1637Research Article

IntroductionThe maintenance of tissue architecture requires that mammaliancells continuously and appropriately respond to extracellular matrixsignals and mechanical stimuli (Geeves et al., 2005; Wakatsuki etal., 2003). These responses include the generation of propulsiveand contractile forces, generated primarily by actin assembly andmyosin motor activity (Dobereiner et al., 2005; Richards andCavalier-Smith, 2005; Yamaguchi and Condeelis, 2007). Fifteenclasses of myosin have been identified, all of which have a motordomain (Richards and Cavalier-Smith, 2005). The motor domaininteracts with polymerized actin, hydrolyzes ATP and enablescellular movement. Non-muscle myosin IIA (NMHC-IIA; alsoknown as MYH9) is a hexameric enzyme composed of two heavychains, each with a regulatory chain and an essential light chain.The ATPase function of NMHC-IIA allows transient binding to theactin cytoskeleton, thereby generating mechanical forces that canmaintain cellular architecture or initiate cell movement. Inconjunction with actin filaments and microtubules, NMHC-IIApowers cell adhesion and migration in a diverse range of culturedcells (Even-Ram et al., 2007; Giannone et al., 2007; Vicente-Manzanares et al., 2007). Mouse embryos lacking NMHC-IIA dieat day 6.5-7 of development, highlighting a fundamental role forthis particular myosin in cellular function (Conti et al., 2004).Autosomal dominant mutations of NMHC-IIA in patients manifestas May-Hegglin syndrome, a disorder characterized by an array ofsymptoms including abnormal maturation of platelets andleukocytes, hearing loss, lens defects and renal failure (Pecci et al.,2005).

Discoidin domain receptor 1 (DDR1) is an unusual receptortyrosine kinase as it responds to extracellular matrix componentssuch as fibrillar collagens, but not to soluble growth factors(Shrivastava et al., 1997; Vogel et al., 1997). DDR1 is over-expressed in several human cancers and is a direct transcriptional

target of the p53 tumor suppressor gene, highlighting a possiblerole in cellular transformation (Alves et al., 1995; Ongusaha et al.,2003). The kinetics of DDR1 activation are slow and sustainedcompared with other receptor tyrosine kinases, suggesting that thisreceptor may be involved in mediating longer-term signals in non-transformed cells (Faraci-Orf et al., 2006). DDR1 phosphorylationis stimulated by fibrillar and basement membrane collagens(currently, types I-VI and type VIII are known to activate), implyingbroad importance for cell adhesion and migration in various tissues.DDR1 can regulate migration of leukocytes and kidney epithelialcells in collagen-rich microenvironments in vitro (Kamohara et al.,2001; Wang et al., 2005). The adaptor molecules ShcA, Nck2 andShp-2 bind to activated DDR1 in a phosphotyrosine-dependentmanner (Kamohara et al., 2001; Koo et al., 2006; Vogel et al., 1997).The interaction of DDR1 with Nck2 is mediated by the SH2 domainof Nck2, and this interaction is implicated in triggering DDR1-induced downstream responses (Koo et al., 2006). In macrophagesand T-cells, collagen-induced DDR1 activation mediates activationof the p38 MAPK and NFκB pathways (Kim et al., 2007;Matsuyama et al., 2004; Yoshimura et al., 2005). Althoughactivation of DDR1 by collagen is independent of β1 integrincollagen receptors, activated DDR1 might inhibit α2β1-integrin-dependent signaling through the Stat1-Stat3 complex (Vogel et al.,2000; Wang et al., 2006). Immunohistochemistry has shown thatDDR1 localizes to basement membrane contacts but it is not partof the focal adhesion complex (Sakamoto et al., 2001; Vogel et al.,2000) and it has not been shown so far to interact with cytoskeletalmotor proteins.

The cellular functions of DDR1 identified from cell culture assaysare supported by in vivo data. For example, DDR1-knockout miceshow reduced post-natal skeletal growth (Alves et al., 2001) andadult females are unable to nourish their litters because themammary gland epithelium fails to secrete milk (Alves et al., 2001;

The spreading and migration of cells on adhesive substrates isregulated by the counterbalance of contractile and protrusiveforces. Non-muscle myosin IIA, an ubiquitously expressedcontractile protein and enzyme, is implicated in the regulationof cell spreading and directional migration in response tovarious stimuli. Here we show that discoidin domain receptor1 (DDR1), a tyrosine kinase receptor activated by type Icollagen, associates with the non-muscle myosin IIA heavy chain(NMHC-IIA) upon ligand stimulation. An association was alsoindicated by coimmunoprecipitation of NMHC-IIA with full-length DDR1, but not with the truncated DDR1d-isoform

lacking the kinase domain. DDR1 was important for assemblyof NMHC-IIA into filaments on cells plated on collagen. DDR1expression inhibited cell spreading over collagen but promotedcell migration. By contrast, blockade of non-muscle myosin IIactivity by blebbistatin enhanced cell spreading but inhibitedmigration over collagen. We propose that myosin and DDR1impact cell spreading and migration by regulating adhesivecontacts with collagen.

Key words: Actin, Discoidin domain receptor, Integrin, Migration,Non-muscle myosin IIA, Cell spreading

Summary

The collagen receptor DDR1 regulates cell spreadingand motility by associating with myosin IIAYun Huang1, Pamela Arora2, Christopher A. McCulloch2,* and Wolfgang F. Vogel1,‡

1Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada2Canadian Institutes of Health Research Group in Matrix Dynamics, University of Toronto, Toronto, Ontario M5S 3E2, Canada*Author for correspondence (e-mail: christopher.mcculloch@utoronto.ca)‡Deceased December 2007

Accepted 9 February 2009Journal of Cell Science 122, 1637-1646 Published by The Company of Biologists 2009doi:10.1242/jcs.046219

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Faraci-Orf et al., 2006). Vascular smooth muscle and mesangialcells derived from knockout mice confirm that DDR1 is of criticalimportance for cell migration (Curat and Vogel, 2002; Hou et al.,2001). However, the mechanisms involved in triggering cell motilitydownstream of activated DDR1 are not understood. In the workreported here have examined the association and functionaloutcomes involving DDR1 and non-muscle myosin IIA aftercollagen stimulation.

