Microtubules mediate changes in membrane cortical elasticity during contractile activation

The mechanical properties of living cells are highly regulated by remodeling dynamics of the cytoarchitecture, and are linked to a wide variety of physiological and pathological processes. Microtubules (MT) and actomyosin contractility are both involved in regulating focal adhesion (FA) size and cortical elasticity in living cells. Although several studies have examined the effects of MT depolymerization or actomyosin activation on biological processes, very few have investigated the influence of both on the mechanical properties, FA assembly, and spreading of fibroblast cells. Here, we examine how activation of both processes modulates cortical elasticity as a function of time. Enhancement of contractility (calyculin A treatment) or the depolymerization of MTs (nocodazole treatment) individually caused a time-dependent increase in FA size, decrease in cell height and an increase in cortical elasticity. Surprisingly, sequentially stimulating both processes led to a decrease in cortical elasticity, loss of intact FAs and a concomitant increase in cell height. Our results demonstrate that loss of MTs disables the ability of fibroblast cells to maintain increased contractility and cortical elasticity upon activation of myosin-II. We speculate that in the absence of an intact MT network, a large amount of contractile tension is transmitted directly to FA sites resulting in their disassembly. This implies that tension-mediated FA growth may have an upper bound, beyond which disassembly takes place. The interplay between cytoskeletal remodeling and actomyosin contractility modulates FA size and cell height, leading to dynamic time-dependent changes in the cortical elasticity of fibroblast cells.     

Calpain-mediated proteolysis of paxillin negatively regulates focal adhesion dynamics and cell migration

The dynamic turnover of integrin-mediated adhesions is important for cell migration. Paxillin is an adaptor protein that localizes to focal adhesions and has been implicated in cell motility. We previously reported that calpain-mediated proteolysis of talin1 and focal adhesion kinase mediates adhesion disassembly in motile cells. To determine whether calpain-mediated paxillin proteolysis regulates focal adhesion dynamics and cell motility, we mapped the preferred calpain proteolytic site in paxillin. The cleavage site is between the paxillin LD1 and LD2 motifs and generates a C-terminal fragment that is similar in size to the alternative product paxillin delta. The calpain-generated proteolytic fragment, like paxillin delta, functions as a paxillin antagonist and impairs focal adhesion disassembly and migration. We generated mutant paxillin with a point mutation (S95G) that renders it partially resistant to calpain proteolysis. Paxillin-deficient cells that express paxillin S95G display increased turnover of zyxin-containing adhesions using time-lapse microscopy and also show increased migration. Moreover, cancer-associated somatic mutations in paxillin are common in the N-terminal region between the LD1 and LD2 motifs and confer partial calpain resistance. Taken together, these findings suggest a novel role for calpain-mediated proteolysis of paxillin as a negative regulator of focal adhesion dynamics and migration that may function to limit cancer cell invasion.     

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.     

The explorations of dynamic interactions of paxillin at the focal adhesions

Paxillin is one of the most important adapters in integrin-mediated adhesions that performs numerous crucial functions relying on its dynamic interactions. Its structural behavior serves different purposes, providing a base for several activities. The various domains of paxillin display different functions in the whole process of cell movements and have a significant role in cell adhesion, migration, signal transmission, and protein-protein interactions. On the other hand, some paxillin-associated proteins provide a unique spatiotemporal mechanism for regulating its dynamic characteristics in the tissue homeostasis and make it a more complex and decisive protein at the focal adhesions. This review briefly describes the structural adaptations and molecular mechanisms of recruitment of paxillin into adhesions, explains paxillin’s binding dynamics and impact on adhesion stability and turnover, and reveals a variety of paxillin-associated regulatory mechanisms and how paxillin is embedded into the signaling networks.     

SNARE-mediated membrane traffic modulates RhoA-regulated focal adhesion formation

In the present study, we examined the role of soluble NSF attachment protein receptor (SNARE)-mediated membrane traffic in the formation of focal adhesions during cell spreading. CHO-K1 cells expressing a dominant-negative form of N-ethylmaleimide-sensitive factor (E329Q-NSF) were unable to spread as well as control cells and they formed focal adhesions (FAs) that were larger than those in control cells. FA formation was impaired in cells transfected with a dominant-negative form of RhoA, but, significantly, not in cells simultaneously expressing dominant-negative NSF. Treatment of E329Q-NSF-expressing cells with the ROCK inhibitor Y-27632 did inhibit FA formation. The results are consistent with a model of cell adhesion in which SNARE-mediated membrane traffic is required for both the elaboration of lamellipodia and the modulation of biochemical signals that control RhoA-mediated FA assembly.     

