Mechanisms Controlling Cell Size and Shape during Isotropic Cell Spreading

Cell motility is important for many developmental and physiological processes. Motility arises from interactions between physical forces at the cell surface membrane and the biochemical reactions that control the actin cytoskeleton. To computationally analyze how these factors interact, we built a three-dimensional stochastic model of the experimentally observed isotropic spreading phase of mammalian fibroblasts. The multiscale model is composed at the microscopic levels of three actin filament remodeling reactions that occur stochastically in space and time, and these reactions are regulated by the membrane forces due to membrane surface resistance (load) and bending energy. The macroscopic output of the model (isotropic spreading of the whole cell) occurs due to the movement of the leading edge, resulting solely from membrane force-constrained biochemical reactions. Numerical simulations indicate that our model qualitatively captures the experimentally observed isotropic cell-spreading behavior. The model predicts that increasing the capping protein concentration will lead to a proportional decrease in the spread radius of the cell. This prediction was experimentally confirmed with the use of Cytochalasin D, which caps growing actin filaments. Similarly, the predicted effect of actin monomer concentration was experimentally verified by using Latrunculin A. Parameter variation analyses indicate that membrane physical forces control cell shape during spreading, whereas the biochemical reactions underlying actin cytoskeleton dynamics control cell size (i.e., the rate of spreading). Thus, during cell spreading, a balance between the biochemical and biophysical properties determines the cell size and shape. These mechanistic insights can provide a format for understanding how force and chemical signals together modulate cellular regulatory networks to control cell motility.     

Morphology of the lamellipodium and organization of actin filaments at the leading edge of crawling cells

Lamellipodium extension, incorporating actin filament dynamics and the cell membrane, is simulated in three dimensions. The actin filament network topology and the role of actin-associated proteins such as Arp2/3 are examined. We find that the orientational pattern of the filaments is in accord with the experimental data only if the spatial orientation of the Arp2/3 complex is restricted during each branching event. We hypothesize that branching occurs when Arp2/3 is bound to Wiskott-Aldrich syndrome protein (WASP), which is in turn bound to Cdc42 signaling complex; Arp2/3 binding geometry is restricted by the membrane-bound complex. Using mechanical and energetic arguments, we show that any membrane protein that is conical or trapezoidal in shape preferentially resides at the curved regions of the plasma membrane. We hypothesize that the transmembrane receptors involved in the recruitment of Cdc42/WASP complex has this property and concentrate at the leading edge. These features, combined with the mechanical properties of the cell membrane, explain why lamellipodium is a flat organelle.     

VASP governs actin dynamics by modulating filament anchoring

Actin filament dynamics at the cell membrane are important for cell-matrix and cell-cell adhesions and the protrusion of the leading edge. Since actin filaments must be connected to the cell membrane to exert forces but must also detach from the membrane to allow it to move and evolve, the balance between actin filament tethering and detachment at adhesion sites and the leading edge is key for cell shape changes and motility. How this fine tuning is performed in cells remains an open question, but possible candidates are the Drosophila enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family of proteins, which localize to dynamic actin structures in the cell. Here we study VASP-mediated actin-related proteins 2/3 (Arp2/3) complex-dependent actin dynamics using a substrate that mimics the fluid properties of the cell membrane: an oil-water interface. We show evidence that polymerization activators undergo diffusion and convection on the fluid surface, due to continual attachment and detachment to the actin network. These dynamics are enhanced in the presence of VASP, and we observe cycles of catastrophic detachment of the actin network from the surface, resulting in stop-and-go motion. These results point to a role for VASP in the modulation of filament anchoring, with implications for actin dynamics at cell adhesions and at the leading edge of the cell.     

