spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

doi: 10.1242/10.1242/jcs.00278

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Coutts, A. S.
Right arrow Articles by Black, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Coutts, A. S.
Right arrow Articles by Black, D. M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

TES is a novel focal adhesion protein with a role in cell spreading

Amanda S. Coutts, Elaine MacKenzie, Elen Griffith and Donald M. Black*

Cancer Research UK Laboratories, Beatson Institute for Cancer Research, Switchback Road, Bearsden, Glasgow, G61 1BD, UK



View larger version (66K):

[in a new window]
  		Fig. 1. TES is a component of cell adhesions. (A) Schematic representation of TES constructs. The deletion constructs used in these studies are demonstrated. Full-length TES contained amino acids 1-421. LIM-only TES, pEGFP-C1 contained amino acids 231-421 and pAS2-1 and pACT2 contained amino acids 228-421. LIM-less TES, pEGFP-C1 contained amino acids 1-246 and pGEX-2T contained amino acids 1-233. LIM 1 contained amino acids 230-297. LIM 1 and 2 contained amino acids 230-360. LIM three contained amino acids 351-421. (B) Localisation of full-length TES in Rat-1 fibroblasts. pEGFP-C1 vector (i) or full-length GFP-tagged TES (ii-ix) were stably expressed in Rat-1 fibroblasts as described in Materials and Methods. Cells were stained with anti-paxillin antibody and TRITC-labelled mouse secondary antibody to visualise endogenous paxillin (viii and ix). Solid arrows point to focal adhesion structures, dotted arrows to areas of cell-cell contact, and arrowheads to actin stress fibres. Inset is enlarged region. Bar, 10 µm.

 


View larger version (78K):

[in a new window]
  		Fig. 2. Localisation of LIM-less and LIM-only regions of TES. Rat-1 fibroblasts stably expressing GFP-tagged LIM-only TES (A-F) or GFP-tagged LIM-less TES (G-I). Cells were stained with anti-paxillin antibody and TRITC-labelled mouse secondary antibody to visualise endogenous paxillin (E,F,N,O) or TRITC-labelled phalloidin to visualise actin stress fibres (K,L). Solid arrows point to focal adhesions, dotted arrows to areas of cell-cell contact and arrowheads to actin stress fibres. Bar, 10 µm.

 


View larger version (106K):

[in a new window]
  		Fig. 3. TES localises to the tips of actin filaments. (A) Rat-1 fibroblasts stably expressing GFP-tagged full-length TES were stained with TRITC-labelled phalloidin to visualise actin stress fibres. Solid arrows indicate focal adhesions; dotted arrows indicate areas of cell-cell contact. Insets are enlarged regions as denoted. (B) Actin stress fibres were disrupted by treating Rat-1 fibroblasts stably expressing GFP-tagged full-length TES with 2 µM (top panel) or 10 µM (lower panel) latrunculin B for 24 hours. Cells were then fixed and stained with TRITC-labelled phalloidin.

 


View larger version (56K):

[in a new window]
  		Fig. 4. TES interacts with adhesion proteins. (A) Rat-1 fibroblasts stably overexpressing full-length GFP-tagged TES were stained with mouse zyxin hybridoma supernatant (164D4) and TRITC-labelled mouse secondary antibody to visualise endogenous zyxin. (B) Rat-1 fibroblasts stably expressing full-length GFP-tagged TES were stained with anti-mena antibody and TRITC-labelled mouse secondary antibody to visualise endogenous mena. (C) HeLa WCE incubated with GST protein alone or various GST-tagged TES constructs are shown. Complexes were separated by 7.5% SDS-PAGE and blots probed with an anti-zyxin antibody. (D) GST-alone or GST-tagged TES proteins were incubated with in-vitro-transcribed/translated zyxin extract. Complexes were separated by 7.5% SDS-PAGE and blots probed with anti-zyxin antibody. HeLa WCE were incubated with GST protein alone or GST-tagged full-length TES and complexes separeated using 7.5% SDS-PAGE before transfer to a PVDF membrane and probing with (E) an anti-mena antibody, (F) an anti-VASP antibody, (G) anti-talin antibody or (H) an anti-actin antibody. (I) GST and GST-tagged proteins used in the pull-down assays were run on 10% SDS-PAGE and stained with Coomassie brilliant blue to visualise protein bands. Positions of molecular weight markers (Amersham) are shown. GST, GST control; FL, GST-tagged full-length TES; LL, GST-tagged LIM-less TES; LIM1, GST-tagged 1st LIM domain; LIM1/2, GST-tagged 1st and 2nd LIM domains; LIM3, GST-tagged 3rd LIM domain; I, input (10% of input WCE was run alongside as a comparison). B, bound protein; U, unbound.

 


View larger version (49K):

[in a new window]
  		Fig. 5. TES affects the rate of cell spreading. Rat-1 fibroblasts stably expressing GFP control or GFP-tagged full-length TES or were replated onto fibronectin-coated coverslips and allowed to adhere and spread for the times indicated. (A) Cells were fixed and direct fluorescence confocal microscopy used to visualise GFP or GFP-tagged full length TES. (B) Cells were fixed and stained with TRITC-labelled phalloidin to visualise endogenous actin stress fibres. (C) Histogram comparing the percentage of enlarged cells in GFP and GFP-TES-expressing Rat-1 fibroblasts. To determine the significance of the enhanced spreading in the GFP-TES-expressing cells this effect was quantified in three independent experiments at 15, 30 and 60 minutes after plating onto fibronectin. The largest GFP-expressing cells were of similar size to smaller GFP-TES-expressing cells and were scored as enlarged and the remaining scored as small. The smaller of the GFP-TES-expressing cells, which were similar in size to the larger GFP-expressing cells, were scored as small and the remaining scored as enlarged. The results represent the percentage enlarged at each time point. *P<0.002, Student's t-test, n=3 independent experiments

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2003