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First published online 24 July 2007
doi: 10.1242/jcs.03477

Journal of Cell Science 120, 2828-2837 (2007)
Published by The Company of Biologists 2007
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ZRP-1 controls Rho GTPase-mediated actin reorganization by localizing at cell-matrix and cell-cell adhesions

Chen-Yu Bai1,*, Miho Ohsugi1,*, Yoshinori Abe2 and Tadashi Yamamoto1,{ddagger}

1 Division of Oncology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
2 Department of Molecular Biology, Institute of Gerontology, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki 211-8533, Japan


Figure 1
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  		Fig. 1. ZRP-1 is localized to focal adhesions and also to cell-cell adhesions. (A) Characterization of anti-ZRP-1 polyclonal antibody. HeLa cell lysates were analyzed by western blotting with anti-ZRP-1 antibody. (B) Localization of ZRP-1 at focal adhesions. HeLa cells were stained with antibodies against ZRP-1 (left) and vinculin (middle). A merged image is shown on the right. Bars, 15 µm. (C) Localization of ZRP-1 at cell-cell contacts. Semi-confluent HeLa cells (upper panel) or saturation-density HeLa cells (lower panel) were stained with antibodies against ZRP-1 (left) and N-cadherin (middle). Merged images are shown on the right. Bars, 15 µm.

 

Figure 2
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  		Fig. 2. ZRP-1 appears at the leading edges of migrating cells. (A) Xenopus A6 cells constitutively expressing EGFP-ZRP-1 (left) were fixed and stained with anti-vinculin antibody (middle). A merged image is shown on the right. (B) Behavior of ZRP-1 in migrating living cells. A6 cells constitutively expressing EGFP-ZRP-1 were grown to confluence and wounded. After 6 hours, cells were imaged at 5-minute intervals. The arrowheads indicate the appearance of EGFP-ZRP-1 at the tip of the leading edge. Bars, 15 µm.

 

Figure 3
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  		Fig. 3. ZRP-1-depleted cells lack actin stress fibers and mature focal adhesions. (A) siRNA-mediated suppression of ZRP-1 expression. Lysates of HeLa cells treated with control-siRNA or ZRP-1-siRNA for 72 hours were immunoblotted with anti-ZRP-1 antibody (upper panel) or anti-{alpha}-tubulin antibody (lower panel, loading control). (B) HeLa cells transfected with control-siRNA or ZRP-1-siRNA were fixed and stained with anti-ZRP-1 antibody and Rhodamine-phalloidin. The lower panels show magnified views. (C,D) HeLa cells transfected with or without ZRP-1 siRNA were grown to confluence and wounded. Ten hours later, cells at wound sites were fixed and stained with anti-ZRP-1 and anti-paxillin (C) or anti-FAK (D) antibodies. Bars, 15 µm.

 

Figure 4
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  		Fig. 4. ZRP-1-depleted cells exhibit aberrant actin dynamics. (B) HeLa cells were treated with control siRNA (upper panel) or ZRP-1 siRNA (lower panel) for 24 hours. Cells were then transfected with EGFP-actin expression vector. Forty-eight hours after vector transfection, cells were imaged at 1-minute intervals (A). Bars, 15 µm. (B) Percentages of cells lacking actin stress fibers. (C) Among the cells lacking actin stress fibers, the emergence rates of cells displaying aberrant actin polymerization were measured. n indicates the number of cells examined.

 

Figure 5
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  		Fig. 5. ZRP-1-depleted cells fail to form N-cadherin-based cell-cell adhesions. (A) Actin dynamics at cell-cell contacts. Control cells (lower panel) and ZRP-1-depleted cells (upper panel) were transfected with EGFP-actin expression vector as described in Fig. 4. Cell-cell contact regions were then imaged at 1-minute intervals. Arrowheads indicate parallel actin filaments that connect neighboring cells. (B) ZRP-1-depleted cells transfected with EGFP-actin were imaged at 1-minute intervals. Red and yellow dashed lines outline two moving cells. (C,D) Three hours after switching the Ca2+ concentration of cell growth medium from low to high, both control cells (upper panels in C,D) and ZRP-1-depleted cells (lower panels in C,D) were fixed and stained with antibodies against ZRP-1 and phalloidin (C) or N-cadherin (D). Merged images are shown on the right of C and D. Bars, 15 µm. (E,F) Percentages of cell-cell adhesion with aligned actin filaments and N-cadherin signals. n indicates the number of cell-cell adhesions examined.

 

Figure 6
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  		Fig. 6. ZRP-1-depleted cells show altered collective cell migration. HeLa cells treated with control siRNA or ZRP-1 siRNA were subjected to wound-healing assay as described in Materials and Methods. Four hours after wounding, cells were imaged at 1-minute intervals for 12 hours. (A) Images of ZRP-1-depleted cells and control cells at 4, 7, 10 and 16 hours after wounding. (B) Percentages of the wound area that has been filled in by migrating cells at each time point. The mean percentages of three independent experiments are shown. Black and white circles indicate ZRP-1-depleted cells and control cells, respectively; error bars indicate standard deviations.

 

Figure 7
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  		Fig. 7. Phosphorylation of focal adhesion kinase (FAK) and paxillin is suppressed in ZRP-1-depleted cells. (A) Endogenous FAK was immunoprecipitated from control or ZRP-1-depleted HeLa cell lysates. Tyr phosphorylation of FAK was detected by a phosphotyrosine antibody (4G10). (B) Endogenous FAK immunoprecipitated from control or ZRP-1-depleted HeLa cells was analyzed with the indicated phosphorylation site-specific antibodies. (C) The relative levels of total and site-specific (Tyr397, Tyr576, Tyr577, Tyr861) Tyr phosphorylation of FAK in ZRP-1-depleted cells as compared with those in control cells. (D) Endogenous paxillin was immunoprecipitated and analyzed as in (A). (E) The relative levels of total tyrosine phosphorylation of paxillin in ZRP-1-depleted cells as compared with those in control cells.

 

Figure 8
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  		Fig. 8. Rac1 activity is enhanced by ZRP-1 depletion. (A) Rac1 pull-down assay. Lysates from HeLa cells treated with or without ZRP-1 siRNA were immunoblotted with anti-ZRP-1 antibody (lower panel) and anti-Rac1 antibody (middle panel). The upper panel shows GTP-bound Myc-Rac1 precipitated with GST-PAK CRIB. (B) Effects of expression of the dominant-negative mutant of Rac1. HeLa cells were transfected with or without Cy3-labeled ZRP-1-siRNA. Twenty-four hours later, these HeLa cells were transfected with expression vectors for Myc-DN-Rac1 and EGFP-actin at a ratio of 9:1. Beginning 48 hours after vector transfection, cells were imaged at 1-minute intervals. Bars, 15 µm. (C) Exogenously expressed Flag-tagged-p1130Cas from control or ZRP-1-depleted HeLa cells was analyzed with a phosphotyrosine antibody (4G10). (D) Endogenous Crk II was immunoprecipitated and analyzed as in (C). (E) The relative levels of total Tyr phosphorylation of Flag-p130Cas and Crk II in ZRP-1-depleted cells as compared with those in control cells. (F) Effects of overexpression of wild-type RhoA. HeLa cells were transfected with or without Cy3-labeled ZRP-1-siRNA. Twenty-four hours later, these HeLa cells were transfected with expression vectors for Myc-WT-RhoA and EGFP-actin at a ratio of 9:1. Beginning 48 hours after vector transfection, cells were imaged at 1-minute intervals. Bars, 15 µm.

 

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© The Company of Biologists Ltd 2007