Differential roles of WAVE1 and WAVE2 in dorsal and peripheral ruffle formation for fibroblast cell migration

Cell migration is driven by actin polymerization at the leading edge of lamellipodia, where WASP family verprolin-homologous proteins (WAVEs) activate Arp2/3 complex. When fibroblasts are stimulated with PDGF, formation of peripheral ruffles precedes that of dorsal ruffles in lamellipodia. Here, we show that WAVE2 deficiency impairs peripheral ruffle formation and WAVE1 deficiency impairs dorsal ruffle formation. During directed cell migration in the absence of extracellular matrix (ECM), cells migrate with peripheral ruffles at the leading edge and WAVE2, but not WAVE1, is essential. In contrast, both WAVE1 and WAVE2 are essential for invading migration into ECM, suggesting that the leading edge in ECM has characteristics of both ruffles. WAVE1 is colocalized with ECM-degrading enzyme MMP-2 in dorsal ruffles, and WAVE1-, but not WAVE2-, dependent migration requires MMP activity. Thus, WAVE2 is essential for leading edge extension for directed migration in general and WAVE1 is essential in MMP-dependent migration in ECM.     

New insights into the formation and the function of lamellipodia and ruffles in mesenchymal cell migration

ABSTRACT

Lamellipodia and ruffles are veil-shaped cell protrusions composed of a highly branched actin filament meshwork assembled by the Arp2/3 complex. These structures not only hallmark the leading edge of cells adopting the adhesion-based mesenchymal mode of migration but are also thought to drive cell movement.

Although regarded as textbook knowledge, the mechanism of formation of lamellipodia and ruffles has been revisited in the last years leveraging new technologies. Furthermore, recent observations have also challenged our current view of the function of lamellipodia and ruffles in mesenchymal cell migration.

Here, I review this literature and compare it with older studies to highlight the controversies and the outstanding open issues in the field. Moreover, I outline simple and plausible explanations to reconcile conflicting results and conclusions. Finally, I integrate the mechanisms regulating actin-based protrusion in a unifying model that accounts for random and ballistic mesenchymal cell migration.     

The Rho GTPases in Macrophage Motility and Chemotaxis

The GTP-binding proteins, Rho, Rac and Cdc42 are known to regulate actin organisation. Rho induces the assembly of contractile actin-based microfilaments such as stress fibres, Rac regulates the formation of membrane ruffles and lamellipodia, and Cdc42 activation is necessary for the formation of filopodia. In addition, all three proteins can also regulate the assembly of integrin-containing focal adhesion complexes. The orchestration of these distinct cytoskeletal changes is thought to form the basis of the co-ordination of cell motility and we have investigated the roles of Rho family proteins in migration using a model system. We have found that in the macrophage cell line Bacl, the cytokine CSF-1 rapidly induces actin reorganisation: it stimulates the formation of filopodia, lamellipodia and membrane ruffles, as well as the appearance of fine actin cables within the cell. We have shown that Cdc42, Rac and Rho regulate the CSF-1 induced formation of these distinct actin filament-based structures. Using a cell tracking procedure we found that both Rho and Rac were required for CSF-1 stimulated cell translocation. In contrast, inhibition of Cdc42 does not prevent macrophages migrating in response to CSF-1, but does prevent recognition of a CSF-1 concentration gradient, so that cells now migrate randomly rather than up the gradient of this chemotactic cytokine. This implies that Cdc42, and thus probably filopodia, are required for gradient sensing and cell polarisation in macrophages.     

