Actin-based Cell Motility Research Articles

Enhanced microglial dynamics and a paucity of tau seeding in the amyloid plaque microenvironment contribute to cognitive resilience in Alzheimer’s disease

Asymptomatic Alzheimer’s disease (AsymAD) describes the status of individuals with preserved cognition but identifiable Alzheimer’s disease (AD) brain pathology (i.e., beta-amyloid (Aβ) deposits, neuritic plaques, and neurofibrillary tangles) at autopsy. In this study, we investigated the postmortem brains of a cohort of AsymAD subjects to gain insight into the mechanisms underlying resilience to AD pathology and cognitive decline. Our results showed that AsymAD cases exhibit enrichment in core plaques, decreased filamentous plaque accumulation, and increased plaque-surrounding microglia. Less pathological tau aggregation in dystrophic neurites was found in AsymAD brains than in AD brains, and tau seeding activity was comparable to that in healthy brains. We used spatial transcriptomics to characterize the plaque niche further and revealed autophagy, endocytosis, and phagocytosis as the pathways associated with the genes upregulated in the AsymAD plaque niche. Furthermore, the levels of ARP2 and CAP1, which are actin-based motility proteins that participate in the dynamics of actin filaments to allow cell motility, were increased in the microglia surrounding amyloid plaques in AsymAD cases. Our findings suggest that the amyloid-plaque microenvironment in AsymAD cases is characterized by the presence of microglia with highly efficient actin-based cell motility mechanisms and decreased tau seeding compared with that in AD brains. These two mechanisms can potentially protect against the toxic cascade initiated by Aβ, preserving brain health, and slowing AD pathology progression.

Identification of MYH9 as a key regulator for synoviocyte migration and invasion through secretome profiling

Objectives‘Invasive pannus’ is a pathological hallmark of rheumatoid arthritis (RA). This study aimed to investigate secretome profile of synovial fibroblasts of patients with RA (RA-FLSs), a major cell type comprising...

The role of neuroplastin65 in macrophage against E. coli infection in mice

BackgroundInnate immune response constitutes the first line of defense against pathogens. Inflammatory responses involve close contact between different populations of cells. These adhesive interactions mediate migration of cells to sites of infection leading the effective action of cells within the lesions. Cell adhesion molecules are critical to controlling immune response mediating cell adhesion or chemotaxis, as well as coordinating actin-based cell motility during phagocytosis and chemotaxis. Recently, a newly discovered neuroplastin (Np) adhesion molecule is found to play an important role in the nervous system. However, there is limited information on Np functions in immune response. To understand how Np is involved in innate immune response, a mouse model of intraperitoneal infection was established to investigate the effect of Np on macrophage-mediated clearance of E. coli infection and its possible molecular mechanisms. MethodsSpecific deficiency mice with Nptn gene controlling Np65 isoform were employed in this study. The expression levels of mRNA and proteins were detected by qPCR and western blot, or evaluated by flow cytometry. The expression level of NO and ROS were measured with their specific indicators. Cell cycle and apoptosis were detected by specific detection kits. Acid phosphatase activity was measured by flow cytometry after labelling with LysoRed fluorescent probe. Bone marrow derived macrophages (BMDMs) were isolated from bone marrow of mice hind legs. Cell proliferation was detected by CCK8 assay. Cell migration was measured by wound healing assay or transwell assay. ResultsThe lethal dose of E. coli infection in Np65–/– mice dropped to the half of lethal dose in WT mice. The bacterial load in the spleen, kidney and liver from Np65–/– mice were significantly higher than that from WT mice, which were due to the dramatic reduction of NO and ROS production in phagocytes from Np65–/– mice. Np65 gene deficiency remarkably impaired phagocytosis and function of lysosome in macrophage. Furthermore, Np65 molecule was involved in maturation and proliferation, even in migration and chemotaxis of BMDM in vitro. ConclusionThis study for the first time demonstrates that Np is involved in multi-function of phagocytes during bacterial infection, proposing that Np adhesion molecule plays a critical role in clearing pathogen infection in innate immunity.

SUN-MKL1 Crosstalk Regulates Nuclear Deformation and Fast Motility of Breast Carcinoma Cells in Fibrillar ECM Microenvironment.

