342
A Molecular Model for How Increased pH Activates Focal Adhesion Kinase.
C-H. Choi1, D. L. Barber1; 1Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA
Activity of focal adhesion kinase (FAK), a key regulator of focal adhesion turnover and cell migration, is dependent on autophosphorylation of Tyr397. The recently identified FAK crystal structure revealed that FAK activity is inhibited in cis by an interaction between its N-terminal FERM and C-terminal kinase domains. Release of this auto-inhibited conformation allows autophosphorylation of Tyr397 in a linker region between FERM and kinase domains, which docks Src kinases that phosphorylate Tyr576/577 in the kinase domain for full activation of FAK. However, how the initial step of releasing an auto-inhibited conformation is regulated remains unknown. We previously showed that increased intracellular pH from activation of the Na-H exchanger NHE1 is necessary for increased FAK-pY397, focal adhesion turnover, and directed cell migration. Our current data suggest that pH > 7.2 disrupts an auto-inhibited conformation of FAK to increase FAK-pY397. The FERM domain of FAK contains an unusually high number of seven conserved histidines compared with FERM domains in other proteins, and two histidines, His41 and His58 are at the interface of the FERM and kinase domains. We reasoned that pH- dependent charges on histidines in the FERM domain might regulate an electrostatic interaction
with the kinase domain. In support of this prediction, we used in vitro kinase assays with recombinant full-length FAK to show increased pY397 from pH 6.5 to 7.5. Increased pY397 at higher pH is dependent on the FERM domain because a truncated FAK lacking the FERM domain has constitutively high pY397 that is pH-independent. We also used separate FERM and kinase domain constructs to show binding in trans is markedly greater at pH 6.5 compared with pH 7.5, further suggesting that increased pH releases an auto-inhibited conformation. To test the role of His58 in pH-dependent pTyr397, we used a mutant full-length FAK-H58A to show constitutively high and pH-independent pY397 similar to a truncated FAK lacking the FERM domain. Our data suggest a molecular mechanism for pH-dependent activation of FAK that predicts increased pH > 7.2 and deprotonation of His58 releases an auto-inhibited charge interaction between FERM and kinase domains.
343
A mechanochemistry model of focal adhesion dynamics in cell migration.
Z. Wu1, S. Plotnikov1, C. M. Waterman1, J. Liu1; 1National Heart, Lung and Blood Institute, National Institute of Health, Bethesda, MD
Focal adhesions are essential to mediate cell extracellular matrix (ECM) adhesion and force transmission during cell motilities, which involve the crosstalk between physical signals such as contractile forces or membrane dynamics, and chemical signaling events such as focal adhesion kinase related regulation pathways. However, the underline mechanism of the biophysical regulations of force transmission among actin cytoskeleton, cell membrane, focal complex and ECM remains poorly understood. We constructed a mathematical model to understand the behavior of focal adhesion complex under different experimental conditions. By integrating the cell membrane dynamics, actin network fluid dynamics, and the mechanochemistry of focal complex, the model reveals itself the capability to capture the essential characteristics of focal adhesions in cell motility. In particular, the model explains the focal adhesion growth pattern at different ECM stiffness. The model thus provides a comprehensive vision of the focal adhesion dynamics.
344
Matrix-properties dependent cell migration speed: characterization of the driving force of cell migration.
B. Ji1; 1Beijing Institute of Technology, Beijing, China
It was shown that cell migration exhibits strong mechanosensitivity behaviors, e.g., the migration speed biphasically depends on the matrix rigidity. However, these behaviors have not been quantitatively understood. In this study, a mechano-chemical coupling model was developed for studying the cell migration behaviors by modeling the dynamics of focal adhesion and effect of cell shape on cell traction force distribution, in which not only the rigidity of matrix but also the concentration of ligands on matrix and the activity of myosin were considered. We showed that the cell migration behaviors depended on not only rigidity of matrix, but also the concentration of ligands in a biphasic manner. The underlying mechanism behind these biphasic behaviors is that these parameters can influence the stability of focal adhesions which are crucial for the creation of pulling force by stable focal adhesions at cell front and the cell detachment and retraction triggered by the destabilization of focal adhesion at cell rear. These results agree with the experimental observations. Furthermore, a motility factor was suggested for characterizing the driving force of cell migration which was mainly determined by the cell shape, matrix stiffness and concentration of ligands on matrix. This study provided a quantitative understanding of how cells control their migration behaviors.