The T3SS Effector EspT Defines a New Category of Invasive Enteropathogenic E. coli (EPEC) Which Form Intracellular Actin Pedestals

Enteropathogenic Escherichia coli (EPEC) strains are defined as extracellular pathogens which nucleate actin rich pedestal-like membrane extensions on intestinal enterocytes to which they intimately adhere. EPEC infection is mediated by type III secretion system effectors, which modulate host cell signaling. Recently we have shown that the WxxxE effector EspT activates Rac1 and Cdc42 leading to formation of membrane ruffles and lamellipodia. Here we report that EspT-induced membrane ruffles facilitate EPEC invasion into non-phagocytic cells in a process involving Rac1 and Wave2. Internalized EPEC resides within a vacuole and Tir is localized to the vacuolar membrane, resulting in actin polymerization and formation of intracellular pedestals. To the best of our knowledge this is the first time a pathogen has been shown to induce formation of actin comets across a vacuole membrane. Moreover, our data breaks the dogma of EPEC as an extracellular pathogen and defines a new category of invasive EPEC.     

Functions and Regulation of Circular Dorsal Ruffles

Cells construct a number of plasma membrane structures to meet a range of physiological demands. Driven by juxtamembrane actin machinery, these actin-based membrane protrusions are essential for the operation and maintenance of cellular life. They are required for diverse cellular functions, such as directed cell motility, cell spreading, adhesion, and substrate/matrix degradation. Circular dorsal ruffles (CDRs) are one class of such structures characterized as F-actin-rich membrane projections on the apical cell surface. CDRs commence their formation minutes after stimulation as flat, open, and immature ruffles and progressively develop into fully enclosed circular ruffles. These “rings” then mature and contract centrifugally before subsiding. Serving a critical function in receptor internalization and cell migration, CDRs are thus highly dynamic but transient formations. Here, we review the current state of knowledge concerning the regulation of circular dorsal ruffles. We focus specifically on the biochemical pathways leading to CDR formation in order to better define the roles and functions of these enigmatic structures.     

The making of filopodia

Filopodia are rod-like cell surface projections filled with bundles of parallel actin filaments. They are found on a variety of cell types and have been ascribed sensory or exploratory functions. Filopodium formation is frequently associated with protrusion of sheet-like actin filament arrays called lamellipodia and membrane ruffles, but, in comparison to these structures, the molecular details underpinning the initiation and maintenance of filopodia are only just beginning to emerge. Recent advances have improved our understanding of the molecular requirements for filopodium protrusion and have yielded insights into the inter-relationships between lamellipodia and filopodia, the two 'sub-compartments' of the protrusive actin cytoskeleton.     

Pointed-end capping by tropomodulin3 negatively regulates endothelial cell motility

Actin filament pointed-end dynamics are thought to play a critical role in cell motility, yet regulation of this process remains poorly understood. We describe here a previously uncharacterized tropomodulin (Tmod) isoform, Tmod3, which is widely expressed in human tissues and is present in human microvascular endothelial cells (HMEC-1). Tmod3 is present in sufficient quantity to cap pointed ends of actin filaments, localizes to actin filament structures in HMEC-1 cells, and appears enriched in leading edge ruffles and lamellipodia. Transient overexpression of GFP-Tmod3 leads to a depolarized cell morphology and decreased cell motility. A fivefold increase in Tmod3 results in an equivalent decrease in free pointed ends in the cells. Unexpectedly, a decrease in the relative amounts of F-actin, free barbed ends, and actin-related protein 2/3 (Arp2/3) complex in lamellipodia are also observed. Conversely, decreased expression of Tmod3 by RNA interference leads to faster average cell migration, along with increases in free pointed and barbed ends in lamellipodial actin filaments. These data collectively demonstrate that capping of actin filament pointed ends by Tmod3 inhibits cell migration and reveal a novel control mechanism for regulation of actin filaments in lamellipodia.     

Subcellular Localization of RhoA and Ezrin at Membrane Ruffles of Human Endothelial Cells: Differential Role of Collagen and Fibronectin