RIAM, an Ena/VASP and Profilin ligand, interacts with Rap1-GTP and mediates Rap1-induced adhesion

The small GTPase Rap1 induces integrin-mediated adhesion and changes in the actin cytoskeleton. The mechanisms that mediate these effects of Rap1 are poorly understood. We have identified RIAM as a Rap1-GTP-interacting adaptor molecule. RIAM defines a family of adaptor molecules that contain a RA-like (Ras association) domain, a PH (pleckstrin homology) domain, and various proline-rich motifs. RIAM also interacts with Profilin and Ena/VASP proteins, molecules that regulate actin dynamics. Overexpression of RIAM induced cell spreading and lamellipodia formation, changes that require actin polymerization. In contrast, RIAM knockdown cells had reduced content of polymerized actin. RIAM overexpression also induced integrin activation and cell adhesion. RIAM knockdown displaced Rap1-GTP from the plasma membrane and abrogated Rap1-induced adhesion. Thus, RIAM links Rap1 to integrin activation and plays a role in regulating actin dynamics.     

Assembly of actin filaments induced by sequestration of membrane cholesterol in transformed cells

Cholesterol is a major lipid component of the plasma membrane that plays an important role in various signaling processes in mammalian cells. Our study is focused on the role of membrane cholesterol in the organization and dynamics of actin cytoskeleton. Experiments were performed on cultured transformed cells characterized by a poorly developed actin network and less prominent stress fibers: human embryonic kidney HEK293, human epidermoid larynx carcinoma HEp-2, and mouse fibroblasts 3T3-SV40. Using Factin labeling with rhodamine phalloidin, actin cytoskeleton rearrangements were analyzed after sequestration of membrane cholesterol by cyclic oligosaccharide methyl-beta-cyclodextrin and polyene macrolide antibiotic filipin. The cells treated with these agents displayed similar reorganization of actin cytoskeleton involving filament assembly. In HEp-2 carcinoma cells and 3T3-SV40 fibroblasts, cholesterol-sequestering reagents induced intense stress fiber formation and enhanced cell spreading; i.e., features of transformed phenotype reversion were observed. The cytoskeleton rearrangements are probably initiated by disruption of lipid raft integrity that is critically dependent on the level of the membrane cholesterol.     

Effects of CapG overexpression on agonist-induced motility and second messenger generation

Actin modulating proteins that bind polyphosphoinositides, such as phosphatidylinositol 4, 5-bisphosphate (PIP2), can potentially participate in receptor signaling by restructuring the membrane cytoskeleton and modulating second messenger generation through the phosphoinositide cycle. We examined these possibilities by overexpressing CapG, an actin filament end capping, Ca(2+)- and polyphosphoinositide-binding protein of the gelsolin family. High level transient overexpression decreased actin filament staining in the center of the cells but not in the cell periphery. Moderate overexpression in clonally selected cell lines did not have a detectible effect on actin filament content or organization. Nevertheless, it promoted a dose-dependent increase in rates of wound healing and chemotaxis. The motile phenotype was similar to that observed with gelsolin overexpression, which in addition to capping, also severs and nucleates actin filaments. CapG overexpressing clones are more responsive to platelet-derived growth factor than control- transfected clones. They form more circular dorsal membrane ruffles, have higher phosphoinositide turnover, inositol 1,4,5-trisphosphate generation and Ca2+ signaling. These responses are consistent with enhanced PLC gamma activity. Direct measurements of PIP2 mass showed that the CapG effect on PLC gamma was not due primarily to an increase in the PIP2 substrate concentration. The observed changes in cell motility and membrane signaling are consistent with the hypothesis that PIP(2)-binding actin regulatory proteins modulate phosphoinositide turnover and second messenger generation in vivo. We infer that CapG and related proteins are poised to coordinate membrane signaling with actin filament dynamics following cell stimulation.     

Actin-based motility: stop and go with Ena/VASP proteins

Proteins of the Ena/VASP (Enabled/vasodilator-stimulated phosphoprotein) family are involved in Abl and/or cyclic nucleotide-dependent protein kinase signaling pathways. These proteins are also crucial factors in regulating actin dynamics and associated processes such as cell-cell adhesion, platelet function and actin-based motility of both cytopathogenic Listeria and their eukaryotic host cells. Although biochemical mechanisms have emerged depicting Ena/VASP proteins as enhancers of actin filament formation, increasing evidence also suggests that these proteins have inhibitory functions in integrin regulation, cell motility and axon guidance.     