Cell shape provides global control of focal adhesion assembly

Cell spreading was controlled independently of the amount and density of immobilized integrin ligand by culturing cells on single adhesive islands of different sizes (100-2500 microm(2)) and shapes (squares, circles, and lines) or on many smaller (3-5 microm diameter) circular islands that were coated with a saturating density of fibronectin and separated by non-adhesive regions. The amount of focal adhesions (FAs) containing vinculin and phosphotyrosine increased in direct proportion to cell spreading under all conditions. FAs localized asymmetrically along the periphery of the small islands that experienced highest tensional stress, and FA staining increased when cytoskeletal tension was stimulated with thrombin, whereas inhibitors of contractility promoted FA disassembly. Thus, these findings demonstrate the existence of an "inside-out" mechanism whereby global cell distortion produces increases in cytoskeletal tension that feed back to drive local changes in FA assembly. This complex interplay between cell morphology, mechanics, and adhesion may be critical to how cells integrate from and function in living tissues.     

Integrin-mediated adhesion regulates membrane order

The properties of cholesterol-dependent domains (lipid rafts) in cell membranes have been controversial. Because integrin-mediated cell adhesion and caveolin both regulate trafficking of raft components, we investigated the effects of adhesion and caveolin on membrane order. The fluorescent probe Laurdan and two-photon microscopy revealed that focal adhesions are highly ordered; in fact, they are more ordered than caveolae or domains that stain with cholera toxin subunit B (CtxB). Membrane order at focal adhesion depends partly on phosphorylation of caveolin1 at Tyr14, which localizes to focal adhesions. Detachment of cells from the substratum triggers a rapid, caveolin-independent decrease in membrane order, followed by a slower, caveolin-dependent decrease that correlates with internalization of CtxB-stained domains. Endocytosed CtxB domains also become more fluid. Thus, membrane order is highly dependent on caveolae and focal adhesions. These results show that lipid raft properties are conferred by assembly of specific protein complexes. The ordered state within focal adhesions may have important consequences for signaling at these sites.     

Regulation of cell adhesion strength by peripheral focal adhesion distribution

Cell adhesion to extracellular matrices is a tightly regulated process that involves the complex interplay between biochemical and mechanical events at the cell-adhesive interface. Previous work established the spatiotemporal contributions of adhesive components to adhesion strength and identified a nonlinear dependence on cell spreading. This study was designed to investigate the regulation of cell-adhesion strength by the size and position of focal adhesions (FA). The cell-adhesive interface was engineered to direct FA assembly to the periphery of the cell-spreading area to delineate the cell-adhesive area from the cell-spreading area. It was observed that redistributing the same adhesive area over a larger cell-spreading area significantly enhanced cell-adhesion strength, but only up to a threshold area. Moreover, the size of the peripheral FAs, which was interpreted as an adhesive patch, did not directly govern the adhesion strength. Interestingly, this is in contrast to the previously reported functional role of FAs in regulating cellular traction where sizes of the peripheral FAs play a critical role. These findings demonstrate, to our knowledge for the first time, that two spatial regimes in cell-spreading area exist that uniquely govern the structure-function role of FAs in regulating cell-adhesion strength.     

Direct observation of α-actinin tension and recruitment at focal adhesions during contact growth

Adherent cells interact with extracellular matrix via cell–substrate contacts at focal adhesions. The dynamic assembly and disassembly of focal adhesions enables cell attachment, migration and growth. While the influence of mechanical forces on the formation and growth of focal adhesions has been widely observed, the force loading on specific proteins at focal adhesion complex is not clear. By co-expressing force sensitive α-actinin FRET probes and fluorescence labeled paxillin in MDCK cells, we have simultaneously observed the time-dependent changes in tension in α-actinin and the dynamics of focal adhesion during cell migration. We show that increase in tension in α-actinin at the focal adhesion coincides with elongation of the adhesion in its growth phase. The enlargement of focal adhesion is through a force sensitive recruitment of α-actinin and paxillin to the adhesion sites. Changes in α-actinin tension and correlated relocation of α-actinin in an active adhesion also guide the growth direction of the adhesion. The results support the model that cytoskeletal tension is coupled to focal adhesion via the linking protein, α-actinin at the adhesion complex. Lysophosphatidic acid caused an immediate increase in α-actinin tension followed by drastic focal adhesion formation and elongation. Application of Rho-ROCK inhibitor, Y27632, resulted in reversible reduction in tension in α-actinin and disassociation of focal adhesion, suggesting the involvement of myosin-II mediated contractile force in the focal adhesion dynamics. These findings suggest that α-actinin not only serves as a physical linker between cytoskeleton and integrin, but also participates in force transmission at adhesion sites to facilitate adhesion?s growth.     

