Adhesion Disassembly Research Articles

Lipid rafts mediate internalization of β1-integrin in migrating intestinal epithelial cells

Intestinal mucosal inflammation is associated with epithelial wounds that rapidly reseal by migration of intestinal epithelial cells (IECs). Cell migration involves cycles of cell-matrix adhesion/deadhesion that is mediated by dynamic turnover (assembly and disassembly) of integrin-based focal adhesions. Integrin endocytosis appears to be critical for deadhesion of motile cells. However, mechanisms of integrin internalization during remodeling of focal adhesions of migrating IECs are not understood. This study was designed to define the endocytic pathway that mediates internalization of beta(1)-integrin in migrating model IECs. We observed that, in SK-CO15 and T84 colonic epithelial cells, beta(1)-integrin is internalized in a dynamin-dependent manner. Pharmacological inhibition of clathrin-mediated endocytosis or macropinocytosis and small-interfering RNA (siRNA)-mediated knock down of clathrin did not prevent beta(1)-integrin internalization. However, beta(1)-integrin internalization was inhibited following cholesterol extraction and after overexpression of lipid raft protein, caveolin-1. Furthermore, internalized beta(1)-integrin colocalized with the lipid rafts marker cholera toxin, and siRNA-mediated knockdown of caveolin-1 and flotillin-1/2 increased beta(1)-integrin endocytosis. Our data suggest that, in migrating IEC, beta(1)-integrin is internalized via a dynamin-dependent lipid raft-mediated pathway. Such endocytosis is likely to be important for disassembly of integrin-based cell-matrix adhesions and therefore in regulating IEC migration and wound closure.

Semaphorin-3B Is an Angiogenesis Inhibitor That Is Inactivated by Furin-Like Pro-Protein Convertases

Semaphorin-3B (sema3B) and semaphorin-3F (sema3F) are secreted tumor suppressors of lung cancer. Sema3F functions as an antiangiogenic factor that repels endothelial cells and compromises their proliferation/survival. However, tumor cells expressing either endogenous or recombinant sema3B fail to repel endothelial cells efficiently. Sema3B found in the conditioned medium of such cells is almost completely cleaved by furin-like pro-protein convertases, generating inactive 61- and 22-kDa fragments. We have generated a sema3B variant that was point mutated at the cleavage site (sema3B-m), thereby conferring partial resistance to cleavage. Conditioned medium from HEK293 cells expressing sema3b-m and conditioned medium of HEK293 cells expressing sema3B contained similar concentrations of semaphorin but sema3B-m was cleaved much less than sema3B. In contrast to HEK293 cells expressing native sema3B, cells expressing sema3b-m strongly repel endothelial cells. Conditioned medium from sema3B-m-expressing cells rapidly caused disassembly of focal adhesions and a collapse of the actin cytoskeleton of endothelial cells, inhibited vascular endothelial growth factor-induced phosphorylation of extracellular signal-regulated kinase 1/2, induced apoptosis of endothelial cells, and inhibited the formation of tubes from endothelial cells in an in vitro angiogenesis assay more potently than conditioned medium from cells expressing sema3B. Furthermore, HEK293 cells expressing sema3B-m inhibited basic fibroblast growth factor-induced angiogenesis in vivo much more potently than cells expressing sema3B. Repulsion of human umbilical vascular endothelial cells by sema3B-m was mediated primarily by the neuropilin-1 (np1) receptor but sema3B-m was also able to transduce signals via neuropilin-2 (np2). These results suggest that up-regulation of furin-like pro-protein convertases in malignant cells may enable tumors to evade the antiangiogenic effects of sema3B.

