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First published online 16 December 2003
doi: 10.1242/jcs.00863

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Dispersal of Golgi matrix proteins during mitotic Golgi disassembly

Department of Biological Sciences, Carnegie Mellon University, 4400 5th Avenue, Pittsburgh, PA 15213, USA

View larger version (64K): [in a new window]   Fig. 1. Comparison of a matrix protein and a non-matrix protein in interphase and metaphase cells. Interphase (A-C) and metaphase (D-F) NRK cells were costained for mannosidase II (A,D) and GM130 (B,E). Single-channel images are also overlayed (C,F) with mannosidase II in green and GM130 in red. The images shown are 2D projections from the deconvolved 3D data set using summation of pixel values in each plane. Bar: 10 µm.

View larger version (117K): [in a new window]   Fig. 2. Presence of Golgi markers in mitotic Golgi clusters and vesicular haze. Metaphase NRK cells were stained using antibodies against giantin (A), GRASP65 (B), GRASP55 (C) and GPP130 (D). Images shown are 2D projections using summation of pixel values. Bar: 10 µm.

View larger version (19K): [in a new window]   Fig. 3. Matrix proteins are extensively dispersed in vesicular haze rather than selectively retained in mitotic Golgi clusters. Untreated interphase (open bars) and metaphase NRK cells were stained for different proteins as indicated, and the percentage fluorescence in objects of each was calculated as in Materials and Methods (±s.e.m.). The results are averages from 10 cells per condition.

View larger version (14K): [in a new window]   Fig. 4. Coincidence of Golgi proteins in mitotic Golgi clusters. Metaphase NRK cells were costained with antibodies against mannosidase II and GRASP65 and a merged image is shown with mannosidase II in green and GRASP65 in red. Metaphase cells were also costained with antibodies against TGN38 and giantin, and the merge shows TGN38 in green and giantin in red. All images shown are summed projections that were thresholded to isolate mitotic Golgi cluster staining from vesicular haze. Note that markers were frequently adjacent in clusters rather than truly coincident. Bar: 10 µm.

View larger version (46K): [in a new window]   Fig. 5. Matrix proteins are selectively, but incompletely, retained in BFA remnants. NRK cells, either untreated (A,B) or treated with BFA for 30 minutes (C,D), were costained for mannosidase II (A,C) and GM130 (B,D). Summed projections are shown. Note that for an unexplained reason, nucleolar-like staining was observed when the anti-mannosidase II antibody was used to stain interphase cells, particularly at the exposure setting necessary to visualize BFA-treated cells. This non-specific staining was excluded in quantitative analyses. Bar: 10 µm. (E) Untreated interphase (open bars) and BFA-treated NRK cells were stained for different proteins as indicated, and the percentage fluorescence in objects of each was calculated as in Materials and Methods (±s.e.m.). The results are averages from 10 cells per condition.

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