ResultsIdentification of NMHC-IIA as DDR1 binding partnerWe examined the protein expression levels of DDR1, NMHC-IIAand β1 integrin in the principal cell types that were used as in vitromodels for the studies described below. There were high levels ofDDR1 expression in MCF-7 cells and in NIH3T3 cells stablytransfected with DDR1 (Fig. 1A). By contrast, DDR1 was notdetected in NIH3T3 cells transfected with a control plasmid(pLXSN) and was present at very low levels in fibroblasts fromDDR1 null mice. Notably, the levels of β1 integrin were relativelysimilar for all of the cell types except for the MCF-7 cells, whichexhibited very low levels of β1 integrin. We confirmed that theNIH3T3 cells stably transfected with DDR1 (3T3-DDR1) wereligand-responsive by plating overnight on 10 μg/ml type I collagenand immunoblotting for tyrosine phosphorylation of DDR1 (Fig.1B).

We next identified proteins associating with DDR1. Collagen-stimulated cells were lysed, DDR1 was immunoprecipitated andco-precipitating bands were analyzed by mass spectrometry. ADDR1-binding protein of approximately 200 kDa was detected bygel electrophoresis; mass spectrometry of tryptic peptides of thisprotein identified it as NMHC-IIA (Table 1). The relative amountof NMHC-IIA that coimmunoprecipitated with DDR1 was increasedafter 8 hours of plating on collagen, suggesting that the association

between NMHC-IIA and DDR1 depends upon collagen-inducedDDR1 activation (Fig. 2A). When similar procedures were appliedto NIH3T3 cells, there was no detectable 200 kDa band (Fig. 2A,lower gel). We confirmed a DDR1–NMHC-IIA association byreciprocal coimmunoprecipitation with an antibody directed againstNMHC-IIA, followed by detection with an anti-DDR1 antibody(Fig. 2B). Immunoprecipitations conducted with pre-immune serumshowed no detectable DDR1 or NMHC-IIA. An association betweenNMHC-IIA and DDR1 was verified in human breast cancer cells(MDA-MB-231) that lack endogenous DDR1 after transfection withfull-length DDR1b, but not with the truncated DDR1d isoformlacking the kinase domain (Fig. 2C). These cells exhibited strongexpression of DDR1 when transfected with plasmids, whichrationalized their use in these experiments. Knockdown ofendogenous DDR1 with siRNA in MCF-7 cells eliminated NMHC-IIA from the DDR1 immunoprecipitates, supporting the specificityof this association (Fig. 2D). Immunoprecipitations prepared usingantibody to NMHC-IIA effectively pulled down DDR1 in controlsiRNA-treated MCF-7 cells but not in siRNA knockdown cells (Fig.2D, middle blots).

Although the association between DDR1 and NMHC-IIA wasevidently stimulated by plating cells on collagen, the associationbetween these two proteins occurred independently of collagen-binding integrin receptors since coimmunoprecipitation ofendogenously expressed proteins isolated from integrin β1-deficient GD25 cellular extracts yielded results similar to thosefrom wild-type fibroblasts (Fig. 2E). The strength of theassociation between DDR1 and NMHC-IIA was relatively high

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Fig. 1. DDR1 and NMHC-IIA expression. (A) Immunoblots of indicatedproteins were prepared from lysates of MCF-7 cells, fibroblasts from DDR1-null (MEF–/–) and wild-type (MEF+/+) mice, NIH3T3 cells, NIH3T3 cellsstably transfected with DDR1, MDA-MB-231, GD25 and HEK293 cells.(B) DDR1 was immunoprecipitated from the lysates of untreated (–) orcollagen-stimulated (+; 18 hours) NIH3T3-DDR1 cells or from controlNIH3T3 cells. Tyrosine phosphorylation of DDR1 immunoprecipitates wasexamined by immunoblotting (upper panel). No DDR1 expression wasdetectable in control NIH3T3 cells (lower panel).

Table 1. Peptides from the 200 kDa protein that associatedwith DDR1*

p200 peptide sequence Location

VVFQEFR HeadNWQWWR NeckVKPLLNSIR NeckGDLPFVVTRR RodRGDLPFVVTR RodALELDSNLYR NeckAGVLAHLEEER NeckLDPHLVLDQLR HeadDLEGLSQRLEEK RodTDLLLEPYNKYR HeadQRYEILTPNSIPK HeadVSHLLGINVTDFTR HeadAGVLAHLEEERDLK NeckIMGIPEDEQMGLLR HeadQGFPNRVVFQEFR HeadALEEAMEQKAELER RodLMATLRNTNPNFVR HeadSFVEKVVQEQGTHPK HeadIAQLEEQLDNETKER RodANLQIDQINTDLNLER RodHEMPPHIYAITDTAYR HeadLQQELDDLLVDLDHQR RodHSQAVEELADQLEQTKR RodKANLQIDQINTDLNLER RodTLEDEAKTHEAQIQEMR RodMQQNIQELEEQLEEEESAR NeckDFSALESQLQDTQELLQEENR Rod

*Peptides were isolated from SDS gels, identified by mass spectrometry,and their relationship with respect to various NMHC-IIA domains weredetermined. None of the 27 peptides matched the related proteins, non-musclemyosin IIB or IIC.

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as the complex could be recovered either in buffer containing 1%Triton X-100 or in immunoprecipitation buffer, which containsmore stringent detergents (data not shown). These more stringentbuffer conditions also ruled out the possibility thatcoimmunoprecipitation of NHHC-IIA with DDR1 was caused bythe high overall protein-protein binding affinity of cytoskeletalmolecules. Furthermore, we did not find actin in the NMHC-IIA–DDR1 immunocomplexes.

We examined whether DDR1 associates with a specific domainof NMHC-IIA. The C-terminal rod domains from NMHC-IIA or-IIB were expressed as His-tagged recombinant proteins, purifiedand assembled into intact rod domains as described previously(Li et al., 2003). In pull-down experiments with rod domains boundto an affinity matrix and incubation with lysates from 3T3-DDR1cells, DDR1 selectively bound to NMHC-IIA assembled into rods,but not to unassembled myosin or to NMHC-IIB (Fig. 2F). Inseparate pull-down experiments using lysates prepared from

transfected HEK293 cells (which strongly expressed NMHC-IIAand -IIB when transfected) and performed in the same manner,NMHC-IIA rods bound to full-length DDR1b, independent of pre-treatment of the HEK293 cells with collagen (Fig. 2G). There wasno binding to the truncated d-isoform or to DDR2 (Fig. 2G). Thesedata suggest that DDR1 probably associates with the rod domainof NMHC-IIA in living cells and that intact DDR1 is required forthis association.