Thrombospondin 1 binding to calreticulin‐LRP1 signals resistance to anoikis

Anoikis, apoptotic cell death due to loss of cell adhesion, is critical for regulation of tissue homeostasis in tissue remodeling. Fibrogenesis is associated with reduced fibroblast apoptosis. The matricellular protein thrombospondin 1 (TSP1) regulates cell adhesion and motility during tissue remodeling and in fibrogenesis. The N-terminal domain of TSP1 binds to the calreticulin-LRP1 receptor co-complex to signal down-regulation of cell adhesion and increased cell motility through focal adhesion disassembly. TSP1 signaling through calreticulin-LRP1 activates cell survival signals such as PI3-kinase. Therefore, we tested the hypothesis that TSP1 supports cell survival under adhesion-independent conditions to facilitate tissue remodeling. Here, we show that platelet TSP1, its N-terminal domain (NoC1) as a recombinant protein, or a peptide comprising the calreticulin-LRP1 binding site [amino acids 17-35 (hep I)] in the N-terminal domain promotes fibroblast survival under anchorage-independent conditions. TSP1 activates Akt and decreases apoptotic signaling through caspase 3 and PARP1 in suspended fibroblasts. Inhibition of PI3K/Akt activity blocks TSP1-mediated anchorage-independent survival. Fibroblasts lacking LRP1 or expressing calreticulin lacking the TSP1 binding site do not respond to TSP1 with anchorage-independent survival. These data define a novel role for TSP1 signaling through the calreticulin/LRP1 co-complex in tissue remodeling and fibrotic responses through stimulation of anoikis resistance.-Pallero, M. A., Elzie, C. A., Chen, J., Mosher, D. F., Murphy-Ullrich, J. E. Thrombospondin 1 binding to calreticulin-LRP1 signals resistance to anoikis.

CaMK‐II promotes focal adhesion turnover and cell motility by inducing tyrosine dephosphorylation of FAK and paxillin

Transient elevations in Ca2+ have previously been shown to promote focal adhesion disassembly and cell motility through an unknown mechanism. In this study, evidence is provided to show that CaMK-II, a Ca2+/calmodulin dependent protein kinase, influences fibroblast adhesion and motility. TIRF microscopy reveals a dynamic population of CaMK-II at the cell surface in migrating cells. Inhibition of CaMK-II with two mechanistically distinct, membrane permeant inhibitors (KN-93 and myr-AIP) freezes lamellipodial dynamics, accelerates spreading on fibronectin, enlarges paxillin-containing focal adhesions and blocks cell motility. In contrast, constitutively active CaMK-II is not found at the cell surface, reduces cell attachment, eliminates paxillin from focal adhesions and decreases the phospho-tyrosine levels of both FAK and paxillin; all of these events can be reversed with myr-AIP. Thus, both CaMK-II inhibition and constitutive activation block cell motility through over-stabilization or destabilization of focal adhesions, respectively. Coupled with the existence of transient Ca2+ elevations and a dynamic CaMK-II population, these findings provide the first direct evidence that CaMK-II enables cell motility by transiently and locally stimulating tyrosine dephosphorylation of focal adhesion proteins to promote focal adhesion turnover.

FAK-MAPK-dependent adhesion disassembly downstream of L1 contributes to semaphorin3A-induced collapse.

Axonal receptors for class 3 semaphorins (Sema3s) are heterocomplexes of neuropilins (Nrps) and Plexin-As signalling coreceptors. In the developing cerebral cortex, the Ig superfamily cell adhesion molecule L1 associates with Nrp1. Intriguingly, the genetic removal of L1 blocks axon responses of cortical neurons to Sema3A in vitro despite the expression of Plexin-As in the cortex, suggesting either that L1 substitutes for Plexin-As or that L1 and Plexin-A are both required and mediate distinct roles. We report that association of Nrp1 with L1 but not Plexin-As mediates the recruitment and activation of a Sema3A-induced focal adhesion kinase-mitogen-activated protein kinase cascade. This signalling downstream of L1 is needed for the disassembly of adherent points formed in growth cones and subsequently their collapse response to Sema3A. Plexin-As and L1 are coexpressed and present in common complexes in cortical neurons and both dominant-negative forms of Plexin-A and L1 impair their response to Sema3A. Consistently, Nrp1-expressing cortical projections are defective in mice lacking Plexin-A3, Plexin-A4 or L1. This reveals that specific signalling activities downstream of L1 and Plexin-As cooperate for mediating the axon guidance effects of Sema3A.

Podosome-type adhesions and focal adhesions, so alike yet so different

Cell–matrix adhesions are essential for cell migration, tissue organization and differentiation, therefore playing central roles in embryonic development, remodeling and homeostasis of tissues and organs. Matrix adhesion-dependent signals cooperate with other pathways to regulate biological functions such as cell survival, cell proliferation, wound healing, and tumorigenesis. Cell migration and invasion are integrated processes requiring the continuous, coordinated assembly and disassembly of integrin-mediated adhesions. An understanding of how integrins regulate cell migration and invasiveness through the dynamic regulation of adhesions is fundamental to both physiological and pathological situations. A variety of cell–matrix adhesions has been identified, namely, focal complexes, focal adhesions, fibrillar adhesions, podosomes, and invadopodia (podosome-type adhesions). These adhesion sites contain integrin clusters able to develop specialized structures, which are different in their architecture and dynamics although they share almost the same proteins. Here we compare recent advances and developments in the elucidation of the organization and dynamics of focal adhesions and podosome-type adhesions, in order to understand how such subcellular sites – though closely related in their composition – can be structurally and functionally different. The underlying question is how their respective physiological or pathological roles are related to their distinct organization.