Aligned collagen fibers provide topography for the rapid migration of single tumor cells (streaming migration) to invade the surrounding stroma, move within tumor nests towards blood vessels to intravasate and form distant metastases. Mechanisms of tumor cell motility have been studied extensively in the 2D context, but the mechanistic understanding of rapid single tumor cell motility in the in vivo context is still lacking. Here, we show that streaming tumor cells in vivo use collagen fibers with diameters below 3 µm. Employing 1D migration assays with matching in vivo fiber dimensions, we found a dependence of tumor cell motility on 1D substrate width, with cells moving the fastest and the most persistently on the narrowest 1D fibers (700 nm–2.5 µm). Interestingly, we also observed nuclear deformation in the absence of restricting extracellular matrix pores during high speed carcinoma cell migration in 1D, similar to the nuclear deformation observed in tumor cells in vivo. Further, we found that actomyosin machinery is aligned along the 1D axis and actomyosin contractility synchronously regulates cell motility and nuclear deformation. To further investigate the link between cell speed and nuclear deformation, we focused on the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex proteins and SRF-MKL1 signaling, key regulators of mechanotransduction, actomyosin contractility and actin-based cell motility. Analysis of The Cancer Genome Atlas dataset showed a dramatic decrease in the LINC complex proteins SUN1 and SUN2 in primary tumor compared to the normal tissue. Disruption of LINC complex by SUN1 + 2 KD led to multi-lobular elongated nuclei, increased tumor cell motility and concomitant increase in F-actin, without affecting Lamin proteins. Mechanistically, we found that MKL1, an effector of changes in cellular G-actin to F-actin ratio, is required for increased 1D motility seen in SUN1 + 2 KD cells. Thus, we demonstrate a previously unrecognized crosstalk between SUN proteins and MKL1 transcription factor in modulating nuclear shape and carcinoma cell motility in an in vivo relevant 1D microenvironment.

Actin-Membrane Release Initiates Cell Protrusions.

Despite the well-established role of actin polymerization as a driving mechanism for cell protrusion, upregulated actin polymerization alone does not initiate protrusions. Using a combination of theoretical modeling and quantitative live-cell imaging experiments, we show that local depletion of actin-membrane links is needed for protrusion initiation. Specifically, we show that the actin-membrane linker ezrin is depleted prior to protrusion onset and that perturbation of ezrin's affinity for actin modulates protrusion frequency and efficiency. We also show how actin-membrane release works in concert with actin polymerization, leading to a comprehensive model for actin-driven shape changes. Actin-membrane release plays a similar role in protrusions driven by intracellular pressure. Thus, our findings suggest that protrusion initiation might be governed by a universal regulatory mechanism, whereas the mechanism of force generation determines the shape and expansion properties of the protrusion.

Monitoring Palladin's Effect on Actin Dynamics and Organization with TIRF Microscopy

The actin-associated human protein palladin plays a critical role in cytoskeletal organization in both normal and cancerous cells. In breast and pancreatic cancer cell lines, palladin expression levels have been shown to correlate with metastatic potential. We have recently established that palladin contributes to actin dynamics in three distinct ways: nucleation of actin, crosslink formation, and filament stabilization. We hypothesize that palladin directly influences cell motility through simultaneous regulation of actin polymerization and organization. In this work we build upon our kinetic assays of actin polymerization to directly visualize actin assembly and protein dynamics with total internal reflection fluorescence microscopy (TIRFM). Bulk co-sedimentation assays can only monitor an increase in the total amount of actin polymerized, whereas the TIRF assay allows one to follow polymerization dynamics and filament topology simultaneously. To gain insight into the role of palladin in the assembly process and in the dynamics of supramolecular actin structures, we imaged the polymerization of monomeric actin and palladin in real time with TIRFM. Our data show that palladin mediates the formation of junctions between filaments. Analysis of filament morphology reveals that palladin promotes side branching in addition to bundles. Through these methods, we hope to understand how palladin contributes to actin-based cell motility in metastatic cells, which will be important for developing new therapies to specifically target this step in cancer progression.

