Autophagy promotes cell motility by driving focal adhesion turnover

In eukaryotic cells, cell migration is a dynamic and complex process that involves finely tuned orchestration of a multitude of proteins including, for example, those involved in focal adhesions (FAs). Cell migration plays an indispensable role in particular stages of development and its proper regulation is crucial in various biological processes, from wound healing to the immune response. FAs are transmembrane protein complexes that traverse cytoskeletal infrastructures all the way to the extracellular matrix, producing traction at the leading edge of the cell, thus allowing for motility. The assembly of FAs has been extensively studied, whereas disassembly remains poorly understood. Here, we highlight 2 recent studies (see the corresponding puncta in the previous and current issues of the journal) that demonstrate a requirement for macroautophagy/autophagy in FA disassembly. These studies also provide a deeper understanding of how autophagy can contribute to cell migration among multiple cell types.     

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.     

Role of Grb7 targeting to focal contacts and its phosphorylation by focal adhesion kinase in regulation of cell migration

We have previously described Grb7 association with focal adhesion kinase (FAK) and its possible roles in cell migration. In this paper, we investigated the mechanisms by which Grb7 and its association with FAK regulate cell migration. We found that deletion of the Grb7 SH2 domain eliminated partial Grb7 localization to focal contacts and its ability to stimulate cell migration. Replacement of the SH2 domain with the focal adhesion targeting sequence from FAK resulted in the focal contacts localization of the chimeric molecule and restored its activity to stimulate cell migration. We also found that Grb7 could be phosphorylated by FAK, which was dependent on the FAK kinase activity but not the presence of the Src family kinases. Cell adhesion also enhanced Grb7 phosphorylation in FAK+/+ cells but not FAK-/- cells, suggesting that Grb7 is a physiological substrate of FAK. Furthermore, both Grb7 and the chimeric molecule did not increase migration of FAK-/- cells, although the chimeric molecule was targeted to the focal contacts. Last, we showed that other Grb7 family members could not stimulate cell migration under similar experimental conditions. Together, these results demonstrate a role for Grb7 targeting to focal contacts and its phosphorylation by FAK in the regulation of cell migration.     

Redox regulation of cell migration and adhesion

Reactive oxygen species (ROS), particularly hydrogen peroxide, and the proteins that regulate them play important roles in the migration and adhesion of cells. Stimulation of cell surface receptors with growth factors and chemoattractants generates ROS, which relay signals from the cell surface to key signaling proteins inside the cell. ROS act within cells to promote migration and also in nonmigrating cells to influence the behavior of migrating cells. Hydrogen peroxide has also been suggested to act as a chemoattractant in its own right, drawing immune cells to wounds. We discuss recent progress made towards understanding how organisms use ROS, and to what degree they depend on them, during the related processes of cell migration and adhesion.     

Roles of nectins in cell adhesion, migration and polarization

Nectins are Ca2+-independent immunoglobulin (Ig)-like cell-cell adhesion molecules, which comprise a family consisting of four members. Nectins have five activities: (1) they show Ca2+-independent cell-cell adhesion activity by homo- and hetero-trans-interactions through their extracellular regions; (2) they bind afadin, an actin filament (F-actin)-binding protein, through their cytoplasmic tails and are connected to the actin cytoskeleton; (3) they induce activation of Cdc42 and Rac small G proteins through their cytoplasmic tails; (4) they bind Par-3, a cell polarity protein, through their cytoplasmic tails; and (5) they heterophilically trans-interact with Necls, nectin-like molecules, through their extracellular regions. Through these activities, nectins regulate a variety of cellular functions, including adhesion, migration, and polarization. Here we describe these activities and functions of nectins.     

