Regulation of cell migration by dynamic microtubules

Microtubules define the architecture and internal organization of cells by positioning organelles and activities, as well as by supporting cell shape and mechanics. One of the major functions of microtubules is the control of polarized cell motility. In order to support the asymmetry of polarized cells, microtubules have to be organized asymmetrically themselves. Asymmetry in microtubule distribution and stability is regulated by multiple molecular factors, most of which are microtubule-associated proteins that locally control microtubule nucleation and dynamics. At the same time, the dynamic state of microtubules is key to the regulatory mechanisms by which microtubules regulate cell polarity, modulate cell adhesion and control force-production by the actin cytoskeleton. Here, we propose that even small alterations in microtubule dynamics can influence cell migration via several different microtubule-dependent pathways. We discuss regulatory factors, potential feedback mechanisms due to functional microtubule-actin crosstalk and implications for cancer cell motility.     

MEGAP impedes cell migration via regulating actin and microtubule dynamics and focal complex formation

Over the past several years, it has become clear that the Rho family of GTPases plays an important role in various aspects of neuronal development including cytoskeleton dynamics and cell adhesion processes. We have analysed the role of MEGAP, a GTPase-activating protein that acts towards Rac1 and Cdc42 in vitro and in vivo, with respect to its putative regulation of cytoskeleton dynamics and cell migration. To investigate the effects of MEGAP on these cellular processes, we have established an inducible cell culture model consisting of a stably transfected neuroblastoma SHSY-5Y cell line that endogenously expresses MEGAP albeit at low levels. We can show that the induced expression of MEGAP leads to the loss of filopodia and lamellipodia protrusions, whereas constitutively activated Rac1 and Cdc42 can rescue the formation of these structures. We have also established quantitative assays for evaluating actin dynamics and cellular migration. By time-lapse microscopy, we show that induced MEGAP expression reduces cell migration by 3.8-fold and protrusion formation by 9-fold. MEGAP expressing cells also showed impeded microtubule dynamics as demonstrated in the TC-7 3x-GFP epithelial kidney cells. In contrast to the wild type, overexpression of MEGAP harbouring an artificially introduced missense mutation R542I within the functionally important GAP domain did not exert a visible effect on actin and microtubule cytoskeleton remodelling. These data suggest that MEGAP negatively regulates cell migration by perturbing the actin and microtubule cytoskeleton and by hindering the formation of focal complexes.     

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.     

One step ahead: Role of filopodia in adhesion formation during cell migration of keratinocytes

Cell adhesion is an essential prerequisite for cell function and movement. It depends strongly on focal adhesion complexes connecting the extracellular matrix to the actin cytoskeleton. Especially in moving cells focal adhesions are highly dynamic and believed to be formed closely behind the leading edge. Filopodia were thought to act mainly as guiding cues using their tip complexes for elongation. Here we show for keratinocytes a strong dependence of lamellipodial adhesion sites on filopodia. Upon stable contact of the VASP-containing tip spot to the substrate, a filopodial focal complex (filopodial FX) is formed right behind along the filopodia axis. These filopodial FXs are fully assembled, yet small adhesions containing all adhesion markers tested. Filopodial FXs when reached by the lamellipodium are just increased in size resulting in classical focal adhesions. At the same time most filopodia regain their elongation ability. Blocking filopodia inhibits development of new focal adhesions in the lamellipodium, while focal adhesion maturation in terms of vinculin exchange dynamics remains active. Our data therefore argue for a strong spatial and temporal dependence of focal adhesions on filopodial focal complexes in keratinocytes with filopodia not permanently initiated via new clustering of actin filaments to induce elongation.     

