motility assay consisting of a-polar actin bundles subjected to the action of myosin II motors where no external loading is applied. In this work we focus on those bundles undergoing fusion with other nearby bundles. Specifically, we investigate the role of the bundles' dimension on the transition from bidirectional to directional motion and on the properties of their motion during fusion. Our experimental data reveal that only small bundles exhibit dynamic transition to directional motion, implying that the forces acting on them exceed the threshold value necessary to induce the transition. Moreover, these bundles accelerate along their trajectory, suggesting that the forces acting on them increase while approaching each other. We show that these forces do not originate from external loading but rather arise from the action of the motors on the bundles. These forces are transmitted through the medium over micron-scale distances without being cut off. Moreover, we show that the forces propagate to distances that are proportional to the size of the bundles, or equivalently, to the number of motors, which they interact with.

The fusion of actin bundles driven by interacting motor proteins.

Cortactin is involved in invadopodia and podosome formation [1], pathogens and endosome motility [2], and persistent lamellipodia protrusion [3, 4]; its overexpression enhances cellular motility and metastatic activity [5-8]. Several mechanisms have been proposed to explain cortactin's role in Arp2/3-driven actin polymerization [9, 10], yet its direct role in cell movement remains unclear. We use a biomimetic system to study the mechanism of cortactin-mediated regulation of actin-driven motility [11]. We tested the role of different cortactin variants that interact with Arp2/3 complex and actin filaments distinctively. We show that wild-type cortactin significantly enhances the bead velocity at low concentrations. Single filament experiments show that cortactin has no significant effect on actin polymerization and branch stability, whereas it strongly affects the branching rate driven by Wiskott-Aldrich syndrome protein (WASP)-VCA fragment and Arp2/3 complex. These results lead us to propose that cortactin plays a critical role in translating actin polymerization at a bead surface into motion, by releasing WASP-VCA from the new branching site. This enhanced release has two major effects: it increases the turnover rate of branching per WASP molecule, and it decreases the friction-like force caused by the binding of the moving surface with respect to the growing actin network.

Cortactin releases the brakes in actin- based motility by enhancing WASP-VCA detachment from Arp2/3 branches.

The structural reorganization of the actin cytoskeleton is facilitated through the action of motor proteins that crosslink the actin filaments and transport them relative to each other. Here, we present a combined experimental-computational study that probes the dynamic evolution of mixtures of actin filaments and clusters of myosin motors. While on small spatial and temporal scales the system behaves in a very noisy manner, on larger scales it evolves into several well distinct patterns such as bundles, asters and networks. These patterns are characterized by junctions with high connectivity, whose formation is possible due to the organization of the motors in 'oligoclusters' (intermediate-size aggregates). The simulations reveal that the self-organization process proceeds through a series of hierarchical steps, starting from local microscopic moves and ranging up to the macroscopic large scales where the steady-state structures are formed. Our results shed light on the mechanisms involved in processes such as cytokinesis and cellular contractility, where myosin motors organized in clusters operate cooperatively to induce the structural organization of cytoskeletal networks.

Hierarchical self-organization of cytoskeletal active networks.

Actin polymerization plays a major role in many cellular processes, including cell motility, vesicle trafficking, and pathogen propulsion. The transformation of the (protrusive) polymerization forces into directed motion requires that the growing filaments are positioned next to the surface. This is achieved by localization of surface actin nucleators (WASP), which then activate Arp2/3 complex to form new actin branches. Yet, the same surface-bound WASP molecule which initiates the nucleation of new actin branches, also inherently prevents the translation of the polymerization forces into motion, essentially because the WASP molecule has to be in contact with the network during the formation of the new branch. In our recent paper we show that cortactin relaxes this internal inhibition by enhancing the release of WASP-VCA molecule from the new branching site after nucleation is initiated. We show that this enhanced release has two major effects; it increases the turnover rate of branching per WASP molecule, and it decreases the friction-like force caused by the binding of the moving surface with respect to the growing actin network.

Releasing the brakes while hanging on: Cortactin effects on actin-driven motility.

