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doi: 10.1242/10.1242/jcs.00278

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

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