Endothelial cells and the regulation of their migration are of prime importance in many physiological and pathological processes such as angiogenesis. RhoA, an important Rho family member known to trigger actin reorganization, has been shown to mediate the formation of focal adhesions and stress fibers in quiescent fibroblasts. However, recent studies have emphasized its functional diversity and its implication in migration or metastatic processes in different cell types other than fibroblasts. Its role in endothelial cells is little known. In this study, we were interested by analyzing in human endothelial cells the subcellular redistribution of endogenous RhoA and the reorganization of cytoskeletal actin induced by two important extracellular matrix proteins, collagen and fibronectin. This paper shows a translocation of RhoA and its association with cortical actin in focal contact domains at membrane ruffles and at lamellipodia of spread or migrating endothelial cells, in the absence of any soluble mitogen stimulation. Furthermore, RhoA was found colocalized with ezrin, a member of the ERM family proteins newly described as important membrane-actin cytoskeleton linkers, at early membrane ruffles of endothelial cells spread on collagen but not on fibronectin. The present study points out that extracellular matrix, depending on the nature of its components, may promote distinct assemblies of focal contact constitutive proteins and strongly suggests that endothelial RhoA, like Rac1, may be an important mediator of matrix signaling pathway regulating endothelial cell adhesiveness and motility, independently of growth factor stimulation.     

Regulation of cell contraction and membrane ruffling by distinct signals in migratory cells.

Cell migration and wound contraction requires assembly of actin into a functional myosin motor unit capable of generating force. However, cell migration also involves formation of actin-containing membrane ruffles. Evidence is provided that actin-myosin assembly and membrane ruffling are regulated by distinct signaling pathways in the migratory cell. Interaction of cells with extracellular matrix proteins or cytokines promote cell migration through activation of the MAP kinases ERK1 and ERK2 as well as the molecular coupling of the adaptor proteins p130CAS and c-CrkII. ERK signaling is independent of CAS/Crk coupling and regulates myosin light chain phosphorylation leading to actin-myosin assembly during cell migration and cell-mediated contraction of a collagen matrix. In contrast, membrane ruffling, but not cell contraction, requires Rac GTPase activity and the formation of a CAS/Crk complex that functions in the context of the Rac activating protein DOCK180. Thus, during cell migration ERK and CAS/Crk coupling operate as components of distinct signaling pathways that control actin assembly into myosin motors and membrane ruffles, respectively.     

Gradient of rigidity in the lamellipodia of migrating cells revealed by atomic force microscopy

Changes in mechanical properties of the cytoplasm have been implicated in cell motility, but there is little information about these properties in specific regions of the cell at specific stages of the cell migration process. Fish epidermal keratocytes with their stable shape and steady motion represent an ideal system to elucidate temporal and spatial dynamics of the mechanical state of the cytoplasm. As the shape of the cell does not change during motion and actin network in the lamellipodia is nearly stationary with respect to the substrate, the spatial changes in the direction from the front to the rear of the cell reflect temporal changes in the actin network after its assembly at the leading edge. We have utilized atomic force microscopy to determine the rigidity of fish keratocyte lamellipodia as a function of time/distance from the leading edge. Although vertical thickness remained nearly constant throughout the lamellipodia, the rigidity exhibited a gradual but significant decrease from the front to the rear of the lamellipodia. The rigidity profile resembled closely the actin density profile, suggesting that the dynamics of rigidity are due to actin depolymerization. The decrease of rigidity may play a role in facilitating the contraction of the actin-myosin network at the lamellipodium/cell body transition zone.     

Interaction of SPIN90 with the Arp2/3 complex mediates lamellipodia and actin comet tail formation

The appropriate regulation of the actin cytoskeleton is essential for cell movement, changes in cell shape, and formation of membrane protrusions like lamellipodia and filopodia. Moreover, several regulatory proteins affecting actin dynamics have been identified in the motile regions of cells. Here, we provide evidence for the involvement of SPIN90 in the regulation of actin cytoskeleton and actin comet tail formation. SPIN90 was distributed throughout the cytoplasm in COS-7 cells, but exposing the cells to platelet-derived growth factor (PDGF) caused a redistribution of SPIN90 to the cell cortex and the formation of lamellipodia (or membrane ruffles), both of which were dramatically inhibited in SPIN90-knockdown cells. In addition, the binding of the C terminus of SPIN90 with both the Arp2/3 complex (actin-related proteins Arp 2 and Arp 3) and G-actin activates the former, leading to actin polymerization in vitro. And when coexpressed with phosphatidylinositol 4-phosphate 5 kinase, SPIN90 was observed within actin comet tails. Taken these findings suggest that SPIN90 participates in reorganization of the actin cytoskeleton and in actin-based cell motility.     