Vasodilator-stimulated phosphoprotein (VASP) is active in many filopodium-based and cytoskeleton reorganization processes. It is not fully understood how VASP directly functions in actin-based motility and how regulatory proteins affect its function. Here, we combine bead motility assay and single filament experiments. In the presence of a bundling component, actin bundles that grow from the surface of WT-VASP-coated beads induced movement of the beads. VASP promotes actin-based movement alone, in the absence of other actin nucleators. We propose that at physiological salt conditions VASP nucleation activity is too weak to promote motility and bundle formation. Rather, VASP recruits F-actin seeds from the solution and promotes their elongation. Cofilin has a crucial role in the nucleation of these F-actin seeds, notably under conditions of unfavorable spontaneous actin nucleation. We explored the role of multiple VASP variants. We found that the VASP-F-actin binding domain is required for the recruitment of F-actin seeds from the solution. We also found that the interaction of profilin-actin complexes with the VASP-proline-rich domain and the binding of the VASP-F-actin binding domain to the side of growing filaments is critical for transforming actin polymerization into motion. At the single filament level, profilin mediates both filament elongation rate and VASP anti-capping activity. Binding of profilin-actin complexes increases the polymerization efficiency by VASP but decreases its efficiency as an anti-capper; binding of free profilin creates the opposite effect. Finally, we found that an additional component such as methylcellulose or fascin is required for actin bundle formation and motility mediated by VASP.

Reconstitution of actin-based motility by vasodilator-stimulated phosphoprotein (VASP) depends on the recruitment of F-actin seeds from the solution produced by cofilin.

Cellular motility is a fundamental process essential for embryonic development, wound healing, immune responses, and tissues development. Cells are mostly moving by crawling on external, or inside, substrates which can differ in their surface composition, geometry, and dimensionality. Cells can adopt different migration phenotypes, e.g., bleb-based and protrusion-based, depending on myosin contractility, surface adhesion, and cell confinement. In the few past decades, research on cell motility has focused on uncovering the major molecular players and their order of events. Despite major progresses, our ability to infer on the collective behavior from the molecular properties remains a major challenge, especially because cell migration integrates numerous chemical and mechanical processes that are coupled via feedbacks that span over large range of time and length scales. For this reason, reconstituted model systems were developed. These systems allow for full control of the molecular constituents and various system parameters, thereby providing insight into their individual roles and functions. In this review we describe the various reconstituted model systems that were developed in the past decades. Because of the multiple steps involved in cell motility and the complexity of the overall process, most of the model systems focus on very specific aspects of the individual steps of cell motility. Here we describe the main advancement in cell motility reconstitution and discuss the main challenges toward the realization of a synthetic motile cell.

Toward the reconstitution of synthetic cell motility.

Cells require actin nucleation factors to catalyze the formation of actin networks and elongation factors to control the rate and extent of actin polymerization. Earlier models suggested that the different factors assemble actin networks independently. However, recent evidence indicates that the assembly of most cellular networks involves multiple nucleation and elongation factors that work in concert. Here, we describe how these different factors cooperate, directly or indirectly, to promote the assembly of functional actin network in cells, both in the cytoplasm and nucleoplasm. We show that, in many cases, multiple factors collaborate to initiate network assembly and growth. The selection of specific sets of key players enables the cells to fine-tune network structure and dynamics, optimizing them for particular cellular functions.

Functional Actin Networks under Construction: The Cooperative Action of Actin Nucleation and Elongation Factors.

Shape transitions in developing organisms can be driven by active stresses, notably, active contractility generated by myosin motors. The mechanisms generating tissue folding are typically studied in epithelia. There, the interaction between cells is also coupled to an elastic substrate, presenting a major difficulty for studying contraction induced folding. Here we study the contraction and buckling of active, initially homogeneous, thin elastic actomyosin networks isolated from bounding surfaces. The network behaves as a poroelastic material, where a flow of fluid is generated during contraction. Contraction starts at the system boundaries, proceeds into the bulk, and eventually leads to spontaneous buckling of the sheet at the periphery. The buckling instability resulted from system self-organization and from the spontaneous emergence of density gradients driven by the active contractility. The buckling wavelength increases linearly with sheet thickness. Our system offers a well-controlled way to study mechanically induced, spontaneous shape transitions in active matter.

