Skip to main content
SpringerLink
Log in

VASP-dependent regulation of actin cytoskeleton rigidity, cell adhesion, and detachment

  • Original paper
  • Published:
Histochemistry and Cell Biology Aims and scope Submit manuscript

Abstract

Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are established regulators of actin-based motility, platelet aggregation, and growth cone guidance. However, the molecular mechanisms involved essentially remain elusive. Here we report on a novel mechanism of VASP action, namely the regulation of tensile strength, contractility, and rigidity of the actin cytoskeleton. Compared to wild-type cells fibroblasts derived from VASP-deficient mice have thicker and more stable actin stress fibres. Furthermore focal adhesions are enlarged, myosin light chain phosphorylation is increased, and the rigidity of the filament-supported plasma membrane is elevated about three- to fourfold, as is evident from atomic force microscopy. Moreover, fibronectin-coated beads adhere stronger to the surface of VASP-deficient cells. The resistance of these beads to mechanical displacement by laser tweezers is dramatically increased in an F-actin-dependent mode. Cytoskeletal stabilization coincides with slower cell adhesion and detachment, while overall adhesion is increased. Interestingly, many of these effects observed in VASP (−/−) cells are recapitulated in VASP-overexpressing cells, hinting towards a balanced stoichiometry necessary for appropriate VASP function. Taken together, our results suggest that VASP regulates surface protrusion formation and cell adhesion through modulation of the mechanical properties of the actin cytoskeleton.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11

Similar content being viewed by others

References

  • Abassi YA, Vuori K (2002) Tyrosine 221 in Crk regulates adhesion-dependent membrane localization of Crk and Rac and activation of Rac signaling. Embo J 21:4571–4582

    Google Scholar 

  • Anderson SI, Behrendt B, Machesky LM, Insall RH, Nash GB (2003) Linked regulation of motility and integrin function in activated migrating neutrophils revealed by interference in remodelling of the cytoskeleton. Cell Motil Cytoskeleton 54:135–146

    Google Scholar 

  • Arthur WT, Noren NK, Burridge K (2002) Regulation of Rho family GTPases by cell-cell and cell-matrix adhesion. Biol Res 35:239–246

    Google Scholar 

  • Aszodi A, Pfeifer A, Ahmad M, Glauner M, Zhou XH, Ny L, Andersson KE, Kehrel B, Offermanns S, Fassler R (1999) The vasodilator-stimulated phosphoprotein (VASP) is involved in cGMP- and cAMP-mediated inhibition of agonist-induced platelet aggregation, but is dispensable for smooth muscle function. Embo J 18:37–48

    Google Scholar 

  • Auerbuch V, Loureiro JJ, Gertler FB, Theriot JA, Portnoy DA (2003) Ena/VASP proteins contribute to Listeria monocytogenes pathogenesis by controlling temporal and spatial persistence of bacterial actins-based motility. Mol Microbiol 49:1361–1375

    Google Scholar 

  • Bachmann C, Fischer L, Walter U, Reinhard M (1999) The EVH2 domain of the vasodilator-stimulated phosphoprotein mediates tetramerization, F-actin binding, and actin bundle formation. J Biol Chem 274:23549–23557

    Google Scholar 

  • Balaban NQ, Schwarz US, Riveline D, Goichberg P, Tzur G, Sabanay I, Mahalu D, Safran S, Bershadsky A, Addadi L, Geiger B (2001) Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates. Nat Cell Biol 3:466–472

    Google Scholar 

  • Bamburg JR (1999) Proteins of the ADF/cofilin family: essential regulators of actin dynamics. Annu Rev Cell Dev Biol 15:185–230

    Google Scholar 

  • Bashaw GJ, Kidd T, Murray D, Pawson T, Goodman CS (2000) Repulsive axon guidance: abelson and enabled play opposing roles downstream of the roundabout receptor. Cell 101:703–715

    Google Scholar 

  • Baumgartner W, Schutz GJ, Wiegand J, Golenhofen N, Drenckhahn D (2003) Cadherin function probed by laser tweezer and single molecule fluorescence in vascular endothelial cells. J Cell Sci 116:1001–1011

    Google Scholar 

  • Bear JE, Loureiro JJ, Libova I, Fassler R, Wehland J, Gertler FB (2000) Negative regulation of fibroblast motility by Ena/VASP proteins. Cell 101:717–728