FAK/src-Family Dependent Activation of the Ste20-Like Kinase SLK Is Required for Microtubule-Dependent Focal Adhesion Turnover and Cell Migration

Cell migration involves a multitude of signals that converge on cytoskeletal reorganization, essential for development, immune responses and tissue repair. Using knockdown and dominant negative approaches, we show that the microtubule-associated Ste20-like kinase SLK is required for focal adhesion turnover and cell migration downstream of the FAK/c-src complex. Our results show that SLK co-localizes with paxillin, Rac1 and the microtubules at the leading edge of migrating cells and is activated by scratch wounding. SLK activation is dependent on FAK/c-src/MAPK signaling, whereas SLK recruitment to the leading edge is src-dependent but FAK independent. Our results show that SLK represents a novel focal adhesion disassembly signal.

Paxillin Dynamics Measured during Adhesion Assembly and Disassembly by Correlation Spectroscopy

Paxillin is an adaptor molecule involved in the assembly of focal adhesions. Using different fluorescence fluctuation approaches, we established that paxillin-EGFP is dynamic on many timescales within the cell, ranging from milliseconds to seconds. In the cytoplasmic regions, far from adhesions, paxillin is uniformly distributed and freely diffusing as a monomer, as determined by single-point fluctuation correlation spectroscopy and photon-counting histogram analysis. Near adhesions, paxillin dynamics are reduced drastically, presumably due to binding to protein partners within the adhesions. The photon-counting histogram analysis of the fluctuation amplitudes reveals that this binding equilibrium in new or assembling adhesions is due to paxillin monomers binding to quasi-immobile structures, whereas in disassembling adhesions or regions of adhesions, the equilibrium is due to exchange of large aggregates. Scanning fluctuation correlation spectroscopy and raster-scan image correlation spectroscopy analysis of laser confocal images show that the environments within adhesions are heterogeneous. Relatively large adhesions appear to slide transversally due to a treadmilling mechanism through the addition of monomeric paxillin at one side and removal of relatively large aggregates of proteins from the retracting edge. Total internal reflection microscopy performed with a fast acquisition EM-CCD camera completes the overall dynamic picture and adds details of the heterogeneous dynamics across single adhesions and simultaneous bursts of activity at many adhesions across the cell.

Calpain 2 and PTP1B function in a novel pathway with Src to regulate invadopodia dynamics and breast cancer cell invasion

Invasive cancer cells form dynamic adhesive structures associated with matrix degradation called invadopodia. Calpain 2 is a calcium-dependent intracellular protease that regulates adhesion turnover and disassembly through the targeting of specific substrates such as talin. Here, we describe a novel function for calpain 2 in the formation of invadopodia and in the invasive abilities of breast cancer cells through the modulation of endogenous c-Src activity. Calpain-deficient breast cancer cells show impaired invadopodia formation that is rescued by expression of a truncated fragment of protein tyrosine phosphatase 1B (PTP1B) corresponding to the calpain proteolytic fragment, which indicates that calpain modulates invadopodia through PTP1B. Moreover, PTP1B activity is required for efficient invadopodia formation and breast cancer invasion, which suggests that PTP1B may modulate breast cancer progression through its effects on invadopodia. Collectively, our experiments implicate a novel signaling pathway involving calpain 2, PTP1B, and Src in the regulation of invadopodia and breast cancer invasion.