Spontaneous buckling of contractile poroelastic actomyosin sheets.

An introduction to the physical properties of living active matter at the mesoscopic scale (tens of nanometers to micrometers) and their unique features compared with "dead," nonactive matter is presented. This field of research is increasingly denoted as "biological physics" where physics includes chemical physics, soft matter physics, hydrodynamics, mechanics, and the related engineering sciences. The focus is on the emergent properties of these systems and their collective behavior, which results in active self-organization and how they relate to cellular-level biological function. These include locomotion (cell motility and migration) forces that give rise to cell division, the growth and form of cellular assemblies in development, the beating of heart cells, and the effects of mechanical perturbations such as shear flow (in the bloodstream) or adhesion to other cells or tissues. An introduction to the fundamental concepts and theory with selected experimental examples related to the authors' own research is presented, including red-blood-cell membrane fluctuations, motion of the nucleus within an egg cell, self-contracting acto-myosin gels, and structure and beating of heart cells (cardiomyocytes), including how they can be driven by an oscillating, mechanical probe.

Living Matter: Mesoscopic Active Materials.

The deposition of cellulose nanocrystals (CNCs) on a supported lipid bilayer (SLB) was investigated at different length scales. Quartz crystal microbalance with dissipation monitoring (QCM-D) was used to probe the bilayer formation and to show for the first time the CNC deposition onto the SLB. Specifically, classical QCM-D measurements gave estimation of the adsorbed hydrated mass and the corresponding film thickness, whereas complementary experiments using DO as the solvent allowed the quantitative determination of the hydration of the CNC layer, showing a high hydration value. Scanning force microscopy (SFM) and total internal reflection fluorescence microscopy (TIRF) were used to probe the homogeneity of the deposited layers, revealing the structural details at the particle and film length scales, respectively, thus giving information on the effect of CNC concentration on the surface coverage. The results showed that the adsorption of CNCs on the supported lipid membrane depended on lipid composition, CNC concentration, and pH conditions, and that the binding process was governed by electrostatic interactions. Under suitable conditions, a uniform film was formed, with thickness corresponding to a CNC monolayer, which provides the basis for a relevant 2D model of a primary plant cell wall.

Deposition of Cellulose Nanocrystals onto Supported Lipid Membranes.

Finger-like protrusions in cells are mostly generated by an active actin cytoskeleton pushing against the cell membrane. Conventional filopodia, localized at the leading edge of the cells, are long and thin protrusions composed of parallel actin filaments that emanate from a branched actin network. In contrast, dendritic filopodia, precursors of dendritic spines in neurons, are entirely filled in with a branched actin network. Here, we investigate in vitro how the dynamics of branched actin structures, polymerized at a membrane surface, trigger the formation of both protrusion types. Using supported bilayers and liposomes, we show that a decrease in the amount of activation sites at the membrane surface leads to the appearance of heterogeneities in the actin network coverage. Such heterogeneities promote the formation of membrane protrusions, and the size of heterogeneity patches matches the one of the protrusion base. Protrusion shape, cylindrical or conical, directly correlates with the absence or the presence of actin branches, respectively.

Finger-like membrane protrusions are favored by heterogeneities in the actin network.

Actin is one of the most studied cytoskeleton proteins showing a very rich span of structures and functions. For example, adenosine triphosphate (ATP)-assisted polymerization of actin is used to push protrusions forward in a mechanism that enables cells to crawl on a substrate. In this process, the chemical energy released from the hydrolysis of ATP is what enables force generation. We study a minimal model system comprised of actin monomers in an excess of ATP concentration. In such a system polymerization proceeds in three stages: nucleation of actin filaments, elongation, and network formation. While the kinetics of filament growth was characterized previously, not much is known about the kinetics of network formation and the evolution of networks towards a steady-state structure. In particular, it is not clear how the non-equilibrium nature of this ATP-assisted polymerization manifests itself in the kinetics of self-assembly. Here, we use time-resolved microrheology to follow the kinetics of the three stages of self-assembly as a function of initial actin monomer concentration. Surprisingly, we find that at high enough initial monomer concentrations the effective elastic modulus of the forming actin networks overshoots and then relaxes with a -2/5 power law. We attribute the overshoot to the non-equilibrium nature of the polymerization and the relaxation to rearrangements of the network into a steady-state structure.

