The role of actin turnover in retrograde actin network flow in neuronal growth cones

The balance of actin filament polymerization and depolymerization maintains a steady state network treadmill in neuronal growth cones essential for motility and guidance. Here we have investigated the connection between depolymerization and treadmilling dynamics. We show that polymerization-competent barbed ends are concentrated at the leading edge and depolymerization is distributed throughout the peripheral domain. We found a high-to-low G-actin gradient between peripheral and central domains. Inhibiting turnover with jasplakinolide collapsed this gradient and lowered leading edge barbed end density. Ultrastructural analysis showed dramatic reduction of leading edge actin filament density and filament accumulation in central regions. Live cell imaging revealed that the leading edge retracted even as retrograde actin flow rate decreased exponentially. Inhibition of myosin II activity before jasplakinolide treatment lowered baseline retrograde flow rates and prevented leading edge retraction. Myosin II activity preferentially affected filopodial bundle disassembly distinct from the global effects of jasplakinolide on network turnover. We propose that growth cone retraction following turnover inhibition resulted from the persistence of myosin II contractility even as leading edge assembly rates decreased. The buildup of actin filaments in central regions combined with monomer depletion and reduced polymerization from barbed ends suggests a mechanism for the observed exponential decay in actin retrograde flow. Our results show that growth cone motility is critically dependent on continuous disassembly of the peripheral actin network.     

Arp2/3 complex-dependent actin networks constrain myosin II function in driving retrograde actin flow

The Arp2/3 complex nucleates actin filaments to generate networks at the leading edge of motile cells. Nonmuscle myosin II produces contractile forces involved in driving actin network translocation. We inhibited the Arp2/3 complex and/or myosin II with small molecules to investigate their respective functions in neuronal growth cone actin dynamics. Inhibition of the Arp2/3 complex with CK666 reduced barbed end actin assembly site density at the leading edge, disrupted actin veils, and resulted in veil retraction. Strikingly, retrograde actin flow rates increased with Arp2/3 complex inhibition; however, when myosin II activity was blocked, Arp2/3 complex inhibition now resulted in slowing of retrograde actin flow and veils no longer retracted. Retrograde flow rate increases induced by Arp2/3 complex inhibition were independent of Rho kinase activity. These results provide evidence that, although the Arp2/3 complex and myosin II are spatially segregated, actin networks assembled by the Arp2/3 complex can restrict myosin II-dependent contractility with consequent effects on growth cone motility.     

Pak1 regulates focal adhesion strength, myosin IIA distribution, and actin dynamics to optimize cell migration

Cell motility requires the spatial and temporal coordination of forces in the actomyosin cytoskeleton with extracellular adhesion. The biochemical mechanism that coordinates filamentous actin (F-actin) assembly, myosin contractility, adhesion dynamics, and motility to maintain the balance between adhesion and contraction remains unknown. In this paper, we show that p21-activated kinases (Paks), downstream effectors of the small guanosine triphosphatases Rac and Cdc42, biochemically couple leading-edge actin dynamics to focal adhesion (FA) dynamics. Quantitative live cell microscopy assays revealed that the inhibition of Paks abolished F-actin flow in the lamella, displaced myosin IIA from the cell edge, and decreased FA turnover. We show that, by controlling the dynamics of these three systems, Paks regulate the protrusive activity and migration of epithelial cells. Furthermore, we found that expressing Pak1 was sufficient to overcome the inhibitory effects of excess adhesion strength on cell motility. These findings establish Paks as critical molecules coordinating cytoskeletal systems for efficient cell migration.     

Myosin II functions in actin-bundle turnover in neuronal growth cones

Retrograde actin flow works in concert with cell adhesion to generate traction forces that are involved in axon guidance in neuronal growth cones. Myosins have been implicated in retrograde flow, but identification of the specific myosin subtype(s) involved has been controversial. Using fluorescent speckle microscopy (FSM) to assess actin dynamics, we report that inhibition of myosin II alone decreases retrograde flow by 51% and the remaining flow can be almost fully accounted for by the 'push' of plus-end actin assembly at the leading edge of the growth cone. Interestingly, actin bundles that are associated with filopodium roots elongated by approximately 83% after inhibition of myosin II. This unexpected result was due to decreased rates of actin-bundle severing near their proximal (minus or pointed) ends which are located in the transition zone of the growth cone. Our study reveals a mechanism for the regulation of actin-bundle length by myosin II that is dependent on actin-bundle severing, and demonstrate that retrograde flow is a steady state that depends on both myosin II contractility and actin-network treadmilling.     