Dynamics of membranes driven by actin polymerization

A motile cell, when stimulated, shows a dramatic increase in the activity of its membrane, manifested by the appearance of dynamic membrane structures such as lamellipodia, filopodia, and membrane ruffles. The external stimulus turns on membrane bound activators, like Cdc42 and PIP2, which cause increased branching and polymerization of the actin cytoskeleton in their vicinity leading to a local protrusive force on the membrane. The emergence of the complex membrane structures is a result of the coupling between the dynamics of the membrane, the activators, and the protrusive forces. We present a simple model that treats the dynamics of a membrane under the action of actin polymerization forces that depend on the local density of freely diffusing activators on the membrane. We show that, depending on the spontaneous membrane curvature associated with the activators, the resulting membrane motion can be wavelike, corresponding to membrane ruffling and actin waves, or unstable, indicating the tendency of filopodia to form. Our model also quantitatively explains a variety of related experimental observations and makes several testable predictions.     

Actin cytoskeleton remodelling in the anterior pituitary folliculostellate cell line TtT/GF: participation of the actin-binding protein cortactin

We have previously shown that the folliculostellate (FS) cells of the anterior pituitary change their shape from stellate (type I) to polygonal (type II) coincidently with variations in the secretory activity of the pituitary. To elucidate the mechanisms involved in this switch in phenotypes, here we studied the impact of serum factors on the morphology of the FS cell line TtT/GF. TtT/GF cells cultured in serum-containing medium displayed elongated shapes and membrane ruffles similarly to type I cells. Serum deprivation caused the loss of plasma membrane activity and the acquisition by the cells of a sedentary phenotype and of a polygonal shape typical of type II FS cells. Addition of serum to the starved cells induced the reappearance of membrane raffles and lamellipodia. The switch in phenotypes and the maintenance of a motile phenotype depended on tyrosine kinase but not on Erk activity. Because the transition between phenotypes involved the tyrosine kinase-dependent reorganization of cortical actin filaments, we studied the participation of the actin-binding protein, cortactin, a tyrosine kinase substrate. Cortactin and its tyrosine-phosphorylated form, pY421-cortactin, localized to membrane ruffles and lamellipodia in serum-cultured TtT/GF cells, while they were evenly distributed over the whole cell cortex in serum-starved cells. Serum treatment of starved cells induced a transient increase in pY421-cortactin levels and the clustering of pY421-cortactin in membrane regions where protrusions were developing. Both serum responses were blocked by a tyrosine kinase inhibitor. Together, the results indicate that the transition from a polygonal to an elongated shape entails the acquisition of a dynamic cortical actin cytoskeleton that involves the tyrosine kinase-dependent phosphorylation of cortactin and the translocation of cortical pY421-cortactin to sites of ruffle formation at the plasma membrane.     

Physiological role of the interaction between CARMIL1 and capping protein

The regulation of free barbed ends is central to the control of dynamic actin assembly and actin-based motility in cells. Capping protein (CP) is known to regulate barbed ends and control actin assembly in cells. The CARMIL family of proteins can bind and inhibit CP in vitro, but the physiological significance of the interaction of CARMIL with CP in cells is poorly understood. Mammalian cells lacking CARMIL1 have defects in lamellipodia, macropinocytosis, cell migration, and Rac1 activation. Here we investigate the physiological significance of the CARMIL1–CP interaction, using a point mutant with a well-defined biochemical defect. We find that the CARMIL1–CP interaction is essential for the assembly of lamellipodia, the formation of ruffles, and the process of macropinocytosis. In contrast, the interaction of CARMIL1 with CP shows little to no importance for other functions of CARMIL1, including localization of CARMIL1 to the membrane, activation of Rac1, and cell migration. One implication is that lamellipodia are only marginally important for cell migration in a wound-healing model. The results also suggest that the ability of CARMIL1 to inhibit CP in cells may be regulated.     