START-GAP3/DLC3 is a GAP for RhoA and Cdc42 and is localized in focal adhesions regulating cell morphology

In the human genome there are three genes encoding RhoGAPs that contain the START (steroidogenic acute regulatory protein (StAR)-related lipid transfer)—domain. START-GAP3/DLC3 is a tumor suppressor gene similar to two other human START-GAPs known as DLC1 or DLC2. Although expression of START-GAP3/DLC3 inhibits the proliferation of cancer cells, its molecular function is not well understood. In this study we carried out biochemical characterization of START-GAP3/DLC3, and explored the effects of its expression on cell morphology and intracellular localization. We found that START-GAP3/DLC3 serves as a stimulator of PLCδ1 and as a GAP for both RhoA and Cdc42 in vitro. Moreover, we found that the GAP activity is responsible for morphological changes. The intracellular localization of endogenous START-GAP3/DLC3 was explored by immunocytochemistry and was revealed in focal adhesions. These results indicate that START-GAP3/DLC3 has characteristics similar to other START-GAPs and the START-GAP family seems to share common characteristics.     

Assembly and mechanosensory function of focal adhesions: experiments and models

Initial integrin-mediated cell-matrix adhesions (focal complexes) appear underneath the lamellipodia, in the regions of the "fast" centripetal flow driven by actin polymerization. Once formed, these adhesions convert the flow behind them into a "slow", myosin II-driven mode. Some focal complexes then turn into elongated focal adhesions (FAs) associated with contractile actomyosin bundles (stress fibers). Myosin II inhibition does not suppress formation of focal complexes but blocks their conversion into mature FAs and further FA growth. Application of external pulling force promotes FA growth even under conditions when myosin II activity is blocked. Thus, individual FAs behave as mechanosensors responding to the application of force by directional assembly. We proposed a thermodynamic model for the mechanosensitivity of FAs, taking into account that an elastic molecular aggregate subject to pulling forces tends to grow in the direction of force application by incorporating additional subunits. This simple model can explain a variety of processes typical of FA behavior. Assembly of FAs is triggered by the small G-protein Rho via activation of two major targets, Rho-associated kinase (ROCK) and the formin homology protein, Dia1. ROCK controls creation of myosin II-driven forces, while Dia1 is involved in the response of FAs to these forces. Expression of the active form of Dia1, allows the external force-induced assembly of mature FAs, even in conditions when Rho is inhibited. Conversely, downregulation of Dia1 by siRNA prevents FA maturation even if Rho is activated. Dia1 and other formins cap barbed (fast growing) ends of actin filaments, allowing insertion of the new actin monomers. We suggested a novel mechanism of such "leaky" capping based on an assumption of elasticity of the formin/barbed end complex. Our model predicts that formin-mediated actin polymerization should be greatly enhanced by application of external pulling force. Thus, the formin-actin complex might represent an elementary mechanosensing device responding to force by enhancement of actin assembly. In addition to its role in actin polymerization, Dia1 seems to be involved in formation of links between actin filaments and microtubules affecting microtubule dynamics. Alpha-tubulin deacetylase HDAC6 cooperates with Dia1 in formation of such links. Since microtubules are known to promote FA disassembly, the Dia1-mediated effect on microtubule dynamics may possibly play a role in the negative feedback loop controlling size and turnover of FAs.     

Mechanical properties of individual focal adhesions probed with a magnetic microneedle

A permanent magnetic microneedle was developed to apply tensional forces to integrin receptors via ligand-coated magnetic microbeads while optically analyzing the mechanical properties of individual focal adhesions. Force application (130 pN for 3 s) through activated beta1 integrins produced less bead displacement than when unligated integrins were stressed. This strengthening response differed markedly on a bead-by-bead basis, correlated directly with local focal adhesion assembly, and was similar when analyzed at 4 degrees C, indicating that it was due to passive material properties of the cell. Viscoelastic analysis clarified that recruitment of focal adhesion proteins increased the local elastic stiffness of the adhesion complex without changing its viscous behavior. These data indicate that individual focal adhesions exhibit distinct mechanical properties that depend upon local focal adhesion assembly, and that these local variations in micromechanics can be detected and analyzed within living cells using the permanent magnetic microneedle technique.     