    Google Scholar 

  • Bear JE, Svitkina TM, Krause M, Schafer DA, Loureiro JJ, Strasser GA, Maly IV, Chaga OY, Cooper JA, Borisy GG, Gertler FB (2002) Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility. Cell 109:509–521

    Google Scholar 

  • Bearer EL, Prakash JM, Manchester RD, Allen PG (2000) VASP protects actin filaments from gelsolin: an in vitro study with implications for platelet actin reorganizations. Cell Motil Cytoskeleton 47:351–364

    Google Scholar 

  • Bennett JS, Zigmond S, Vilaire G, Cunningham ME, Bednar B (1999) The platelet cytoskeleton regulates the affinity of the integrin alpha(IIb)beta(3) for fibrinogen. J Biol Chem 274:25301–25307

    Google Scholar 

  • Binnig G, Quate CF, Gerber C (1986) Atomic force microscope. Physical Review Letters 56:930–933

    Google Scholar 

  • Bishop AL, Hall A (2000) Rho GTPases and their effector proteins. Biochem J 348 (Pt 2):241–255

    Google Scholar 

  • Blanchoin L, Pollard TD, Mullins RD (2000) Interactions of ADF/cofilin, Arp2/3 complex, capping protein and profilin in remodeling of branched actin filament networks. Curr Biol 10:1273–1282

    Google Scholar 

  • Burack WR, Shaw AS (2000) Signal transduction: hanging on a scaffold. Curr Opin Cell Biol 12:211–216

    Google Scholar 

  • Burridge K, Chrzanowska-Wodnicka M (1996) Focal adhesions, contractility, and signaling. Annu Rev Cell Dev Biol 12:463–518

    Google Scholar 

  • Burwen SJ, Satir BH (1977) Plasma membrane folds on the mast cell surface and their relationship to secretory activity. J Cell Biol 74:690–697

    Google Scholar 

  • Calderwood DA, Shattil SJ, Ginsberg MH (2000) Integrins and actin filaments: reciprocal regulation of cell adhesion and signaling. J Biol Chem 275:22607–22610

    Google Scholar 

  • Castellano F, Le Clainche C, Patin D, Carlier MF, Chavrier P (2001) A WASp-VASP complex regulates actin polymerization at the plasma membrane. Embo J 20:5603–5614

    Google Scholar 

  • Chew TL, Masaracchia RA, Goeckeler ZM, Wysolmerski RB (1998) Phosphorylation of non-muscle myosin II regulatory light chain by p21- activated kinase (gamma-PAK). J Muscle Res Cell Motil 19:839–854

    Google Scholar 

  • Choquet D, Felsenfeld DP, Sheetz MP (1997) Extracellular matrix rigidity causes strengthening of integrin–cytoskeleton linkages. Cell 88:39–48

    Google Scholar 

  • Colavita A, Culotti JG (1998) Suppressors of ectopic UNC-5 growth cone steering identify eight genes involved in axon guidance in Caenorhabditis elegans. Dev Biol 194:72–85

    Google Scholar 

  • del Pozo MA, Price LS, Alderson NB, Ren XD, Schwartz MA (2000) Adhesion to the extracellular matrix regulates the coupling of the small GTPase Rac to its effector PAK. Embo J 19:2008–2014

    Google Scholar 

  • del Pozo MA, Kiosses WB, Alderson NB, Meller N, Hahn KM, Schwartz MA (2002) Integrins regulate GTP-Rac localized effector interactions through dissociation of Rho-GDI. Nat Cell Biol 4:232–239

    Google Scholar 

  • del Pozo MA, Alderson NB, Kiosses WB, Chiang HH, Anderson RG, Schwartz MA (2004) Integrins regulate Rac targeting by internalization of membrane domains. Science 303:839–842

    Google Scholar 

  • Escolar G, Leistikow E, White JG (1989) The fate of the open canalicular system in surface and suspension-activated platelets. Blood 74:1983–1988

    Google Scholar 

  • Fukata M, Nakagawa M, Itoh N, Kawajiri A, Yamaga M, Kuroda S, Kaibuchi K (2001) Involvement of IQGAP1, an effector of Rac1 and Cdc42 GTPases, in cell–cell dissociation during cell scattering. Mol Cell Biol 21:2165–2183