A fruitful tree: developing the dendritic nucleation model of actin-based cell motility

A fundamental question in cell biology concerns how cells move, and this has been the subject of intense research for decades. In the 1990s, a major leap forward was made in our understanding of cell motility, with the proposal of the dendritic nucleation model. This essay describes the events leading to the development of the model, including findings from many laboratories and scientific disciplines. The story is an excellent example of the scientific process in action, with the combination of multiple perspectives leading to robust conclusions.

Cell Migration and Invadopodia Formation Require a Membrane-binding Domain of CARMIL2

CARMILs regulate capping protein (CP), a critical determinant of actin assembly and actin-based cell motility. Vertebrates have three conserved CARMIL genes with distinct functions. In migrating cells, CARMIL2 is important for cell polarity, lamellipodial assembly, ruffling, and macropinocytosis. In cells, CARMIL2 localizes with a distinctive dual pattern to vimentin intermediate filaments and to membranes at leading edges and macropinosomes. The mechanism by which CARMIL2 localizes to membranes has not been defined. Here, we report that CARMIL2 has a conserved membrane-binding domain composed of basic and hydrophobic residues, which is necessary and sufficient for membrane localization, based on expression studies in cells and on direct binding of purified protein to lipids. Most important, we find that the membrane-binding domain is necessary for CARMIL2 to function in cells, based on rescue expression with a set of biochemically defined mutants. CARMIL1 and CARMIL3 contain similar membrane-binding domains, based on sequence analysis and on experiments, but other CPI motif proteins, such as CD2AP, do not. Based on these results, we propose a model in which the membrane-binding domain of CARMIL2 tethers this multidomain protein to the membrane, where it links dynamic vimentin filaments with regulation of actin assembly via CP.

Extending the molecular clutch beyond actin-based cell motility

Many cell movements occur via polymerization of the actin cytoskeleton beneath the plasma membrane at the front of the cell, forming a protrusion called a lamellipodium, while myosin contraction squeezes forward the back of the cell. In what is known as the ‘molecular clutch’ description of cell motility, forward movement results from the engagement of the acto-myosin motor with cell-matrix adhesions, thus transmitting force to the substrate and producing movement. However during cell translocation, clutch engagement is not perfect, and as a result, the cytoskeleton slips with respect to the substrate, undergoing backward (retrograde) flow in the direction of the cell body. Retrograde flow is therefore inversely proportional to cell speed and depends on adhesion and acto-myosin dynamics. Here we asked whether the molecular clutch was a general mechanism by measuring motility and retrograde flow for the Caenorhabditis elegans sperm cell in different adhesive conditions. These cells move by adhering to the substrate and emitting a dynamic lamellipodium, but the sperm cell does not contain an acto-myosin cytoskeleton. Instead the lamellipodium is formed by the assembly of major sperm protein, which has no biochemical or structural similarity to actin. We find that these cells display the same molecular clutch characteristics as acto-myosin containing cells. We further show that retrograde flow is produced both by cytoskeletal assembly and contractility in these cells. Overall this study shows that the molecular clutch hypothesis of how polymerization is transduced into motility via adhesions is a general description of cell movement regardless of the composition of the cytoskeleton.

Dynamic Localization of G-Actin during Membrane Protrusion in Neuronal Motility

Actin-based cell motility is fundamental for development, function, and malignant events in eukaryotic organisms. During neural development, axonal growth cones depend on rapid assembly and disassembly of actin filaments (F-actin) for their guided extension to specific targets for wiring. Monomeric globular actin (G-actin) is the building block for F-actin but is not considered to play a direct role in spatiotemporal control of actin dynamics in cell motility. Here we report that a pool of G-actin dynamically localizes to the leading edge of growth cones and neuroblastoma cells to spatially elevate the G-/F-actin ratio that drives membrane protrusion and cell movement. Loss of G-actin localization leads to the cessation and retraction of membrane protrusions. Moreover, G-actin localization occurs asymmetrically in growth cones during attractive turning. Finally, we identify the actin monomer-binding proteins profilin and thymosin β4 as key molecules that localize actin monomers to the leading edge of lamellipodia for their motility. Our results suggest that dynamic localization of G-actin provides a novel mechanism to regulate the spatiotemporal actin dynamics underlying membrane protrusion in cell locomotion and growth cone chemotaxis.

