Marching at the front and dragging behind: differential alphaVbeta3-integrin turnover regulates focal adhesion behavior.

Integrins are cell-substrate adhesion molecules that provide the essential link between the actin cytoskeleton and the extracellular matrix during cell migration. We have analyzed alphaVbeta3-integrin dynamics in migrating cells using a green fluorescent protein-tagged beta3-integrin chain. At the cell front, adhesion sites containing alphaVbeta3-integrin remain stationary, whereas at the rear of the cell they slide inward. The integrin fluorescence intensity within these different focal adhesions, and hence the relative integrin density, is directly related to their mobility. Integrin density is as much as threefold higher in sliding compared with stationary focal adhesions. High intracellular tension under the control of RhoA induced the formation of high-density contacts. Low-density adhesion sites were induced by Rac1 and low intracellular tension. Photobleaching experiments demonstrated a slow turnover of beta3-integrins in low-density contacts, which may account for their stationary nature. In contrast, the fast beta3-integrin turnover observed in high-density contacts suggests that their apparent sliding may be caused by a polarized renewal of focal contacts. Therefore, differential acto-myosin-dependent integrin turnover and focal adhesion densities may explain the mechanical and behavioral differences between cell adhesion sites formed at the front, and those that move in the retracting rear of migrating cells.     

Differential dynamics of alpha 5 integrin, paxillin, and alpha-actinin during formation and disassembly of adhesions in migrating cells.

To investigate the mechanisms by which adhesions form and disperse in migrating cells, we expressed alpha 5 integrin, alpha-actinin, and paxillin as green fluorescent protein (GFP) fusions. All localized with their endogenous counterparts and did not perturb migration when expressed at moderate levels. alpha 5-GFP also rescued the adhesive defects in CHO B2 cells, which are alpha 5 integrin deficient. In ruffling cells, alpha 5-GFP and alpha-actinin--GFP localized prominently at the leading edge in membrane protrusions. Of the three GFP fusion proteins that we examined, paxillin was the first component to appear visibly organized in protrusive regions of the cell. When a new protrusion formed, the paxillin appeared to remodel from older to newer adhesions at the leading edge. alpha-Actinin subsequently entered adhesions, which translocated toward the cell center, and inhibited paxillin turnover. The new adhesions formed from small foci of alpha-actinin--GFP and paxillin-GFP, which grew in size. Subsequently, alpha 5 integrin entered the adhesions to form visible complexes, which served to stabilize the adhesions. alpha 5-GFP also resided in endocytic vesicles that emanated from the leading edge of protrusions. Integrin vesicles at the cell rear moved toward the cell body. As cells migrated, alpha 5 vesicles also moved from a perinuclear region to the base of the lamellipodium. The alpha 5 vesicles colocalized with transferrin receptor and FM 4-64 dye. After adhesions broke down in the rear, alpha 5-GFP was found in fibrous structures behind the cell, whereas alpha-actinin--GFP and paxillin-GFP moved up the lateral edge of retracting cells as organized structures and then dissipated.     

Novel insights into how autophagy regulates tumor cell motility

Metastasis requires tumor cells to overcome a series of challenges to successfully travel to and colonize new microenvironments. As an adaptive (or maladaptive) response to stress, macroautophagy/autophagy has garnered increasing interest with respect to cancer metastasis, supported by clinical observations of increased autophagic flux in distant metastases relative to primary tumors. Recently, we identified a new role for autophagy in tumor cell motility through the turnover of focal adhesions, large multi-protein structures that link extracellular matrix-bound integrins to the cytoskeleton. The disassembly of focal adhesions at the cell rear is critical to forward movement and successful migration/invasion. We demonstrated that the focal adhesion protein PXN (paxillin), which serves as a crucial scaffolding and signal integrator, binds directly to LC3B through a conserved LC3-interacting region (LIR) motif to stimulate focal adhesion disassembly and metastasis and that this interaction is further promoted by oncogenic SRC.     

Microtubule targeting of substrate contacts promotes their relaxation and dissociation.