The Role of PKC delta and p190RhoGAP in Actin Filament Stabilization

Protein kinase C delta (PKCδ) belongs to the PKC family of serine/threonine kinases whose involvement with the actin cytoskeleton and associated protein complexes mediates a myriad of cellular functions, including monolayer permeability. Our published data showed disassembly of focal adhesions (FA), disruption of stress fibers (SF), increased monolayer permeability and decreased RhoA GTPase (RhoA) activity upon inhibition of PKCδ (Harrington E.O. et al, 2005). We also observed binding of PKCδ to p190RhoGAP (p190), a negative regulator of RhoA function. In the current study, we investigated the role of p190 in PKCδ modulation of SF, FA, and monolayer permeability. We found that inhibition of PKCδ in endothelial cells (EC) led to increase in p190 activity. Suppression of p190 in these cells caused a partial loss of SF and reduction in FA, effects which were further accentuated upon inhibition of PKCδ. Interestingly, however, permeability in these cells was not affected by p190 suppression. Examination of p190‐deficient fibroblasts revealed a similar phenotype to that noted in EC treated with PKCδ inhibitors, with a loss of SF and diminished FA formation. Additionally, we observed that RhoA activity is further reduced in p190‐deficient fibroblasts when PKCδ activity is inhibited. Our findings suggest that both p190 and PKCδ may participate in common signaling pathways important in actin filament stabilization.

FAK, PDZ-RhoGEF and ROCKII cooperate to regulate adhesion movement and trailing-edge retraction in fibroblasts

A key step in cell migration is the dynamic formation and disassembly of adhesions at the front and the concomitant movement and release of adhesions in the rear of the cell. Fibroblasts maintained in the absence of serum have stable adhesions within the rear of the cell and exhibit reduced trailing-edge retraction resulting in an elongated cell phenotype. Addition of lysophosphatidic acid (LPA) induced the movement of adhesions and retraction of the trailing edge, thus mimicking tail retraction in a migrating cell. Focal adhesion kinase (FAK), guanine nucleotide exchange factors (GEF) for Rho and the Rho effector Rho kinase II (ROCKII) are crucial for the regulation of adhesion movement and trailing-edge retraction. Downregulation of FAK by small interfering RNAs or small hairpin RNAs blocked LPA-induced adhesion movement and restoration of cell shape. This phenotype was rescued by the ectopic expression of PDZ-RhoGEF or a RhoA-effector-domain mutant that activates ROCK. Knockdown of PDZ-RhoGEF or ROCKII inhibited LPA-induced trailing-edge retraction and adhesion movement. Moreover, overexpressed PDZ-RhoGEF co-immunoprecipitated with FAK and localized to FAK-containing adhesions. These studies support a model in which FAK and PDZ-RhoGEF cooperate to induce Rho/ROCKII-dependent focal adhesion movement and trailing-edge retraction in response to LPA.

445. Focal adhesions disassemble during early pregnancy in rat uterine epithelial cells

Successful blastocyst implantation require uterine epithelial cells (UECs) to undergo the 'plasma membrane transformation' followed by the removal of these cells around the implantation sites. The present study investigated the distribution and expression of two principal focal adhesion proteins talin and paxillin in rat UECs during early pregnancy and their role in the loss of these cells at the time of implantation. Results from immunofluorescence microscopy have demonstrated a major distributional change of talin and paxillin in UECs where an intense basal staining of these proteins on day 1 of pregnancy was lost at time of implantation. This was consistent with the significant decrease in paxillin seen through western blotting analysis. Interestingly the amount of talin was not significantly different between day 1 of pregnancy and at the time of implantation with a calpain 2 mediated proteolytic fragment of talin seen on both of these days of pregnancy. Calpain 2 activity was further investigated through western blotting analysis and increases in the active form of calpain 2 at the time of implantation. These observations suggest that talin and paxillin have disassembled from the site of focal adhesions where talin is undergoing cleavage by active calpain 2 at the time of implantation. This allows UECs to become less adherent to the underlying basal lamina facilitating their removal during blastocyst invasion. Hence, disassembly of focal adhesions at the time of implantation is a critical event for successful implantation and placentation.

Phosphorylation of Paxillin LD4 Destabilizes Helix Formation and Inhibits Binding to Focal Adhesion Kinase

Cell migration is a dynamic process that requires the coordinated formation and disassembly of focal adhesions (FAs). Several proteins such as paxillin, focal adhesion kinase (FAK), and G protein-coupled receptor kinase-interacting protein 1 (GIT1) are known to play a regulatory role in FA disassembly and turnover. However, the mechanisms by which this occurs remain to be elucidated. Paxillin has been shown to bind the C-terminal domain of FAK in FAs, and an increasing number of studies have linked paxillin association with GIT1 during focal adhesion disassembly. It has been reported recently that phosphorylation of serine 273 in the LD4 motif of paxillin leads to an increased association with Git1 and focal adhesion turnover. In the present study, we examined the effects of phosphorylation of the LD4 peptide on its binding affinity to the C-terminal domain of FAK. We show that phosphorylation of LD4 results in a reduction of binding affinity to FAK. This reduction in binding affinity is not due to the introduction of electrostatic repulsion or steric effects but rather by a destabilization of the helical propensity of the LD4 motif. These results further our understanding of the focal adhesion turnover mechanism as well as identify a novel process by which phosphorylation can modulate intracellular signaling.