Consistent with the immunoprecipitation studies of 3T3-DDR1cells indicating an association between DDR1 and NMHC-IIA,confocal images of immunostained 3T3-DDR1 cells that had beenallowed to spread for 1 hour on collagen showed colocalization ofNMHC-IIA with DDR1 that was restricted to a central ring pattern(Fig. 3A). Actin filaments colocalized with NMHC-IIA alonglamellipodial margins (Fig. 3B). In 3T3-DDR1 cells that migratedinto an in vitro wound, DDR1 and NMHC-IIA colocalized at theleading cell edge (Fig. 3C). Quantitative assessment of the extent

Fig. 2. Non-muscle myosin heavy chain IIA associationwith DDR1. (A) Time course experiment of cellsactivated by collagen. DDR1 immunoprecipitates wereseparated by SDS-PAGE and stained with Coomassieblue to show NMHC-IIA (IIA) bound to DDR1.(B) NMHC-IIA was immunoprecipitated from DDR1-expressing NIH3T3 cells after stimulation with type Icollagen for the times indicated. Western blot analysisshowed time-dependent increase of DDR1 co-precipitation from stimulated cells (top panel). The blotwas re-probed with anti-NMHC-IIA antibodies (lowerpanel). Western blot analysis of total cell lysatesconfirmed DDR1 and NMHC-IIA expression. Controlantibody did not immunoprecipitate DDR1 or NMHC-IIA. (C) Human mammary carcinoma MDA-MB-231cells lacking endogenous DDR1 expression weretransfected with full-length DDR1 (b-isoform) or the C-terminally truncated d-isoform lacking the kinase andmost of the juxta-membrane region (Alves et al., 2001).Immunoprecipitation of NMHC-IIA and western blotanalysis with an N-terminal DDR1 antibody showed co-precipitation of DDR1b but not DDR1d (upper blot).Comparable amounts of NMHC-IIA were isolated byimmunoprecipitation (lower blot). (D) Small-interferingRNA (siRNA) against DDR1 or control siRNA wasused in MCF-7 cells to knock down DDR1 expression(lower left panel). Cells were plated on collagen for 8hours before the experiment. Western blot analysis forNMHC-IIA confirmed that DDR1 suppressionabolished complex formation with NMHC-IIA (top leftpanel). Center panels show immunoprecipitations usingantibody to NMHC-IIA. Top panels are immunoblottedfor NMHC-IIA and lower panels show loss of DDR1expression in siRNA-treated cells. Right panel showsimmunoblots of total cell lysate.(E) Immunoprecipitation of endogenous DDR1.Collagen-stimulated, integrin β1-deficient GD25 cellswere immunoblotted for NMHC-IIA (left 4 lanes).Plating of cells on collagen induced progressivelyincreasing amounts of immunoprecipitated NMHC-IIA(top panel). Equal amounts of DDR1 wereimmunoprecipitated in each reaction (lower panel).(F) The C-terminal domains of NMHC-IIA or NMHC-IIB were purified from bacteria as His-tagged fusionproteins and assembled into rods as describedpreviously (Li et al., 2003). Non-assembled (NMHC)and assembled (rods) proteins were analyzed on a Coomassie blue stained gel (left panel) or mixed with equal amounts of 3T3-DDR1 cell lysates and subjected toanti-DDR1 western blotting (right panel). (G) The assembled rod domain of NMHC-IIA was incubated with equal amounts of HEK293 cell lysates expressing HA-tagged DDR1b, DDR2 or DDR1d. Anti-HA immunoblotting revealed binding of full-length DDR1, but not truncated DDR1 or DDR2 to the NMHC-IIA roddomain (left panel). Expression of HA-tagged proteins in HEK293 lysates (5% of protein input used in binding assays) was confirmed by anti-HA western analysisof total cell lysates (right panel).

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of colocalization in DDR1-expressing cells (by calculation of thecorrelation coefficient) showed significantly higher correlations ofDDR1 and NMHC-IIA immunostaining for cells migrating oncollagen than for cells migrating on tissue culture plastic orfibronectin (Fig. 3C, histogram). In spreading 3T3-DDR1 cells thathad been immunostained, there was marked colocalization ofDDR1 and β1 integrin at the cell peripheries, although the stainingfor DDR1 was more punctate than that of the β1 integrin (Fig. 3D).

Cellular distribution of NMHC-IIA is regulated by DDR1We investigated the subcellular localization of NMHC-IIA in thecontext of DDR1 function by labeling 3T3-DDR1 and control cellswith a NMHC-IIA-specific antibody (Fig. 4A). As previouslyreported, NMHC-IIA formed largely parallel arrays of filamentsthroughout the cell body but these were not present withinlamellipodia (Even-Ram et al., 2007; Takubo et al., 2003). To assessthe influence of collagen on the NMHC-IIA–DDR1 complex, we

quantified changes in myosin filament bundling in 3T3-DDR1 andNIH3T3 cells (which do not express DDR1) after collagenstimulation. For NIH3T3 cells, plating on collagen caused no changein the percentage of myosin-filament-forming cells whereas NIH3T3cells, expressing high levels of DDR1, exhibited much higherpercentage of myosin-filament-forming cells (Fig. 4A), independentof substrate coating. There was a 20% increase in the number of3T3-DDR1 forming myosin filaments when they were plated oncollagen compared with those plated on tissue culture plastic(P<0.001). In mouse embryonic fibroblasts (MEF) derived fromDDR1-knockout and wild-type animals, there was no difference inthe percentage of myosin-filament-forming cells when plated ontissue culture plastic (Fig. 4B) but there was a significant increasein myosin-filament-forming wild-type cells when plated on collagen(20%; P<0.001).

We wondered whether plating cells on collagen would affectmyosin function in relation to filament formation, specifically

phosphorylation of serine 19 in the myosin light chain(Bresnick, 1999). 3T3-DDR1 cells were plated oncollagen or tissue culture plastic and phosphoserine 19of myosin light chain and β-actin (as loading control)were evaluated by immunoblotting (Fig. 4C). In cellsplated on tissue culture plastic, there was moderateincrease in phosphorylation at 10 minutes but there wasmore abundant phosphorylation, when adjusted forloading, at 30 and 60 minutes in cells plated on collagen.Phosphorylation of myosin light chain was much lessin NIH3T3 cells, which do not express DDR1 (Fig. 4C,lower panels).