Germinal center-specific protein human germinal center associated lymphoma directly interacts with both myosin and actin and increases the binding of myosin to actin

Human germinal center associated lymphoma (HGAL) is a germinal center-specific gene whose expression correlates with a favorable prognosis in patients with diffuse large B-cell and classic Hodgkin lymphomas. HGAL is involved in negative regulation of lymphocyte motility. The movement of lymphocytes is directly driven by actin polymerization and actin-myosin interactions. We demonstrate that HGAL interacts directly and independently with both actin and myosin and delineate the HGAL and myosin domains responsible for the interaction. Furthermore, we show that HGAL increases the binding of myosin to F-actin and inhibits the ability of myosin to translocate actin by reducing the maximal velocity of myosin head/actin movement. No effects of HGAL on actomyosin ATPase activity and the rate of actin polymerization from G-actin to F-actin were observed. These findings reveal a new mechanism underlying the inhibitory effects of germinal center-specific HGAL protein on lymphocyte and lymphoma cell motility.     

Growth cone collapse through coincident loss of actin bundles and leading edge actin without actin depolymerization

Repulsive guidance cues can either collapse the whole growth cone to arrest neurite outgrowth or cause asymmetric collapse leading to growth cone turning. How signals from repulsive cues are translated by growth cones into this morphological change through rearranging the cytoskeleton is unclear. We examined three factors that are able to induce the collapse of extending Helisoma growth cones in conditioned medium, including serotonin, myosin light chain kinase inhibitor, and phorbol ester. To study the cytoskeletal events contributing to collapse, we cultured Helisoma growth cones on polylysine in which lamellipodial collapse was prevented by substrate adhesion. We found that all three factors that induced collapse of extending growth cones also caused actin bundle loss in polylysine-attached growth cones without loss of actin meshwork. In addition, actin bundle loss correlated with specific filamentous actin redistribution away from the leading edge that is characteristic of repulsive factors. Finally, we provide direct evidence using time-lapse studies of extending growth cones that actin bundle loss paralleled collapse. Taken together, these results suggest that actin bundles could be a common cytoskeletal target of various collapsing factors, which may use different signaling pathways that converge to induce growth cone collapse.     

Myosin VI stabilizes an actin network during Drosophila spermatid individualization

Here, we demonstrate a new function of myosin VI using observations of Drosophila spermatid individualization in vivo. We find that myosin VI stabilizes a branched actin network in actin structures (cones) that mediate the separation of the syncytial spermatids. In a myosin VI mutant, the cones do not accumulate F-actin during cone movement, whereas overexpression of myosin VI leads to bigger cones with more F-actin. Myosin subfragment 1-fragment decoration demonstrated that the actin cone is made up of two regions: a dense meshwork at the front and parallel bundles at the rear. The majority of the actin filaments were oriented with their pointed ends facing in the direction of cone movement. Our data also demonstrate that myosin VI binds to the cone front using its motor domain. Fluorescence recovery after photobleach experiments using green fluorescent protein-myosin VI revealed that myosin VI remains bound to F-actin for minutes, suggesting its role is tethering, rather than transporting cargo. We hypothesize that myosin VI protects the actin cone structure either by cross-linking actin filaments or anchoring regulatory molecules at the cone front. These observations uncover a novel mechanism mediated by myosin VI for stabilizing long-lived actin structures in cells.     

Modeling cell protrusion predicts how myosin II and actin turnover affect adhesion-based signaling

Orchestration of cell migration is essential for development, tissue regeneration, and the immune response. This dynamic process integrates adhesion, signaling, and cytoskeletal subprocesses across spatial and temporal scales. In mesenchymal cells, adhesion complexes bound to extracellular matrix mediate both biochemical signal transduction and physical interaction with the F-actin cytoskeleton. Here, we present a mathematical model that offers insight into both aspects, considering spatiotemporal dynamics of nascent adhesions, active signaling molecules, mechanical clutching, actin treadmilling, and nonmuscle myosin II contractility. At the core of the model is a positive feedback loop, whereby adhesion-based signaling promotes generation of barbed ends at, and protrusion of, the cell’s leading edge, which in turn promotes formation and stabilization of nascent adhesions. The model predicts a switch-like transition and optimality of membrane protrusion, determined by the balance of actin polymerization and retrograde flow, with respect to extracellular matrix density. The model, together with new experimental measurements, explains how protrusion can be modulated by mechanical effects (nonmuscle myosin II contractility and adhesive bond stiffness) and F-actin turnover.     

