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First published online 28 April 2009
doi: 10.1242/jcs.046219

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The collagen receptor DDR1 regulates cell spreading and motility by associating with myosin IIA

Yun Huang¹, Pamela Arora², Christopher A. McCulloch^2,* and Wolfgang F. Vogel^1,

¹ Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada
² Canadian Institutes of Health Research Group in Matrix Dynamics, University of Toronto, Toronto, Ontario M5S 3E2, Canada

View larger version (46K): [in this window] [in a new window]   Fig. 1. DDR1 and NMHC-IIA expression. (A) Immunoblots of indicated proteins were prepared from lysates of MCF-7 cells, fibroblasts from DDR1-null (MEF–/–) and wild-type (MEF+/+) mice, NIH3T3 cells, NIH3T3 cells stably transfected with DDR1, MDA-MB-231, GD25 and HEK293 cells. (B) DDR1 was immunoprecipitated from the lysates of untreated (–) or collagen-stimulated (+; 18 hours) NIH3T3-DDR1 cells or from control NIH3T3 cells. Tyrosine phosphorylation of DDR1 immunoprecipitates was examined by immunoblotting (upper panel). No DDR1 expression was detectable in control NIH3T3 cells (lower panel).

View larger version (51K): [in this window] [in a new window]   Fig. 2. Non-muscle myosin heavy chain IIA association with DDR1. (A) Time course experiment of cells activated by collagen. DDR1 immunoprecipitates were separated by SDS-PAGE and stained with Coomassie blue to show NMHC-IIA (IIA) bound to DDR1. (B) NMHC-IIA was immunoprecipitated from DDR1-expressing NIH3T3 cells after stimulation with type I collagen for the times indicated. Western blot analysis showed time-dependent increase of DDR1 co-precipitation from stimulated cells (top panel). The blot was re-probed with anti-NMHC-IIA antibodies (lower panel). Western blot analysis of total cell lysates confirmed DDR1 and NMHC-IIA expression. Control antibody did not immunoprecipitate DDR1 or NMHC-IIA. (C) Human mammary carcinoma MDA-MB-231 cells lacking endogenous DDR1 expression were transfected with full-length DDR1 (b-isoform) or the C-terminally truncated d-isoform lacking the kinase and most of the juxta-membrane region (Alves et al., 2001). Immunoprecipitation of NMHC-IIA and western blot analysis with an N-terminal DDR1 antibody showed co-precipitation of DDR1b but not DDR1d (upper blot). Comparable amounts of NMHC-IIA were isolated by immunoprecipitation (lower blot). (D) Small-interfering RNA (siRNA) against DDR1 or control siRNA was used in MCF-7 cells to knock down DDR1 expression (lower left panel). Cells were plated on collagen for 8 hours before the experiment. Western blot analysis for NMHC-IIA confirmed that DDR1 suppression abolished complex formation with NMHC-IIA (top left panel). Center panels show immunoprecipitations using antibody to NMHC-IIA. Top panels are immunoblotted for NMHC-IIA and lower panels show loss of DDR1 expression in siRNA-treated cells. Right panel shows immunoblots of total cell lysate. (E) Immunoprecipitation of endogenous DDR1. Collagen-stimulated, integrin β1-deficient GD25 cells were immunoblotted for NMHC-IIA (left 4 lanes). Plating of cells on collagen induced progressively increasing amounts of immunoprecipitated NMHC-IIA (top panel). Equal amounts of DDR1 were immunoprecipitated in each reaction (lower panel). (F) The C-terminal domains of NMHC-IIA or NMHC-IIB were purified from bacteria as His-tagged fusion proteins and assembled into rods as described previously (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 to anti-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 rod domain (left panel). Expression of HA-tagged proteins in HEK293 lysates (5% of protein input used in binding assays) was confirmed by anti-HA western analysis of total cell lysates (right panel).

View larger version (40K): [in this window] [in a new window]   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. Scale bars: 10 µm. (B) Confocal microscopy images of immunostained NIH3T3-DDR1 cells spread for 2 hours on collagen. Actin filaments and NMHC-IIA colocalize particularly at the cell periphery. (C) Confocal microscopy of 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). Cells spreading 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 culture plastic (TC) for 18 hours and then immunostained for DDR1 and β1 integrin.

View larger version (41K): [in this window] [in a new window]   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 percentage of filament-forming cells (mean ± s.d.) was quantified by calculating the number of cells with myosin filaments extending over more than half of the cell surface area. Collagen-stimulated 3T3-DDR1 cells showed a significant increase (P<0.001) of myosin filaments compared with cells on TC (–Col). (B) Myosin filaments in wild-type and DDR1-null mouse embryonic fibroblasts (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 cells showed increased myosin filament formation when plated on collagen but not DDR1-null cells (mean ± s.d., P<0.001). (C) Top panels: 3T3-DDR1 cell suspensions were analyzed or plated 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 for β-actin. Densitometry of blots were measured and the ratio of MLC to β-actin densities was computed. Lower panels: NIH3T3 cells were plated on collagen (lanes 1, 3) or tissue culture plastic (lanes 2, 4) for 10 minutes (lanes 1, 2) or for 60 minutes (lanes 3, 4). Cell lysates were immunoblotted for pMLC or for NMHC-IIA as indicated.

