JCS00748 Supplemental Data

Files in this Data Supplement:

• Movie A
• Movie B
• Movie C Back
• Movie C Front -

  Movies. Transport, docking and fusion of vesicles in stationary and migrating NRK fibroblasts. NRK fibroblasts were microinjected with cDNA encoding LDLR-GFP and imaged in TIR-FM with a temporal resolution of ~5 frames per second between 10 minutes and 60 minutes after the release of the Golgi block. Each video shows only a region of the cell because of the limited file size. The fusions are indicated by white arrows appearing during the ten frames before the fusion start. (A) Untreated stationary cell: vesicles fuse in the periphery of the cell. (B) Nocodazole-treated stationary cell: an NRK fibroblast was treated with 10 mM nocodazole during the last hour of the Golgi block. Under nocodazole treatment, large, highly fluorescent static spots are typically seen rather than small moving vesicles (compare A and C). These large spots might be miniature Golgi stacks in the evanescent field. They appear to fuse partially with the plasma membrane in multiple rounds. Some small vesicles are transported over short distance, which could be due to nocodazole-resistant microtubules. (C) Untreated migrating cell. (C, back) Many vesicles are visible under TIR at the back of the migrating cell but very few fuse with the plasma membrane in this region of the cell surface. (C, front) Many vesicles are transported to the leading edge before they dock and fuse with the plasma membrane just a few micrometres before the cell edge.

• Supplemental Figure 1 -

  Fig. S1. Microtubule depolymerization and Golgi dispersal in nocodazole treated NRK fibroblasts. NRK fibroblasts were left untreated (a) or were treated with 10 mM nocodazole for various times (b,c), were then extracted in 0.1% Triton-X100 for 3 minutes, fixed in methanol and stained with antibodies against b-tubulin (red) and Gos28 (green). Untreated cells show dense microtubule arrays and a perinuclear Golgi. After nocodazole treatment for 1 hour at 20°C, most microtubules are depolymerized and the Golgi is partially dispersed. After nocodazole treatment for 30 minutes at 37°C followed by 3 hours at 20°C, very few microtubules are left fully depolymerized and the Golgi was fully dispersed.