α-Actinin: Immunofluorescent localization of a muscle structural protein in nonmuscle cells

Antibodies specific for the skeletal muscle structural protein α-actinin are used to localize this protein by indirect immunofluorescence in nonmuscle cells. In cultured nonmuscle cells, α-actinin is localized along or between actin filament bundles producing an almost regular periodicity. The protein is also detected in the form of fluorescent plaques at some ends of actin filament bundles, as well as in a filamentous form in some overlap areas of cells. In spreading rat embryo cells, α-actinin assumes a focal distribution which corresponds to the vertices of a highly regular actin filament network. The results suggest that α-actinin may be involved in the organization of actin filament bundles, in the attachment of actin filaments to the plasma membrane, and in the assembly of actin filaments in areas of cell to cell contact.     

Analysis of turnover dynamics of the submembranous actin cortex

The cell cortex is a thin network of actin, myosin motors, and associated proteins that underlies the plasma membrane in most eukaryotic cells. It enables cells to resist extracellular stresses, perform mechanical work, and change shape. Cortical structural and mechanical properties depend strongly on the relative turnover rates of its constituents, but quantitative data on these rates remain elusive. Using photobleaching experiments, we analyzed the dynamics of three classes of proteins within the cortex of living cells: a scaffold protein (actin), a cross-linker (α-actinin), and a motor (myosin). We found that two filament subpopulations with very different turnover rates composed the actin cortex: one with fast turnover dynamics and polymerization resulting from addition of monomers to free barbed ends, and one with slow turnover dynamics with polymerization resulting from formin-mediated filament growth. Our data suggest that filaments in the second subpopulation are on average longer than those in the first and that cofilin-mediated severing of formin-capped filaments contributes to replenishing the filament subpopulation with free barbed ends. Furthermore, α-actinin and myosin minifilaments turned over significantly faster than F-actin. Surprisingly, only one-fourth of α-actinin dimers were bound to two actin filaments. Taken together, our results provide a quantitative characterization of essential mechanisms under­lying actin cortex homeostasis.     

Modeling actin filament reorganization in endothelial cells subjected to cyclic stretch

Hemodynamic forces affect endothelial cell morphology and function. In particular, circumferential cyclic stretch of blood vessels, due to pressure changes during the cardiac cycle, is known to affect the endothelial cell shape, mediating the alignment of the cells in the direction perpendicular to stretch. This change in cell shape proceeds a drastic reorganization at the internal level. The cellular scaffolding, mainly composed of actin filaments, reorganize in the direction which later becomes the cell’s long axis. How this external mechanical stimulus is ’sensed’ and transduced into the cell is still unknown. Here, we develop a mathematical model depicting the dynamics of actin filaments, and the influence of the cyclic stretch of the substratum based on the experimental evidence that external stimuli may be transduced inside the cell via transmembrane proteins which are coupled with actin filaments on the cytoplasmic side. Based on this view, we investigate two approaches describing the formulation of the transduction mechanisms involving the coupling between filaments and the membrane proteins. As a result, we find that the mechanical stimulus could cause the experimentally observed reorganization of the entire cytoskeleton simply by altering the dynamics of the filaments connected with the integral membrane proteins, as described in our model. Comparison of our results with previous studies of cytoskeletal dynamics reveals that the cytoskeleton, which, in the absence of the effect of stretch would maintain its isotropic distribution, slowly aligns with the precise direction set by the external stimulus. It is found that even a feeble stimulus, coupled with a strong internal dynamics, is sufficient to align actin filaments perpendicular to the direction of stretch.     