Pro-migratory function of the DDR1–NMHC-IIAcomplexNMHC-IIA generates mechanical forces essential forcell migration (Even-Ram et al., 2007; Zeng et al.,2004). To determine whether DDR1 contributed to thisprocess confluent cell monolayers were wounded andcell migration was measured 24 hours later. Migrationof 3T3-DDR1 cells was more rapid than that of controlNIH3T3 cells (Fig. 5A). The distance of the leading edgeof migrating cells into in vitro wounds was quantifiedin cultures of 3T3-DDR1 and NIH3T3 cells, as well asMCF-7 cells treated with control or DDR1 siRNA (Fig.5B). These data showed that in cells plated on collagen(but not on tissue culture plastic or fibronectin),expression of DDR1 was associated with significantly(P<0.05) greater migration distances. This migratory-enhancing function of DDR1 was confirmed with aBoyden-chamber cell migration assay employinguncoated or collagen-coated membranes and NIH3T3and 3T3-DDR1 cells (Fig. 5C). Both NIH3T3 and 3T3-

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Fig. 3. DDR1 and NMHC-IIA in spreading cells. (A) Confocal microscopy images of immunostained NIH3T3-DDR1 cells that had spread for 1 hour on collagen. DDR1 and NMHC-IIA colocalize in a ring-like pattern. Scalebars: 10 μm. (B) Confocal microscopy images of immunostained NIH3T3-DDR1 cells spread for 2 hours oncollagen. Actin filaments and NMHC-IIA colocalize particularly at the cell periphery. (C) Confocal microscopyof 3T3-DDR1 cells migrating into an in vitro wound on collagen-coated substrate and tissue culture plastic (TC)18 hours after wounding. NMHC-IIA and DDR1 colocalized in the cell periphery. The correlation coefficient (±s.d.) of immunostaining for NMHC-IIA and DDR1 was computed from confocal microscopy images of 3T3-DDR1 cells spreading for 18 hours on tissue culture plastic (TC), fibronectin (FN) or collagen (Col). Cellsspreading on collagen exhibited significantly greater colocalization (P<0.02) than cells spreading on TC or FN.(D) Confocal microscopy of 3T3-DDR1 cells spreading on collagen (Col), fibronectin (FN) or tissue cultureplastic (TC) for 18 hours and then immunostained for DDR1 and β1 integrin.

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DDR1 cells migrated much more quickly on collagen than onuncoated membranes but the 3T3-DDR1 cells migrated morequickly than the NIH3T3 cells on collagen (P<0.05). Furthermore,there was increased migration of DDR1-expressing cells in an invitro wounding assay using monolayer cultures of wild-type andDDR1-knockout mouse embryonic fibroblasts (Fig. 5D,E).

Blebbistatin, a specific inhibitor of the non-muscle myosin IIATPase (Straight et al., 2003), retards directional locomotion of avariety of cell types (Even-Ram et al., 2007). In migration assaysof NIH3T3 and MCF-7 cells, as well as MEFs, we found thatinhibition of NMHC-II ATPase activity with blebbistatin (25 μM)retarded migration only in those cells that expressed DDR1 (Fig.6A; P<0.01). We visualized the assembly of myosin filaments bytransfecting cells with a plasmid coding for a GFP-tagged NMHC-IIA, as previously described (Nizak et al., 2003). Fibroblasts fromwild-type and DDR1-null mice, NIH3T3 and 3T3-DDR1 cells, andMCF-7 cells treated with control or DDR1 siRNA, were examinedafter plating on collagen and then the cell layers were wounded.GFP-NMHC-IIA-labeled filaments were readily imaged (Fig. 6B,C)and quantitative studies were conducted to estimate the velocity oflabeled filament elongation as a measure of the rate of assemblyof myosin filaments. Measurements were made in the leading edgeof the cell and in the cell body. In cells expressing DDR1, the

estimated filament elongation velocities were higher (P<0.05) nearthe leading edge of the cell (facing the in vitro wound) whereas inthe cell bodies the velocities were higher in those cells expressinglow or no DDR1 (P<0.01; Fig. 6D). These findings suggest a rolefor DDR1 in regulating the assembly of myosin IIA filaments.

We examined the role of actin filaments in the NMHC-IIA–DDR1 interaction by treating cells with cytochalasin D, atoxin that caps actin barbed ends and prevents actin filamentassembly. In coimmunoprecipitation experiments, cytochalasin Denhanced interactions between NMHC-IIA and DDR1 (Fig. 6E)whereas cell lysates immunoprecipitated with pre-immuneantibody showed no reactive NMHC-IIA or DDR1 (Fig. 6E). Weexamined the binding of collagen to DDR1 by incubating collagen-coated beads (with or without cytochalasin D or blebbistatin) withNIH3T3 and 3T3-DDR1 cells for 1 hour followed byimmunoblotting of the collagen-bead-associated proteins. Thesedata showed that blebbistatin and cytochalasin D treatments didnot affect the relative abundance of DDR1 that was bound to thecollagen beads (Fig. 6F). These findings are consistent with thenotion that only a relatively small proportion of leading edge-localized NMHC-IIA associates with DDR1 during migrationwhereas after actin filament disassembly and disruption of actin-myosin interactions, additional NMHC-IIA can associate with

Fig. 4. DDR1 regulates NMHC-IIA filament formation. (A) NIH3T3 control and 3T3-DDR1-expressing cells were stimulated by plating on type I collagen-coated glass (+Col; 18 hours)or plated on tissue culture plastic (–Col) and immunostained for NMHC-IIA. The percentageof filament-forming cells (mean ± s.d.) was quantified by calculating the number of cells withmyosin filaments extending over more than half of the cell surface area. Collagen-stimulated3T3-DDR1 cells showed a significant increase (P<0.001) of myosin filaments compared withcells on TC (–Col). (B) Myosin filaments in wild-type and DDR1-null mouse embryonicfibroblasts (MEF) were stained and quantified as in A. Fibroblasts from wild-type or DDR-null mice were plated on collagen or tissue culture plastic (–Col; TC). DDR-expressing cellsshowed increased myosin filament formation when plated on collagen but not DDR1-nullcells (mean ± s.d., P<0.001). (C) Top panels: 3T3-DDR1 cell suspensions were analyzed orplated on tissue culture plastic (TC) or collagen (Col) for the indicated times (10–60 minutes).Cell lysates were immunoblotted for phosphoserine 19 of myosin light chain (pMLC) or forβ-actin. Densitometry of blots were measured and the ratio of MLC to β-actin densities wascomputed. Lower panels: NIH3T3 cells were plated on collagen (lanes 1, 3) or tissue cultureplastic (lanes 2, 4) for 10 minutes (lanes 1, 2) or for 60 minutes (lanes 3, 4). Cell lysates wereimmunoblotted for pMLC or for NMHC-IIA as indicated.

