T H Etel-00435843, J O U R N A Lversion O F C1 E- 24 L LNov B I 2009 O L O G Y Published January 28, 2008 Cell adaptive response to extracellu<strong>la</strong>r matrix <strong>de</strong>nsity is controlled by <strong>ICAP</strong>-1 – <strong>de</strong>pen<strong>de</strong>nt � 1 -integrin affi nity Ang é lique Millon-Fr é millon , 1,2,3 Daniel Bouvard , 1,2,3 Alexei Grichine , 2,4 Sandra Man<strong>et</strong>-Dup é , 1,2,3 Marc R. Block , 1,2,3 and Corinne Albiges-Rizo 1,2,3 1 Institut National <strong>de</strong> <strong>la</strong> Sant é <strong>et</strong> <strong>de</strong> <strong>la</strong> Recherche M é dicale U823, 2 Universit é Joseph Fourier, 3 Centre National <strong>de</strong> <strong>la</strong> Recherche Scientifi que, Equipe <strong>de</strong> Recherche Labellis é e 3148, and 4 Cell Imaging P<strong>la</strong>tform, Institut Albert Bonniot, 38042 Grenoble Ce<strong>de</strong>x 9, France Cell migration is an integrated process requiring the continuous coordinated assembly and disassembly of adhesion structures. How cells orchestrate adhesion turnover is only partially un<strong>de</strong>rstood. We provi<strong>de</strong> evi<strong>de</strong>nce for a novel mechanistic insight into focal adhesion (FA) dynamics by <strong>de</strong>monstrating that integrin cytop<strong>la</strong>smic domain – associated protein 1 (<strong>ICAP</strong>-1) slows down FA assembly. Live cell imaging, which was performed in both Icap-1 – <strong>de</strong>fi cient mouse embryonic fi brob<strong>la</strong>sts and cells expressing active � 1 integrin, shows that the integrin high Introduction Adhesion to ECM is primarily mediated by integrins, a family of h<strong>et</strong>erodimeric receptors ( Hynes, 2002 ) that cluster into adhesion sites. Focal adhesions (FAs) have been shown to form a scaffold that � rst sense and then transform extracellu<strong>la</strong>r cues into cellu<strong>la</strong>r responses and, in turn, transmit the contractile intracellu<strong>la</strong>r tensions to the ECM ( Bershadsky <strong>et</strong> al., 2003 ; Ingber, 2003; Chen <strong>et</strong> al., 2004 ). Because of their involvement in cell motility and matrix remo<strong>de</strong>ling, adhesion sites are necessarily dynamic structures that are able to assemble and disassemble. FA assembly takes p<strong>la</strong>ce at the leading edge of membrane protrusions, whereas disassembly occurs both at the cell rear and at the base of protrusions. The molecu<strong>la</strong>r mechanisms controlling FA turnover have been partly characterized. They require Rho family GTPases, integrin engagement, and coordinated interaction b<strong>et</strong>ween integrins and structural/signaling molecules as well as actin-binding proteins, actin micro � <strong>la</strong>ments, and microtubules ( Raftopoulou and Hall, 2004 ; Webb Correspon<strong>de</strong>nce to Corinne Albiges-Rizo: corinne.albiges-rizo@ujf-grenoble.fr Abbreviations used in this paper: FA, focal adhesion; FN, fi bronectin; <strong>ICAP</strong>-1, integrin cytop<strong>la</strong>smic domain – associated protein 1; MEF, mouse embryonic fi brob<strong>la</strong>st; MFI, mean fl uorescence intensity; VASP, vasodi<strong>la</strong>tor-stimu<strong>la</strong>ted phosphoprotein; VN, vitronectin; WT, wild type. The online version of this article contains supplemental material. © The Rockefeller University Press $30.00 The Journal of Cell Biology, Vol. 180, No. 2, January 28, 2008 427–441 http://www.jcb.org/cgi/doi/ 10.1083/jcb.200707142 JCB: ARTICLE affi nity state favored by talin is antagonistically controlled by <strong>ICAP</strong>-1. This affi nity switch results in modu<strong>la</strong>tion in the speed of FA assembly and, consequently, of cell spreading and migration. Unexpectedly, the <strong>ICAP</strong>-1 – <strong>de</strong>pen<strong>de</strong>nt <strong>de</strong>crease in integrin affi nity allows cell sensing of matrix surface <strong>de</strong>nsity, suggesting that integrin conformational changes are important in mechanotransduction. Our results c<strong>la</strong>rify the function of <strong>ICAP</strong>-1 in cell adhesion and highlight the central role it p<strong>la</strong>ys in the cell ’ s integrated response to the extracellu<strong>la</strong>r microenvironment. <strong>et</strong> al., 2004; Ezratty <strong>et</strong> al., 2005 ). Talin, a direct cytop<strong>la</strong>smic � integrin – binding protein, is involved in adhesion site for ma - tion, reinforcement, and stabilization ( Albiges-Rizo <strong>et</strong> al., 1995 ; Priddle <strong>et</strong> al., 1998; Giannone <strong>et</strong> al., 2003 ; Jiang <strong>et</strong> al., 2003 ) as well as in integrin activation and local PIP 2 generation ( Martel <strong>et</strong> al., 2001 ; Cal<strong>de</strong>rwood <strong>et</strong> al., 2002 ; Tadokoro <strong>et</strong> al., 2003 ). Integrin clustering requires the formation of complexes composed of activated integrins, immobilized ligands, talin, and PIP 2 ( Cluzel <strong>et</strong> al., 2005 ). Additionally, the calpain family regu<strong>la</strong>tes cell motility, partly by down-regu<strong>la</strong>ting integrinmediated adhesion complexes ( Bhatt <strong>et</strong> al., 2002 ) and through talin cleavage, a rate-limiting step during adhesion turnover, and is critical for FA disassembly ( Franco <strong>et</strong> al., 2004 ). The current un<strong>de</strong>rstanding is that integrin-containing adhesion sites function as signal transduction centers. Consequently, tight regu<strong>la</strong>tion of integrin af� nity is crucial for adhesion signaling ( Benn<strong>et</strong>t, 2005 ). Integrins adopt high and low af� nity conformations, and ligand – integrin binding is often prece<strong>de</strong>d by intracellu<strong>la</strong>r changes, resulting in increased af� nity ( Ginsberg <strong>et</strong> al., 2005 ). Crystal structure analysis suggests a bent, hooklike conformation for the extracellu<strong>la</strong>r domain in the inactive state and an exten<strong>de</strong>d conformation in the active state ( Liddington and Ginsberg, 2002 ; Takagi and Springer, 2002 ). Talin binding Supplemental Material can be found at: http://jcb.rupress.org/cgi/content/full/jcb.200707142/DC1 Downloa<strong>de</strong>d from jcb.rupress.org on March 30, 2009 JCB 427
tel-00435843, version 1 - 24 Nov 2009 428 Published January 28, 2008 Figure 1. <strong>ICAP</strong>-1 loss induces the central redistribution of FAs and increases � 1 -integrin clustering. (A and B) Confocal images obtained with Icap-1 +/+ and Icap-1 � / � MEF cells (A) or Icap-1 +/+ , Icap-1 � / � , and Icap-1 rescue osteob<strong>la</strong>sts (B). Cells were cultured overnight on 10 µ g/ml FN and processed for JCB • VOLUME 180 • NUMBER 2 • 2008 Downloa<strong>de</strong>d from jcb.rupress.org on March 30, 2009
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