Paxillin-dependent stimulation of microtubule catastrophes at focal adhesion sites

An organized microtubule array is essential for the polarized motility of fibroblasts. Dynamic microtubules closely interact with focal adhesion sites in migrating cells. Here, we examined the effect of focal adhesions on microtubule dynamics. We observed that the probability of microtubule catastrophes (transitions from growth to shrinkage) was seven times higher at focal adhesions than elsewhere. Analysis of the dependence between the microtubule growth rate and catastrophe probability throughout the cytoplasm revealed that a nonspecific (mechanical or spatial) factor provided a minor contribution to the catastrophe induction by decreasing microtubule growth rate at adhesions. Strikingly, at the same growth rate, the probability of catastrophes was significantly higher at adhesions than elsewhere, indicative of a site-specific biochemical trigger. The observed catastrophe induction occurred at adhesion domains containing the scaffolding protein paxillin that has been shown previously to interact with tubulin. Furthermore, replacement of full-length paxillin at adhesion sites by microinjected paxillin LIM2-LIM3 domains suppressed microtubule catastrophes exclusively at adhesions. We suggest that paxillin influences microtubule dynamics at focal adhesions by serving as a scaffold for a putative catastrophe factor and/or regulating its exposure to microtubules.

[1]  N. Balaban,et al.  Adhesion-dependent cell mechanosensitivity. , 2003, Annual review of cell and developmental biology.

[2]  P. Janmey,et al.  Tissue Cells Feel and Respond to the Stiffness of Their Substrate , 2005, Science.

[3]  E. Zamir,et al.  Molecular complexity and dynamics of cell-matrix adhesions. , 2001, Journal of cell science.

[4]  A. Hyman,et al.  Microtubule polymerases and depolymerases. , 2007, Current opinion in cell biology.

[5]  K. Rottner,et al.  Interplay between Rac and Rho in the control of substrate contact dynamics , 1999, Current Biology.

[6]  H. Gradin,et al.  Control of microtubule dynamics by oncoprotein 18: dissection of the regulatory role of multisite phosphorylation during mitosis , 1997, Molecular and cellular biology.

[7]  J. Swedlow,et al.  Aurora B regulates MCAK at the mitotic centromere. , 2004, Developmental cell.

[8]  J. Resau,et al.  The adaptor protein Tks5/Fish is required for podosome formation and function, and for the protease-driven invasion of cancer cells. , 2005, Cancer cell.

[9]  P. Polakis.  The many ways of Wnt in cancer. , 2007, Current opinion in genetics & development.

[10]  Daniel Axelrod.  Total internal reflection fluorescence microscopy. , 1989, Methods in cell biology.

[11]  C. Turner,et al.  Paxillin-dependent paxillin kinase linker and p21-activated kinase localization to focal adhesions involves a multistep activation pathway. , 2002, Molecular biology of the cell.

[12]  Irina Kaverina,et al.  Microtubules meet substrate adhesions to arrange cell polarity. , 2003, Current opinion in cell biology.

[13]  R. Milo,et al.  A paxillin tyrosine phosphorylation switch regulates the assembly and form of cell-matrix adhesions , 2006, Journal of Cell Science.

[14]  B. Danowski.  Microtubule dynamics in serum-starved and serum-stimulated Swiss 3T3 mouse fibroblasts: implications for the relationship between serum-induced contractility and microtubules. , 1998, Cell motility and the cytoskeleton.

[15]  S. Étienne-Manneville.  Actin and Microtubules in Cell Motility: Which One is in Control? , 2004, Traffic.

[16]  T. Mitchison,et al.  Differentiation of cytoplasmic and meiotic spindle assembly MCAK functions by Aurora B-dependent phosphorylation. , 2004, Molecular biology of the cell.

[17]  K. Rottner,et al.  Zyxin is not colocalized with vasodilator-stimulated phosphoprotein (VASP) at lamellipodial tips and exhibits different dynamics to vinculin, paxillin, and VASP in focal adhesions. , 2001, Molecular biology of the cell.

[18]  K. Wennerberg,et al.  Integrin signaling to the actin cytoskeleton. , 2003, Current opinion in cell biology.

[19]  Y. Kawasaki,et al.  Asef, a link between the tumor suppressor APC and G-protein signaling. , 2000, Science.

[20]  C. Cabañas,et al.  Paxillin Localizes to the Lymphocyte Microtubule Organizing Center and Associates with the Microtubule Cytoskeleton* , 2000, The Journal of Biological Chemistry.

[21]  C. Turner,et al.  Roles for the tubulin- and PTP-PEST-binding paxillin LIM domains in cell adhesion and motility. , 2002, The international journal of biochemistry & cell biology.

[22]  C. Fagerstrom,et al.  Cell cycle dependent changes in microtubule dynamics in living cells expressing GFP-alpha tubulin , 2000 .

[23]  B. Geiger,et al.  Contact formation during fibroblast locomotion: involvement of membrane ruffles and microtubules , 1988, The Journal of cell biology.

[24]  M. Brown,et al.  Serine and threonine phosphorylation of the paxillin LIM domains regulates paxillin focal adhesion localization and cell adhesion to fibronectin. , 1998, Molecular biology of the cell.

[25]  C. Waterman-Storer,et al.  Cell motility: can Rho GTPases and microtubules point the way? , 2001, Journal of cell science.