Tropomyosin isoform modulation of focal adhesion structure and cell migration

Orderly cell migration is essential for embryonic development, efficient wound healing and a functioning immune system and the dysregulation of this process leads to a number of pathologies. The speed and direction of cell migration is critically dependent on the structural organization of focal adhesions in the cell. While it is well established that contractile forces derived from the acto-myosin filaments control the structure and growth of focal adhesions, how this may be modulated to give different outcomes for speed and persistence is not well understood. The tropomyosin family of actin-associating proteins are emerging as important modulators of the contractile nature of associated actin filaments. The multiple non-muscle tropomyosin isoforms are differentially expressed between tissues and across development and are thought to be major regulators of actin filament functional specialization. In the present study we have investigated the effects of two splice variant isoforms from the same α-tropomyosin gene, TmBr1 and TmBr3, on focal adhesion structure and parameters of cell migration. These isoforms are normally switched on in neuronal cells during differentiation and we find that exogenous expression of the two isoforms in undifferentiated neuronal cells has discrete effects on cell migration parameters. While both isoforms cause reduced focal adhesion size and cell migration speed, they differentially effect actin filament phenotypes and migration persistence. Our data suggests that differential expression of tropomyosin isoforms may coordinate acto-myosin contractility and focal adhesion structure to modulate cell speed and persistence.Key words: focal adhesion, tropomyosin, actin, migration, persistence, speed, mesenchymal     

The focal adhesion kinase--a regulator of cell migration and invasion

Cell migration plays an important role in embryonic development, wound healing, immune responses, and in pathological phenomena such as tissue invasion and metastasis formation. In this review, we summarize recent reports that connect the focal adhesion kinase (FAK) to cell migration and invasion. FAK is a nonreceptor protein tyrosine kinase involved in signal transduction from integrin-enriched focal adhesion sites that mediate cell contact with the extracellular matrix. Multiple protein-protein interaction sites allow FAK to associate with adapter and structural proteins allowing for the modulation of mitogen-activated protein (MAP) kinases, stress-activated protein (SAP) kinases, and small GTPase activity. FAK-enhanced signals have been shown to mediate the survival of anchorage-dependent cells and are critical for efficient cell migration in response to growth factor receptor and integrin stimulation. Elevated expression of FAK in human tumors has been correlated with increased malignancy and invasiveness. Because recent findings show that FAK contributes to the secretion of matrix-metalloproteinases, FAK may represent an important checkpoint in coordinating the dynamic processes of cell motility and extracellular matrix remodeling during tumor cell invasion.     

Integrin-specific signaling pathways controlling focal adhesion formation and cell migration

The fibronectin (FN)-binding integrins alpha4beta1 and alpha5beta1 confer different cell adhesive properties, particularly with respect to focal adhesion formation and migration. After analyses of alpha4+/alpha5+ A375-SM melanoma cell adhesion to fragments of FN that interact selectively with alpha4beta1 and alpha5beta1, we now report two differences in the signals transduced by each receptor that underpin their specific adhesive properties. First, alpha5beta1 and alpha4beta1 have a differential requirement for cell surface proteoglycan engagement for focal adhesion formation and migration; alpha5beta1 requires a proteoglycan coreceptor (syndecan-4), and alpha4beta1 does not. Second, adhesion via alpha5beta1 caused an eightfold increase in protein kinase Calpha (PKCalpha) activation, but only basal PKCalpha activity was observed after adhesion via alpha4beta1. Pharmacological inhibition of PKCalpha and transient expression of dominant-negative PKCalpha, but not dominant-negative PKCdelta or PKCzeta constructs, suppressed focal adhesion formation and cell migration mediated by alpha5beta1, but had no effect on alpha4beta1. These findings demonstrate that different integrins can signal to induce focal adhesion formation and migration by different mechanisms, and they identify PKCalpha signaling as central to the functional differences between alpha4beta1 and alpha5beta1.     

RhoB regulates cell migration through altered focal adhesion dynamics

The Rho GTPase RhoB has been shown to affect cell migration, but how it does this is not clear. Here we show that cells depleted of RhoB by RNAi are rounded and have defects in Rac-mediated spreading and lamellipodium extension, although they have active membrane ruffling around the periphery. Depletion of the exchange factor GEF-H1 induces a similar phenotype. RhoB-depleted cells migrate faster, but less persistently in a chemotactic gradient, and frequently round up during migration. RhoB-depleted cells have similar numbers of focal adhesions to control cells during spreading and migration, but show more diffuse and patchy contact with the substratum. They have lower levels of surface β1 integrin, and β1 integrin activity is reduced in actin-rich protrusions. We propose that RhoB contributes to directional cell migration by regulating β1 integrin surface levels and activity, thereby stabilizing lamellipodial protrusions.     