Immunocytochemical evidence for colocalization in neurite growth cones of actin and myosin and their relationship to cell-substratum adhesions

Sensory neurons from 8- to 11-day chick embryos were cultured on polyornithine-treated coverslips, fixed with glutaraldehyde, and stained for immunofluorescent localization of actin. Actin was distributed in a fibrous form in the growth cones, extending into filopodia and lamellipodial expansions of the growth cone margin. Often, these actin fibers were located at sites of linear adhesions to the glass substratum, as viewed by interference reflection optics. Our antisera to myosin did not recognize myosin in glutaraldehyde-fixed cells, and paraformaldehyde, which preserves the antigenicity of myosin, did not fix embryonic neurons well. Thus, myosin was localized in NGF-stimulated PC12 cells, whose morphology is better preserved by paraformaldehyde. Within the growth cones of PC12 neurites, actin and myosin are distributed into fibrous arrays which resemble the actin fibers seen in the growth cones of sensory neurons. Thus, actomyosin-like contractile forces may be exerted in neurite growth cones. These forces may act in concert with cell-substratum adhesive bonds to move the growth cone across the substratum or move organelles within the growth cone.     

Coordination of actin filament and microtubule dynamics during neurite outgrowth

Although much evidence suggests that axon growth and guidance depend on well-coordinated cytoskeletal dynamics, direct characterization of the corresponding molecular events has remained a challenge. Here, we address this outstanding problem by examining neurite outgrowth stimulated by local application of cell adhesion substrates. During acute outgrowth, the advance of organelles and underlying microtubules was correlated with regions of attenuated retrograde actin network flow in the periphery. Interestingly, as adhesion sites matured, contractile actin arc structures, known to be regulated by the Rho/Rho Kinase/myosin II signaling cascade, became more robust and coordinated microtubule movements in the growth cone neck. When Rho Kinase was inhibited, although growth responses occurred with less of a delay, microtubules failed to consolidate into a single axis of growth. These results reveal a role for Rho Kinase and myosin II contractility in regulation of microtubule behavior during neuronal growth.     

Modelling actin polymerization: the effect on confined cell migration

The aim of this work is to model cell motility under conditions of mechanical confinement. This cell migration mode may occur in extravasation of tumour and neutrophil-like cells. Cell migration is the result of the complex action of different forces exerted by the interplay between myosin contractility forces and actin processes. Here, we propose and implement a finite element model of the confined migration of a single cell. In this model, we consider the effects of actin and myosin in cell motility. Both filament and globular actin are modelled. We model the cell considering cytoplasm and nucleus with different mechanical properties. The migration speed in the simulation is around 0.1 μm/min, which is in agreement with existing literature. From our simulation, we observe that the nucleus size has an important role in cell migration inside the channel. In the simulation the cell moves further when the nucleus is smaller. However, this speed is less sensitive to nucleus stiffness. The results show that the cell displacement is lower when the nucleus is stiffer. The degree of adhesion between the channel walls and the cell is also very important in confined migration. We observe an increment of cell velocity when the friction coefficient is higher.

     

Activation of heavy meromyosin adenosine triphosphatase by various states of actin.

F-actin monomer (F-monomer) is formed upon the addition of neutral salt to G-actin. Since F-monomer has a digestibility similar to that of F-actin and much lower than that of G-actin, it has been proposed that F-monomer has a conformation different from that of G-actin and similar to the conformation of the subunits in F-actin. To examine whether F-monomer will enhance the magnesium-activated myosin adenosine triphosphatase (Mg2+-ATPase) as much as F-actin, the ability of partially polymerized actin populations at equilibrium to activate the Mg2+-ATPase of heavy meromyosin was investigated. Correlations were made between ATPase activities and the polymerization state of actin as determined by measurements of viscosity and digestibility. No significant activation of the heavy meromyosin ATPase was observed under conditions where G-actin or mixtures of G-actin and F-monomer were present. As polymer formation occurred at higher actin concentrations, or with increased KCl concentrations, substantial activation characteristic of F-actin was observed. The data suggest that F-monomer may undergo a further conformational change as it forms nuclei or joins onto polymers. Alternatively, the site of actin which activates the myosin ATPase may involve the crevice between two adjacent actin subunits.     