We recently showed that substrate contact sites in living fibroblasts are specifically targeted by microtubules (Kaverina, I., K. Rottner, and J.V. Small. 1998. J. Cell Biol. 142:181-190). Evidence is now provided that microtubule contact targeting plays a role in the modulation of substrate contact dynamics. The results are derived from spreading and polarized goldfish fibroblasts in which microtubules and contact sites were simultaneously visualized using proteins conjugated with Cy-3, rhodamine, or green fluorescent protein.For cells allowed to spread in the presence of nocodazole the turnover of contacts was retarded, as compared with controls and adhesions that were retained under the cell body were dissociated after microtubule reassembly. In polarized cells, small focal complexes were found at the protruding cell front and larger adhesions, corresponding to focal adhesions, at the retracting flanks and rear. At retracting edges, multiple microtubule contact targeting preceded contact release and cell edge retraction. The same effect could be observed in spread cells, in which microtubules were allowed to reassemble after local disassembly by the application of nocodazole to one cell edge. At the protruding front of polarized cells, focal complexes were also targeted and as a result remained either unchanged in size or, more rarely, were disassembled. Conversely, when contact targeting at the cell front was prevented by freezing microtubule growth with 20 nM taxol and protrusion stimulated by the injection of constitutively active Rac, peripheral focal complexes became abnormally enlarged. We further found that the local application of inhibitors of myosin contractility to cell edges bearing focal adhesions induced the same contact dissociation and edge retraction as observed after microtubule targeting.Our data are consistent with a mechanism whereby microtubules deliver localized doses of relaxing signals to contact sites to retard or reverse their development. We propose that it is via this route that microtubules exert their well-established control on cell polarity.     

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.     

Integrin adhesion and force coupling are independently regulated by localized PtdIns(4,5)2 synthesis

The 90-kDa isoform of the lipid kinase PIP kinase Type I γ (PIPKIγ) localizes to focal adhesions (FAs), where it provides a local source of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). Although PtdIns(4,5)P(2) regulates the function of several FA-associated molecules, the role of the FA-specific pool of PtdIns(4,5)P(2) is not known. We report that the genetic ablation of PIPKIγ specifically from FAs results in defective integrin-mediated adhesion and force coupling. Adhesion defects in cells deficient in FAPtdIns(4,5)P(2) synthesis are corrected within minutes while integrin-actin force coupling remains defective over a longer period. Talin and vinculin, but not kindlin, are less efficiently recruited to new adhesions in these cells. These data demonstrate that the specific depletion of PtdIns(4,5)P(2) from FAs temporally separates integrin-ligand binding from integrin-actin force coupling by regulating talin and vinculin recruitment. Furthermore, it suggests that force coupling relies heavily on locally generated PtdIns(4,5)P(2) rather than bulk membrane PtdIns(4,5)P(2).     

FAK is required for tension-dependent organization of collective cell movements in Xenopus mesendoderm

Collective cell movements are integral to biological processes such as embryonic development and wound healing and also have a prominent role in some metastatic cancers. In migrating Xenopus mesendoderm, traction forces are generated by cells through integrin-based adhesions and tension transmitted across cadherin adhesions. This is accompanied by assembly of a mechanoresponsive cadherin adhesion complex containing keratin intermediate filaments and the catenin-family member plakoglobin. We demonstrate that focal adhesion kinase (FAK), a major component of integrin adhesion complexes, is required for normal morphogenesis at gastrulation, closure of the anterior neural tube, axial elongation and somitogenesis. Depletion of zygotically expressed FAK results in disruption of mesendoderm tissue polarity similar to that observed when expression of keratin or plakoglobin is inhibited. Both individual and collective migrations of mesendoderm cells from FAK depleted embryos are slowed, cell protrusions are disordered, and cell spreading and traction forces are decreased. Additionally, keratin filaments fail to organize at the rear of cells in the tissue and association of plakoglobin with cadherin is diminished. These findings suggest that FAK is required for the tension-dependent assembly of the cadherin adhesion complex that guides collective mesendoderm migration, perhaps by modulating the dynamic balance of substrate traction forces and cell cohesion needed to establish cell polarity.     

Spatial and temporal coordination of traction forces in one-dimensional cell migration

ABSTRACT

Migration of a fibroblast along a collagen fiber can be regarded as cell locomotion in one-dimension (1D). In this process, a cell protrudes forward, forms a new adhesion, produces traction forces, and releases its rear adhesion in order to advance itself along a path. However, how a cell coordinates its adhesion formation, traction forces, and rear release in 1D migration is unclear. Here, we studied fibroblasts migrating along a line of microposts. We found that when the front of a cell protruded onto a new micropost, the traction force produced at its front increased steadily, but did so without a temporal correlation in the force at its rear. Instead, the force at the front coordinated with a decrease in force at the micropost behind the front. A similar correlation in traction forces also occurred at the rear of a cell, where a decrease in force due to adhesion detachment corresponded to an increase in force at the micropost ahead of the rear. Analysis with a bio-chemo-mechanical model for traction forces and adhesion dynamics indicated that the observed relationship between traction forces at the front and back of a cell is possible only when cellular elasticity is lower than the elasticity of the cellular environment.     