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DDR1. Collectively these data support the hypothesis thatfunctional interactions between DDR1 and NMHC-IIA filamentsare involved in cell migration.

Role of DDR1 and NMHC-IIA during cell spreadingWe investigated the roles of DDR1 and NMHC-IIA in formationof initial cell-substratum contacts. We first determined whetherNIH3T3 and 3T3-DDR1 cells would attach to collagen. Initial celladhesion (10 minutes) to collagen was not affected by DDR1expression (Fig. 7A). Cell spreading assays in time courseexperiments (10 minutes to 4 hours) were performed on collagen-coated plates with 3T3-DDR1 cells and mock-transfected NIH3T3control cells, which do not have detectable levels of DDR1 (Fig.1A,B). Compared with control NIH3T3 cells there was reduced(P<0.001) spreading of 3T3-DDR1 cells up to 2 hour; this differencein spreading dissipated by 4 hours (Fig. 7B,C). Similarly, inspreading assays using MEFs and MCF-7 cells treated with siRNAto DDR1, there was significantly (P<0.001) less spreading by thosecells expressing DDR1 (Fig. 7B).

In experiments in which β1 integrin function was inhibited withan integrin-blocking antibody, NIH3T3 and 3T3-DDR1 cells were

allowed to spread on collagen in the presence or absence of antibodyfor 1 or 4 hours (Fig. 7D). The integrin-blocking antibody inhibitedcell spreading of NIH3T3 cells at 1 and 4 hours (P<0.001) whereasinhibition of spreading was only detectable in 3T3-DDR1 cells at4 hours. In 3T3-DDR1 cells plated on collagen for 1 hour, inhibitionof the NMHC-II ATPase by blebbistatin enhanced spreading(P<0.05) whereas there was no significant effect (P>0.2) onNIH3T3 cells (Fig. 7E). If blebbistatin treatment was followed byinhibition of β1 integrin function, with an integrin blocking antibody(1 hour), there was no further reduction of spreading of 3T3-DDR1cells (Fig. 6E) whereas there was a marked inhibition of spreadingof NIH3T3 cells.

Conceivably, the presence or absence of DDR1 may affect β1integrin function. Accordingly, in non-permeabilized cells platedfor 1 hour on collagen, fibronectin or tissue culture plastic, wemeasured β1 integrin activation with 9EG7, an activation-specific,neo-epitope antibody (Lenter et al., 1993) that binds to activatedβ1 integrins. By quantitative fluorescence photometry of individualcells we found no effect of DDR1 expression on β1 integrinactivation on cells plated on collagen, fibronectin or tissue cultureplastic (P>0.2; Fig. 7F), which is consistent with earlier data (Vogel

et al., 2000) but is at variance with a morerecent report (Yeh et al., 2009).

DiscussionThe discoidin domain receptors, DDR1and DDR2, are a sub-family of receptortyrosine kinases that function as collagenreceptors and may act independently of theβ1 integrin (Vogel et al., 2000). BothDDRs are important regulators of a widevariety of developmental processes andcan modify cell adhesion, the migration

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Fig. 5. Role of DDR1 and NMHC-IIA in cellmigration. (A) Glass slides were coated withfibrillar collagen and confluent monolayers ofNIH3T3-DDR1 or control NIH3T3 fibroblastswere wounded in vitro, leaving a wide gap withintact collagen on the glass slide. Therepopulation of the denuded area was measuredafter 18 hours. (B) The indicated cell types wereplated on collagen (Col), tissue culture plastic(TC) or fibronectin (FN) and migration of theleading edge of the cell front after in vitrowounding was measured after 18 hours. Notethat cells expressing DDR1 migratedsignificantly faster than cells null for DDR1(mean ± s.d., *P<0.05), but only when plated oncollagen. (C) 3T3-DDR1 and control NIH3T3cells were seeded on top of a Transwell insertcoated with type I collagen or left uncoated.Collagen was added to the lower reservoir as achemoattractant. Cells migrating to the undersideof the filter were counted following Diff-Quikstaining (mean ± s.d., *P<0.05). (D) Fibroblastsfrom wild-type or DDR1-null mice were growninto confluent monolayers and then injured byscraping with a 200 μl pipette tip. Representativeimages at 0–6 hours are shown. Scale bar:300 μm. (E) Closing of the in vitro wound bycell migration was quantified at ten differentpositions in each culture and the data areexpressed as the width of the gap (mean ± s.d.,*P<0.05 at 6 hours).

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of cells over various substrates, proliferation and the remodelingof the extracellular matrix (Vogel et al., 2006; Leitinger andHohenester, 2007). The proteins that interact with DDRs to mediatethese processes are poorly defined. The main findings of this studyare that DDR1 and NMHC-IIA associate with one another, and thatDDR1 and non-muscle myosin activity regulate cell spreading andmigration on collagen.

The association between DDR1 and NMHC-IIA wasdemonstrated by coimmunoprecipitation of cell lysates and by invitro binding assays using recombinant rod domains of NMHC-IIAwith DDR1 prepared from cell lysates. This association increasedafter cell binding to collagen and occurred prior to collagen-inducedphosphorylation of DDR1 (Vogel et al., 1997). Consistent with thesedata, two-color confocal microscopy and correlation analyses ofintact cells showed that DDR1 and non-muscle myosin IIAcolocalized. The regulation of the DDR1–NMHC-IIA associationapparently relies on intact NMHC-IIA, since we did not detectcollagen-induced changes in the affinity of activated DDR1 for theisolated rod domain of NMHC-IIA. Notably, a direct interactionhas been demonstrated between the NMHC-IIA rod domain andS100A4 (Li et al., 2003) and with the small GTPase Rap1 inregulation of collagen binding to the β1 integrin (Arora et al., 2008),but it is not known how the association of NMHC-IIA with DDR1is regulated by collagen adhesion.