Discrete mechanical model of lamellipodial actin network implements molecular clutch mechanism and generates arcs and microspikes

Mechanical forces, actin filament turnover, and adhesion to the extracellular environment regulate lamellipodial protrusions. Computational and mathematical models at the continuum level have been used to investigate the molecular clutch mechanism, calculating the stress profile through the lamellipodium and around focal adhesions. However, the forces and deformations of individual actin filaments have not been considered while interactions between actin networks and actin bundles is not easily accounted with such methods. We develop a filament-level model of a lamellipodial actin network undergoing retrograde flow using 3D Brownian dynamics. Retrograde flow is promoted in simulations by pushing forces from the leading edge (due to actin polymerization), pulling forces (due to molecular motors), and opposed by viscous drag in cytoplasm and focal adhesions. Simulated networks have densities similar to measurements in prior electron micrographs. Connectivity between individual actin segments is maintained by permanent and dynamic crosslinkers. Remodeling of the network occurs via the addition of single actin filaments near the leading edge and via filament bond severing. We investigated how several parameters affect the stress distribution, network deformation and retrograde flow speed. The model captures the decrease in retrograde flow upon increase of focal adhesion strength. The stress profile changes from compression to extension across the leading edge, with regions of filament bending around focal adhesions. The model reproduces the observed reduction in retrograde flow speed upon exposure to cytochalasin D, which halts actin polymerization. Changes in crosslinker concentration and dynamics, as well as in the orientation pattern of newly added filaments demonstrate the model’s ability to generate bundles of filaments perpendicular (actin arcs) or parallel (microspikes) to the protruding direction.     

Interactions between G-actin and myosin subfragment 1: immunochemical probing of the NH2-terminal segment on actin

The role of the N-terminal segment of actin in myosin-induced polymerization of G-actin was studied by using peptide antibodies directed against the first seven N-terminal residues of alpha-skeletal actin. Light scattering, fluorescence, and analytical ultracentrifugation experiments showed that the Fab fragments of these antibodies inhibited the polymerization of G-actin by myosin subfragment 1 (S-1) by inhibiting the binding of these proteins to each other. Fluorescence measurements using actin labeled with pyrenyliodoacetamide revealed that Fab inhibited the initial step in the binding of S-1 to G-actin. It is deduced from these results and from other literature data that the initial contact between G-actin and S-1 involves residues 1-7 on actin and residues 633-642 on the S-1 heavy chain. This interaction appears to be of major importance for the binding of S-1 and G-actin. The presence of additional myosin contact sites on G-actin was indicated by concentration-dependent recovery of S-1 binding to G-actin without displacement of Fab. The reduced Fab inhibition of S-1 binding to polymerizing and polymerized actin is consistent with the tightening of acto-S-1 binding at these sites or the creation of new sites upon formation of F-actin.     

Mechanical coupling between transsynaptic N-cadherin adhesions and actin flow stabilizes dendritic spines

The morphology of neuronal dendritic spines is a critical indicator of synaptic function. It is regulated by several factors, including the intracellular actin/myosin cytoskeleton and transcellular N-cadherin adhesions. To examine the mechanical relationship between these molecular components, we performed quantitative live-imaging experiments in primary hippocampal neurons. We found that actin turnover and structural motility were lower in dendritic spines than in immature filopodia and increased upon expression of a nonadhesive N-cadherin mutant, resulting in an inverse relationship between spine motility and actin enrichment. Furthermore, the pharmacological stimulation of myosin II induced the rearward motion of actin structures in spines, showing that myosin II exerts tension on the actin network. Strikingly, the formation of stable, spine-like structures enriched in actin was induced at contacts between dendritic filopodia and N-cadherin–coated beads or micropatterns. Finally, computer simulations of actin dynamics mimicked various experimental conditions, pointing to the actin flow rate as an important parameter controlling actin enrichment in dendritic spines. Together these data demonstrate that a clutch-like mechanism between N-cadherin adhesions and the actin flow underlies the stabilization of dendritic filopodia into mature spines, a mechanism that may have important implications in synapse initiation, maturation, and plasticity in the developing brain.     