RACK1 Targets the Extracellular Signal-Regulated Kinase/Mitogen-Activated Protein Kinase Pathway To Link Integrin Engagement with Focal Adhesion Disassembly and Cell Motility

The extracellular signal-regulated kinase (ERK) cascade is activated in response to a multitude of extracellular signals and converts these signals into a variety of specific biological responses, including cell differentiation, cell movement, cell division, and apoptosis. The specificity of the biological response is likely to be controlled in large measure by the localization of signaling, thus enabling ERK activity to be directed towards specific targets. Here we show that the RACK1 scaffold protein functions specifically in integrin-mediated activation of the mitogen-activated protein kinase/ERK cascade and targets active ERK to focal adhesions. We found that RACK1 associated with the core kinases of the ERK pathway, Raf, MEK, and ERK, and that attenuation of RACK1 expression resulted in a decrease in ERK activity in response to adhesion but not in response to growth factors. RACK1 silencing also caused a reduction of active ERK in focal adhesions, an increase in focal adhesion length, a decreased rate of focal adhesion disassembly, and decreased motility. Our data further suggest that focal adhesion kinase is an upstream activator of the RACK1/ERK pathway. We suggest that RACK1 tethers the ERK pathway core kinases and channels signals from upstream activation by integrins to downstream targets at focal adhesions.

Focal adhesion disassembly is an essential early event in hepatic stellate cell chemotaxis

Chemotaxis (i.e., directed migration) of hepatic stellate cells to areas of inflammation is a requisite event in the liver's response to injury. Previous studies of signaling pathways that regulate stellate cell migration suggest a key role for focal adhesions, but the exact function of these protein complexes in motility remains unclear. Focal adhesions attach a cell to its substrate and therefore must be regulated in a highly coordinated manner during migration. To test the hypothesis that focal adhesion turnover is an essential early event for chemotaxis in stellate cells, we employed a live-cell imaging technique in which chemotaxis was induced by locally stimulating the tips of rat stellate cell protrusions with platelet-derived growth factor-BB (PDGF). Focal adhesions were visualized with an antibody directed against vinculin, a structural component of the focal adhesion complex. PDGF triggered rapid disassembly of adhesions within 6.25 min, subsequent reassembly by 12.5 min, and continued adhesion assembly in concert with the spreading protrusion until the completion of chemotaxis. Blockade of adhesion disassembly by growing cells on fibronectin or treatment with nocodazole prevented a chemotactic response to PDGF. Augmentation of adhesion disassembly with ML-7 enhanced the chemotactic response to PDGF. These data suggest that focal adhesion disassembly is an essential early event in stellate cell chemotaxis in response to PDGF.

Focal Adhesion Disassembly Using Electrochemically Programmed Sub-Cellular Release

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Numb Cells Keep Moving

STKE Integrins are heterodimeric transmembrane receptors that bind to components of the extracellular matrix and are important for both cellular adhesion and migration. The clustering of activated integrins on the substrate-facing surface of the leading edge of a cell results in the recruitment of various proteins, including actin stress fibers, to form a focal adhesion complex (FAC). Cells move, in part, through the coordinated assembly and disassembly of focal adhesions at the leading edge of the cell. Numb is a cargo-specific adaptor protein that binds to several endocytic proteins, and Nishimura et al. examined the role of Numb in endothelial and epithelial cell cultures. In a wound-healing assay, Numb polarized toward the leading edge of migrating cells (just behind the lamellipodium), and immunostaining demonstrated that Numb and β-integrin colocalized at focal adhesions. Coimmunoprecipitation experiments revealed that Numb bound to the PAR (for partitioning defective) polarization complex PAR-3. This complex also localizes to the leading edge of polarized migrating cells. One component of this complex, atypical protein kinase C (aPKC) phosphorylated Numb in HeLa cells and, as a consequence, Numb no longer bound to integrins. The authors propose that Numb binds to free integrin molecules (rather than disrupting FACs) and recruits them to clathrin-coated structures to initiate integrin recycling, and that the localization and function of Numb are negatively regulated by aPKC. — JFF Dev. Cell 13 , 15 (2007).