Serine palmitoyltransferase subunit 1 is present in the endoplasmic reticulum,nucleus and focal adhesions,and functions in cell morphology

Serine palmitoyltransferase (SPT) has been localized to the endoplasmic reticulum (ER) by subcellular fractionation and enzymatic assays, and fluorescence microscopy of epitope-tagged SPT; however, our studies have suggested that SPT subunit 1 might be present also in focal adhesions and the nucleus. These additional locations have been confirmed by confocal microscopy using HEK293 and HeLa cells, and for focal adhesions by the demonstration that SPT1 co-immunoprecipitates with vinculin, a focal adhesion marker protein. The focal adhesion localization of SPT1 is associated with cell morphology, and possibly cell migration, because it is seen in most cells before they reach confluence but disappears when they become confluent, and is restored by a standard scratch-wound healing assay. Conversely, elimination of SPT1 using SPTLC1 siRNA causes cell rounding. Thus, in addition to its “traditional” localization in the ER for de novo sphingolipid biosynthesis, SPT1 is present in other cellular compartments, including focal adhesions where it is associated with cell morphology.     

Angiotensin II induces focal adhesion kinase/paxillin phosphorylation and cell migration in human umbilical vein endothelial cells

In the present study, we demonstrated that Ang II provokes a transitory enhancement of focal adhesion kinase (FAK) and paxillin phosphorylation in human umbilical endothelial cells (HUVEC). Moreover, Ang II induces a time- and dose-dependent augmentation in cell migration, but does not affect HUVEC proliferation. The effect of Ang II on FAK and paxillin phosphorylation was markedly attenuated in cells pretreated with wortmannin and LY294002, indicating that phosphoinositide 3-kinase (PI3K) plays an important role in regulating FAK activation. Similar results were observed when HUVEC were pretreated with genistein, a non-selective tyrosine kinases inhibitor, or with the specific inhibitor PP2 for Src family kinases, demonstrating the involvement of protein tyrosine kinases, and particularly Src family of tyrosine kinases, in the downstream signalling pathway of Ang II receptors. Furthermore, FAK and paxillin phosphorylation was markedly blocked after treatment of HUVEC with AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) phosphorylation. Pretreatment of cells with inhibitors of PI3K, Src family tyrosine kinases, and EGFR also decreased HUVEC migration. In conclusion, these results suggest that Ang II mediates an increase in FAK and paxillin phosphorylation and induces HUVEC migration through signal transduction pathways dependent on PI3K and Src tyrosine kinase activation and EGFR transactivation.     

The snake venom disintegrin salmosin induces apoptosis by disassembly of focal adhesions in bovine capillary endothelial cells

Salmosin, a disintegrin purified from a Korean snake (Agkistrodon halys brevicaudus) venom, interacts with integrin alpha(v)beta(3) and inhibits the proliferation of bovine capillary endothelial (BCE) cells induced by basic fibroblast growth factor (bFGF). We investigated salmosin's mechanism of inhibition of BCE cell proliferation by examining changes in the cytoskeleton and activation of integrin-mediated signaling molecules. Salmosin disassembled cortical actins at focal adhesions and induced cells to be rounded and detached, but it did not alter microtubule structures in the early stage of cells being rounded. Immunolocalization of paxillin also demonstrated that focal adhesions were disassembled by salmosin. In salmosin-treated BCE cells, focal adhesion kinase (FAK) was dephosphorylated and expression of paxillin and p130(CAS) was decreased, but PI3 kinase, ILK, and beta-catenin were not expressed in decreased amounts or modified, suggesting that salmosin inactivated FAK-dependent integrin signaling pathways. While BCE cells proliferated normally on plates coated with salmosin, cells treated with salmosin eventually underwent apoptosis. These observations strongly suggest that salmosin disorganizes focal contacts to detach cells by competing with the extracellular matrix (ECM) for direct binding to integrin alpha(v)beta(3) on the cell surface, eventually leading to apoptosis.     