The last but not the least: the origin and significance of trailing adhesions in fibroblastic cells

Mature adhesions in a motile fibroblast can be classified as stationary "towing" adhesions in the front and sliding trailing adhesions that resist the traction force. Adhesions formed at the front of motile fibroblasts rarely reach the trailing zone, due to disassembly promoted by intensive microtubule targeting. Here, we show that the majority of adhesions found at the trailing edge originate within small short-lived protrusions that extend laterally and backwards from the cell edge. These adhesions enlarge by sliding and by fusion with neighboring adhesions. A further subset of trailing adhesions is initiated at a novel site proximal to trailing stress fibre termini. Following tail retraction, trailing adhesions are actively regenerated and the stress fibre system is remodeled accordingly; the tensile forces elaborated by the contractile actin system are consequently redirected according to trailing adhesion location. We conclude that persistent and dynamic anchorage of the cell rear is needed for the maintenance of continuous unidirectional movement of fibroblasts.     

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.     

GNrep mouse: A reporter mouse for front–rear cell polarity

Cell migration is essential during development, regeneration, homeostasis, and disease. Depending on the microenvironment, cells use different mechanisms to migrate. Yet, all modes of migration require the establishment of an intracellular front–rear polarity axis for directional movement. Although front–rear polarity can be easily identified in in vitro conditions, its assessment in vivo by live‐imaging is challenging due to tissue complexity and lack of reliable markers. Here, we describe a novel and unique double fluorescent reporter mouse line to study front–rear cell polarity in living tissues, called GNrep. This mouse line simultaneously labels Golgi complexes and nuclei allowing the assignment of a nucleus‐to‐Golgi axis to each cell, which functions as a readout for cell front–rear polarity. As a proof‐of‐principle, we validated the efficiency of the GNrep line using an endothelial‐specific Cre mouse line. We show that the GNrep labels the nucleus and the Golgi apparatus of endothelial cells with very high efficiency and high specificity. Importantly, the features of fluorescent intensity and localization for both mCherry and eGFP fluorescent intensity and localization allow automated segmentation and assignment of polarity vectors in complex tissues, making GNrep a great tool to study cell behavior in large‐scale automated analyses. Altogether, the GNrep mouse line, in combination with different Cre recombinase lines, is a novel and unique tool to study of front–rear polarity in mice, both in fixed tissues or in intravital live imaging. This new line will be instrumental to understand cell migration and polarity in development, homeostasis, and disease.     

Mechanotransduction in endothelial cell migration

The migration of endothelial cells (ECs) plays an important role in vascular remodeling and regeneration. EC migration can be regulated by different mechanisms such as chemotaxis, haptotaxis, and mechanotaxis. This review will focus on fluid shear stress-induced mechanotransduction during EC migration. EC migration and mechanotransduction can be modulated by cytoskeleton, cell surface receptors such as integrins and proteoglycans, the chemical and physical properties of extracellular matrix (ECM) and cell-cell adhesions. The shear stress applied on the luminal surface of ECs can be sensed by cell membrane and associated receptor and transmitted throughout the cell to cell-ECM adhesions and cell-cell adhesions. As a result, shear stress induces directional migration of ECs by promoting lamellipodial protrusion and the formation of focal adhesions (FAs) at the front in the flow direction and the disassembly of FAs at the rear. Persistent EC migration in the flow direction can be driven by polarized activation of signaling molecules and the positive feedback loops constituted by Rho GTPases, cytoskeleton, and FAs at the leading edge. Furthermore, shear stress-induced EC migration can overcome the haptotaxis of ECs. Given the hemodynamic environment of the vascular system, mechanotransduction during EC migration has a significant impact on vascular development, angiogenesis, and vascular wound healing.     

Glycogen synthase kinase 3 in the world of cell migration

Glycogen synthase kinase 3 (GSK3) is one of the few master switch kinases that regulate many aspects of cell functions. Recent studies on cell polarization and migration have shown that GSK3 is also essential for proper regulation of these processes. GSK3 influences cell migration as one of the regulators of the spatiotemporally controlled dynamics of the actin cytoskeleton, microtubules, and cell‐to‐matrix adhesions. In this mini‐review, the effects of GSK3 on these three aspects of cell migration will be discussed.     