    Google Scholar 

  • Gambaryan S, Hauser W, Kobsar A, Glazova M, Walter U (2001) Distribution, cellular localization, and postnatal development of VASP and Mena expression in mouse tissues. Histochem Cell Biol 116:535–543

    Google Scholar 

  • García Arguinzonis MI, Galler AB, Walter U, Reinhard M, Simm A (2002) Increased spreading, Rac/p21-activated kinase (PAK) activity, and compromised cell motility in cells deficient in vasodilator-stimulated phosphoprotein (VASP). J Biol Chem 277:45604–45610

    Google Scholar 

  • Geiger B, Bershadsky A (2001) Assembly and mechanosensory function of focal contacts. Curr Opin Cell Biol 13:584–592

    Google Scholar 

  • Geiger B, Bershadsky A, Pankov R, Yamada KM (2001) Transmembrane crosstalk between the extracellular matrix–cytoskeleton crosstalk. Nat Rev Mol Cell Biol 2:793–805

    Google Scholar 

  • Geiger B, Bershadsky A (2002) Exploring the neighborhood: adhesion-coupled cell mechanosensors. Cell 110:139–142

    Google Scholar 

  • Gitai Z, Yu TW, Lundquist EA, Tessier-Lavigne M, Bargmann CI (2003) The netrin receptor UNC-40/DCC stimulates axon attraction and outgrowth through enabled and, in parallel, Rac and UNC-115/AbLIM. Neuron 37:53–65

    Google Scholar 

  • Griffiths EK, Penninger JM (2002) Communication between the TCR and integrins: role of the molecular adapter ADAP/Fyb/Slap. Curr Opin Immunol 14:317–322

    Google Scholar 

  • Grosse R, Copeland JW, Newsome TP, Way M, Treisman R (2003) A role for VASP in RhoA-Diaphanous signalling to actin dynamics and SRF activity. Embo J 22:3050–3061

    Google Scholar 

  • Han YH, Chung CY, Wessels D, Stephens S, Titus MA, Soll DR, Firtel RA (2002) Requirement of a vasodilator-stimulated phosphoprotein family member for cell adhesion, the formation of filopodia, and chemotaxis in dictyostelium. J Biol Chem 277:49877–49887

    Google Scholar 

  • Harbeck B, Huttelmaier S, Schluter K, Jockusch BM, Illenberger S (2000) Phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) regulates its interaction with actin. J Biol Chem 275:30817–30825

    Google Scholar 

  • Hauser W, Knobeloch KP, Eigenthaler M, Gambaryan S, Krenn V, Geiger J, Glazova M, Rohde E, Horak I, Walter U, Zimmer M (1999) Megakaryocyte hyperplasia and enhanced agonist-induced platelet activation in vasodilator-stimulated phosphoprotein knockout mice. Proc Natl Acad Sci USA 96:8120–8125

    Google Scholar 

  • Heidemann SR, Kaech S, Buxbaum RE, Matus A (1999) Direct observations of the mechanical behaviors of the cytoskeleton in living fibroblasts. J Cell Biol 145:109–122

    Google Scholar 

  • Hüttelmaier S, Harbeck B, Steffens O, Messerschmidt T, Illenberger S, Jockusch BM (1999) Characterization of the actin binding properties of the vasodilator-stimulated phosphoprotein VASP. FEBS Lett 451:68–74

    Google Scholar 

  • Juliano RL (2002) Signal transduction by cell adhesion receptors and the cytoskeleton: functions of integrins, cadherins, selectins, and immunoglobulin-superfamily members. Annu Rev Pharmacol Toxicol 42:283–323

    Google Scholar 

  • Kiosses WB, Daniels RH, Otey C, Bokoch GM, Schwartz MA (1999) A role for p21-activated kinase in endothelial cell migration. J Cell Biol 147:831–844

    Google Scholar 

  • Krause M, Bear JE, Loureiro JJ, Gertler FB (2002) The Ena/VASP enigma. J Cell Sci 115:4721–4726

    Google Scholar 

  • Krugmann S, Jordens I, Gevaert K, Driessens M, Vandekerckhove J, Hall A (2001) Cdc42 induces filopodia by promoting the formation of an IRSp53:Mena complex. Curr Biol 11:1645–1655