[26]  C. Waterman-Storer,et al.  Conserved microtubule–actin interactions in cell movement and morphogenesis , 2003, Nature Cell Biology.

[27]  Yoshiharu Kawaguchi,et al.  HDAC6 deacetylation of tubulin modulates dynamics of cellular adhesions , 2007, Journal of Cell Science.

[28]  T. Akiyama,et al.  Interaction with IQGAP1 links APC to Rac1, Cdc42, and actin filaments during cell polarization and migration. , 2004, Developmental cell.

[29]  Michael P. Sheetz,et al.  The relationship between force and focal complex development , 2002, The Journal of cell biology.

[30]  M. Brown,et al.  Identification of LIM3 as the principal determinant of paxillin focal adhesion localization and characterization of a novel motif on paxillin directing vinculin and focal adhesion kinase binding , 1996, The Journal of cell biology.

[31]  J. Parsons,et al.  Focal adhesion kinase: the first ten years , 2003, Journal of Cell Science.

[32]  Irina Kaverina,et al.  Microtubule Targeting of Substrate Contacts Promotes Their Relaxation and Dissociation , 1999, The Journal of cell biology.

[33]  C. Nobes,et al.  Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia , 1995, Cell.

[34]  Joe Tien,et al.  Mechanotransduction at cell-matrix and cell-cell contacts. , 2004, Annual review of biomedical engineering.

[35]  C. Turner,et al.  JCB Article , 2001 .

[36]  Marileen Dogterom,et al.  Dynamic instability of microtubules is regulated by force , 2003, The Journal of cell biology.

[37]  L. Cassimeris.  The oncoprotein 18/stathmin family of microtubule destabilizers. , 2002, Current opinion in cell biology.

[38]  Nasser M. Rusan,et al.  Cell Cycle-Dependent Changes in Microtubule Dynamics in Living Cells Expressing Green Fluorescent Protein-α Tubulin , 2001 .

[39]  C. Turner,et al.  Paxillin: adapting to change. , 2004, Physiological reviews.

[40]  S. Hanks,et al.  Focal adhesion kinase signaling activities and their implications in the control of cell survival and motility. , 2003, Frontiers in bioscience : a journal and virtual library.

[41]  E. Salmon,et al.  Actomyosin-based Retrograde Flow of Microtubules in the Lamella of Migrating Epithelial Cells Influences Microtubule Dynamic Instability and Turnover and Is Associated with Microtubule Breakage and Treadmilling , 1997, The Journal of cell biology.

[42]  Klemens Rottner,et al.  Targeting, Capture, and Stabilization of Microtubules at Early Focal Adhesions , 1998, The Journal of cell biology.

[43]  L. Amos,et al.  Microtubules and maps. , 2005, Advances in protein chemistry.

[44]  J. McIntosh,et al.  Spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching , 1984, The Journal of cell biology.

[45]  Masaaki Yoshigi,et al.  Mechanical force mobilizes zyxin from focal adhesions to actin filaments and regulates cytoskeletal reinforcement , 2005, The Journal of cell biology.

[46]  G. Gundersen,et al.  Distribution of detyrosinated microtubules in motile NRK fibroblasts is rapidly altered upon cell-cell contact: implications for contact inhibition of locomotion. , 1992, Cell motility and the cytoskeleton.

[47]  Yu-Li Wang,et al.  Tensile stress stimulates microtubule outgrowth in living cells. , 2001, Journal of cell science.

[48]  M. Kirschner,et al.  A microtubule-binding Rho-GEF controls cell morphology during convergent extension of Xenopus laevis , 2005, Development.

[49]  P Wadsworth,et al.  Regional regulation of microtubule dynamics in polarized, motile cells. , 1999, Cell motility and the cytoskeleton.

[50]  P Wadsworth,et al.  Observation and quantification of individual microtubule behavior in vivo: microtubule dynamics are cell-type specific , 1993, The Journal of cell biology.

[51]  J. Bulinski,et al.  GFP chimeras of E-MAP-115 (ensconsin) domains mimic behavior of the endogenous protein in vitro and in vivo. , 1999, Cell structure and function.

[52]  E. Salmon,et al.  E-MAP-115 (ensconsin) associates dynamically with microtubules in vivo and is not a physiological modulator of microtubule dynamics. , 1999, Journal of cell science.

[53]  G. Bokoch,et al.  Nucleotide exchange factor GEF-H1 mediates cross-talk between microtubules and the actin cytoskeleton , 2002, Nature Cell Biology.

[54]  G. von Dassow,et al.  MCAK associates with the tips of polymerizing microtubules , 2005, The Journal of cell biology.

[55]  M. Kirschner,et al.  Dynamic instability of microtubule growth , 1984, Nature.

[56]  P Wadsworth,et al.  Cell cycle-dependent changes in microtubule dynamics in living cells expressing green fluorescent protein-alpha tubulin. , 2001, Molecular biology of the cell.

[57]  Z. Kam,et al.  Early molecular events in the assembly of matrix adhesions at the leading edge of migrating cells , 2003, Journal of Cell Science.

[58]  Eleni Bazigou,et al.  Cell signaling and cancer , 2007, Genome Biology.

[59]  M. Gimona,et al.  Podosome formation in cultured A7r5 vascular smooth muscle cells requires Arp2/3-dependent de-novo actin polymerization at discrete microdomains , 2003, Journal of Cell Science.