QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 15 May 2007
doi: 10.1242/jcs.03456

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chuang, Y.-y. Articles by Symons, M. Search for Related Content PubMed PubMed Citation Articles by Chuang, Y.-y. Articles by Symons, M.

The atypical Rho family GTPase Wrch-1 regulates focal adhesion formation and cell migration

Ya-yu Chuang^1,*, Aline Valster^1,*,, Salvatore J. Coniglio^1,2, Jonathan M. Backer³ and Marc Symons^1,2,4,

¹ Center for Oncology and Cell Biology, The Feinstein Institute for Medical Research at North Shore-LIJ, North Shore University Hospital, Manhasset, NY 11030, USA
² Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
³ Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
⁴ Department of Surgery, North Shore University Hospital, Manhasset, NY 11030, USA

View larger version (136K): [in this window] [in a new window]   Fig. 1. Wrch-1 localizes to focal adhesions. HelaS3 cells were plated and transfected with GFP-Wrch-1 as described in Materials and Methods. Sixteen hours after transfection, cells were plated on laminin-coated coverslips for 24 hours, fixed and processed for indirect immunofluorescence using a vinculin antibody and Texas-Red-labeled secondary antibody. Micrographs depict cells with low GFP-Wrch-1 expression level. Punctate staining in the center of the cell that does not correspond to focal adhesions is indicated with an asterisk. Bar, 10 µm.

View larger version (60K): [in this window] [in a new window]   Fig. 2. Wrch-1 depletion increases focal adhesions. (A) Fluorescence micrographs of Wrch-1-depleted cells and control cells. Cells were transfected with siRNA targeting luciferase (control) or Wrch-1 (W-1 and W-2) as described in Materials and Methods. Twenty-four hours after transfection, cells were re-plated onto laminin-coated coverslips for another 48 hours. Subsequently, cells were fixed and processed for indirect immunofluorescence using a anti-vinculin antibody and a Texas-Red-labeled secondary antibody, and co-stained for F-actin (FITC-phalloidin). Bar, 10 µm. (B) Quantitative PCR analysis of Wrch-1 knockdown. Cells were transfected as described for A. Seventy-two hours after transfection, total RNA was isolated and analyzed for Wrch-1 mRNA levels. Shown is the average of seven independent experiments ± s.e.m. (C) Wrch-1 depletion diminishes cell spreading. Cells were processed as described for A. For each treatment, the average cell area and s.e.m. was determined for 20 to 25 cells using Esee software (Inovision). Data shown are representative of three independent experiments. (D-F) Quantification of focal adhesions (D), average focal adhesion length (E) and focal adhesion brightness (F) was performed as described in Materials and Methods. For each treatment, the average number of focal adhesions per cell, the average focal adhesion length and brightness (± s.e.m.) was determined for eight cells, randomly selected. Data shown are representative of three independent experiments. *P<0.05, two-tailed t-test.

View larger version (41K): [in this window] [in a new window]   Fig. 3. Wrch-1 overexpression promotes focal adhesion disassembly. (A) Fluorescence micrographs of Wrch-1-overexpressing cells and control cells. HelaS3 cells were transfected with GFP-Wrch-1 or GFP and processed as described in Fig. 1. Bars, 10 µm. (B) Quantification of focal adhesions was performed as in Fig. 2D. *P<10–5, two-tailed t-test. Data are representative of two independent experiments. (C) Wrch-1 overexpression induces cell rounding. The average cell-spread area and s.e.m. were determined for ten cells for each condition as in Fig. 2C. The same micrographs as shown in Fig. 3B were used. *P<0.02, two-tailed t-test.

View larger version (9K): [in this window] [in a new window]   Fig. 4. Wrch-1 regulates cell migration. (A) Wrch-1 depletion inhibits HelaS3 cell migration. Cells were transfected with the indicated siRNAs for 24 hours and subsequently re-plated into a 12-well plate to obtain a confluent layer in 24 hours. Monolayer wound healing assays were performed as described in Materials and Methods. Migration was quantified 24 hours after wounding. Shown is the average ± s.e.m. made over ten to 12 sites. Data shown are representative of three independent experiments. (B) Wounding-induced Wrch-1 mRNA induction was performed as described in Materials and Methods. Shown are the mean and s.e.m. of three independent experiments. Some s.e.m. are smaller than the size of the data points.