    Google Scholar 

  • Legg JA, Machesky LM (2004) MRL proteins: leading Ena/VASP to Ras GTPases. Nat Cell Biol 6:1015–1017

    Google Scholar 

  • Levchenko A, Bruck J, Sternberg PW (2000) Scaffold proteins may biphasically affect the levels of mitogen-activated protein kinase signaling and reduce its threshold properties. Proc Natl Acad Sci USA 97:5818–5823

    Google Scholar 

  • Liddington RC, Bankston LA, de Pereda JM (2003) Cell adhesion: a FERM grasp of membrane dynamics. Curr Biol 13:R94–R95

    Google Scholar 

  • Massberg S, Gruener S, Konrad I, García Arguinzonis MI, Eigenthaler M, Hemler K, Kersting J, Schulz C, Mueller I, Besta F, Nieswandt B, Heinzmann U, Walter U, Gawaz M (2004) Enhanced in vivo platelet adhesion in vasodilator-stimulated phosphoprotein (VASP)-deficient mice. Blood 103:136–142

    Google Scholar 

  • Nishizaka T, Shi Q, Sheetz MP (2000) Position-dependent linkages of fibronectin-integrin-cytoskeleton. Proc Natl Acad Sci USA 97:692–697

    Google Scholar 

  • Nobes CD, Hall A (1995) Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81:53–62

    Google Scholar 

  • Pollard TD, Borisy GG (2003) Cellular motility driven by assembly and disassembly of actin filaments. Cell 112:453–465

    Google Scholar 

  • Price CJ, Brindle NP (2000) Vasodilator-stimulated phosphoprotein is involved in stress-fiber and membrane ruffle formation in endothelial cells. Arterioscler Thromb Vasc Biol 20:2051–2016

    Google Scholar 

  • Price LS, Leng J, Schwartz MA, Bokoch GM (1998) Activation of Rac and Cdc42 by integrins mediates cell spreading. Mol Biol Cell 9:1863–1871

    Google Scholar 

  • Raucher D, Sheetz MP (1999) Characteristics of a membrane reservoir buffering membrane tension. Biophys J 77:1992–2002

    Google Scholar 

  • Raucher D, Sheetz MP (2000) Cell spreading and lamellipodial extension rate is regulated by membrane tension. J Cell Biol 148:127–136

    Google Scholar 

  • Raucher D, Stauffer T, Chen W, Shen K, Guo S, York JD, Sheetz MP, Meyer T (2000) Phosphatidylinositol 4,5-bisphosphate functions as a second messenger that regulates cytoskeleton-plasma membrane adhesion. Cell 100:221–228

    Google Scholar 

  • Reinhard M, Halbrügge M, Scheer U, Wiegand C, Jockusch BM, Walter U (1992) The 46/50 kDa phosphoprotein VASP purified from human platelets is a novel protein associated with actin filaments and focal contacts. Embo J 11:2063–2070

    Google Scholar 

  • Reinhard M, Jarchau T, Walter U (2001) Actin-based motility: stop and go with Ena/VASP proteins. Trends Biochem Sci 26:243–249

    Google Scholar 

  • Ridley AJ (2001) Rho GTPases and cell migration. J Cell Sci 114:2713–2722

    Google Scholar 

  • Samarin S, Romero S, Kocks C, Didry D, Pantaloni D, Carlier MF (2003) How VASP enhances actin-based motility. J Cell Biol 163:131–142

    Google Scholar 

  • Sheetz MP (2001) Cell control by membrane–cytoskeleton adhesion. Nat Rev Mol Cell Biol 2:392–396

    Google Scholar 

  • Sheikh S, Gratzer WB, Pinder JC, Nash GB (1997) Actin polymerisation regulates integrin-mediated adhesion as well as rigidity of neutrophils. Biochem Biophys Res Commun 238:910–915

    Google Scholar 

  • Simon A, Cohen-Bouhacina T, Porte MC, Aime JP, Amedee J, Bareille R, Baquey C (2003) Characterization of dynamic cellular adhesion of osteoblasts using atomic force microscopy. Cytometry 54A:36–47

    Google Scholar 

  • Skoble J, Auerbuch V, Goley ED, Welch MD, Portnoy DA (2001) Pivotal role of VASP in Arp2/3 complex-mediated actin nucleation, actin branch-formation, and Listeria monocytogenes motility. J Cell Biol 155:89–100