Actin,microtubules and focal adhesion dynamics during cell migration

Cell migration is a complex cellular behavior that results from the coordinated changes in the actin cytoskeleton and the controlled formation and dispersal of cell-substrate adhesion sites. While the actin cytoskeleton provides the driving force at the cell front, the microtubule network assumes a regulatory function in coordinating rear retraction. The polarity within migrating cells is further highlighted by the stationary behavior of focal adhesions in the front and their sliding in trailing ends. We discuss here the cross-talk of the actin cytoskeleton with the microtubule network and the potential mechanisms that control the differential behavior of focal adhesions sites during cell migration.     

Spatial activation of ezrin by epidermal growth factor receptor and focal adhesion kinase co-ordinates epithelial cell migration

Epidermal growth factor receptor (EGFR) plays a critical role in the promotion of epithelial cell proliferation and migration. Previous studies have suggested a cooperative role between EGFR and integrin signalling pathways that enable efficient adhesion and migration but the mechanisms controlling this remain poorly defined. Here, we show that EGFR forms a complex with focal adhesion kinase in epithelial cells. Surprisingly, this complex enhances local Src activity at focal adhesions to promote phosphorylation of the cytoskeletal adaptor protein ezrin at Y478, leading to actomyosin contractility, suppression of focal adhesion dynamics and slower migration. We further demonstrate this regulation of Src is due to the suppression of PTP1B activity. Our data provide new insight into EGF-independent cooperation between EGFR and integrins and suggest transient interactions between these kinases at the leading edge of cells act to spatially control signalling to permit efficient motility.     

Spatial and temporal regulation of focal adhesion kinase activity in living cells

Focal adhesion kinase (FAK) is an essential kinase that regulates developmental processes and functions in the pathology of human disease. An intramolecular autoinhibitory interaction between the FERM and catalytic domains is a major mechanism of regulation. Based upon structural studies, a fluorescence resonance energy transfer (FRET)-based FAK biosensor that discriminates between autoinhibited and active conformations of the kinase was developed. This biosensor was used to probe FAK conformational change in live cells and the mechanism of regulation. The biosensor demonstrates directly that FAK undergoes conformational change in vivo in response to activating stimuli. A conserved FERM domain basic patch is required for this conformational change and for interaction with a novel ligand for FAK, acidic phospholipids. Binding to phosphatidylinositol 4,5-bisphosphate (PIP2)-containing phospholipid vesicles activated and induced conformational change in FAK in vitro, and alteration of PIP2 levels in vivo changed the level of activation of the conformational biosensor. These findings provide direct evidence of conformational regulation of FAK in living cells and novel insight into the mechanism regulating FAK conformation.     

S100P dissociates myosin IIA filaments and focal adhesion sites to reduce cell adhesion and enhance cell migration

S100 proteins promote cancer cell migration and metastasis. To investigate their roles in the process of migration we have constructed inducible systems for S100P in rat mammary and human HeLa cells that show a linear relationship between its intracellular levels and cell migration. S100P, like S100A4, differentially interacts with the isoforms of nonmuscle myosin II (NMIIA, K(d) = 0.5 μM; IIB, K(d) = 8 μM; IIC, K(d) = 1.0 μM). Accordingly, S100P dissociates NMIIA and IIC filaments but not IIB in vitro. NMIIA knockdown increases migration in non-induced cells and there is no further increase upon induction of S100P, whereas NMIIB knockdown reduces cell migration whether or not S100P is induced. NMIIC knockdown does not affect S100P-enhanced cell migration. Further study shows that NMIIA physically interacts with S100P in living cells. In the cytoplasm, S100P occurs in discrete nodules along NMIIA-containing filaments. Induction of S100P causes more peripheral distribution of NMIIA filaments. This change is paralleled by a significant drop in vinculin-containing, actin-terminating focal adhesion sites (FAS) per cell. The induction of S100P, consequently, causes significant reduction in cellular adhesion. Addition of a focal adhesion kinase (FAK) inhibitor reduces disassembly of FAS and thereby suppresses S100P-enhanced cell migration. In conclusion, this work has demonstrated a mechanism whereby the S100P-induced dissociation of NMIIA filaments leads to a weakening of FAS, reduced cell adhesion, and enhanced cell migration, the first major step in the metastatic cascade.     