Deletion of the cell adhesion adaptor protein vinculin disturbs the localization of GFAP in Bergmann glial cells

Astrocytes operate in close spatial relationship to other cells including neurons. Structural interaction is controlled by a dynamic interplay between actin-based cell motility and contact formation via cell-cell and cell-extracellular matrix adhesions. A central player in the control of cell adhesion is the cytoskeletal adaptor protein Vinculin. Incorporation of Vinculin affects mechanical properties and turnover of cell adhesion sites. To study the in vivo function of Vinculin in astrocytes, a mouse line with astrocyte specific and inducible deletion of vinculin was generated. Deletion of vinculin decreased the expression of the glial acidic fibrillary protein (GFAP) in Bergmann glial cells in the cerebellum. In addition, localization of GFAP to Bergmann glial endfeet was disturbed, indicating a role for vinculin in controlling its expression and localization. In contrast, vimentin expression, morphology, activation state and polarity of the targeted cells as well as the localization of the extracellular matrix protein laminin was not compromised. Furthermore, stab wound lesions were performed in the cerebellar cortex. In both wildtype and vinculin knockout mice GFAP expression was upregulated in Bergmann glial cells of the lesioned area with no differences observed between genotypes in expression and localization of GFAP. These results propose a selective requirement for vinculin in cellular events related to cell adhesion in vivo. As in vitro data suggested a major role for vinculin in the control of the cytoskeletal connection affecting mechanical stability and cell motility, our data add a note of caution to the extrapolation of in vitro data to in vivo function.

Role of km23-1 in RhoA/actin-based cell migration

km23-1 was originally identified as a TGFß receptor-interacting protein that plays an important role in TGFß signaling. Moreover, km23-1 is actually part of an ancient superfamily of NTPase-regulatory proteins, widely represented in archaea and bacteria. To further elucidate the function of km23-1, we identified novel protein interacting partners for km23-1 by using tandem affinity purification (TAP) and tandem mass spectrometry (MS). Here we show that km23-1 interacted with a class of proteins involved in actin-based cell motility and modulation of the actin cytoskeleton. We further showed that km23-1 modulates the formation of a highly organized stress fiber network. More significantly, we demonstrated that knockdown (KD) of km23-1 decreased RhoA activation in Mv1Lu epithelial cells. Finally, our results demonstrated for the first time that depletion of km23-1 inhibited cell migration of human colon carcinoma cells (HCCCs) in wound-healing assays. Overall, our findings demonstrate that km23-1 regulates RhoA and motility-associated actin modulating proteins, suggesting that km23-1 may represent a novel target for anti-metastatic therapy.

Nexilin is a dynamic protein at Listeria monocytogenes comet tails and enteropathogenic E. coli pedestals

The extracellular pathogen enteropathogenic Escherichia coli (EPEC) and the intracellular bacterium Listeria monocytogenes (Listeria) control the actin cytoskeletal machinery during their infections as part of their disease processes. Through the subversion of this cytoskeletal system, motile actin‐rich structures (EPEC pedestals and Listeria comet tails) are generated. Nexilin, an actin‐binding protein, is thought to facilitate actin‐based cell motility. Consequently, we tested the hypothesis that nexilin is recruited to EPEC pedestals as well as Listeria comet tails and is needed for proper functioning of those structures. Using a combination of immunolocalization, RNA interference and time‐lapse imaging, we demonstrate that nexilin co‐localizes with actin in EPEC pedestals and Listeria comet tails. As stationary EPEC and Listeria becomes motile, nexilin focuses distally in these long actin‐rich structures. When nexilin expression was perturbed, EPEC pedestals remain unaltered, however Listeria comet tails had aberrant morphologies. Our data suggests that nexilin is redistributed to the dissociating end of actin filaments where it may act as a platform for bacterial propulsion when infecting host cells.Grant Funding Source: CIHR, NSERC, Institut Pasteur, Institut National de la Santé et de la Recherche Médicale, Institut National de la Recherche Agronomique, European Research Council, Howard Hughes Medical Institute