Substrate-attached materials are enriched with tetraspanins and are analogous to the structures associated with rear-end retraction in migrating cells

Substrate-attached materials (SAMs) are cellular feet that remain on substrates after the treatment of adherent cells with EGTA. SAMs are thought to contain cell adhesion machineries, but their biochemical properties have not been addressed in detail. To gain insight into the molecular mechanisms operating in cell adhesions, we comprehensively identified the protein components of SAMs by liquid chromatography coupled with tandem mass spectrometry, followed by immunoblot analysis. We found that the tetraspanins CD9, CD81, and CD151 were enriched in SAMs along with other transmembrane proteins that are known to associate with tetraspanins. Notably, integrins were detected in SAMs, but the components of focal adhesions were scarcely detected. These observations are reminiscent of the “footprints” that remain on substrates when the retraction fibers at the rear of migrating cells are released, because such footprints have been reported to contain tetraspanins and integrins but not focal adhesion proteins. In support of this hypothesis, the formation of SAMs was attenuated by inhibitors of ROCK, myosin II and dynamin, all of which are known to participate in rear-end retraction in migrating cells. Furthermore, SAMs left on collagen-coated substrates were found by electron microscopy to be fewer and thinner than those on laminin-coated substrates, reflecting the thin and fragile retraction fibers of cells migrating on collagen. Collectively, these results indicate that SAMs closely resemble the footprints and retraction fibers of migrating cells in their protein components, and that they are yielded by similar mechanisms.     

Myosin IIA/IIB restrict adhesive and protrusive signaling to generate front-back polarity in migrating cells

Migratory front-back polarity emerges from the cooperative effect of myosin IIA (MIIA) and IIB (MIIB) on adhesive signaling. We demonstrate here that, during polarization, MIIA and MIIB coordinately promote localized actomyosin bundling, which generates large, stable adhesions that do not signal to Rac and thereby form the cell rear. MIIA formed dynamic actomyosin proto-bundles that mark the cell rear during spreading; it also bound to actin filament bundles associated with initial adhesion maturation in protrusions. Subsequent incorporation of MIIB stabilized the adhesions and actomyosin filaments with which it associated and formed a stable, extended rear. These adhesions did not turn over and no longer signal to Rac. Microtubules fine-tuned the polarity by positioning the front opposite the MIIA/MIIB-specified rear. Decreased Rac signaling in the vicinity of the MIIA/MIIB-stabilized proto-bundles and adhesions was accompanied by the loss of Rac guanine nucleotide exchange factor (GEFs), like βPIX and DOCK180, and by inhibited phosphorylation of key residues on adhesion proteins that recruit and activate Rac GEFs. These observations lead to a model for front-back polarity through local GEF depletion.     

FAK dimerization controls its kinase‐dependent functions at focal adhesions

Focal adhesion kinase (FAK) controls adhesion‐dependent cell motility, survival, and proliferation. FAK has kinase‐dependent and kinase‐independent functions, both of which play major roles in embryogenesis and tumor invasiveness. The precise mechanisms of FAK activation are not known. Using x‐ray crystallography, small angle x‐ray scattering, and biochemical and functional analyses, we show that the key step for activation of FAK's kinase‐dependent functions—autophosphorylation of tyrosine‐397—requires site‐specific dimerization of FAK. The dimers form via the association of the N‐terminal FERM domain of FAK and are stabilized by an interaction between FERM and the C‐terminal FAT domain. FAT binds to a basic motif on FERM that regulates co‐activation and nuclear localization. FAK dimerization requires local enrichment, which occurs specifically at focal adhesions. Paxillin plays a dual role, by recruiting FAK to focal adhesions and by reinforcing the FAT:FERM interaction. Our results provide a structural and mechanistic framework to explain how FAK combines multiple stimuli into a site‐specific function. The dimer interfaces we describe are promising targets for blocking FAK activation.     

Coronin 1C negatively regulates cell-matrix adhesion and motility of intestinal epithelial cells

Coronins, WD-repeat actin-binding proteins, are known to regulate cell motility by coordinating actin filament turnover in lamellipodia of migrating cell. Here we report a novel mechanism of Coronin 1C-mediated cell motility that involves regulation of cell-matrix adhesion. RNAi silencing of Coronin 1C in intestinal epithelial cells enhanced cell migration and modulated lamellipodia dynamics by increasing the persistence of lamellipodial protrusion. Coronin 1C-depleted cells showed increased cell-matrix adhesions and enhanced cell spreading compared to control cells, while over-expression of Coronin 1C antagonized cell adhesion and spreading. Enhanced cell-matrix adhesion of coronin-deficient cells correlated with hyperphosphorylation of focal adhesion kinase (FAK) and paxillin, and an increase in number of focal adhesions and their redistribution at the cell periphery. siRNA depletion of FAK in coronin-deficient cells rescued the effects of Coronin 1C depletion on motility, cell-matrix adhesion, and spreading. Thus, our findings provide the first evidence that Coronin 1C negatively regulates epithelial cell migration via FAK-mediated inhibition of cell-matrix adhesion.     