MicroRNA-8 promotes robust motor axon targeting by coordinate regulation of cell adhesion molecules during synapse development

Neuronal connectivity and specificity rely upon precise coordinated deployment of multiple cell-surface and secreted molecules. MicroRNAs have tremendous potential for shaping neural circuitry by fine-tuning the spatio-temporal expression of key synaptic effector molecules. The highly conserved microRNA miR-8 is required during late stages of neuromuscular synapse development in Drosophila. However, its role in initial synapse formation was previously unknown. Detailed analysis of synaptogenesis in this system now reveals that miR-8 is required at the earliest stages of muscle target contact by RP3 motor axons. We find that the localization of multiple synaptic cell adhesion molecules (CAMs) is dependent on the expression of miR-8, suggesting that miR-8 regulates the initial assembly of synaptic sites. Using stable isotope labelling in vivo and comparative mass spectrometry, we find that miR-8 is required for normal expression of multiple proteins, including the CAMs Fasciclin III (FasIII) and Neuroglian (Nrg). Genetic analysis suggests that Nrg and FasIII collaborate downstream of miR-8 to promote accurate target recognition. Unlike the function of miR-8 at mature larval neuromuscular junctions, at the embryonic stage we find that miR-8 controls key effectors on both sides of the synapse. MiR-8 controls multiple stages of synapse formation through the coordinate regulation of both pre- and postsynaptic cell adhesion proteins.     

Retrograde fluxes of focal adhesion proteins in response to cell migration and mechanical signals

Recent studies suggest that mechanical signals mediated by the extracellular matrix play an essential role in various physiological and pathological processes; yet, how cells respond to mechanical stimuli remains elusive. Using live cell fluorescence imaging, we found that actin filaments, in association with a number of focal adhesion proteins, including zyxin and vasodilator-stimulated phosphoprotein, undergo retrograde fluxes at focal adhesions in the lamella region. This flux is inversely related to cell migration, such that it is amplified in fibroblasts immobilized on micropatterned islands. In addition, the flux is regulated by mechanical signals, including stretching forces applied to flexible substrates and substrate stiffness. Conditions favoring the flux share the common feature of causing large retrograde displacements of the interior actin cytoskeleton relative to the substrate anchorage site, which may function as a switch translating mechanical input into chemical signals, such as tyrosine phosphorylation. In turn, the stimulation of actin flux at focal adhesions may function as part of a feedback mechanism, regulating structural assembly and force production in relation to cell migration and mechanical load. The retrograde transport of associated focal adhesion proteins may play additional roles in delivering signals from focal adhesions to the interior of the cell.     

Membrane-type 1 matrix metalloproteinase modulates focal adhesion stability and cell migration

Membrane-type 1 matrix metalloproteinase (MT1-MMP) plays an important role in extracellular matrix-induced cell migration and the activation of extracellular signal-regulated kinase (ERK). We showed here that transfection of the MT1-MMP gene into HeLa cells promoted fibronectin-induced cell migration, which was accompanied by fibronectin degradation and reduction of stable focal adhesions, which function as anchors for actin-stress fibers. MT1-MMP expression attenuated integrin clustering that was induced by adhesion of cells to fibronectin. The attenuation of integrin clustering was abrogated by MT1-MMP inhibition with a synthetic MMP inhibitor, BB94. When cultured on fibronectin, HT1080 cells, which endogenously express MT1-MMP, showed so-called motile morphology with well-organized focal adhesion formation, well-oriented actin-stress fiber formation, and the lysis of fibronectin through trails of cell migration. Inhibition of endogenous MT1-MMP by BB94 treatment or expression of the MT1-MMP carboxyl-terminal domain, which negatively regulates MT1-MMP activity, resulted in the suppression of fibronectin lysis and cell migration. BB94 treatment promoted stable focal adhesion formation concomitant with enhanced phosphorylation of tyrosine 397 of focal adhesion kinase (FAK) and reduced ERK activation. These results suggest that lysis of the extracellular matrix by MT1-MMP promotes focal adhesion turnover and subsequent ERK activation, which in turn stimulates cell migration.     