345
Direct observation of catch bonds in focal adhesions of living cells.
N. Bonakdar1, A. Schilling1, C. Metzner1, B. Fabry1; 1Biophysics Group, Center for Medical Physics and Technology, Erlangen, Germany
Force spectroscopy measurements on isolated integrins revealed the counterintuitive effect that integrin binding with fibronectin strengthened with force. This so-called catch bond behavior has never been observed in living cells, however. The anchorage of cells to the extracellular matrix is established through a force-transmitting molecular chain of integrin-type adhesion receptors, proteins of the focal adhesion complex, and the cytoskeleton. The resulting adhesion strength is determined by a collective binding energy landscape which arises from the superposition of the energy landscapes of the individual molecular bonds along the force transmission chain. To measure this collective energy landscape, we used high-force magnetic tweezers to apply forces of up to 80 nN to 5 µm RGD coated beads bound to the cell. The forces were applied as a linear ramp (loading rates of 1...40 nN/s) or as staircase-like force steps. The average force at which the beads detached from the cell was recorded and was taken as a measure of bond stability. Bond stability increased with higher loading rates, as expected for thermally activated molecular bonds. Surprisingly, a staircase-like loading protocol further increased bond stability and bond lifetimes. Such behavior cannot be explained by active mechanotransduction processes or by thermally activated molecular bonds, but is consistent with the existence of force-strengthening catch-bonds. A possible molecular mechanism for catch-bond behavior are hidden binding domains in focal adhesion proteins that become available during force-induced unfolding.
346
Lasp-2 Binds Vinculin and Paxillin in Focal Adhesions and Affects Cell Adhesion and Spreading.
K. T. Bliss1, C. M. Jones-Weinert1, C. C. Gregorio1; 1Cellular and Molecular Medicine, University of Arizona, Tuscon, AZ
Focal adhesions are intricate protein complexes that facilitate cell attachment, migration and cellular communication. Lasp-2 (LIM-nebulette) is a recently identified member of the nebulin family of actin-binding proteins and appears to be an integral component of focal adhesions. The function of lasp-2 is currently viewed as a molecular scaffold capable of organizing and bundling actin filaments. To gain further insights into the functional role of lasp-2 at focal adhesions, we set out to identify additional binding partners of lasp-2 using yeast two-hybrid screens. Two new focal adhesion interacting partners, vinculin and paxillin, were identified. These novel interactions were confirmed using solid phase binding assays. Immunofluorescence microscopy demonstrated that Lasp-2 co-localizes with vinculin and paxillin in focal adhesions. Interestingly, overexpression of GFP-lasp-2 decreases cell adhesion and the number of vinculin-containing focal adhesions. Reduction of lasp-2 protein levels via siRNA knockdown reduces the ability for cells to spread. Taken together, these data suggest that lasp-2 has an important role in coordinating and regulating the composition and dynamics of focal adhesions.
347
β-actin mRNA compartmentalization by ZBP1 controls focal adhesion stability and directed cell motility.