Release of integrin macroaggregates as a mechanism of rear detachment during keratinocyte migration

Cell-substrate adhesion can be mediated by the relatively short-lived focal complexes and focal adhesions and by the more stable hemidesmosomes. During cell migration both types of cell-substrate adhesions must be disrupted allowing the cell rear to detach. Rear detachment has been described to be accompanied by membrane ripping and the loss of cellular material in a variety of cell types including fibroblasts and chondrocytes, but also in fast moving cells such as keratinocytes. Here we show that migrating keratinocytes leave behind "migration tracks" of cellular remnants that can be classified due to their size, distribution and molecular composition. Type I macroaggregates appeared as spherical and tubular structures with a diameter of about 50-100 nm that were arranged like "pearls on a string". These structures apparently derived from fragmentation of long tubular extensions, the retracting fibers, at the cell rear and contained high amounts of beta1 integrin and different alpha integrins that are components of fibronectin and laminin receptors in migrating keratinocytes usually found in focal adhesions. Type II macroaggregates were recognized as spherical structures with a diameter of about 30 - 50 nm that were arranged in clusters scattered over the gaps between type I, macroaggregates. In contrast to type I type II macroaggregates contained high amounts of beta4 integrin and seemed to derive from former hemidesmosomes. Both types of macroaggregates were completely membrane covered, impermeable compartments devoid of cytosolic proteins. Our observations strongly support the concept that the release of macroaggregates represents a distinct cellular mechanism of rear detachment based on the loss of adhesive receptors embedded in membrane-covered cellular remnants.     

Focal adhesion kinase localizes to sites of cell‐to‐cell contact in vivo and increases apically in rat uterine luminal epithelium and the blastocyst at the time of implantation

Focal adhesions play an important role in promoting embryo invasion; in particular, focal adhesions disassemble at the time of implantation in the rat, facilitating the detachment of the uterine luminal epithelium to allow the embryo to invade the endometrium. This study investigated focal adhesion protein, focal adhesion kinase (FAK) in the rat uterine luminal, and glandular epithelial cells to understand the dynamics of focal adhesions during early pregnancy. FAK undergoes extensive distributional change during early pregnancy, and surprisingly, FAK was not localized at the site of focal adhesions, instead being localized to the site of cell‐to‐cell contact and colocalizing with ZO‐1 on day 1 of pregnancy. At the time of implantation, FAK increases in the apical region of the uterine luminal epithelial cells which was regulated by progesterone. Using an in vitro co‐culture model of rat blastocysts attached to Ishikawa cells, FAK was present apically both in the rat blastocyst and the Ishikawa cells, suggesting a role in attachment andin mediating signal transduction between these two genetically different cell types. J. Morphol., 2012. © 2012 Wiley Periodicals, Inc.     

The effect of elevated extracellular glucose on adherens junction proteins in cultured rat heart endothelial cells

Vascular permeability is a proof of vascular endothelial cell dysfunction induced by diabetes. Vascular permeability is directly related to the width of intercellular endothelial cells junctions, which may become permeable to macromolecules as a result of a change in endothelial cell shape. To determine the role of hyperglycemia in endothelial cell shape, the study examined the effect of high concentrations of glucose on the shape of cultured rat heart endothelial cells. This result indicated that the high-glucose-induced changes in the morphology of endothelial cells, via the glucose-mediated reorganization of F-actin. In endothelial cells, the actin cytoskeleton is tethered to the zonula adherens and focal adhesions, which mediate cell-cell and cell-matrix interactions respectively. The present study demonstrated that the high-glucose-induced changes in the actin-binding protein such as filamin, zonula adherens proteins such as alpha-, beta-, and gamma-catenin, focal adhesions proteins such as focal adhesion kinase, paxillin, and tyrosine phosphorylation of paxillin. It appears that differences in expression of adherens junctions molecules on rat heart endothelial cells in response to high glucose reflect endothelial glucose toxicity, which may also induce endothelial dysfunction in diabetes.