Endothelin-1 promotes migration of endothelial cells through the activation of ARF6 and the regulation of FAK activity

Several proteins act in concert to promote remodeling of the actin cytoskeleton during migration. This process is highly regulated by small GTP-binding proteins of the ADP-ribosylation factor (ARF) family of proteins. Here, we show that endothelin-1 (ET-1) can promote the activation of ARF6 and migration of endothelial cells through the activation of ET_B receptors. Inhibition of ARF6 expression using RNA interference markedly impairs basal and ET-1 stimulated cell migration. In contrast, depletion of ARF1 has no significant effect. In order to delineate the underlying mechanism, we examined the signaling events activated in endothelial cells following ET-1 stimulation. Here, we show that this hormone promotes the phosphorylation of focal adhesion kinase (FAK), Erk1/2, and the association of FAK to Src, as well as of FAK to GIT1. These have been shown to be important for the formation and turnover of focal adhesions. In non-stimulated cells, depletion of ARF6 leads to increased FAK and Erk1/2 phosphorylation, similar to what is observed in ET-1 treated cells. In these conditions, FAK is found constitutively associated with the soluble tyrosine kinase, Src. In contrast, depletion of ARF6 impairs the ability of GIT1 to form an agonist promoted complex with FAK, thereby preventing disassembly of focal adhesions. As a consequence, ARF6 depleted endothelial cells are impaired in their ability to form capillary tubes. Taken together, our data suggest that ARF6 is central in regulating focal adhesion turnover in endothelial cells. Our study provides a molecular mechanism by which, this small GTPase regulates cell motility, and ultimately angiogenesis.     

Analysis of directional cell migration on defined FN gradients: role of intracellular signaling molecules

Directional cell migration on extracellular matrix (ECM) plays important roles in embryonic development and adult organisms. To study the mechanisms and signaling pathways involved in the regulation of directional cell migration, we created defined fibronectin (FN) gradients by using microfluidic systems. We found that fibroblasts exhibited haptotaxis towards higher FN concentration on the gradient. Furthermore, the net movements in the direction of FN gradients correlated with the increase in the slope of the gradient although the overall rate of migration was not correlated. Consistent with previous observations on the uniformly coated surface, local higher FN concentration led to reduced migration rate due to increased spreading. Upon transfection of N-WASP or activated Cdc42, but not FAK or Grb7, the cells showed increased directional migration. However, transfection of FAK, but not the other signaling molecules, led to an increase in the persistence of directional cell migration, which is dependent on the slope of the gradient as well as FAK interaction with PI3K. Together, these studies reveal some novel properties of directional cell migration on defined FN gradient and suggested a role for FAK signaling and N-WASP and Cdc42 in the differential regulation of the persistence and rate of directional cell migration.     

Growth factors stimulate kidney proximal tubule cell migration independent of augmented tyrosine phosphorylation of focal adhesion kinase

Migration of human proximal tubule cells (HKC-5) was stimulated by epidermal growth factor (EGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1). Integrin signaling via phosphorylation of focal adhesion kinase (FAK) appears to play a central role in cell migration. Once stimulated, FAK undergoes autophosphorylation at tyrosine (Y) 397, followed by phosphorylation of several sites including Y576/Y577 which increases FAK's kinase activity, as well as at Y407, Y861, and Y925. EGF, HGF, and IGF-1 stimulate FAK phosphorylation in various cells. We showed that endothelin stimulated phosphorylation of Y397 in fibroblasts but not HKC-5 cells. After EGF stimulation, HKC-5 cells showed no change in tyrosine phosphorylation at FAK Y397, 407, 576, 861, or 925. Similarly, HGF and IGF-1 did not stimulate the phosphorylation of FAK Y397 in HKC-5 cells. Further, after inhibition of FAK expression by siRNA, cell migration was similar to cells treated with non-target siRNA and responded to EGF with increased migration. Thus, in proximal tubule cells, stimulation of cell migration by growth factors was independent of augmented FAK tyrosine phosphorylation.     

