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doi: 10.1242/10.1242/jcs.00109

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The integrin ß tail is required and sufficient to regulate adhesion signaling to Rac1

¹ The Center for Cell Biology and Cancer Research, Albany Medical College, Albany, NY 12208, USA
² Department of Immunology and Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA

View larger version (34K): [in a new window]   Fig. 1. Comparison of Rac1 activatino in A5 cells and ETC12 cells adhered to fibrinogen. (A) A5 cells stably expressing IIbß3 and ETC12 cells stably expressing IIbß3728 were incubated in suspension for 2.5 hours and then aliquots of these cells were adhered to fibrinogen (15 µg/ml) for 15, 30 or 45 minutes as indicated. The amount of Rac1 GTP-loading was compared in cell lysates from A5 and ETC12 cells. The levels of Rac1 were detected by western blot in the GST-PAK pull-down assay (top panel) and the lysate (bottom panel) are shown. The average fold change in Rac1-GTP±s.e.m. from five trials is shown in the bar graph (A5 solid bars, ETC12 open bars). The level of Rac1-GTP in the suspended cells was normalized to 1. Significant differences are indicated (*). (B) Surface levels of IIbß3 and IIbß3728 on A5 and ETC12 cells were analyzed by flow cytometry. The x-axis is the FITC fluorescence intensity of A5 or ETC12 cells stained with the directly conjugated P2-FITC monoclonal antibody that recognizes the llb subunit (solid line) or with an IgG-FITC control (dotted line). The y-axis is cell counts. In control samples, human fibroblasts were stained with P2-FITC or IgG-FITC.

View larger version (24K): [in a new window]   Fig. 2. Analysis of Rac1 activation in ETC12 cells adhered to fibronectin. (A) ETC12 cells were incubated in suspension and then aliquots of these cells were adhered to fibronectin (10 µg/ml) for 1 or 2 hours as indicated and the amount of Rac1 GTP-loading was compared in cell lysates. The levels of Rac1 detected by western blot in the GST-PAK pull-down assay (top panel) and the lysate (bottom panel) are shown. (B) The average fold change in Rac1 GTP-loading±s.e.m. from three trials is shown in the bar graph. The level of Rac1-GTP in the suspended cells was normalized to 1. Significant differences are indicated (*).

View larger version (30K): [in a new window]   Fig. 3. Adhesion to collagen I increases the GTP-loading of endogenous Rac1 and transfected myc-tagged Rac1 in primary human fibroblasts. (A) Primary human fibroblasts were incubated in suspension for 2.5 hours and then aliquots of these cells were adhered to collagen I (50 µg/ml) for 15, 30 or 45 minutes as indicated. The amount of Rac1 GTP-loading was compared in lysates from suspended and adhered cells. The levels of Rac1 detected by western blot in the GST-PAK pull-down assay (top panel) and the lysate (bottom panel) are shown. (B) The average fold change in endogenous Rac1 GTP-loading±s.e.m. from three trials is shown in the bar graph. The level of Rac1-GTP in suspended cells was normalized to 1. (C) Primary human fibroblasts were transiently transfected with myc-tagged wild-type Rac1 and the amount of Rac1 GTP-loading was compared in lysates from suspended cells and cells adhered to collagen I for 15 and 30 minutes. The average fold change in Rac1-GTP loading±s.e.m. from three trials is shown in the bar graph. The level of Rac1-GTP in the suspended transfected cells was normalized to 1. Significant differences are indicated (*).

View larger version (35K): [in a new window]   Fig. 4. Clustering integrin ß1 or ß3 tails is sufficient to activate Rac1 in primary human fibroblasts. (A) Tac-, tac-ß1- and tac-ß3-transfected primary human fibroblasts were analyzed for surface expression of the tac epitope by flow cytometry. The x-axis is the FITC fluorescence intensity of transfected cells stained with the directly conjugated anti-CD25-FITC monoclonal antibody (Becton Dickinson) that recognizes the tac epitope of the IL-2-receptor. Transfected cells were also stained with IgG-FITC (Becton Dickinson) as a negative control. (B) Primary human fibroblasts co-transfected with tac-ß1 and myctagged Rac1 were placed in suspension and one fraction of these cells was stained for expression of the tac epitope at the cell surface, permeabilized and then stained for expression of the myc epitope to demonstrate the distribution of co-transfected cells (a). The second fraction was incubated in a clustering assay with anti-tac antibody-coated magnetic beads (b). The remaining cells that were not selected magnetically for Rac activity assays were stained for tac and myc expression to demonstrate the depletion of tac positive and Rac co-transfected cells. Two-color flow cytometry was performed to analyze the distribution of tac and myc expression for the starting and negatively sorted cells. The x-axis is the FITC fluorescence intensity of cells stained with the directly conjugated anti-CD25-FITC monoclonal antibody. The y-axis is the PE fluorescence intensity of cells stained with anti-myc primary antibody and PE conjugated secondary antibody. (C) Normal human fibroblasts were co-transfected with wild-type Rac1 and either the control tac receptor (C), tac-ß1 (ß1) or tac-ß3 (ß3). Equal numbers of tac-expressing cells were incubated in clustering assays for 15 minutes. The levels of transfected Rac1 in the lysates of the clustered cells detected by western blot of the GST-PAK pull-down assay (top panel) and the lysate (bottom panel) are shown. (D) The average fold change in Rac1 GTP-loading±s.e.m. from five clustering trials is shown in the bar graph. The fold change in Rac1 GTP-loading was normalized relative to the levels in control tac-transfected cells. Significant differences are indicated (*).