    Google Scholar 

  • Smolenski A, Poller W, Walter U, Lohmann SM (2000) Regulation of human endothelial cell focal adhesion sites and migration by cGMP-dependent protein kinase I. J Biol Chem 275:25723–25732

    Google Scholar 

  • Suter DM, Forscher P (2000) Substrate–cytoskeletal coupling as a mechanism for the regulation of growth cone motility and guidance. J Neurobiol 44:97–113

    Google Scholar 

  • von Wichert G, Haimovich B, Feng GS, Sheetz MP (2003) Force-dependent integrin-cytoskeleton linkage formation requires downregulation of focal complex dynamics by Shp2. Embo J 22:5023–5035

    Google Scholar 

  • Walders-Harbeck B, Khaitlina SY, Hinssen H, Jockusch BM, Illenberger S (2002) The vasodilator-stimulated phosphoprotein promotes actin polymerisation through direct binding to monomeric actin. FEBS Lett 529:275–280

    Google Scholar 

  • Wessel D, Flugge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138:141–143

    Google Scholar 

  • Yu TW, Hao JC, Lim W, Tessier-Lavigne M, Bargmann CI (2002) Shared receptors in axon guidance: SAX-3/Robo signals via UNC-34/Enabled and a netrin-independent UNC-40/DCC function. Nat Neurosci 5:1147–1154

    Google Scholar 

Download references

Acknowledgments

The authors thank Elke Baumeister, Agnes Weth, Elke Butt, and Stepan Gambaryan for help and reagents, and Ulrich Walter for continuous support and discussions. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 487 B4 & B5, SFB 355 B4 & C8, and Re1011/3-2).

Author information

Author notes

  1. Matthias Reinhard

    Present address: immunoGlobe Antikoerpertechnik GmbH, Rudolf-Diesel-Str. 8A, 97267, Himmelstadt, Germany

  2. Annette B. Galler

    Present address: Onkologie, Merck Pharma GmbH, Location: AL17/145, Alsfelder Str. 17, 64289, Darmstadt, Germany

  3. Maísa I. García Arguinzonis

    Present address: Centro de Investigaciones Cardiovasculares CSIC-ICCC, Hospital de la Santa Creu i Sant Pau - Barcelona, Av. Antoni Maria Claret 167, 08025, Barcelona, Spain

  4. Werner Baumgartner

    Present address: Department of Cellular Neurobionics, Institute of Biology 2, RWTH-Aachen, Kopernikusstr. 16, 52056, Aachen, Germany

  5. Monika Kuhn

    Present address: Rudolf-Virchow-Center and Department of Dermatology, Molecular Cell Dynamics Group, University of Würzburg, Josef-Schneider-Str. 2, 97080, Würzburg, Germany

  6. Albert Smolenski

    Present address: Institute for Biochemistry II, University Clinic, Bldg. 75, Theodor-Stern-Kai 7, 60590, Frankfurt, Germany

  7. Andreas Simm

    Present address: Clinic for Heart and Thorax Surgery, University of Halle, Ernst-Grube-Str. 40, 06120, Halle, Germany

Authors and Affiliations

  1. Institute for Clinical Biochemistry and Pathobiochemistry, University of Würzburg, Josef-Schneider-Str. 2, 97080, Würzburg, Germany

    Annette B. Galler, Maísa I. García Arguinzonis, Monika Kuhn, Albert Smolenski, Andreas Simm & Matthias Reinhard

  2. Institute of Anatomy and Cell Biology, University of Würzburg, Koellikerstr. 6, 97070, Würzburg, Germany

    Werner Baumgartner

Authors
  1. Annette B. Galler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maísa I. García Arguinzonis
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Werner Baumgartner
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Monika Kuhn
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Albert Smolenski
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Andreas Simm
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Matthias Reinhard
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Matthias Reinhard.

Additional information

Annette B. Galler, Maísa I. García Arguinzonis these authors contributed equally to this work

Rights and permissions

Reprints and permissions

About this article

Cite this article

Galler, A.B., García Arguinzonis, M.I., Baumgartner, W. et al. VASP-dependent regulation of actin cytoskeleton rigidity, cell adhesion, and detachment. Histochem Cell Biol 125, 457–474 (2006). https://doi.org/10.1007/s00418-005-0091-z

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00418-005-0091-z

Keywords

Search

Navigation