Distinct roles of MLCK and ROCK in the regulation of membrane protrusions and focal adhesion dynamics during cell migration of fibroblasts

We examined the role of regulatory myosin light chain (MLC) phosphorylation of myosin II in cell migration of fibroblasts. Myosin light chain kinase (MLCK) inhibition blocked MLC phosphorylation at the cell periphery, but not in the center. MLCK-inhibited cells did not assemble zyxin-containing adhesions at the periphery, but maintained focal adhesions in the center. They generated membrane protrusions all around the cell, turned more frequently, and migrated less effectively. In contrast, Rho-associated kinase (ROCK) inhibition blocked MLC phosphorylation in the center, but not at the periphery. ROCK-inhibited cells assembled zyxin-containing adhesions at the periphery, but not focal adhesions in the center. They moved faster and more straight. On the other hand, inhibition of myosin phosphatase increased MLC phosphorylation and blocked peripheral membrane ruffling, as well as turnover of focal adhesions and cell migration. Our results suggest that myosin II activated by MLCK at the cell periphery controls membrane ruffling, and that the spatial regulation of MLC phosphorylation plays critical roles in controlling cell migration of fibroblasts.     

Annexin A6 contributes to the invasiveness of breast carcinoma cells by influencing the organization and localization of functional focal adhesions

The interaction of annexin A6 (AnxA6) with membrane phospholipids and either specific extracellular matrix (ECM) components or F-actin suggests that it may influence cellular processes associated with rapid plasma membrane reorganization such as cell adhesion and motility. Here, we examined the putative roles of AnxA6 in adhesion-related cellular processes that contribute to breast cancer progression. We show that breast cancer cells secrete annexins via the exosomal pathway and that the secreted annexins are predominantly cell surface-associated. Depletion of AnxA6 in the invasive BT-549 breast cancer cells is accompanied by enhanced anchorage-independent cell growth but cell–cell cohesion, cell adhesion/spreading onto collagen type IV or fetuin-A, cell motility and invasiveness were strongly inhibited. To explain the loss in adhesion/motility, we show that vinculin-based focal adhesions in the AnxA6-depleted BT-549 cells are elongated and randomly distributed. These focal contacts are also functionally defective because the activation of focal adhesion kinase and the phosphoinositide-3 kinase/Akt pathway were strongly inhibited while the MAP kinase pathway remained constitutively active. Compared with normal human breast tissues, reduced AnxA6 expression in breast carcinoma tissues correlates with enhanced cell proliferation. Together this suggests that reduced AnxA6 expression contributes to breast cancer progression by promoting the loss of functional cell–cell and/or cell–ECM contacts and anchorage-independent cell proliferation.     

A computational study of the Warburg effect identifies metabolic targets inhibiting cancer migration

Over the last decade, the field of cancer metabolism has mainly focused on studying the role of tumorigenic metabolic rewiring in supporting cancer proliferation. Here, we perform the first genome‐scale computational study of the metabolic underpinnings of cancer migration. We build genome‐scale metabolic models of the NCI‐60 cell lines that capture the Warburg effect (aerobic glycolysis) typically occurring in cancer cells. The extent of the Warburg effect in each of these cell line models is quantified by the ratio of glycolytic to oxidative ATP flux (AFR), which is found to be highly positively associated with cancer cell migration. We hence predicted that targeting genes that mitigate the Warburg effect by reducing the AFR may specifically inhibit cancer migration. By testing the anti‐migratory effects of silencing such 17 top predicted genes in four breast and lung cancer cell lines, we find that up to 13 of these novel predictions significantly attenuate cell migration either in all or one cell line only, while having almost no effect on cell proliferation. Furthermore, in accordance with the predictions, a significant reduction is observed in the ratio between experimentally measured ECAR and OCR levels following these perturbations. Inhibiting anti‐migratory targets is a promising future avenue in treating cancer since it may decrease cytotoxic‐related side effects that plague current anti‐proliferative treatments. Furthermore, it may reduce cytotoxic‐related clonal selection of more aggressive cancer cells and the likelihood of emerging resistance.     