Z. B. Katz1, A. L. Wells1, B. Wu1, H. Y. Park1, S. M. Shenoy1, R. H. Singer1; 1Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY
Localization of β-actin mRNA is facilitated by the zipcode binding protein, ZBP1. Binding to the cognate zipcode within the 3’UTR of β-actin mRNA enables ZBP1 to asymmetrically distribute mRNA and control translation of its target, a process known to be necessary for directed cell motility. This study utilized a ZBP1 KO fibroblast cell line to compare β-actin mRNA dynamics to wild-type cells. TIRF microscopy and mRNA particle tracking in live cells enabled us to discover a specific compartment near focal adhesions where β-actin mRNA dwells for periods greater than one minute. In ZBP1 KO fibroblasts, the probability of tracking β-actin mRNA within the adhesion environment is significantly reduced. Consequently, adhesion lifetimes are reduced in ZBP1 KO cells. This supports the hypothesis that ZBP1 facilitates mRNA localization to strengthen focal adhesions and therefore direct cell migration. To test directly whether mRNA localization to adhesions can alter adhesion dynamics and cell motility we utilized a novel mRNA tethering technique. We tethered β-actin mRNA to focal adhesion complexes through MS2 stem loops in the 3’UTR of β-actin mRNA and the MS2 coat protein fused to vinculin. This produced a significant increase in adhesion lifetime and adhesion size in cells expressing MS2- β-actin mRNA. Additionally, cell velocity was significantly reduced in cells with tethered β-actin mRNA. These experimental results lead us to conclude that β-actin mRNA compartmentalization to adhesions, mediated by ZBP1, strengthen adhesions and therefore produce an asymmetric force distribution within the cell to control its directionality.
Supported by: GM84364 to RHS; T32 GM007491 to ZBK 348
β1- and β3-integrins move and function as distinct adhesion units inside cell adhesions. G. Giannone1, O. Rossier1, V. Octeau2, J-B. Sibarita1, C. Leduc2, D. Nair1, L. Duchesne3, O. Destaing4, V. Gatterdam5, C. Albiges-Rizo4, R. Tampé5, D. Fernig3, L. Cognet2, D. Choquet1, B. Lounis2; 1Interdisciplinary Institute for NeuroScience, UMR5297, Université de Bordeaux and CNRS, 33077 Bordeaux, France, Bordeaux, France, 2Laboratoire Photonique, Numérique et Nanosciences (LP2N) Institut d'Optique Graduate School, CNRS and Université Bordeaux, France, 3Institute of Integrative Biology, Crown Street, University of Liverpool, Liverpool L69 7ZB, UK, 4Institut Albert Bonniot, Université Joseph Fourier; INSERM U823; CNRS ERL 3148 Grenoble, France, 5Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Frankfurt a.M., Germany
Focal adhesions (FAs) are adhesive structures linking the cell to the extracellular matrix (ECM) and constitute molecular platforms for biochemical and mechanical signals that control cell adhesion, migration, growth, differentiation and apoptosis. Integrin receptors are core components of FAs, where they trigger signaling and connect the ECM to the actin cytoskeleton (F-actin) via recruitment of intracellular scaffolds such as talin. Different classes of αβ-integrin heterodimers perform distinct functions and are simultaneously present in a FA. The static nanoscale organization of FAs has been recently described, with integrins being at the base of a vertical multilamelar protein organization ending with F-actin connection. However, the nanoscale dynamics of integrins within FAs is still unknown. Here we show, using super- resolution microscopy and single particle tracking, that integrins could move and function as nanoclusters within FAs. β3- and β1-integrins form distinct homotypic nanoclusters displaying specific dynamics within the same FA. β3-integrins undergo repeated activation cycles within a FA, seen as rapid switches between levels of free-diffusion, confinement, and immobilization
lasting less than a hundred seconds. Talin binding triggers immobilization more than interaction with the ECM, while both control immobilization duration, suggesting that talin and ECM cooperate to fully activate integrins inside FAs. Comparing β1- and β3-integrins, we showed an increased density of β3-integrin immobilization in FAs, supporting specific functions for each type of β-integrin in the same FA. Thus, this dynamic nanoscale partitioning of homotypic β- integrin nanoclusters within FAs could control local forces and signaling necessary for integrin- mediated cellular processes such as adhesion and migration.
349
The Endosomal Signaling Adaptor APPL1 Impairs Cell Migration by Inhibiting the Turnover of Adhesions at the Leading Edge.