Localized RhoA activation as a requirement for the induction of membrane ruffling

We examined the spatio-temporal activity of RhoA in migrating cells and growth factor-stimulated cells by using probes based on the principle of fluorescence resonance energy transfer. In HeLa cells migrating at a low cell density, RhoA was activated both at the contractile tail and at the leading edge. However, RhoA was activated only at the leading edge in MDCK cells migrating as a monolayer sheet. In growth factor-stimulated Cos1 and NIH3T3 cells, the activity of RhoA was greatly decreased at the plasma membrane, but remained high at the membrane ruffles in nascent lamellipodia. These observations are in agreement with the proposed role played by RhoA in stress fiber formation, but they also implicated RhoA in the regulation of membrane ruffling, the induction of which is a typical phenotype of activated Rac. In agreement with this view, dominant negative RhoA was found to inhibit membrane ruffling induced by active Rac. Furthermore, we found that Cdc42 activity was also required for high RhoA activity in membrane ruffles. Finally, we found that mDia1, but not ROCK, was stably associated with membrane ruffles. In conclusion, these results suggested that RhoA cooperates with Rac1 and Cdc42 to induce membrane ruffles via the recruitment of mDia.     

Actin-dependent lamellipodia formation and microtubule-dependent tail retraction control-directed cell migration

Migrating cells are polarized with a protrusive lamella at the cell front followed by the main cell body and a retractable tail at the rear of the cell. The lamella terminates in ruffling lamellipodia that face the direction of migration. Although the role of actin in the formation of lamellipodia is well established, it remains unclear to what degree microtubules contribute to this process. Herein, we have studied the contribution of microtubules to cell motility by time-lapse video microscopy on green flourescence protein-actin- and tubulin-green fluorescence protein-transfected melanoma cells. Treatment of cells with either the microtubule-disrupting agent nocodazole or with the stabilizing agent taxol showed decreased ruffling and lamellipodium formation. However, this was not due to an intrinsic inability to form ruffles and lamellipodia because both were restored by stimulation of cells with phorbol 12-myristate 13-acetate in a Rac-dependent manner, and by stem cell factor in melanoblasts expressing the receptor tyrosine kinase c-kit. Although ruffling and lamellipodia were formed without microtubules, the microtubular network was needed for advancement of the cell body and the subsequent retraction of the tail. In conclusion, we demonstrate that the formation of lamellipodia can occur via actin polymerization independently of microtubules, but that microtubules are required for cell migration, tail retraction, and modulation of cell adhesion.     

Inhibition of SNARE-mediated membrane traffic impairs cell migration

Cell migration occurs as a highly-regulated cycle of cell polarization, membrane extension at the leading edge, adhesion, contraction of the cell body, and release from the extracellular matrix at the trailing edge. In this study, we investigated the involvement of SNARE-mediated membrane trafficking in cell migration. Using a dominant-negative form of the enzyme N-ethylmaleimide-sensitive factor as a general inhibitor of SNARE-mediated membrane traffic and tetanus toxin as a specific inhibitor of VAMP3/cellubrevin, we conducted transwell migration assays and determined that serum-induced migration of CHO-K1 cells is dependant upon SNARE function. Both VAMP3-mediated and VAMP3-independent traffic were involved in regulating this cell migration. Inhibition of SNARE-mediated membrane traffic led to a decrease in the protrusion of lamellipodia at the leading edge of migrating cells. Additionally, the reduction in cell migration resulting from the inhibition of SNARE function was accompanied by perturbation of a Rab11-containing alpha(5)beta(1) integrin compartment and a decrease in cell surface alpha(5)beta(1) without alteration to total cellular integrin levels. Together, these observations suggest that inhibition of SNARE-mediated traffic interferes with the intracellular distribution of integrins and with the membrane remodeling that contributes to lamellipodial extension during cell migration.     