Interaction of paxillin with poly(A)-binding protein 1 and its role in focal adhesion turnover and cell migration

We have previously identified poly(A)-binding protein 1 (PABP1) as a ligand for paxillin and shown that the paxillin-PABP1 complex undergoes nucleocytoplasmic shuttling. By targeting the paxillin-binding subdomain sequences in PABP1, we have generated mutants of PABP1 that do not bind to cellular paxillin. Here we report that paxillin association is necessary for efficient nuclear export of PABP1 and that RNA interference of paxillin drives the nuclear accumulation of PABP1. Furthermore, ablation of paxillin-PABP1 association impeded a number of indices of cell motility including spreading on fibronectin, cell migration on two-dimensional matrices, and transmigration in Boyden chambers. These data indicate that PABP1 must associate with paxillin in order to be efficiently transported from the nucleus to the cytoplasm and that this event is necessary for cells to remodel their focal adhesions during cell migration.     

Autocrine netrin function inhibits glioma cell motility and promotes focal adhesion formation

Deregulation of mechanisms that control cell motility plays a key role in tumor progression by promoting tumor cell dissemination. Secreted netrins and their receptors, Deleted in Colorectal Cancer (DCC), neogenin, and the UNC5 homologues, regulate cell and axon migration, cell adhesion, and tissue morphogenesis. Netrin and netrin receptor expression have previously been shown to be disrupted in invasive tumors, including glioblastoma. We determined that the human glioblastoma cell lines U87, U343, and U373 all express neogenin, UNC5 homologues, and netrin-1 or netrin-3, but only U87 cells express DCC. Using transfilter migration assays, we demonstrate DCC-dependent chemoattractant migration of U87 cells up a gradient of netrin-1. In contrast, U343 and U373 cells, which do not express DCC, were neither attracted nor repelled. Ectopic expression of DCC by U343 and U373 cells resulted in these cells becoming competent to respond to a gradient of netrin-1 as a chemoattractant, and also slowed their rate of spontaneous migration. Here, in addition to netrins' well-characterized chemotropic activity, we demonstrate an autocrine function for netrin-1 and netrin-3 in U87 and U373 cells that slows migration. We provide evidence that netrins promote the maturation of focal complexes, structures associated with cell movement, into focal adhesions. Consistent with this, netrin, DCC, and UNC5 homologues were associated with focal adhesions, but not focal complexes. Disrupting netrin or DCC function did not alter cell proliferation or survival. Our findings provide evidence that DCC can slow cell migration, and that neogenin and UNC5 homologues are not sufficient to substitute for DCC function in these cells. Furthermore, we identify a role for netrins as autocrine inhibitors of cell motility that promote focal adhesion formation. These findings suggest that disruption of netrin signalling may disable a mechanism that normally restrains inappropriate cell migration.     

Requirement of phosphatidylinositol 3-kinase in focal adhesion kinase-promoted cell migration

We have previously shown that overexpression of focal adhesion kinase (FAK) in Chinese hamster ovary (CHO) cells promoted their migration on fibronectin. This effect was dependent on the phosphorylation of FAK at Tyr-397. This residue was known to serve as a binding site for both Src and phosphatidylinositol 3-kinase (PI3K), implying that either one or both are required for FAK to promote cell migration. In this study, we have examined the role of PI3K in FAK-promoted cell migration. We have demonstrated that the PI3K inhibitors, wortmannin and LY294002, were able to inhibit FAK-promoted migration in a dose-dependent manner. Furthermore, a FAK mutant capable of binding Src but not PI3K was generated by a substitution of Asp residue 395 with Ala. When overexpressed in CHO cells, this differential binding mutant failed to promote cell migration although its association with Src was retained. Together, these results strongly suggest that PI3K binding is required for FAK to promote cell migration and that the binding of Src and p130(Cas) to FAK may not be sufficient for this event.