J. A. Broussard1, W-H. Lin1, D. Majumdar1, B. Anderson1, D. J. Webb1,2; 1Biological Sciences, Vanderbilt University, Nashville, TN, 2Cancer Biology, Vanderbilt University
Cell migration is a complex process that requires the coordination of signaling events that take place in distinct locations within the cell. Adaptor proteins are emerging as key modulators of spatially integrated processes because of their ability to localize to different subcellular compartments and bring together important signaling proteins at these sites. However, the role that adaptor proteins play in regulating cell migration is not well understood. Here, we show a novel function for the adaptor protein containing a pleckstrin homology (PH), phosphotyrosine binding (PTB), and leucine zipper motif denoted APPL1 in modulating cell migration. APPL1 impairs the turnover of adhesions at the leading edge of cells thereby inhibiting their migration. The ability of APPL1 to impair migration is dependent on its PTB domain, which interacts with the serine/threonine kinase Akt, suggesting the interaction of APPL1 with Akt is important for its effect on migration. Interestingly, APPL1 decreases the amount of active Akt in cells. Using a mutant-based approach, we further show that APPL1 modulates migration and adhesion dynamics via a mechanism that involves regulation of Akt function. An APPL1 mutant which is unable to localize to endosomal membranes no longer has an effect on migration or adhesion turnover, indicating that APPL1 endosomal localization is required for its ability to regulate these processes. Furthermore, APPL1 is found in vesicular structures containing the non-receptor tyrosine kinase Src. Src has been shown to regulate Akt function through the phosphorylation of two Akt tyrosine residues, and intriguingly, we have found that APPL1 reduces the tyrosine phosphorylation of Akt. Therefore, we propose a model in which APPL1 regulates adhesion dynamics and cell migration by altering Src-mediated tyrosine phosphorylation of Akt. Our results further underscore the importance of adaptor proteins in modulating the flow of information through signaling pathways by demonstrating a critical new function for APPL1 in regulating cell migration and adhesion turnover.
350
TKS5 regulates invadopodium membrane anchoring in breast cancer cells.
V. Sharma1, J. Condeelis1; 1Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY
A growing body of research implicates invadopodia in cancer cell invasion and metastasis (Oser et al., 2009, J Cell Biochem.; Stylli et al., 2008, J Clin. Neurosci.). Invadopodia are protrusive structures of cancer cells, 0.5-1 micron in diameter and 1-10 microns long. The primary function of these structures is to degrade ECM and protrude into degraded spaces, which creates a passage (like a tunnel) through the extra-cellular space, which cells utilize to migrate from the site of primary tumor to enter the bloodstream and eventually metastasize at distant sites to make secondary tumors. To understand the signaling pathways during the assembly of invadopodia in breast cancer cells, we did live-cell time-lapse imaging to visualize the order of
arrival of the proteins of the invadopodium precursor, N-WASp, Cortactin and Tks5. Our data indicate that Cortactin and N-WASp arrive together to form the invadopodium precursor core, followed by Tks5 recruitment to the complex. We found that Tks5 is not required for invadopodium precursor formation but is needed for the stabilization and anchoring of invadopodium precursors. Tks5 has been shown to interact with 3’ phosphoinositides (Abram et al., 2003, J Biol. Chem.), and recently PI(3,4,5)P3 was shown to localize at invadopodia (Yamaguchi et al., 2011, J. Cell Biol.). We show that PI(3,4)P2 localizes to invadopodial precursors and appears to be the main binding partner ofTks5 as a link between the precursor core and the cell membrane.
351
A novel focal adhesion TRIM protein regulates focal adhesion disassembly.