VASP-dependent regulation of actin cytoskeleton rigidity, cell adhesion, and detachment

Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are established regulators of actin-based motility, platelet aggregation, and growth cone guidance. However, the molecular mechanisms involved essentially remain elusive. Here we report on a novel mechanism of VASP action, namely the regulation of tensile strength, contractility, and rigidity of the actin cytoskeleton. Compared to wild-type cells fibroblasts derived from VASP-deficient mice have thicker and more stable actin stress fibres. Furthermore focal adhesions are enlarged, myosin light chain phosphorylation is increased, and the rigidity of the filament-supported plasma membrane is elevated about three- to fourfold, as is evident from atomic force microscopy. Moreover, fibronectin-coated beads adhere stronger to the surface of VASP-deficient cells. The resistance of these beads to mechanical displacement by laser tweezers is dramatically increased in an F-actin-dependent mode. Cytoskeletal stabilization coincides with slower cell adhesion and detachment, while overall adhesion is increased. Interestingly, many of these effects observed in VASP (−/−) cells are recapitulated in VASP-overexpressing cells, hinting towards a balanced stoichiometry necessary for appropriate VASP function. Taken together, our results suggest that VASP regulates surface protrusion formation and cell adhesion through modulation of the mechanical properties of the actin cytoskeleton.Annette B. Galler, Maísa I. García Arguinzonis these authors contributed equally to this work     

Lamellipodial actin mechanically links myosin activity with adhesion-site formation

Cell motility proceeds by cycles of edge protrusion, adhesion, and retraction. Whether these functions are coordinated by biochemical or biomechanical processes is unknown. We find that myosin II pulls the rear of the lamellipodial actin network, causing upward bending, edge retraction, and initiation of new adhesion sites. The network then separates from the edge and condenses over the myosin. Protrusion resumes as lamellipodial actin regenerates from the front and extends rearward until it reaches newly assembled myosin, initiating the next cycle. Upward bending, observed by evanescence and electron microscopy, results in ruffle formation when adhesion strength is low. Correlative fluorescence and electron microscopy shows that the regenerating lamellipodium forms a cohesive, separable layer of actin above the lamellum. Thus, actin polymerization periodically builds a mechanical link, the lamellipodium, connecting myosin motors with the initiation of adhesion sites, suggesting that the major functions driving motility are coordinated by a biomechanical process.     

Stretching actin filaments within cells enhances their affinity for the myosin II motor domain

To test the hypothesis that the myosin II motor domain (S1) preferentially binds to specific subsets of actin filaments in vivo, we expressed GFP-fused S1 with mutations that enhanced its affinity for actin in Dictyostelium cells. Consistent with the hypothesis, the GFP-S1 mutants were localized along specific portions of the cell cortex. Comparison with rhodamine-phalloidin staining in fixed cells demonstrated that the GFP-S1 probes preferentially bound to actin filaments in the rear cortex and cleavage furrows, where actin filaments are stretched by interaction with endogenous myosin II filaments. The GFP-S1 probes were similarly enriched in the cortex stretched passively by traction forces in the absence of myosin II or by external forces using a microcapillary. The preferential binding of GFP-S1 mutants to stretched actin filaments did not depend on cortexillin I or PTEN, two proteins previously implicated in the recruitment of myosin II filaments to stretched cortex. These results suggested that it is the stretching of the actin filaments itself that increases their affinity for the myosin II motor domain. In contrast, the GFP-fused myosin I motor domain did not localize to stretched actin filaments, which suggests different preferences of the motor domains for different structures of actin filaments play a role in distinct intracellular localizations of myosin I and II. We propose a scheme in which the stretching of actin filaments, the preferential binding of myosin II filaments to stretched actin filaments, and myosin II-dependent contraction form a positive feedback loop that contributes to the stabilization of cell polarity and to the responsiveness of the cells to external mechanical stimuli.     

On the interaction of muscle actin with gadolinium.

The polymerization of G-actin is prevented by concentrations of gadolinium (GdIII) that exceed the ATP present. Since the susceptibility of G-actin to enzymatic proteolysis is slightly decreased upon addition of GdIII, and the digestibility of F-actin is markedly increased with the same treatment, it appears that actin undergoes GdIII-induced conformational changes. The altered states of actin formed inhibit the GdIII-ATPase activity of myosin, but in all cases, the effect of GdIII on actin is reversed by removal of the trivalent ion with ATP. The reversible changes in conformation induced by GdIII create a state of actin which has properties unlike those of G-actin, F-monomer or F-actin.