The Membrane‐Associated Protein,Supervillin, Accelerates F‐Actin‐Dependent Rapid Integrin Recycling and Cell Motility

In migrating cells, the cytoskeleton coordinates signal transduction and redistribution of transmembrane proteins, including integrins and growth factor receptors. Supervillin is an F‐actin‐ and myosin II‐binding protein that tightly associates with signaling proteins in cholesterol‐rich, ‘lipid raft’ membrane microdomains. We show here that supervillin also can localize with markers for early and sorting endosomes (EE/SE) and with overexpressed components of the Arf6 recycling pathway in the cell periphery. Supervillin tagged with the photoswitchable fluorescent protein, tdEos, moves both into and away from dynamic structures resembling podosomes at the basal cell surface. Rapid integrin recycling from EE/SE is inhibited in supervillin‐knockdown cells, but the rates of integrin endocytosis and recycling from the perinuclear recycling center (PNRC) are unchanged. A lack of synergy between supervillin knockdown and the actin filament barbed‐end inhibitor, cytochalasin D, suggests that both treatments affect actin‐dependent rapid recycling. Supervillin also enhances signaling from the epidermal growth factor receptor (EGFR) to extracellular signal‐regulated kinases (ERKs) 1 and 2 and increases the velocity of cell translocation. These results suggest that supervillin, F‐actin and associated proteins coordinate a rapid, basolateral membrane recycling pathway that contributes to ERK signaling and actin‐based cell motility.     

Non-visual arrestins regulate the focal adhesion formation via small GTPases RhoA and Rac1 independently of GPCRs

Arrestins recruit a variety of signaling proteins to active phosphorylated G protein-coupled receptors in the plasma membrane and to the cytoskeleton. Loss of arrestins leads to decreased cell migration, altered cell shape, and an increase in focal adhesions. Small GTPases of the Rho family are molecular switches that regulate actin cytoskeleton and affect a variety of dynamic cellular functions including cell migration and cell morphology. Here we show that non-visual arrestins differentially regulate RhoA and Rac1 activity to promote cell spreading via actin reorganization, and focal adhesion formation via two distinct mechanisms. Arrestins regulate these small GTPases independently of G-protein-coupled receptor activation.     

Regulation of cell-matrix adhesion by OLA1, the Obg-like ATPase 1

Attachment of cells to the extracellular matrix induces clustering of membrane receptor integrins which in turn triggers the formation of focal adhesions (FAs). The adaptor/scaffold proteins in FAs provide linkage to actin cytoskeleton, whereas focal adhesion kinase (FAK) and other FA-associated kinases and phosphatases transduce integrin-mediated signaling cascades, promoting actin polymerization and progression of cell spreading. In this study, we explored the role of OLA1, a newly identified member of Obg-like ATPases, in regulating cell adhesion processes. We showed that in multiple human cell lines RNAi-mediated downregulation of OLA1 significantly accelerated cell adhesion and spreading, and conversely overexpression of OLA1 by gene transfection resulted in delayed cell adhesion and spreading. We further found that OLA1-deficient cells had elevated levels of FAK protein and decreased Ser3 phosphorylation of cofilin, an actin-binding protein and key regulator of actin filament dynamics, while OLA1-overexpressing cells exhibited the opposite molecular alterations in FAK and cofilin. These findings suggest that OLA1 plays an important negative role in cell adhesion and spreading, in part through the regulation of FAK expression and cofilin phosphorylation, and manipulation of OLA1 may lead to significant changes in cell adhesion and the associated phenotypes.     

A mathematical model of the coupled mechanisms of cell adhesion,contraction and spreading

Recent research has shown that cell spreading is highly dependent on the contractility of its cytoskeleton and the mechanical properties of the environment it is located in. The dynamics of such process is critical for the development of tissue engineering strategy but is also a key player in wound contraction, tissue maintenance and angiogenesis. To better understand the underlying physics of such phenomena, the paper describes a mathematical formulation of cell spreading and contraction that couples the processes of stress fiber formation, protrusion growth through actin polymerization at the cell edge and dynamics of cross-membrane protein (integrins) enabling cell-substrate attachment. The evolving cell’s cytoskeleton is modeled as a mixture of fluid, proteins and filaments that can exchange mass and generate contraction. In particular, besides self-assembling into stress fibers, actin monomers able to polymerize into an actin meshwork at the cell’s boundary in order to push the membrane forward and generate protrusion. These processes are possible via the development of cell-substrate attachment complexes that arise from the mechano-sensitive equilibrium of membrane proteins, known as integrins. After deriving the governing equation driving the dynamics of cell evolution and spreading, we introduce a numerical solution based on the extended finite element method, combined with a level set formulation. Numerical simulations show that the proposed model is able to capture the dependency of cell spreading and contraction on substrate stiffness and chemistry. The very good agreement between model predictions and experimental observations suggests that mechanics plays a strong role into the coupled mechanisms of contraction, adhesion and spreading of adherent cells.     

