Fig. 4.

The role of the cytoskeleton in adhesion-dependent Golgi organization. (A) Stable adherent (SA), detached (5′ SUSP), suspended (120′SUSP) and re-adherent (5′ FN) WT-MEFs immuno-stained for β-tubulin (microtubules), phalloidin (actin) and γ-tubulin (centrosomes). Representative cross-sectional images of cells stained for microtubules (middle and upper plane of cells) or jointly for actin and γ-tubulin (lower panel) for each time point are shown. Images are representative of 30 cells from 3 independent experiments. (B) WT-MEFs surface labeled with GM1-CTxB were left untreated (control), or were treated with 10 µM Nocodazole (NOC) or 0.5 µM latrunculin A (Lat A), detached (5′ SUSP) and held in suspension for 120 min (120′ SUSP). Representative cross-sectional images from 20 cells (NOC) and 30 cells (Lat A), respectively, from 3 independent experiments. (C–F) WT-MEFs expressing ManII or GalTase were suspended (C,D) for 60 min and additional 30 min not treated (60′SUSP+30′CNT) or treated with NOC (60′SUSP+30′NOC) or Lat A (60′SUSP+30′LatA) and re-plated on FN (E,F) for 5 min without drug (5′FN+CNT) or following treatment with with NOC (5′FN+NOC) (D) or Lat A (5′FN+LatA) (F). Representative MIP- and surface-rendered de-convoluted cells (1.5× magnified) are shown. Graphs show discontinuous cis-medial (ManII) or trans-Golgi (GalTase) objects per cell as mean±s.e. of 18–30 cells (as indicated) from 3 independent experiments. Scale bars: 5 µm (A,B), 4 µm (C–F). Statistical analysis was done using the Mann–Whitney U test (***P<0.001).