View larger version (29K): [in this window] [in a new window]   Fig. 5. Role of DDR1 and NMHC-IIA in cell migration. (A) Glass slides were coated with fibrillar collagen and confluent monolayers of NIH3T3-DDR1 or control NIH3T3 fibroblasts were wounded in vitro, leaving a wide gap with intact collagen on the glass slide. The repopulation of the denuded area was measured after 18 hours. (B) The indicated cell types were plated on collagen (Col), tissue culture plastic (TC) or fibronectin (FN) and migration of the leading edge of the cell front after in vitro wounding was measured after 18 hours. Note that cells expressing DDR1 migrated significantly faster than cells null for DDR1 (mean ± s.d., *P<0.05), but only when plated on collagen. (C) 3T3-DDR1 and control NIH3T3 cells were seeded on top of a Transwell insert coated with type I collagen or left uncoated. Collagen was added to the lower reservoir as a chemoattractant. Cells migrating to the underside of the filter were counted following Diff-Quik staining (mean ± s.d., *P<0.05). (D) Fibroblasts from wild-type or DDR1-null mice were grown into confluent monolayers and then injured by scraping with a 200 µl pipette tip. Representative images at 0–6 hours are shown. Scale bar: 300 µm. (E) Closing of the in vitro wound by cell migration was quantified at ten different positions in each culture and the data are expressed as the width of the gap (mean ± s.d., *P<0.05 at 6 hours).

View larger version (44K): [in this window] [in a new window]   Fig. 6. Cell migration and myosin filament assembly. (A) Migration of 3T3-DDR1 cells was inhibited by blebbistatin (Bleb; 25 µM; mean ± s.d., *P<0.01). (B-D) The dynamic rearrangement of NMHC-IIA-containing myosin filaments was recorded in control (B) and DDR1-null (C) MEF cells transfected with a plasmid coding for a GFP-tagged NMHC-IIA. Scale bars: 2 µm (B,C). (D) The velocity of myosin filament assembly at the leading edge was higher in cells expressing DDR1 than cells null for DDR1 (mean ± s.d., *P<0.05), whereas myosin velocity in the cell body was higher in DDR1 null cells than in cells expressing DDR1 (mean ± s.d., P<0.01). (E) 3T3-DDR1 cells were treated with cytochalasin D (CytoD; 200 ng/ml) for 1 hour. DDR1 was immunoprecipitated from the resulting lysates. Immunoblotting showed greatly enhanced NMHC-IIA association with DDR1 in the presence of cytochalasin D. Immunoblots from immunoprecipitations using control antibody are shown in the middle panels, and immunoblots of NMHC-IIA and DDR1 are shown in the right panels. (F) Immunoblots of collagen-bead-associated proteins in NIH3T3 and 3T3-DDR1 cells that were untreated, or treated with cytochalasin D (200 ng/ml) or 25 µM blebbistatin for 1 hour. Cells were incubated with collagen beads for 1 hour before lysis and preparation of collagen bead-associated proteins.

View larger version (32K): [in this window] [in a new window]   Fig. 7. DDR1 inhibits cell spreading via NMHC-IIA. (A) 3T3-DDR1 and control NIH3T3 cells were allowed to adhere to collagen-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 types expressing DDR1 or null for DDR1. Cells were spread on collagen-coated dishes for up to 1 hour after plating (*P<0.001). (C) Comparison of NIH3T3 control and 3T3-DDR1 cell spreading on collagen-coated dishes for 1–4 hours after plating (*P<0.001 for all NIH3T3 cells compared with 3T3-DDR1 cells plated for 1 hour). (D) NIH3T3 cells or 3T3-DDR1 cells were allowed to spread on collagen-coated dishes for the indicated times in the presence or absence of β1-integrin-blocking antibody (Lia1/2; Beckman-Coulter, 20 µg/ml; 30 minutes pre-incubation before spreading assays). (E) Spreading assays (1 hour) of 3T3-DDR1 and control NIH3T3 cells in the presence or absence of blebbistatin and of integrin-β1 blocking antibody (30 minutes incubation before spreading). (F) Confocal microscopy fluorescence intensity measurements of 9EG7 binding to estimate β1 integrin activation were performed on non-permeabilized cells of indicated cell type on the three different substrates. Data are mean ± s.d. of 30 cells per group.

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