A novel role for 14-kDa phosphohistidine phosphatase in lamellipodia formation

Cell migration involves dynamic regulation of the actin cytoskeleton, which exhibits rapid actin polymerization at the leading edge of migrating cells. This process relies on regulated recruitment of actin nucleators and actin-binding proteins to the leading edge to polymerize new actin filaments. Many of these proteins have been identified, including the actin-related protein (Arp) 2/3 complex, which has emerged as the core player in the initiation of actin polymerization. However, the functional coordination of these proteins is unclear. Previously, we have demonstrated that the 14-kDa phosphohistidine phosphatase (PHP14) is involved in cell migration regulation and affects actin cytoskeleton reorganization. Here, we show that PHP14 may regulate actin remodeling directly and play an important role in dynamic regulation of the actin cytoskeleton. We observed a colocalization of PHP14 with Arp3 and F-actin at the leading edge of migrating cells. Moreover, PHP14 was recruited to the actin remodeling sites in parallel with Arp3 during lamellipodia formation. Furthermore, PHP14 knockdown impaired Arp3 localization at the leading edge of lamellipodia, as well as lamellipodia formation. Most importantly, we found that PHP14 was a novel F-actin-binding protein, displaying an Arp2/3-dependent localization to the leading edge. Collectively, our results indicated a crucial role for PHP14 in the dynamic regulation of the actin cytoskeleton and cell migration.     

G proteins G12 and G13 control the dynamic turnover of growth factor-induced dorsal ruffles

Growth factors induce massive actin cytoskeletal remodeling in cells. These reorganization events underlie various cellular responses such as cell migration and morphological changes. One major form of actin reorganization is the formation and disassembly of dorsal ruffles (also named waves, dorsal rings, or circular ruffles). Dorsal ruffles are involved in physiological functions including cell migration, invasion, macropinocytosis, plasma membrane recycling, and others. Growth factors initiate rapid formation (within 5 min) of circular membrane ruffles, and these ruffles move along the dorsal side of the cells, constrict, close, and eventually disassemble ( approximately 20 min). Considerable attention has been devoted to the mechanism by which growth factors induce the formation of dorsal ruffles. However, little is known of the mechanism by which these ruffles are disassembled. Here we have shown that G proteins G(12) and G(13) control the rate of disassembly of dorsal ruffles. In Galpha(12)(-/-)Galpha(13)(-/-) fibroblast cells, dorsal ruffles induced by growth factor treatment remain visible substantially longer ( approximately 60 min) than in wild-type cells, whereas the rate of formation of these ruffles was the same with or without Galpha(12) and Galpha(13). Thus, Galpha(12)/Galpha(13) critically regulate dorsal ruffle turnover.     

The homolog of the five SH3-domain protein (HOFI/SH3PXD2B) regulates lamellipodia formation and cell spreading

Motility of normal and transformed cells within and across tissues requires specialized subcellular structures, e.g. membrane ruffles, lamellipodia and podosomes, which are generated by dynamic rearrangements of the actin cytoskeleton. Because the formation of these sub-cellular structures is complex and relatively poorly understood, we evaluated the role of the adapter protein SH3PXD2B [HOFI, fad49, Tks4], which plays a role in the development of the eye, skeleton and adipose tissue. Surprisingly, we find that SH3PXD2B is requisite for the development of EGF-induced membrane ruffles and lamellipodia, as well as for efficient cellular attachment and spreading of HeLa cells. Furthermore, SH3PXD2B is present in a complex with the non-receptor protein tyrosine kinase Src, phosphorylated by Src, which is consistent with SH3PXD2B accumulating in Src-induced podosomes. Furthermore, SH3PXD2B closely follows the subcellular relocalization of cortactin to Src-induced podosomes, EGF-induced membrane ruffles and lamellipodia. Because SH3PXD2B also forms a complex with the C-terminal region of cortactin, we propose that SH3PXD2B is a scaffold protein that plays a key role in regulating the actin cytoskeleton via Src and cortactin.