On the Modeling of Endocytosis in Yeast

The cell membrane deforms during endocytosis to surround extracellular material and draw it into the cell. Results of experiments on endocytosis in yeast show general agreement that 1) actin polymerizes into a network of filaments exerting active forces on the membrane to deform it, and 2) the large-scale membrane deformation is tubular in shape. In contrast, there are three competing proposals for precisely how the actin filament network organizes itself to drive the deformation. We use variational approaches and numerical simulations to address this competition by analyzing a meso-scale model of actin-mediated endocytosis in yeast. The meso-scale model breaks up the invagination process into three stages: 1) initiation, where clathrin interacts with the membrane via adaptor proteins; 2) elongation, where the membrane is then further deformed by polymerizing actin filaments; and 3) pinch-off. Our results suggest that the pinch-off mechanism may be assisted by a pearling-like instability. We rule out two of the three competing proposals for the organization of the actin filament network during the elongation stage. These two proposals could be important in the pinch-off stage, however, where additional actin polymerization helps break off the vesicle. Implications and comparisons with earlier modeling of endocytosis in yeast are discussed.     

Nanoscale dynamics of actin filaments in the red blood cell membrane skeleton

Red blood cell (RBC) shape and deformability are supported by a planar network of short actin filament (F-actin) nodes (∼37 nm length, 15–18 subunits) interconnected by long spectrin strands at the inner surface of the plasma membrane. Spectrin-F-actin network structure underlies quantitative modeling of forces controlling RBC shape, membrane curvature, and deformation, yet the nanoscale organization and dynamics of the F-actin nodes in situ are not well understood. We examined F-actin distribution and dynamics in RBCs using fluorescent-phalloidin labeling of F-actin imaged by multiple microscopy modalities. Total internal reflection fluorescence and Zeiss Airyscan confocal microscopy demonstrate that F-actin is concentrated in multiple brightly stained F-actin foci ∼200–300 nm apart interspersed with dimmer F-actin staining regions. Single molecule stochastic optical reconstruction microscopy imaging of Alexa 647-phalloidin-labeled F-actin and computational analysis also indicates an irregular, nonrandom distribution of F-actin nodes. Treatment of RBCs with latrunculin A and cytochalasin D indicates that F-actin foci distribution depends on actin polymerization, while live cell imaging reveals dynamic local motions of F-actin foci, with lateral movements, appearance and disappearance. Regulation of F-actin node distribution and dynamics via actin assembly/disassembly pathways and/or via local extension and retraction of spectrin strands may provide a new mechanism to control spectrin-F-actin network connectivity, RBC shape, and membrane deformability.     

Actin dynamics: growth from dendritic branches

The dendritic nucleation model was devised to explain the cycle of actin dynamics resulting in actin filament network assembly and disassembly in two contexts--at the leading edge of motile cells and in the actin comet tails of intracellular pathogenic bacteria and viruses. Due to the detailed nature of its biochemical predictions, the model has provided an excellent focus for subsequent experimentation. This review summarizes recent work on actin dynamics in the context of the dendritic nucleation model. One outcome of this research is the possibility that additional proteins, as well as the six proteins included in the original model, might increase the efficiency of dendritic nucleation or modify the resulting actin network. In addition, actin dynamics at the leading edge might be influenced by a second actin filament network, independent of dendritic nucleation.