Cell Adhesion-dependent Serine 85 Phosphorylation of Paxillin Modulates Focal Adhesion Formation and Haptotactic Migration via Association with the C-terminal Tail Domain of Talin

Integrin-mediated adhesion to extracellular matrix proteins is dynamically regulated during morphological changes and cell migration. Upon cell adhesion, protein-protein interactions among molecules at focal adhesions (FAs) play major roles in the regulation of cell morphogenesis and migration. Although tyrosine phosphorylation of paxillin is critically involved in adhesion-mediated signaling, the significance of paxillin phosphorylation at Ser-85 and the mechanism by which it regulates cell migration remain unclear. In this study, we examined how Ser-85 phosphorylation of paxillin affects FA formation and cell migration. We found that paxillin phosphorylation at Ser-85 occurred during HeLa cell adhesion to collagen I and was concomitant with tyrosine phosphorylation of both focal adhesion kinase and talin. However, the non-phosphorylatable S85A mutant of paxillin impaired cell spreading, FA turnover, and migration toward collagen I but not toward serum. Furthermore, whereas the (presumably indirect) interaction between paxillin and the C-terminal tail of talin led to dynamic FAs at the cell boundary, S85A paxillin did not bind talin and caused stabilized FAs in the central region of cells. Together, these observations suggest that cell adhesion-dependent Ser-85 phosphorylation of paxillin is important for its interaction with talin and regulation of dynamic FAs and cell migration.     

Role of paxillin in the early phase of orientation of the vascular endothelial cells exposed to cyclic stretching

Paxillin, a structural and signaling scaffold molecule in focal adhesions (FAs), is considered to be important in intracellular signaling transduction and the cell shape changes in response to cyclic stretching. However, the detailed role of paxillin in stretch-induced morphological changes of endothelial cells (ECs) has not fully determined until date. In this study, in order to understand the role of paxillin in the orientation of ECs exposed to cyclic stretching, we examined the time course of changes in the shape and distribution of FA proteins of paxillin knockdown ECs. Non-treated ECs subjected to 20% cyclic stretching at 0.5Hz oriented perpendicularly to the direction of stretching after 10min of exposure. On the other hand, the orientation of paxillin knockdown ECs was abolished at 10min, but it was observed after 60min of cyclic stretching exposure. Immunofluorescent microscopy revealed that accumulation and redistribution of FA proteins, including focal adhesion kinase (FAK) and integrin β1, were observed at 10min of exposure to cyclic stretching in non-treated ECs. The accumulation of FAK and integrin β1 was not prominent in paxillin knockdown ECs under static conditions and after 10min of exposure to cyclic stretching. However, we found that accumulation of FA proteins in paxillin knockdown ECs at 30 and 60min was similar to that in non-transfected ECs. Because paxillin is an adaptor protein offering binding sites for FAK and integrin β1, which are critical molecules for the early signaling events of focal adhesion formation in ECs, these results suggest that paxillin is required for the early phase of EC orientation in response to cyclic stretching by scaffolding for accumulation of FA proteins.     

Tyrosine phosphorylation of paxillin alpha is involved in temporospatial regulation of paxillin-containing focal adhesion formation and F-actin organization in motile cells

Temporal and spatial regulation of actin-based cytoskeletal organization and focal adhesion formation play an essential role in cell migration. Here, we show that tyrosine phosphorylation of a focal adhesion protein, paxillin, crucially participates in these regulations. We found that tyrosine phosphorylation of paxillin was a prominent event upon integrin activation during epithelial-mesenchymal trans-differentiation and cell migration. Four major tyrosine phosphorylation sites were identified, and two of them were highly inducible upon integrin activation. Paxillin exhibits three distinct subcellular localizations as follows: localization along the cell periphery colocalized with circumferential actin meshworks, macroaggregation at focal adhesions connected to actin stress fibers, and diffuse cytoplasmic distribution. Tyrosine phosphorylation of paxillin localized at the cell periphery and focal adhesions was shown using phosphorylation site-specific antibodies. Mutations in the phosphorylation sites affected the peripheral localization of paxillin and paxillin-containing focal adhesion formation during cell migration and cell-cell collision, accompanied by altered actin organizations. Our analysis indicates that phosphorylation of multiple tyrosines in paxillin alpha is necessary for the proper function of paxillin and is involved in the temporospatial regulation of focal adhesion formation and actin cytoskeletal organization in motile cells.     