Our finding that depolymerization of actin filaments withcytochalasin D strongly enhanced the association of DDR1 withNMHC-IIA suggests that this association may be regulated in partby the interaction of NMHC-IIA with intact actin filaments.Previous studies have shown that the non-muscle myosininteractions with actin that are required for force-generatingstructures are dissipated by cytochalasin (Kolega and Kumar,1999). Conceivably, in early stages of cell adhesion to collagen,DDR1 does not strongly associate with NMHC-IIA because muchof the myosin is already actin-associated and is not accessible forinteraction with DDR1. As shown by our data, within 6 hours ofcell adhesion to collagen, non-muscle myosin IIA associates withDDR1. Possibly, this association is mediated by remodeling of actinfilaments, a process that is induced by cell adhesion (Mooney etal., 1995) and that may enable dissociation of actin from myosinand thereby enhance DDR1-non-muscle myosin interactions.

Our data showed that NMHC-IIA interacts with full-lengthDDR1, but not with the DDR1d isoform that lacks most of thecytoplasmic region. Five isoforms of DDR1 are generated byalternative splicing, which lead to deletions of the juxta-membraneor kinase domains (Alves et al., 2001). Conceivably, the associationof NMHC-IIA with DDR1 and the possibly resultant effect on cellspreading and migration depend on full-length DDR1. In thiscontext, full-length a- or b-isoforms of DDR1 enhance leukocyteadherence to collagen (Kamohara et al., 2001) and our data showthat full-length DDR1, when transfected into cells, increasesmigration over collagen.

Cognizant of the complexity of the processes regulatingadherence and migration, we examined the effect of DDR1 aloneon cell attachment, spreading and migration. Although DDR1expression had no effect on initial cell attachment, it stronglyinhibited cell spreading. Thus whereas NIH3T3 control cells rapidly

Fig. 6. Cell migration and myosin filamentassembly. (A) Migration of 3T3-DDR1 cellswas inhibited by blebbistatin (Bleb; 25 μM;mean ± s.d., *P<0.01). (B-D) The dynamicrearrangement of NMHC-IIA-containingmyosin filaments was recorded in control (B)and DDR1-null (C) MEF cells transfected witha plasmid coding for a GFP-tagged NMHC-IIA. Scale bars: 2 μm (B,C). (D) The velocityof myosin filament assembly at the leadingedge was higher in cells expressing DDR1than cells null for DDR1 (mean ± s.d.,*P<0.05), whereas myosin velocity in the cellbody was higher in DDR1 null cells than incells expressing DDR1 (mean ± s.d., P<0.01).(E) 3T3-DDR1 cells were treated withcytochalasin D (CytoD; 200 ng/ml) for 1 hour.DDR1 was immunoprecipitated from theresulting lysates. Immunoblotting showedgreatly enhanced NMHC-IIA association withDDR1 in the presence of cytochalasin D.Immunoblots from immunoprecipitationsusing control antibody are shown in the middlepanels, and immunoblots of NMHC-IIA andDDR1 are shown in the right panels.(F) Immunoblots of collagen-bead-associatedproteins in NIH3T3 and 3T3-DDR1 cells thatwere untreated, or treated with cytochalasin D(200 ng/ml) or 25 μM blebbistatin for 1 hour.Cells were incubated with collagen beads for 1hour before lysis and preparation of collagenbead-associated proteins.

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extended cell protrusions on collagen-coated dishes, 3T3-DDR1cells took much longer to become fully spread. A recent report hasindicated that DDR1 inhibits cell spreading by suppressing α2β1-integrin-mediated activation of Cdc42 (Yeh et al., 2009) and that asignaling complex comprising DDR1 and SHP-2 inhibits α2β1-integrin-mediated cell migration (Wang et al., 2006). Our dataindicate that whereas DDR1 and β1 integrins co-distribute at theleading edges of cells, the expression of DDR1 restricts cellspreading, an effect that is relieved by the non-muscle myosin IIinhibitor, blebbistatin, consistent with earlier findings (Even-Ramet al., 2007). Furthermore, our data are consistent with the notionthat following initial adherence of cells to collagen surfaces viaDDR1, myosin IIA-dependent contractility prevents cell spreading(Sandquist et al., 2006). In NIH3T3 cells in which DDR1 was notexpressed and adherence to collagen relied on β1 integrins andpresumably other collagen receptors, blebbistatin had no effect oncell spreading. Therefore the effect of DDR1 expression inrestricting cell spreading on collagen was dependent on non-musclemyosin II-dependent contractility. This contention is consistent withour observation of prolonged myosin light chain phosphorylationat serine 19 in 3T3-DDR1 cells spreading on collagen in comparisonto cells spreading on tissue culture plastic. Indeed, assembly ofmyosin II into cytoskeletal structures, where it can generate andresist forces, is regulated by phosphorylation of myosin light chainsat serine 19 (Kolega and Kumar, 1999). These findings do not ruleout an independent process by which DDR1 may inhibit Cdc42-

induced filopodia formation (Yeh et al., 2009), an important initialstep for cell spreading (Partridge and Marcantonio, 2006).

In contrast to inhibition of non-directional cell spreading, DDR1enhanced directed cell migration on collagen, as we observed inBoyden chamber assays and in in vitro wounds. In all of the celltypes that were examined, expression of DDR1 enhanced directedcell migration for cells plated on collagen but much less so for cellsplated on fibronectin or tissue culture plastic. This effect maydepend, in part, on non-muscle myosin II since treatment of DDR1-expressing cells (but not DDR1 null cells) with blebbistatin reducedcell migration. Furthermore, the rate of assembly of non-musclemyosin filaments was much more rapid at the leading edge of cellsexpressing DDR1 than cells that were null for DDR1. Taken togetherthese data indicate that once DDR1-expressing cells make sufficientnumbers of contacts with the substratum and spread fully, DDR1and NMHC-IIA facilitate the process of directional cell migration.Previous data have shown that blebbistatin enhances cell migration(Even-Ram et al., 2007) but the expression of DDR1 in these cellswas not described.