Boyden chamber-based method for characterizing the distribution of adhesions and cytoskeletal structure in HT1080 fibrosarcoma cells

A 2D model was previously presented that describes the gliding motility of human fibrosarcoma cells. The model was based on the observation that adhesions are present only on the outer rim of the leading lamella of the semicircular cell. The present model describes the organization of adhesions and the cytoskeleton of migrating HT1080 fibrosarcoma and LX2 hepatic stellate cells in three dimensions. The migration assays were performed in a modified Boyden chamber using fibronectin, Matrigel, or collagen I as chemoattractants. The distribution of the adhesions was analyzed by confocal laser scanning microscope, and following decoration with heavy meromyosin, the organization of actin filaments was analyzed by electron microscopy. Double labeling was performed to study the relationship of the actin and vimentin filament network in the moving cells. Vinculin containing adhesions were observed only at the front of the cell in the form of a ring while passing through a filter pore of the Boyden chamber. Actin filaments were present only below the plasma membrane, except the very tip of the leading lamella. Vimentin intermediate filaments were localized around the cell nucleus behind the actin filament-rich lamella.

This paper describes a model of the organization of adhesions and the cytoskeleton of migrating cells in the Boyden chamber. The model is based on the observation that adhesions are present only at the leading edge of the cell. The results extend the earlier 2D model of cell locomotion into 3D.     

Hyaluronic acid induces transglutaminase II to enhance cell motility; role of Rac1 and FAK in the induction of transglutaminase II

The role of transglutaminase II (TGase II) in hyaluronic acid (HA)-promoted melanoma cell motility was investigated. HA induced the expression of TGase II via the nuclear factor κB (NF-kB) in melanoma cells. HA increased the Rac1 activity and phosphorylation of focal adhesion kinase (FAK). Transfection by lipofectamine of dominant-negative Rac1, and FAK-related non-kinase (FRNK), an endogenous inhibitor of FAK, suppressed the induction of TGase II. This suggests that Rac1 and FAK mediate induction of TGase II by HA. HA-promoted melanoma cell motility was inhibited by cystamine, an inhibitor of TGase II, and overexpression of TGase II enhanced melanoma cell motility through reactive oxygen species. Taken together, HA promotes melanoma cell motility through activation of Rac1, FAK, and induction of TGase II.     

Inhibitory role of focal adhesion kinase on anoikis in the lung cancer cell A549

Resistance to anoikis is a characteristic of malignant cells with increased tumorigenesis and metastasis. Altered FAK activity has been strongly implicated in the development, growth, progression, and metastasis of human cancers, but the mechanism of FAK in regulating anoikis is unknown. In this study, the resistance anoikis role of FAK and its downstream mediators was evaluated in the human lung cancer cell line A549. It has been shown that down regulation of FAK stimulates the apoptosis of cells and the down-regulation of p-ERK, p-PI3K, p-Src, and p-p38. Furthermore, in detached A549 cells, increased FAK phosphorylations (Tyr397, Tyr861, Tyr925) were detected in a time-dependent manner, and the specific inhibitors of MEK1, PI3K, and Src (PD98059, LY294002, and PP2) partly abolished the resistance to the anoikis characteristic of cancer cells. Altogether, our data suggested that Src is involved in the progress of detachment-induced FAK activation in lung tumor cells. PI3K/AKT, MAPK-ERK, and perhaps MAPK-p38 but not MAPK-JNK, appear to be the key downstream effectors of FAK in mediating cell survival. The increased FAK activity upon cell detachment may contribute to the metastasis potential of malignant tumors.