Fig. 3.

Integrin-mediated adhesion regulates Golgi organization. (A) WT-MEFs expressing the trans-Golgi marker GalTase were suspended for 30 min and incubated with uncoated beads (CNT), FN-beads or PLL-beads for 15 min. Representative DIC images of cell and attached bead, MIP and surface rendered de-convoluted z-stacks (1.5× magnified) are shown. The graph shows the number of discontinuous trans-Golgi objects per cell as mean±s.e. of 34 cells from 3 independent experiments. (B,C) Representative DIC images and a cross-sectional images of a cell with the attached FN-bead and labeled Golgi (left), and schematic of it (right). The position of the GM130- (B) and GalTase- (C) labeled Golgi was mapped (see Materials and Methods) relative to the FN-bead (FN) for 14 and 20 cells, respectively, (left; from 3 independent experiments) (D) WT-MEFs expressing GalTase (trans-Golgi) and suspended for 120 min (120′ SUSP) were incubated with mock (CNT), RGD peptide (+RGD) or RGE peptide (+RGE) for 15 min. Representative MIP and surface-rendered de-convoluted images (1.5× magnified) of cells are shown. (E) Human fibroblasts (BJ cells) expressing GalTase (trans-Golgi) and suspended for 120 min were incubated with PBS (CNT), anti-mouse (IgG) or β1-integrin function-blocking antibody (4B4 Ab) for 15 min, and re-plated on FN (with respective antibodies) for up to 10 min. Representative MIP and surface-rendered de-convoluted images (1.5× magnified) of cells are shown. Data are representative of 3 independent experiments. Scale bars in all images are 4 µm. Statistical analysis was done using Mann–Whitney U test (***P<0.0001).