Lamellipodial Actin Research Articles

Granger-causal inference of the lamellipodial actin regulator hierarchy by live cell imaging without perturbation.

Many cell regulatory systems implicate nonlinearity and redundancy among components. The regulatory network governing lamellipodial and lamellar actin structures is prototypical of such a system, containing tens of actin-nucleating and -modulating molecules with functional overlap and feedback loops. Due to instantaneous and long-term compensation, phenotyping the system response to perturbation provides limited information on the roles the targeted component plays in the unperturbed system. Accordingly, how individual actin regulators contribute to lamellipodial dynamics remains ambiguous. Here, we present a perturbation-free reconstruction of cause-effect relations among actin regulators by applying Granger-causal inference to constitutive image fluctuations that indicate regulator recruitment as a proxy for activity. Our analysis identifies distinct zones of actin regulator activation and of causal effects on filament assembly and delineates actin-dependent and actin-independent regulator roles in controlling edge motion. We propose that edge motion is driven by assembly of two independently operating actin filament systems.

Forces generated by lamellipodial actin filament elongation regulate the WAVE complex during cell migration.

Actin filaments generate mechanical forces that drive membrane movements during trafficking, endocytosis and cell migration. Reciprocally, adaptations of actin networks to forces regulate their assembly and architecture. Yet, a demonstration of forces acting on actin regulators at actin assembly sites in cells is missing. Here we show that local forces arising from actin filament elongation mechanically control WAVE regulatory complex (WRC) dynamics and function, that is, Arp2/3 complex activation in the lamellipodium. Single-protein tracking revealed WRC lateral movements along the lamellipodium tip, driven by elongation of actin filaments and correlating with WRC turnover. The use of optical tweezers to mechanically manipulate functional WRC showed that piconewton forces, as generated by single-filament elongation, dissociated WRC from the lamellipodium tip. WRC activation correlated with its trapping, dwell time and the binding strength at the lamellipodium tip. WRC crosslinking, hindering its mechanical dissociation, increased WRC dwell time and Arp2/3-dependent membrane protrusion. Thus, forces generated by individual actin filaments on their regulators can mechanically tune their turnover and hence activity during cell migration.

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.

Twinfilin1 controls lamellipodial protrusive activity and actin turnover during vertebrate gastrulation.

The dynamic control of the actin cytoskeleton is a key aspect of essentially all animal cell movements. Experiments in single migrating cells and in vitro systems have provided an exceptionally deep understanding of actin dynamics. However, we still know relatively little of how these systems are tuned in cell-type-specific ways, for example in the context of collective cell movements that sculpt the early embryo. Here, we provide an analysis of the actin-severing and depolymerization machinery during vertebrate gastrulation, with a focus on Twinfilin1 (Twf1) in Xenopus. We find that Twf1 is essential for convergent extension, and loss of Twf1 results in a disruption of lamellipodial dynamics and polarity. Moreover, Twf1 loss results in a failure to assemble polarized cytoplasmic actin cables, which are essential for convergent extension. These data provide an in vivo complement to our more-extensive understanding of Twf1 action in vitro and provide new links between the core machinery of actin regulation and the specialized cell behaviors of embryonic morphogenesis.

Elastic wrinkling of keratocyte lamellipodia driven by myosin-induced contractile stress

During actin-based cell migration, the actin cytoskeleton in the lamellipodium both generates and responds to force, which has functional consequences for the ability of the cell to extend protrusions. However, the material properties of the lamellipodial actin network and its response to stress on the timescale of motility are incompletely understood. Here, we describe a dynamic wrinkling phenotype in the lamellipodium of fish keratocytes, in which the actin sheet buckles upward away from the ventral membrane of the cell, forming a periodic pattern of wrinkles perpendicular to the cell’s leading edge. Cells maintain an approximately constant wrinkle wavelength over time despite new wrinkle formation and the lateral movement of wrinkles in the cell frame of reference, suggesting that cells have a preferred or characteristic wrinkle wavelength. Generation of wrinkles is dependent upon myosin contractility, and their wavelength scales directly with the density of the actin network and inversely with cell adhesion. These results are consistent with a simple physical model for wrinkling in an elastic sheet under compression and suggest that the lamellipodial cytoskeleton behaves as an elastic material on the timescale of cell migration despite rapid actin turnover.

Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks.

Coordinated polymerization of actin filaments provides force for cell migration, morphogenesis and endocytosis. Capping protein (CP) is a central regulator of actin dynamics in all eukaryotes. It binds to actin filament (F-actin) barbed ends with high affinity and slow dissociation kinetics to prevent filament polymerization and depolymerization. However, in cells, CP displays remarkably rapid dynamics within F-actin networks, but the underlying mechanism remains unclear. Here, we report that the conserved cytoskeletal regulator twinfilin is responsible for CP's rapid dynamics and specific localization in cells. Depletion of twinfilin led to stable association between CP and cellular F-actin arrays, as well as to its retrograde movement throughout leading-edge lamellipodia. These were accompanied by diminished F-actin turnover rates. In vitro single-filament imaging approaches revealed that twinfilin directly promotes dissociation of CP from filament barbed ends, while enabling subsequent filament depolymerization. These results uncover a bipartite mechanism that controls how actin cytoskeleton-mediated forces are generated in cells.