P. Uchil1, T. Pawliczek1, T. Reynolds1, S. Ding1, A. Hinz1, R. Floyd1, H. Yang2, Y. Xiong2, W. Mothes1; 1MMPATH, Yale University, New Haven, CT, 2MB&B, Yale University, New Haven, CT The tripartite motif (TRIM) family of proteins play important roles in diverse cellular functions such as cell differentiation, oncogenesis and innate immunity. Here we identify a TRIM protein as a novel component of focal adhesions. This TRIM protein is recruited to focal adhesions by an interaction between its coiled coil domain and the LD2 motif of paxillin. It is recruited to focal adhesions early in a myosin-II-independent manner. But unlike any other focal adhesion component, it remains stably bound, forming a long-lived focal adhesion scaffold. Cells lacking the TRIM protein are impaired in cell migration due to a defect in focal adhesion disassembly. We hypothesize that the TRIM protein senses cues delivered by microtubuli to initiate focal adhesion disassembly because microtubules correctly target focal adhesion, but fail to induce focal adhesion turnover. Given the importance of the TRIM protein in the dynamic turnover of focal adhesions, it is predicted to play a critical role in metastasis and oncogenesis.
352
Fascin phosphorylation is necessary for efficient turnover of focal adhesions. N. Elkhatib1, M. Neu1, D. Louvard1, D. Matic Vignjevic1; 1Institut Curie, Paris, France
Efficient cell migration depends on phospho-regulation of the actin bundling protein fascin. Cells expressing phospho-mimetic fascin mutant, S39E, had difficulties to polarize and protrude, while cells expressing non-phosphorylatable fascin mutant, S39A, had problems retracting the uropod (Hashimoto et al, 2007). Our hypothesis is that cycles of fascin phosphorylation/dephosphorylation are necessary for directional cell motility: non- phosphorylated fascin for filopodia formation and cell guidance and phosphorylated fascin for focal adhesions (FAs) turnover and cell body retraction. Using TIRF microscopy we found that GFP-tagged wt and S39A mutant fascin, but not S39E, were enriched in the FAs marked with Cherry-tagged vinculin, paxillin, α- actinin or zyxin. In fascin-depleted cells, FAs were thinner and longer with slower turnover rate. The FA turnover rate was rescued by expression of S39E mutant, but not with S39A, suggesting that fascin phosphorylation is required for rapid turnover of FAs. Using a FAs disassembly assay (Ezratty et al, 2005), we found that fascin phosphorylation is required for FAs disassembly after microtubule re-growth. Our current model is that fascin plays a role in FAs assembly by bundling actin filaments and its phosphorylation is required for efficient FAs disassembly.
353
An integrin endocytic recycling pathway mediated by FAK and Src controls the polarized reassembly of focal adhesions after their disassembly.
G. Nader1, E. Ezratty1,2, G. Gundersen1; 1Columbia University, New York, NY, 2Rockefeller University,
Cell migration is a multi-step process that involves focal adhesion (FA) turnover and recycling of integrins. FA disassembly is an endocytic process that involves focal adhesion kinase (FAK), dynamin and clathrin (Ezratty, E. et al., NCB, 2005; JCB, 2009; Chao, W. and Kunz, J., FEBS, 2009). Little is known about the recycling of integrin following FA disassembly and whether FA components might play a role in the trafficking of endocytosed integrin. We used microtubule regrowth to synchronously induce FA disassembly and integrin endocytosis to examine whether endocytic recycling is coupled to FA reassembly. Focal adhesion reassembly commenced 30 min after microtubule-induced FA disassembly and occurred preferentially at the leading edge of cells. Integrin and active FAK were detected in both Rab 5- and Rab11-positive endosomes and FA reassembly required both Rab 5 and Rab 11. Src kinase participates in FA reassembly following disassembly induced by microtubules (Yeo, M. et al., MBC, 2006), so we explored the possible link between FAK and Src during the disassembly/reassembly cycle. Notably, both Src inhibitor (PP2) and FAK inhibitor (PF228) reversely blocked FA reassembly (without affecting disassembly), but only PP2 blocked the recycling of integrins to the cell surface. Furthermore, FAK and Src colocalized at the Rab 11 endocytic recycling compartment in either PP2 or PF228 treated cells. FAK mutants FAK-Y397F (autophosphorylation/Src binding site mutant) and FAK- K454R (kinase dead) rescued FA disassembly but not reassembly in FAK-/- cells, further suggesting that FAK kinase activity is essential for FA reassembly but not for disassembly.