We found that DDR1-expressing cells plated on collagengenerated more prominent myosin filaments than DDR1 null cells,therefore DDR1 may enhance the generation of increased contractileforces while simultaneously dampening the protrusive forcesassociated with cell spreading. Conceivably, in the absence ofDDR1-collagen attachments, in fibroblasts plated on tissue cultureplastic or in cells null for DDR1, there is more limited adhesion

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Fig. 7. DDR1 inhibits cell spreading via NMHC-IIA. (A) 3T3-DDR1 and control NIH3T3 cells were allowed to adhere tocollagen-coated plates for 10 minutes. No significant difference(P>0.2) in adhesion between the two cell types was found.(B) Comparison of spreading in the indicated cell typesexpressing DDR1 or null for DDR1. Cells were spread oncollagen-coated dishes for up to 1 hour after plating (*P<0.001).(C) Comparison of NIH3T3 control and 3T3-DDR1 cellspreading on collagen-coated dishes for 1–4 hours after plating(*P<0.001 for all NIH3T3 cells compared with 3T3-DDR1 cellsplated for 1 hour). (D) NIH3T3 cells or 3T3-DDR1 cells wereallowed to spread on collagen-coated dishes for the indicatedtimes in the presence or absence of β1-integrin-blocking antibody(Lia1/2; Beckman-Coulter, 20 μg/ml; 30 minutes pre-incubationbefore spreading assays). (E) Spreading assays (1 hour) of 3T3-DDR1 and control NIH3T3 cells in the presence or absence ofblebbistatin and of integrin-β1 blocking antibody (30 minutesincubation before spreading). (F) Confocal microscopyfluorescence intensity measurements of 9EG7 binding to estimateβ1 integrin activation were performed on non-permeabilized cellsof indicated cell type on the three different substrates. Data aremean ± s.d. of 30 cells per group.

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strength and consequently protrusive forces may exceed contractileforces. Under these conditions relatively low myosin activity wouldbe associated with the generation of weak cytoskeletal tensile forces(isometric contractility) and cell migration would be limited. Bycontrast, fibroblasts plated on collagen use DDR1 to moreeffectively ‘pull’ on the substratum, and stronger myosin-drivencontractile forces overshadow protrusive forces, enabling fastermigration rates. This notion is consistent with our observation thatnon-muscle myosin filament assembly was much more rapid in theleading edge of cells expressing DDR1 than in DDR1 null cells.

In summary, our data provide evidence for a novel associationbetween DDR1 and NMHC-IIA. This association may be importantfor regulating the generation of contractile forces that are importantfor cell migration and spreading. The association between DDR1and NMHC-IIA could constitute part of a larger signaling networkanalogous to the β1 integrin-focal adhesion kinase signalingpathway and may be of fundamental importance for wound healingand organ regeneration.

Materials and MethodsCells, reagents and antibodiesMouse NIH3T3 fibroblasts (from ATCC) and primary mouse embryonic fibroblasts(MEF, from day 12.5 embryos) were cultured in Dulbecco’s modified Eagle’s medium(DMEM) with 10% fetal calf serum and penicillin and streptomycin (100 U/ml and100 μg/ml). The expression of DDR2 was not altered in DDR1-null cells comparedwith control cells. These cells provided a good in vitro system to compare the effectof the presence or absence of DDR1 on cell migration and spreading, and are readilytransfected. NIH3T3 cells were stably transfected with the DDR1 (b-isoform) or acontrol (pLXSN) plasmid (Vogel et al., 2000). Human breast cancer MCF-7 and MDA-MB-231 cells (ATCC) were grown in RPMI or DMEM medium with 10% fetal calfserum, respectively. These cells bind to collagen tightly, can invade collagen and arereadily transfected, thereby facilitating protocols that required the modulation of DDR1levels. Integrin-β1-deficient GD25 cells were provided by Reinhard Fässler (Max-Planck Institute for Biochemistry, Munich, Germany). As these cells do not expressthe β1 integrin and are readily transfected with DDR1-expressing plasmids, theyfacilitate experiments examining the functional importance of DDR1 and the β1integrin. Human embryonic kidney 293 (HEK293) cells were used for expressionstudies of DDR1 and non-muscle myosin IIA interaction experiments. Plasmids codingfor NMHC-IIA and NMHC-IIB rod domains were kindly provided by Anne Bresnick(Albert Einstein College of Medicine, New York). DDR1-specific and control siRNApools were purchased from Dharmacon and transfected according to themanufacturer’s instructions. Cytochalasin D and (±)-blebbistatin were obtained fromCalbiochem. A monoclonal antibody directed against DDR1 (YH1-10D4) wasgenerated by injecting DDR1-expressing C2C12 cells into BALB/c mice, and wasused for immunofluorescence staining. DDR1 was identified by immunofluorescenceusing a biotinylated anti-DDR1 (BAF2396, R&D Systems, Minneapolis, MN). Apolyclonal antibody raised against a peptide corresponding to amino acids 505-523of DDR1b was described previously (Vogel, 2002). The following antibodies wereemployed for immunoblotting and/or immunostaining: anti-DDR1 and anti-β1-integrin(sc-532 and N-20 respectively, Santa Cruz Biotechnology; 9EG7-BD Biosciences),anti-β-actin (clone AC15; Sigma-Aldrich), anti-NMHC-IIA (BiomedicalTechnologies) and anti-phosphoserine 19 of myosin light chain (Cell Signaling).

Immunoprecipitation and immunoblottingCells were lysed on ice in lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1.5 mMMgCl2, 5 mM EGTA, 10% glycerol, 1% Triton X-100, 10 μg/ml aprotinin, 10 mMsodium fluoride, 1 mM PMSF and 1 mM sodium orthovanadate) and lysates werecentrifuged at 14,000 g at 4°C for 10 minutes. For immunoprecipitation, 1 mg ofsolubilized protein was incubated with 0.5 μg polyclonal anti-DDR1 (Vogel, 2002),0.1 μg of biotinylated polyclonal anti-DDR1 antibody (R&D systems) or anti-NMHC-IIA antibody (Biomedical Technologies), along with protein A-Sepharose beads(Amersham Biosciences) at 4°C overnight. Beads were washed three times with buffercontaining 20 mM Hepes pH 7.5, 150 mM NaCl, 0.1% Triton X-100, and 10%glycerol. Proteins were eluted with Laemmli buffer at 95°C for 5 minutes and separatedby SDS-PAGE. Western blots were developed using enhanced chemoluminescence(Amersham). All western blot experiments were repeated at least three times andrepresentative data sets are presented. His-tagged proteins coding for residues 1339-1961 of the human myosin-IIA heavy chain or residues 1346-1976 of the humanmyosin-IIB heavy chain were isolated from bacterial cultures and assembled intofunctional myosin rod domains as described previously (Li et al., 2003). For analysisof collagen-bead-associated proteins, cells were incubated with collagen beads and1 hour later, cells were lysed and collagen beads were separated. Bead-associatedproteins were eluted and immunoblotted (Arora et al., 2008).