Predictive assembling model reveals the self-adaptive elastic properties of lamellipodial actin networks for cell migration

Branched actin network supports cell migration through extracellular microenvironments. However, it is unknown how intracellular proteins adapt the elastic properties of the network to the highly varying extracellular resistance. Here we develop a three-dimensional assembling model to simulate the realistic self-assembling process of the network by encompassing intracellular proteins and their dynamic interactions. Combining this multiscale model with finite element method, we reveal that the network can not only sense the variation of extracellular resistance but also self-adapt its elastic properties through remodeling with intracellular proteins. Such resistance-adaptive elastic behaviours are versatile and essential in supporting cell migration through varying extracellular microenvironments. The bending deformation mechanism and anisotropic Poisson’s ratios determine why lamellipodia persistently evolve into sheet-like structures. Our predictions are confirmed by published experiments. The revealed self-adaptive elastic properties of the networks are also applicable to the endocytosis, phagocytosis, vesicle trafficking, intracellular pathogen transport and dendritic spine formation.

Loss of Ena/VASP interferes with lamellipodium architecture, motility and integrin-dependent adhesion.

Cell migration entails networks and bundles of actin filaments termed lamellipodia and microspikes or filopodia, respectively, as well as focal adhesions, all of which recruit Ena/VASP family members hitherto thought to antagonize efficient cell motility. However, we find these proteins to act as positive regulators of migration in different murine cell lines. CRISPR/Cas9-mediated loss of Ena/VASP proteins reduced lamellipodial actin assembly and perturbed lamellipodial architecture, as evidenced by changed network geometry as well as reduction of filament length and number that was accompanied by abnormal Arp2/3 complex and heterodimeric capping protein accumulation. Loss of Ena/VASP function also abolished the formation of microspikes normally embedded in lamellipodia, but not of filopodia capable of emanating without lamellipodia. Ena/VASP-deficiency also impaired integrin-mediated adhesion accompanied by reduced traction forces exerted through these structures. Our data thus uncover novel Ena/VASP functions of these actin polymerases that are fully consistent with their promotion of cell migration.

Lamellipodin tunes cell migration by stabilizing protrusions and promoting adhesion formation.

Efficient migration on adhesive surfaces involves the protrusion of lamellipodial actin networks and their subsequent stabilization by nascent adhesions. The actin-binding protein lamellipodin (Lpd) is thought to play a critical role in lamellipodium protrusion, by delivering Ena/VASP proteins onto the growing plus ends of actin filaments and by interacting with the WAVE regulatory complex, an activator of the Arp2/3 complex, at the leading edge. Using B16-F1 melanoma cell lines, we demonstrate that genetic ablation of Lpd compromises protrusion efficiency and coincident cell migration without altering essential parameters of lamellipodia, including their maximal rate of forward advancement and actin polymerization. We also confirmed lamellipodia and migration phenotypes with CRISPR/Cas9-mediated Lpd knockout Rat2 fibroblasts, excluding cell type-specific effects. Moreover, computer-aided analysis of cell-edge morphodynamics on B16-F1 cell lamellipodia revealed that loss of Lpd correlates with reduced temporal protrusion maintenance as a prerequisite of nascent adhesion formation. We conclude that Lpd optimizes protrusion and nascent adhesion formation by counteracting frequent, chaotic retraction and membrane ruffling.This article has an associated First Person interview with the first author of the paper.

Control of Growth Cone Polarity, Microtubule Accumulation, and Protrusion by UNC-6/Netrin and Its Receptors in Caenorhabditis elegans.

