Inducible inactivation of GSK3β is disrupted in migrating mDia1^-/-T cells.

<p>(<b>A</b>) Immunofluoresence analysis of wild-type and mDia1^-/- T-lymphoblasts loaded on ICAM-1/CXCL12-coated plates for 30 min and stained with Cy3-labeled anti-phospho-GSK3β antibody (red) and FITC-conjugated anti-α-tubulin antibody. (<b>B</b> & <b>C</b>) mDia1^-/- and WT T cells were stimulated with ICAM-1/CXCL12 for the indicated times and the lysates then either (<b>B</b>) subjected to SDS-PAGE followed by sequential immunoblotting analysis with anti-phospho-GSK3β (S9A) and anti-GSK3β antibodies (<b>C</b>) or subjected to immunoprecipitation with anti-GSK3β antibody or control IgG and the precipitated samples then incubated with [γ-³²P]-ATP and assayed for incorporated radioactivity. Values represent means ± SEM, ***, p<0.001 by two-way ANOVA. (<b>D</b>) Lysates prepared from T-lymphoblasts (2.5x10^-6) were immunoprecipitated with anti-mDia1 antibody or control IgG and the lysates as well as immunoprecipitated proteins then resolved on SDS-PAGE and subjected to sequential immunoblotting with anti-mDia1, anti-GSK3β or anti-LFA-1 antibodies. (<b>E</b>) Representative differential interference contrast images of pEGF-GSK3β or pEGF-expressing T cells migrating on ICAM-1/Mg²⁺-coated substrate. WT T-lymphoblasts were transfected with either pEGFP-GSK3β (S9A) or pEGFP vector for 24 hours and the GFP⁺-cells then isolated by cell sorting, loaded over ICAM-1-coated plates and their movement tracked using time-lapse microscopy. Outlines of the cells at 0 (red), 1 (blue), 2 (green) and 3 minutes (yellow) post plating indicate the cell migration paths. Scale Bars: 10μm. (<b>F</b>) Bar graphs showing the percentages of morphologically polarized (left) and MTOC reorientated (right) EGF-GSK3β-(S9A) or EGF-expressing T cells. Values represent means ± SEM and all data are representative of at least 4 independent experiments. ∗∗∗, <i>p</i><0.001. (<b>G</b>) WT and mDia1^-/- T-lymphoblasts (5x10⁶) were untreated or treated with ICAM-1/CXCL12 or the GSK-3 inhibitor LiCl (20mM) and APC phosphorylation assessed by evaluating mean fluorescence intensity (MFI) of anti-phospho-APC antibody staining. Values represent means ± SEM, ***, <i>p</i><0.001, NS, not significant. (H) Immunofluoresence analysis of wild-type T lymphoblasts transfected with either GSK3β (S9A) or control vector and loaded on ICAM-1/CXCL12-coated plates for 30 min and stained with Cy3-labeled anti-phospho-GSK3β antibody (red) and Cy5-conjugated anti-α-tubulin antibody. All data are representative of at least four independent experiments.