Mammalian Diaphanous-Related Formin 1 Regulates GSK3β-Dependent Microtubule Dynamics Required for T Cell Migratory Polarization
Figure 5
mDia1 is required for induction of MT plus-ends dynamics downstream of LFA-1-ICAM-1 interaction.
WT or mDia1-/- T lymphoblasts were loaded onto ICAM-1(3µg/ml)-coated plates for 30 min, fixed and immunostained using FITC-conjugated anti-α-tubulin and Cy3-conjugated anti-EB1 antibody. (A) Left: Representative images of WT and mDia1-/- T cells migrating on ICAM-1 show EB1 clustering with MTs plus ends in polarized T cells. The images shown in the far right panel are enlarged versions of the boxed areas shown in the middle panels. Middle graph: Quantitation of the fluorescence intensities of EB1 staining across the cell axis from uropod to the leading edge of polarized T cells (depicted by dashed arrow in far left image).Right bar graph: Quantitation of the number of cells among 100 cells assessed showing EB1 binding to the tips of growing MT plus-ends at the leading edge cell periphery. (B) Left: Representative images showing the distribution of APC in wild-type and mDia1-/- T cells migrating on ICAM-1. Right: Quantitation of the number of cells among 100 cells counted showing APC clustering at the leading edge. (C) Lysates prepared from mDia1-/-and WT T cells were subjected to SDS-PAGE and immunoblotted with anti-APC or anti-EB1 antibodies followed by anti-α-tubulin antibody. (D) Wild-type or mDia1-/- T lymphoblasts were treated for the indicated times with 10μM cyclohexamide in the absence or presence of the proteosome inhibitor MG-132 (10μM). The cells were then fixed and permeabilized for immunostaining with anti-APC antibody followed by flow cytometric analysis. Data are expressed as the mean fluorescence intensity (MFI). Values represent mean ± SEM and all data for A-D are representative of 4 independent experiments. ∗∗∗, p<0.001 by two-way ANOVA, NS: not significant.