αV-Integrins Are Required for Mechanotransduction in MDCK Epithelial Cells

The properties of epithelial cells within tissues are regulated by their immediate microenvironment, which consists of neighboring cells and the extracellular matrix (ECM). Integrin heterodimers orchestrate dynamic assembly and disassembly of cell-ECM connections and thereby convey biochemical and mechanical information from the ECM into cells. However, the specific contributions and functional hierarchy between different integrin heterodimers in the regulation of focal adhesion dynamics in epithelial cells are incompletely understood. Here, we have studied the functions of RGD-binding αV-integrins in a Madin Darby Canine Kidney (MDCK) cell model and found that αV-integrins regulate the maturation of focal adhesions (FAs) and cell spreading. αV-integrin-deficient MDCK cells bound collagen I (Col I) substrate via α2β1-integrins but failed to efficiently recruit FA components such as talin, focal adhesion kinase (FAK), vinculin and integrin-linked kinase (ILK). The apparent inability to mature α2β1-integrin-mediated FAs and link them to cellular actin cytoskeleton led to disrupted mechanotransduction in αV-integrin deficient cells seeded onto Col I substrate.     

Recognition of LD motifs by the focal adhesion targeting domains of focal adhesion kinase and proline‐rich tyrosine kinase 2‐beta: Insights from molecular dynamics simulations

The focal adhesion kinase (FAK) and the proline‐rich tyrosine kinase 2‐beta (PYK2) are implicated in cancer progression and metastasis and represent promising biomarkers and targets for cancer therapy. FAK and PYK2 are recruited to focal adhesions (FAs) via interactions between their FA targeting (FAT) domains and conserved segments (LD motifs) on the proteins Paxillin, Leupaxin, and Hic‐5. A promising new approach for the inhibition of FAK and PYK2 targets interactions of the FAK domains with proteins that promote localization at FAs. Advances toward this goal include the development of surface plasmon resonance, heteronuclear single quantum coherence nuclear magnetic resonance (HSQC‐NMR) and fluorescence polarization assays for the identification of fragments or compounds interfering with the FAK‐Paxillin interaction. We have recently validated this strategy, showing that Paxillin mimicking polypeptides with 2 to 3 LD motifs displace FAK from FAs and block kinase‐dependent and independent functions of FAK, including downstream integrin signaling and FA localization of the protein p130Cas. In the present work we study by all‐atom molecular dynamics simulations the recognition of peptides with the Paxillin and Leupaxin LD motifs by the FAK‐FAT and PYK2‐FAT domains. Our simulations and free‐energy analysis interpret experimental data on binding of Paxillin and Leupaxin LD motifs at FAK‐FAT and PYK2‐FAT binding sites, and assess the roles of consensus LD regions and flanking residues. Our results can assist in the design of effective inhibitory peptides of the FAK‐FAT: Paxillin and PYK2‐FAT:Leupaxin complexes and the construction of pharmacophore models for the discovery of potential small‐molecule inhibitors of the FAK‐FAT and PYK2‐FAT focal adhesion based functions.     

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.     

Dephosphorylation of focal adhesion kinase (FAK) and loss of focal contacts precede caspase-mediated cleavage of FAK during apoptosis in renal epithelial cells

The relationship between focal adhesion protein (FAK) activity and loss of cell-matrix contact during apoptosis is not entirely clear nor has the role of FAK in chemically induced apoptosis been studied. We investigated the status of FAK phosphorylation and cleavage in renal epithelial cells during apoptosis caused by the nephrotoxicant dichlorovinylcysteine (DCVC). DCVC treatment caused a loss of cell-matrix contact which was preceded by a dissociation of FAK from the focal adhesions and tyrosine dephosphorylation of FAK. Paxillin was also dephosphorylated at tyrosine. DCVC treatment activated caspase-3 which was associated with cleavage of FAK. However, FAK cleavage occurred after cells had already lost focal adhesions indicating that cleavage of FAK by caspases is not responsible for loss of FAK from focal adhesions. Accordingly, although inhibition of caspase activity with zVAD-fmk blocked activation of caspase-3, FAK cleavage, and apoptosis, it neither affected dephosphorylation nor translocation of FAK or paxillin. However, zVAD-fmk completely blocked the cell detachment caused by DCVC treatment. Orthovanadate prevented DCVC-induced tyrosine dephosphorylation of both FAK and paxillin; however, it did not inhibit DCVC-induced apoptosis and actually potentiated focal adhesion disorganization and cell detachment. Thus, FAK dephosphorylation and loss of focal adhesions are not due to caspase activation; however, caspases are required for FAK proteolysis and cell detachment.     