UNC-6/Netrin has a conserved role in dorsal-ventral axon guidance, but the cellular events in the growth cone regulated by UNC-6/Netrin signaling during outgrowth are incompletely understood. Previous studies showed that, in growth cones migrating away from UNC-6/Netrin, the receptor UNC-5 regulates growth cone polarity, as observed by polarized F-actin, and limits the extent of growth cone protrusion. It is unclear how UNC-5 inhibits protrusion, and how UNC-40 acts in concert with UNC-5 to regulate polarity and protrusion. New results reported here indicate that UNC-5 normally restricts microtubule (MT) + end accumulation in the growth cone. Tubulin mutant analysis and colchicine treatment suggest that stable MTs are necessary for robust growth cone protrusion. Thus, UNC-5 might inhibit protrusion in part by restricting growth cone MT accumulation. Previous studies showed that the UNC-73/Trio Rac GEF and UNC-33/CRMP act downstream of UNC-5 in protrusion. Here, we show that UNC-33/CRMP regulates both growth cone dorsal asymmetric F-actin accumulation and MT accumulation, whereas UNC-73/Trio Rac GEF activity only affects F-actin accumulation. This suggests an MT-independent mechanism used by UNC-5 to inhibit protrusion, possibly by regulating lamellipodial and filopodial actin. Furthermore, we show that UNC-6/Netrin and the receptor UNC-40/DCC are required for excess protrusion in unc-5 mutants, but not for loss of F-actin asymmetry or MT + end accumulation, indicating that UNC-6/Netrin and UNC-40/DCC are required for protrusion downstream of, or in parallel to, F-actin asymmetry and MT + end entry. F-actin accumulation might represent a polarity mark in the growth cone where protrusion will occur, and not protrusive lamellipodial and filopodial actin per se Our data suggest a model in which UNC-6/Netrin first polarizes the growth cone via UNC-5, and then regulates protrusion based upon this polarity (the polarity/protrusion model). UNC-6/Netrin inhibits protrusion ventrally via UNC-5, and stimulates protrusion dorsally via UNC-40, resulting in dorsally-directed migration. The polarity/protrusion model represents a novel conceptual paradigm in which to understand axon guidance and growth cone migration away from UNC-6/Netrin.

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators.

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged β-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged β-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

Vinculin Forms a Directionally Asymmetric Catch Bond with F-Actin

Vinculin is an actin-binding protein thought to reinforce cell-cell and cell-matrix adhesions. However, how mechanical load affects the vinculin–F-actin bond is unclear. Using a single-molecule optical trap assay, we found that vinculin forms a force-dependent catch bond with F-actin through its tail domain, but with lifetimes that depend strongly on the direction of the applied force. Force toward the pointed (–) end of the actin filament resulted in a bond that was maximally stable at 8 piconewtons, with a mean lifetime (12 seconds) 10 times as long as the mean lifetime when force was applied toward the barbed (+) end. A computational model of lamellipodial actin dynamics suggests that the directionality of the vinculin–F-actin bond could establish long-range order in the actin cytoskeleton. The directional and force-stabilized binding of vinculin to F-actin may be a mechanism by which adhesion complexes maintain front-rear asymmetry in migrating cells.

Actin Networks: Adapting to Load through Geometry

Cell migration frequently involves the protrusion of lamellipodial actin networks, the structure and regulation of which have been studied for decades. New work highlights how the geometry of these networks endows cells with the ability to adapt to environmental conditions and load.

Actin Turnover in Lamellipodial Fragments

Actin turnover is the central driving force underlying lamellipodial motility. The molecular components involved are largely known, and their properties have been studied extensively invitro. However, a comprehensive picture of actin turnover invivo is still missing. We focus on fragments from fish epithelial keratocytes, which are essentially stand-alone motile lamellipodia. The geometric simplicity of the fragments and the absence of additional actin structures allow us to characterize the spatiotemporal lamellipodial actin organization with unprecedented detail. We use fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and extraction experiments to show that about two-thirds of the lamellipodial actin diffuses in the cytoplasm with nearly uniform density, whereas the rest forms the treadmilling polymer network. Roughly a quarter of the diffusible actin pool is in filamentous form as diffusing oligomers, indicating that severing and debranching are important steps in the disassembly process generating oligomers as intermediates. The remaining diffusible actin concentration is orders of magnitude higher than the invitro actin monomer concentration required to support the observed polymerization rates, implying that the majority of monomers are transiently kept in a non-polymerizable "reserve" pool. The actin network disassembles and reassembles throughout the lamellipodium within seconds, so the lamellipodial network turnover is local. The diffusible actin transport, on the other hand, is global: actin subunits typically diffuse across the entire lamellipodium before reassembling into the network. This combination of local network turnover and global transport of dissociated subunits through the cytoplasm makes actin transport robust yet rapidly adaptable and amenable to regulation.

Load Adaptation of Lamellipodial Actin Networks

Actin filaments polymerizing against membranes power endocytosis, vesicular traffic, and cell motility. Invitro reconstitution studies suggest that the structure and the dynamics of actin networks respond tomechanical forces. We demonstrate that lamellipodial actin of migrating cells responds to mechanical load when membrane tension is modulated. Inasteady state, migrating cell filaments assume the canonical dendritic geometry, defined byArp2/3-generated 70° branch points. Increased tension triggers a dense network with a broadened range ofangles, whereas decreased tension causes a shift to a sparse configuration dominated by filaments growing perpendicularly to the plasma membrane. We show that these responses emerge from the geometry of branched actin: when load per filament decreases, elongation speed increases and perpendicular filaments gradually outcompete others because they polymerize the shortest distance to the membrane, where they are protected from capping. This network-intrinsic geometrical adaptation mechanism tunes protrusive force in response to mechanical load.