View larger version (33K): [in this window] [in a new window]   Fig. 5. Wrch-1 controls focal adhesions by regulating myosin light-chain phosphorylation. (A) Wrch-1 depletion inhibits myosin light-chain phosphorylation. Cells were transfected with siRNA targeting luciferase (control) or Wrch-1 (W-1) as described in Materials and Methods. Ninety hours after transfection, cells were lysed directly into 2x sample buffer and phosphorylated MLC was analyzed by western blotting. Subsequently, blots were stripped and re-blotted with anti-MLC antibody. Tubulin blot was used as loading control. Levels of phosphorylated MLC from seven independent experiments were quantified as detailed in Materials and Methods and normalized to levels of either MLC or tubulin. *P=10–4, two-tailed t-test. (B) treatment with ML-7 increases stress fiber formation and the number of focal adhesions. Cells were plated onto laminin-coated coverslips for 1 hour and subsequently incubated in the presence of 5 µM ML-7, or DMSO as control. After 48 hours, cells were fixed and processed as described in Fig. 2A. Bars, 10 µm. (C) Quantification of focal adhesions was performed as in Fig. 2D. Data shown are representative of two independent experiments. *P<0.0005, two-tailed t-test. (D) ML-7 inhibits cell migration. HelaS3 cells were transfected with siRNA targeting luciferase (control) or Wrch-1 (W-1) for 72 hours, plated as a confluent monolayer for several hours and subsequently pretreated overnight with either ML-7 (5 µM) or DMSO, and treated with ML-7 or DMSO upon wounding. Migration was quantified 7 hours after wounding, as described in Fig. 4A. Shown is the average ± s.e.m. of eight sites. *P<10–5, two-tailed t-test. Data shown are representative of four independent experiments.

View larger version (43K): [in this window] [in a new window]   Fig. 6. PAK regulates focal adhesions and cell migration. (A) Depletion of PAK kinases increases focal adhesions. Fluorescence micrographs of PAK-depleted cells and control cells. HelaS3 cells were co-transfected with siRNAs against PAK1 and PAK2 for 48 hours and subsequently plated on laminin-coated coverslips for another 48 hours. Coverslips were fixed and stained with anti-vinculin antibody and phalloidin as in Fig. 2A. Bars, 10 µm. (B) Quantification of focal adhesions was performed as in Fig. 2D. *P<0.01, two-tailed t-test. (C) Depletion of PAK kinases inhibits myosin light-chain phosphorylation. Cells were transfected with siRNAs targeting luciferase control (ctrl) or PAK1 and PAK2. Ninety hours after transfection, cells were lysed directly into 2x sample buffer. Levels of PAK1, PAK2 and phosphorylated MLC were analyzed by western blotting. The phosphorylated MLC blot was stripped and re-blotted with an anti-MLC antibody. Data shown are representative of three independent experiments. (D) Depletion of PAK kinases inhibits cell migration. HelaS3 cells were transfected with the indicated siRNAs for 72 hours, plated and pretreated overnight and incubated with 5 µM of ML-7 upon wounding. Migration was quantified 7 hours after wounding, as in Fig. 4A. Shown are the averages ± s.e.m. made over eight sites. *P<10–5, two-tailed t-test. Data shown are representative of two independent experiments.

View larger version (18K): [in this window] [in a new window]   Fig. 7. Wrch-1 regulates JNK activation. (A) Wrch-1 depletion inhibits JNK activation. Wounding-induced JNK activation was determined as described in Materials and Methods. Blot is representative of three independent experiments. Levels of phosphorylated Jun from two independent experiments were quantified and normalized to tubulin levels. *P<0.05, two-tailed t test. (B) JNK activity is necessary for HelaS3 migration. JNK activity was inhibited by 2 µM of SP600125 (with overnight pre-treatment). Other conditions as in Fig. 5D. *P<5x10–7, two-tailed t-test. Data are representative of four independent experiments. (C) Inhibition of JNK activity does not affect focal adhesion formation in HeLaS3 cells. Cells were treated with either DMSO or SP600125 (20 µM) for 24 hours. The number of focal adhesions per cell was determined as in Fig. 2D. Data are representative of two independent experiments.

View larger version (17K): [in this window] [in a new window]   Fig. 8. Wrch-1 binds to the regulatory p85 subunit of PI3K and is necessary for Akt activation. (A) Wrch-1 depletion inhibits wound-induced Akt phosphorylation. Lysates were prepared as in Fig. 7A. Akt phosphorylation was visualized using an against antibody phosphorylated Akt (Ser473). An antibody against dynamin 2 was used to demonstrate equal loading. Levels of phosphorylated Akt from three independent experiments were quantified and normalized to levels of dynamin. *P<0.001, two-tailed t-test. (B) PI3K is necessary for cell migration. PI3K activity was inhibited by LY294002 (4 µM) (with overnight pre-treatment). Other conditions are as described in Fig. 5D. *P<0.0005 and **P<5x10–6, two-tailed t-test. Data are representative of two independent experiments. (C) Wrch-1 binds to p85. Wrch-1 proteins were expressed in HelaS3 cells and pulled down using GST-p85 beads, as described in Materials and Methods.