Kinetics of actin networks formation measured by time resolved particle-tracking microrheology.

Intra-cellular active transport by native cargos is ubiquitous. We investigate the motion of spherical nano-particles (NPs) grafted with flexible polymers that end with a nuclear localization signal peptide. This peptide allows the recruitment of several  dynein motors from cytoplasmic extracts. To determine how motor-motor interactions influenced motility on the single microtubule level, we conducted bead-motility assays incorporating surface adsorbed microtubules and combined them with model simulations that were based on the properties of a single dynein. The experimental and simulation results revealed long time trajectories: when the number of NP-ligated motors  increased, run-times and run-lengths were enhanced and mean velocities were somewhat decreased. Moreover, the dependence of the velocity on run-time followed a universal curve, regardless of the system composition. Model simulations also demonstrated left- and right-handed helical motion and revealed self-regulation of the number of microtubule-bound, actively transporting dynein motors. This number was stochastic along trajectories and was distributed mainly between one, two, and three motors, regardless of . We propose that this self-regulation allows our synthetic NPs to achieve persistent motion that is associated with major helicity. Such a helical motion might affect obstacle bypassing, which can influence active transport efficiency when facing the crowded environment of the cell.

Article	Year
Reconstitution of the transition from lamellipodium to filopodium in a membrane-free system. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 16549794	2006
Thickness distribution of actin bundles in vitro. European biophysics journal : EBJ\| PubMed ID: 18004557	2008
A cytoskeletal demolition worker: myosin II acts as an actin depolymerization agent. Journal of molecular biology\| PubMed ID: 18021803	2008
Arp2/3 branched actin network mediates filopodia-like bundles formation in vitro. PloS one\| PubMed ID: 18820726	2008
The fusion of actin bundles driven by interacting motor proteins. Physical biology\| PubMed ID: 19411736	2009
Cortactin releases the brakes in actin- based motility by enhancing WASP-VCA detachment from Arp2/3 branches. Current biology : CB\| PubMed ID: 22169534	2011
Hierarchical self-organization of cytoskeletal active networks. Physical biology\| PubMed ID: 22476003	2012
Releasing the brakes while hanging on: Cortactin effects on actin-driven motility. Bioarchitecture\| PubMed ID: 22754622	2012
Reconstitution of actin-based motility by vasodilator-stimulated phosphoprotein (VASP) depends on the recruitment of F-actin seeds from the solution produced by cofilin. The Journal of biological chemistry\| PubMed ID: 25246528	2014
Toward the reconstitution of synthetic cell motility. Cell adhesion & migration\| PubMed ID: 27019160	2016
Functional Actin Networks under Construction: The Cooperative Action of Actin Nucleation and Elongation Factors. Trends in biochemical sciences\| PubMed ID: 28372857	2017
Spontaneous buckling of contractile poroelastic actomyosin sheets. Nature communications\| PubMed ID: 29941969	2018
Living Matter: Mesoscopic Active Materials. Advanced materials (Deerfield Beach, Fla.)\| PubMed ID: 30256463	2018
Deposition of Cellulose Nanocrystals onto Supported Lipid Membranes. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 31904979	2020
Finger-like membrane protrusions are favored by heterogeneities in the actin network. Soft matter\| PubMed ID: 32435778	2020
Kinetics of actin networks formation measured by time resolved particle-tracking microrheology. Soft matter\| PubMed ID: 32803212	2020
Nano-Particles Carried by Multiple Dynein Motors Self-Regulate Their Number of Actively Participating Motors. International journal of molecular sciences\| PubMed ID: 34445598	2021

Anne Bernheim-Groswasser

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