Vinculin forms a directionally asymmetric catch bond with F-actin.

Vinculin is an actin-binding protein thought to reinforce cell-cell and cell-matrix adhesions. However, how mechanical load affects the vinculin-F-actin bond is unclear. Using a single-molecule optical trap assay, we found that vinculin forms a force-dependent catch bond with F-actin through its tail domain, but with lifetimes that depend strongly on the direction of the applied force. Force toward the pointed (-) end of the actin filament resulted in a bond that was maximally stable at 8 piconewtons, with a mean lifetime (12 seconds) 10 times as long as the mean lifetime when force was applied toward the barbed (+) end. A computational model of lamellipodial actin dynamics suggests that the directionality of the vinculin-F-actin bond could establish long-range order in the actin cytoskeleton. The directional and force-stabilized binding of vinculin to F-actin may be a mechanism by which adhesion complexes maintain front-rear asymmetry in migrating cells.

Buckle up: Membrane tension drives lamellipodial network compression and adhesion deposition.

Whether to spread on a surface or to crawl, cells must apply traction forces to the underlying substrate via adhesion complexes. In this issue, Pontes et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201611117) shed new light on how the interplay among membrane tension, the lamellipodial actin network, and adhesions coordinate the dynamics of spreading fibroblasts.

Abstract 288: Loss of a branched actin checkpoint in cancer cells

Abstract Cell proliferation depends on multiple signals, such as growth factors, adhesion to the substratum and to neighboring cells. Cancer cells proliferate independently of these signals. The same stimuli also induce actin polymerization. Here we investigate whether the actin cytoskeleton plays a role in transmitting the proliferation signal. We found that inactivation of the Arp2/3 complex by either a chemical inhibitor or by siRNAs blocks S phase entry in untransformed human breast cell lines and in primary breast epithelial cells. In contrast, breast cancer cell lines did not respond to Arp2/3 inhibition. Inactivating the tumor suppressor p21WAF1/CIP1, or Rb, converts sensitive cells into unresponsive cells further highlighting that this response to Arp2/3 inactivation is at the heart of the transformation process. The depletion of the WAVE complex, the Arp2/3 activator that generates branched actin in lamellipodia, but not of other Arp2/3 activators, blocks cell cycle progression. Conversely, activation of WAVE by active Rac, or depletion of Arpin, which antagonizes WAVE, potentiates cell cycle progression. Together, these results show that branched actin networks generated in lamellipodia are sensed and convey an essential signal for S Phase entry. The signal that branched actin delivers integrates stimulation from soluble growth factor with mechanotransduction from cell adhesions to the substratum and to neighboring cells. This signaling pathway qualifies as a checkpoint, which is required in normal cells, but lost in transformed cells, that monitors the integrity of branched actin networks and instructs the cell as a response that the required conditions to proliferate are fulfilled. Citation Format: Nicolas Molinie, Svetlana Rubtsova, Alexis Gautreau. Loss of a branched actin checkpoint in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 288. doi:10.1158/1538-7445.AM2017-288

Human myosin VIIa is a very slow processive motor protein on various cellular actin structures

Human myosin VIIa (MYO7A) is an actin-linked motor protein associated with human Usher syndrome (USH) type 1B, which causes human congenital hearing and visual loss. Although it has been thought that the role of human myosin VIIa is critical for USH1 protein tethering with actin and transportation along actin bundles in inner-ear hair cells, myosin VIIa's motor function remains unclear. Here, we studied the motor function of the tail-truncated human myosin VIIa dimer (HM7AΔTail/LZ) at the single-molecule level. We found that the HM7AΔTail/LZ moves processively on single actin filaments with a step size of 35 nm. Dwell-time distribution analysis indicated an average waiting time of 3.4 s, yielding ∼0.3 s-1 for the mechanical turnover rate; hence, the velocity of HM7AΔTail/LZ was extremely slow, at 11 nm·s-1 We also examined HM7AΔTail/LZ movement on various actin structures in demembranated cells. HM7AΔTail/LZ showed unidirectional movement on actin structures at cell edges, such as lamellipodia and filopodia. However, HM7AΔTail/LZ frequently missed steps on actin tracks and exhibited bidirectional movement at stress fibers, which was not observed with tail-truncated myosin Va. These results suggest that the movement of the human myosin VIIa motor protein is more efficient on lamellipodial and filopodial actin tracks than on stress fibers, which are composed of actin filaments with different polarity, and that the actin structures influence the characteristics of cargo transportation by human myosin VIIa. In conclusion, myosin VIIa movement appears to be suitable for translocating USH1 proteins on stereocilia actin bundles in inner-ear hair cells.