Protein Kinase C δ Activated Adhesion Regulates Vascular Smooth Muscle Cell Migration

Vascular smooth muscle cell (VSMC) migration, fundamental in the pathophysiology of atherogenesis and restenosis, is a coordinated process governed by the formation and disassembly of focal adhesions. Previous studies have demonstrated that VSMC migration is regulated via a signaling network involving protein kinase C delta (PKCdelta). In these studies, we test the hypothesis that PKCdelta regulates VSMC migration through modulation of cell adhesion. Using primary VSMCs isolated from PKCdelta wild type (+/+) and knock-out (-/-) mice, the effects of PKCdelta on VSMC migration and adhesion were assessed by chemotaxis and cell adhesion. In evaluating cell migration, we found a decrease in platelet-derived growth factor-BB (PDGF-BB; 5 ng/mL x 6 h) stimulated migration of PKCdelta-/-VSMCs as compared to PKCdelta+/+VSMCs, by 59.4 +/- 5.9% (P < 0.01). A similar reduction in migration of PKCdelta-/-VSMCs (66.5 +/- 5.7%, P < 0.01) was also observed on collagen-coated (COL) membranes. Next, we examined cell attachment, a critical step of migration. PKCdelta-/-VSMCs exhibited significantly reduced adherence by 50.3 +/- 1.8% (P < 0.01). A similar defect of PKCdelta-/-VSMCs was also observed on the COL surface, 30.7 +/- 2.3% (P < 0.01). Interestingly, PDGF-BB did not stimulate attachment of VSMCs of either genotype. Consistent with these results, Rottlerin (2 microM), a selective inhibitor of PKCdelta, blocked migration and attachment of VSMCs by 56.8 +/- 3.4% (P < 0.01) and 37.7 +/- 1.9% (P < 0.01), respectively. Taken together, our data indicate that PKCdelta activation is necessary for VSMC adhesion, which could, at least in part, contribute to the regulatory function of this kinase in cell migration thus pathogenesis of vascular lesions.

Regulation of protrusion, adhesion dynamics, and polarity by myosins IIA and IIB in migrating cells

We have used isoform-specific RNA interference knockdowns to investigate the roles of myosin IIA (MIIA) and MIIB in the component processes that drive cell migration. Both isoforms reside outside of protrusions and act at a distance to regulate cell protrusion, signaling, and maturation of nascent adhesions. MIIA also controls the dynamics and size of adhesions in central regions of the cell and contributes to retraction and adhesion disassembly at the rear. In contrast, MIIB establishes front–back polarity and centrosome, Golgi, and nuclear orientation. Using ATPase- and contraction-deficient mutants of both MIIA and MIIB, we show a role for MIIB-dependent actin cross-linking in establishing front–back polarity. From these studies, MII emerges as a master regulator and integrator of cell migration. It mediates each of the major component processes that drive migration, e.g., polarization, protrusion, adhesion assembly and turnover, polarity, signaling, and tail retraction, and it integrates spatially separated processes.

EHD1 regulates β1 integrin endosomal transport: effects on focal adhesions, cell spreading and migration

beta1 integrins bind to the extracellular matrix and stimulate signaling pathways leading to crucial cellular functions, including proliferation, apoptosis, cell spreading and migration. Consequently, control of beta1 integrin function depends upon its subcellular localization, and recent studies have begun to unravel the complex regulatory mechanisms involved in integrin trafficking. We report that the C-terminal Eps15-homology (EH) domain-containing protein EHD1 plays an important role in regulating beta1 integrin transport. Initially, we demonstrated that RNAi-knockdown of Ehd1 results in impaired recycling of beta1 integrins and their accumulation in a transferrin-containing endocytic recycling compartment. Mouse embryonic fibroblast (MEF) cells derived from EHD1-knockout mice (Ehd1(-/-) MEF) exhibited lower overall levels of beta1 integrins on the plasma membrane, but higher cell-surface-expressed activated beta1 integrins, and larger, more prominent focal adhesions resulting from slower kinetics of focal adhesion disassembly. In addition, both migration and cell spreading on fibronectin were impaired in Ehd1(-/-) MEF cells, and these defects could be similarly induced by EHD1-RNAi treatment of normal Ehd1(+/+) MEF cells. They could also be rescued by transfection of wild-type EHD1 into Ehd1(-/-) MEF cells. Our data support a role for EHD1 in beta1 integrin recycling, and demonstrate a requirement for EHD1 in integrin-mediated downstream functions.