In vitro anti-metastatic activity of enterolactone, a mammalian lignan derived from flax lignan, and down-regulation of matrix metalloproteinases in MCF-7 and MDA MB 231 cell lines

Actin cytoskeleton is involved in actin-based cell adhesion, cell motility, and matrix metalloproteinases(MMPs) MMP2, MMP9, MMP11 and MMP14 are responsible for cell invasion in breast cancer metastasis. The dietary intake of lignan from flax seed gets converted to enterolactone (EL) and enterodiol in the human system. Here we show that the enterolactone has a very significant anti-metastatic activity as demonstrated by its ability to inhibit adhesion and invasion and migration in MCF-7 and MDA MB231 cell lines. Migration inhibition assay, actin-based cell motility assay along with reverse transcriptase polymerase chain reaction (RT-PCR) for MMP2, MMP9, MMP11 and MMP14 genes were performed in MCF-7 and MDA MB 231 cell lines. Enterolactone seems to inhibit actin-based cell motility as evidenced by confocal imaging and photo documentation of cell migration assay. The results are supported by the observation that the enterolactone in vitro significantly down-regulates the metastasis-related metalloproteinases MMP2, MMP9 and MMP14 gene expressions. No significant alteration in the MMP11 gene expression was found. Therefore we suggest that the anti-metastatic activity of EL is attributed to its ability to inhibit cell adhesion, cell invasion and cell motility. EL affects normal filopodia and lamellipodia structures, polymerization of actin filaments at their leading edges and thereby inhibits actin-based cell adhesion and cell motility. The process involves multiple force-generating mechanisms of actin filaments i.e. protrusion, traction, deadhesion and tail-retraction. By down-regulating the metastasis-related MMP2, MMP9 and MMP14 gene expressions, EL may be responsible for cell invasion step of metastasis.

Abstract B148: The miRNA-200 family regulates cell migration and metastasis in lung adenocarcinoma.

Abstract miRNAs are post-transcriptional regulators of genes involved in such canonical signaling pathways as the TGF-β and EGF pathways, both of which are frequently altered in tumor cells. miRNA downregulation results in upregulation of its target genes and ultimately aberrant signaling of pathways involved in invasion and migration, for example. To better understand how alterations in miRNA expression could lead to metastasis, we used a lung adenocarcinoma mouse model in which a lentivirus expressing the Cre recombinase activates oncogenic K-Ras and deletes p53, giving rise to primary lung tumors (T) and metastases (M). We performed miRNA expression profiling on 30 T and M derived cell lines and identified three members of the miR-200 family - miR-200b, miR-200c, miR-429 - to be significantly downregulated in the M lines compared to their related T line. To explore the functional contribution of the miR-200s to metastasis, we designed a miRNA sponge to stably inhibit endogenous miR-200 levels in T cell lines. We found miR-200 depletion promoted wound healing and cell migration towards either serum or EGF. Transplantation of the sponge-expressing lines via tail vein led to in vivo formation of more invasive, metastatic tumors compared to controls. Previously, the miR-200 family has been shown to inhibit the TGF-β target gene and EMT mediator, Zeb1. We have identified another miR-200 target, Epidermal Growth Factor Receptor Substrate 8 (Eps8), which mediates actin based cell motility. In miR-200 low (M) cells, Eps8 is expressed three-fold higher than in corresponding miR-200 high (T) cells. Additionally, exogenous expression of Eps8 in T lines promotes wound healing and cell migration, relative to control cells. Thus, our findings implicate the miR-200s as a class of metastasis regulators that modulate expression of genes functionally relevant in tumor cell dissemination and provide new targets for therapeutic intervention of late stage lung adenocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B148.

How does the antagonism between capping and anti-capping proteins affect actin networkdynamics?

