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Fig. 5. tGolgin-1 depletion mimics disruption of dynein-dynactin or microtubules in terms of Golgi element position, function and movement. (A-F) HeLa cells transduced with both siRNA-1 and plasmid encoding Sec13-EGFP (A-C) or Sec13-EGFP alone (D-F) were fixed 2 days later without (A-C) or with (D-F) treatment with nocodazole (Noc.) for 2 hours, and analysed by fluorescence microscopy after labelling with anti-TGN46 and RRX-conjugated secondary antibody. Individual labels are shown in A,B,D,E, and the overlay is shown in C,F; the boxed region in C is magnified 2.5 times in the bottom right corner. tGolgin-1-depleted cells were identified based on the labelling pattern for TGN46. Notice the accumulation of TGN46 labelling near sites labelled by Sec13-EGFP. (G-L) HeLa cells transduced with both siRNA-1 and plasmid encoding VSV-G/EGFP were incubated overnight at 39°C and then fixed either immediately (G,H) or after a 30 minute (I,J) or 60 minute (K,L) incubation at 32°C. Cells were analysed by fluorescence microscopy after labelling with anti-TGN46 and RRX-conjugated secondary antibody. tGolgin-1-depleted cells were identified based on the labelling pattern for TGN46. Notice the appearance of cell-surface labelling (particularly in surface spikes) for VSV-G/EGFP by 60 minutes. (M-P) HeLa cells stably expressing GRASP65-EGFP were transduced transiently with siRNA-1 (M) or dynamitin-p50 (n), treated with nocodazole for 2 hours (O), or exposed to brefeldin A for 1 hour, washed, recovered at 16°C for 3 hours and then released at 37°C (P). Cells were analysed by video microscopy and representative images were collected every 60 seconds (M-O) or 20 (P) seconds for 5 minutes. Images from all time points were overlaid, with the initial image labelled in white and all subsequent images labelled in red to indicate movement from the initial position. Notice that very few labelled elements in m-o showed significant movement over the 5 minute time course. (Q) Quantitation of Golgi element movement. 50-150 spots from at least five cells in each experimental sample represented in m-p were analysed for distance moved over the 5 minute time course. The mean, median, maximum (max) and minimum (min) values, and 25th and 75th quartile values for distance moved in each experimental set are shown. Pairs of value sets were analysed by Student's t test, and P values for each paired analysis are indicated.
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