The compass to follow: Focal adhesion turnover

How cells move is a fundamental biological question. The directionality of adherent migrating cells depends on the assembly and disassembly (turnover) of focal adhesions (FAs). FAs are micron-sized actin-based structures that link cells to the extracellular matrix. Traditionally, microtubules have been considered key to triggering FA turnover. Through the years, advancements in biochemistry, biophysics, and bioimaging tools have been invaluable for many research groups to unravel a variety of mechanisms and molecular players that contribute to FA turnover, beyond microtubules. Here, we discuss recent discoveries of key molecular players that affect the dynamics and organization of the actin cytoskeleton to enable timely FA turnover and consequently proper directed cell migration.     

Structure and function of focal adhesions

Integrin-dependent cell adhesions come in different shapes and serve in different cell types for tasks ranging from cell-adhesion, migration, and the remodeling of the extracellular matrix to the formation and stabilization of immunological and chemical synapses. A major challenge consists in the identification of adhesion-specific as well as common regulatory mechanisms, motivating the need for a deeper analysis of protein-protein interactions in the context of intact focal adhesions. Specifically, it is critical to understand how small differences in binding of integrins to extracellular ligands and/or cytoplasmic adapter proteins affect the assembly and function of an entire focal adhesion. By using the talin-integrin pair as a starting point, I would like to discuss how specific protein-protein and protein-lipid interactions can control the behavior and function of focal adhesions. By responding to chemical and mechanical cues several allosterically regulated proteins create a dynamic multifunctional protein network that provides both adhesion to the extracellular matrix as well as intracellular signaling in response to mechanical changes in the cellular environment.     

Bidirectional control of the inner dynamics of focal adhesions promotes cell migration

Focal adhesions (FA) are bidirectional mechanical biosensors that allow cells to integrate intracellular and extracellular cues. Their function is tightly regulated by changes in molecular composition and also by variation in the spatio-temporal dynamics of FA components within this structure. A closely regulated turnover of FA proteins within FA sites allows cells to respond appropriately to their environment, thereby impacting on cell shape and function. FA protein dynamics are linked to FA maturation and rates of assembly and disassembly, and have a significant influence on tumor cell migration. Using the FRAP technique to investigate the hidden internal dynamics of FA, we identified two new regulators of FA dynamics and cell migration: the Mgat5/galectin-3 lattice and tyrosine phosphorylated caveolin-1 (pY14Cav1). In this short review we first introduce FA and their complex dynamic behavior. We then present the Mgat5/galectin-3 lattice and caveolin-1 and discuss their concerted role in FA dynamics, which defines previously unknown, interdependent roles in tumor cell migration. We conclude with a discussion of interesting unexplored avenues that might lead to a better understanding of the complex mechanism of FA dynamics.Key words: focal adhesion, migration, caveolin-1, tyrosine 14, galectin-3, Mgat5, turnover, dynamics     

Stress fibers are generated by two distinct actin assembly mechanisms in motile cells

Stress fibers play a central role in adhesion, motility, and morphogenesis of eukaryotic cells, but the mechanism of how these and other contractile actomyosin structures are generated is not known. By analyzing stress fiber assembly pathways using live cell microscopy, we revealed that these structures are generated by two distinct mechanisms. Dorsal stress fibers, which are connected to the substrate via a focal adhesion at one end, are assembled through formin (mDia1/DRF1)-driven actin polymerization at focal adhesions. In contrast, transverse arcs, which are not directly anchored to substrate, are generated by endwise annealing of myosin bundles and Arp2/3-nucleated actin bundles at the lamella. Remarkably, dorsal stress fibers and transverse arcs can be converted to ventral stress fibers anchored to focal adhesions at both ends. Fluorescence recovery after photobleaching analysis revealed that actin filament cross-linking in stress fibers is highly dynamic, suggesting that the rapid association-dissociation kinetics of cross-linkers may be essential for the formation and contractility of stress fibers. Based on these data, we propose a general model for assembly and maintenance of contractile actin structures in cells.