Actin-based cell motility is essential to many biological processes. We built a simplified,three-dimensional computational model and subsequently performed stochastic simulationsto study the growth dynamics of lamellipodia-like branched networks. In this work, we shedlight on the antagonism between capping and anti-capping proteins in regulatingactin dynamics in the filamentous network. We discuss detailed mechanisms bywhich capping and anti-capping proteins affect the protrusion speed of the actinnetwork and the rate of nucleation of filaments. We computed a phase diagramshowing the regimes of motility enhancement and inhibition by these proteins.Our work shows that the effects of capping and anti-capping proteins are mainlytransmitted by modulation of the filamentous network density and local availability ofmonomeric actin. We discovered that the combination of the capping/anti-cappingregulatory network with nucleation-promoting proteins introduces robustnessand redundancy in cell motility machinery, allowing the cell to easily achievemaximal protrusion speeds under a broader set of conditions. Finally, we discussdistributions of filament lengths under various conditions and speculate on theirpotential implication for the emergence of filopodia from the lamellipodial network.

Advances in Experiments and Modeling in Micro- and Nano-Biomechanics: A Mini Review

Recent advances in micro- and nano-technologies and high-end computing have enabled the development of new experimental and modeling approaches to study biomechanics at the micro- and nano-scales that were previously not possible. These new cutting-edge approaches are contributing toward our understanding in emerging areas such as mechanobiology and mechanochemistry. Another important potential contribution lies in translational medicine, since biomechanical studies at the cellular and molecular levels have direct relevance in areas such disease diagnosis, nano-medicine and drug delivery. Thus, the developed experimental and modeling approaches are critical in elucidating important mechanistic insights in both basic sciences and clinical treatment. While it is hard to cover all the recent advances in this mini-review, we focus on several important approaches. For experimental techniques, we review the assays involving shear flow, cellular imaging, microbead, microcontact printing, and micropillars at the micro-scale, and micropipette aspiration, optical tweezers, parallel flow chamber, and atomic force microscopy at the nano-scale. In modeling and simulations, we outline the theoretical modeling for actin dynamics in migrating cell and actin-based cell motility in cellular mechanics, as well as the receptor–ligand binding in cell adhesion and the application of free, steered, and flow molecular dynamics simulations in molecular biomechanics. Relevant scientific issues and applications are also discussed.

Stochastic Simulation Study of the Role of Capping and Anti-Capping Proteins in Regulating Actin Network Growth

Actin-based cell motility is essential to many biological processes. We build simplified computational model and perform stochastic simulation to study the growth of lamellipodia-like branched filamentous network. Here we investigate how capping proteins and anti-capping proteins affect the speed of protrusion and the nucleation of filaments in the model system. Phase diagram showing the regimes of motility enhancement and motility inhibition is presented. Furthermore, we discuss filament length distribution and its implication in the formation of filopodia from lamellipodial network.

Monitoring the Real-Time Binding of Tropomyosin to Actin using Total Internal Reflection Fluorescence Microscopy

Tropomyosin, an elongated actin binding protein, regulates the access of numerous other proteins onto the thin filament in virtually all cell types. This association, in turn, regulates various functions. Tropomyosin monomers assemble end-to-end to form a continuous polymeric chain that lies along the length of the actin filament. A number of different tropomyosin isoforms are present in all three muscle types: viz. smooth, cardiac, and skeletal. In cardiac and skeletal muscle, tropomyosin and troponin are the primary regulators of contraction. A plethora of tropomyosin isoforms are also present in non-muscle cells where they associate with cytoskeletal actin and play a role in numerous cellular processes including lamellipodia formation and actin-based cell motility. Moreover, various mutations in tropomyosin are associated with diseases such as congenital fiber type distortion (CFTD), ulcerative colitis, nemaline myopathy, and various cardiomyopathies. Tropomyosin is a modular protein, and individually charged residues within its repeating domains are thought to be critical for actin binding. However, the mechanism of tropomyosin binding to actin is still uncertain. In the present study, we have acquired real-time videos of skeletal and smooth tropomyosin isoforms binding to actin filaments using total internal reflection fluorescence microscopy (TIRF). The videos suggest that weak monomer binding facilitates the gradual formation of small “nuclei” of tropomyosin monomers and/or oligomers. Once the nucleus is formed, the affinity of tropomyosin for actin increases exponentially and additional monomers are able to rapidly add to either end of the growing tropomyosin chain lying on F-actin.

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.