Mass spectrometryImmunoprecipitated protein complexes were resolved by SDS-PAGE and visualizedwith Coomassie Blue. Protein bands were excised and digested with trypsin (RocheDiagnostics). Saturated α-cyano-4-hydroxycinnamic acid in 70% acetonitrile/0.1%trifluoroacetic acid was used as the matrix solution. The peptide mix was subjectedto matrix-assisted laser desorption ionization time-of-flight analysis on a Voyager-DE STR mass spectrometer (Applied Biosystems). The mass spectra were externallycalibrated from the molecular mass of a mixture of standard peptides. Results wereanalyzed using ProFound (http://129.85.19.192/profound_bin/WebProFound.exe).

ImmunofluorescenceCells grown on glass coverslips were washed three times with PBS, fixed in 2.5%paraformaldehyde for 10 minutes, washed again with PBS, permeabilized with 0.1%Triton X-100 for 5 minutes and blocked with 1% BSA in PBS at room temperaturefor 1 hour. Cells were incubated with primary antibodies at room temperature for1 hour, extensively washed, and incubated with secondary FITC-labeled anti-rabbitantibody (Sigma-Aldrich), Cy3-labeled anti-mouse antibody (Jackson ImmunoResearch Laboratories, Inc.) or Cy3-labeled streptavidin (Sigma) at room temperaturefor 1 hour. Coverslips were mounted with 33% glycerol and 13% Mowiol 4-88(Calbiochem). Images were captured using a LSM510 Zeiss laser scanningconfocal microscope or a Leica TCS confocal microscope. Colocalization ofimmunofluorescence images was analyzed in six fields per cell using the ImageJplug-in JACoP (Bolte and Cordelieres, 2006). The Pearson’s correlation coefficientwas expressed as the mean ± s.d.

Spreading and migrationGlass coverslips were coated with 10 μg/ml type I collagen (from rat tail; BDBiosciences) that was allowed to polymerize, incubated at 4°C overnight and washedwith PBS twice prior to use. The efficacy of the collagen coating was verified byimmunostaining for collagen and by scratch wounding to visualize the denudedsurface. Cells were harvested by briefly exposing the cells to 0.05% trypsin in EDTA,following extensive washing with medium plus 10% serum. In contrast to stimulationwith collagen, serum-containing medium had no effect on DDR1 activation (Vogelet al., 1997). Cells were loaded onto coverslips in medium with 1% serum for theindicated period of time. Non-adherent cells were removed, and adherent cells werestained with either Diff-Quik (Invitrogen, Canada) or 5 μg/ml Alexa-Fluor-488-conjugated wheat germ agglutinin (WGA; Molecular Probes). Following Diff-Quikstaining, five randomly chosen fields were counted, and spreading cells were definedas those cells with total cell size twice the size of the nucleus. Following WGA staining,cell images in five random fields were captured by confocal microscopy and cellsurface area was measured using ImageJ software (version 1.37, NIH). To measuremigration, 1�104 cells were seeded on top of a Transwell insert (8 μm pore size;Costar) coated with type I collagen or left uncoated, and 50 μg/ml of collagen wasadded as a chemoattractant to the lower reservoir. After 18 hours, cells migrating tothe underside of the filter were counted following Diff-Quik staining. All experimentswere performed in triplicate and repeated at least three times.

AdhesionCells (5�104) were loaded onto 96-well plates in medium with 1% serum for theindicated period of time; unattached cells were removed by washing with PBS. Cellswere cultured with 0.05% MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] in medium with 1% serum for 3 hours. After removing the medium, 200 μlDMSO was added to each well and absorbance measured at 570 nm. All experimentswere performed in triplicate.

Wound closureCells were cultured on tissue culture plates until confluent. An in vitro ‘wound’,generated by scratching with the blunt end of a 200 μl plastic pipette tip, was createdand the edge of the wound was marked. After washing with PBS, cells were culturedin the medium with 10% serum in the absence or the presence of blebbistatin (50μM) or DMSO for 18 hours. The average distance of cell migration from the initialwound edge was measured at ten random loci using Zeiss Axiovision software.Similarly, equal numbers of DDR1-null and control MEFs were plated on collagen-coated dishes and wounded by gently scratching with a pipette tip. Wound closurewas followed by videomicroscopy, taking one frame every 6 minutes for 6 hours(Nikon 200 microscope and SimplePSI software). For imaging of living cells, MEFswere transfected with the plasmid pEGFP-SF9, which encodes an EGFP-taggedNMHC-IIA (Nizak et al., 2003). Confluent cells were wounded and the organizationof endogenous myosin filaments were recorded using a Zeiss 200M microscopeequipped with a Solarmere Yokogawa spinning disk confocal scanning system anda 63� Plan-Apochromat objective, which was placed in an environmental chambermaintained at 37°C, 5% CO2 and at constant humidity. Images were captured every3 seconds for a total duration of 8 minutes using a XR/Mega10 CCD camera (StanfordPhotonics). Deconvolution of the images was performed using AutoDeblur (MediaCybernetics). The rate of assembly of individual GFP-labeled non-muscle myosinfilaments (velocity expressed in μm/seconds) was quantified in the leading edge ofcells and in cell bodies using ImagePro Plus Version 6.1 (Media Cybernetics).Transfection with a pEGFP control plasmid yielded no specific cellular staining.

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Statistical analysisAll data are presented as mean value ± standard deviation of the mean (s.d.). Student’st-test was used for statistical comparison of mean values between two groups.Differences between means with P<0.05 were considered to be statistically significant.

We thank Franck Perez (CNRS UMR 144, Paris, France), AnneBresnick and Reinhard Fässler for generously providing reagents andacknowledge the help of Michelle Bendeck and Caroline Ford inproviding helpful comments on the manuscript. We thank CarolLaschinger for assistance with immunoprecipitations. This work wassupported in part by grants from the Canadian Institutes of HealthResearch (MOP 158698 and 416228) and the Canada Research ChairsProgram (W.F.V. and C.A.M.). Following the completion of theseexperiments, the senior author, Wolfgang Vogel, died in Toronto, Canadaon December 5, 2007. We are grateful to Avrum Gotlieb and MichelleBendeck for their support and consideration during the period ofsubsequent data collection from January 2008 until January 2009.

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