Disruption of Golgi Structure and Function
Disruption of Golgi structure and function in mammalian cells expressing a mutant dynamin
Journal of Cell Science 113, 1993-2002(2000)
The large GTPase dynamin is a mechanoenzyme that participates in the scission of nascent vesicles from the plasma membrane. Recently, dynamin has been demonstrated to associate with the Golgi apparatus in mammalian cells by morphological and biochemical methods. Additional studies using a well characterized, cell-free assay have supported these findings by demonstrating a requirement for dynamin function in the formation of clathrin-coated, and non-clathrin-coated vesicles from the trans-Golgi network (TGN). In this study, we tested if dynamin participates in Golgi function in living cells through the expression of a dominant negative dynamin construct (K44A). Cells co-transfected to express this mutant dynamin and a GFP-tagged Golgi resident protein (TGN38) exhibit Golgi structures that are either compacted, vesiculated, or tubulated. Electron microscopy of these mutant cells revealed large numbers of Golgi stacks comprised of highly tubulated cisternae and an extraordinary number of coated vesicle buds. Cells expressing mutant dynamin and GFP-tagged VSVG demonstrated a marked retention (8- to 11-fold) of the nascent viral G-protein in the Golgi compared to control cells. These observations in living cells are consistent with previous morphological and in vitro studies demonstrating a role for dynamin in the formation of secretory vesicles from the TGN.
Cells expressing the mutant Dyn 2(aa)K44A show altered Golgi morphologies
Fluorescence images of Clone 9 cell expressing Golgi resident marker protein TGN38-GFP. (a) Control cells expressing TGN38-GFP show normal Golgi structures situated to one side of the nucleus (N). (b-e) Cells expressing the Dyn 2(aa)K44A mutant display a variety of altered Golgi structures. These include a tightly compressed mass of Golgi cisternae (b), an enlarged vesiculated Golgi (c), and a highly tubulated Golgi (d) in which TGN38-GFP positive tubules extend out from the Golig cisternae to encircle the nucleus. (e) A higher maginification image of the boxed region in d shows that these tubules vary greatly in length but are of a consistent diameter. Bars, 10 um.
Clone 9 cells expressing Dyn 2(aa)K44A possess many Golgi clusters comprised of tubulated cisternae and an extensive number of associated coated vesicle buds
Electron micrographs of cells expressing the mutant Dyn 2(aa)K44A protein display prominent clusters of Golgi stacks that are comprised of classic cisternae and an extensive array of associated tubules and vesicles. High magnification images clearly show the tubulation of the cisternae. A remarkable number of densely coated vesicle buds with elongated necks (arrows) are intimately associated with the membrane tubules. Bars: (c-f) 0.1 um.
Nascent VSVG-ts045-GFP accumulates in the Golgi region of cells expressing Dyn 2(aa)K44A.
Fluorescence micrographs of cultured BHK cells co-transfected with plasmids encoding the secretory marker protein VSVG-ts045-GFP and either wild-type Dyn 2(aa) or the mutant Dyn 2(aa)K44A. Cells were transfected, then incubated for 16 hours at 40°C during which cells accumulated VSVG-ts045-GFP in the ER. Cells were then treated with 100 mg/ml of cycloheximide for thirty minutes to stop protein synthesis and then shifted to 32°C for 15, 60, or 120 minutes prior to fixation to allow transport of the nascent VSVG through the secretory pathway. (a,e) Co-transfected BHK cells expresssing either wt (a) or mutant (e) dynamin following a 16 hour incubation at 40°C. The VSVG-ts045-GFP protein is distributed in a diffuse pattern thoughout the cytoplasm consistent with the retention in the ER. Some nascent protein can be seen scattered about the nucleus due to the large amount of ER elements in this area and cell thickness. Following a 15 minute incubation at the permissive temperature (32°C) in cycloheximide, both wt (b) and mutant cells (f) have transported nascent VSVG-ts045-GFP to a Golgi-like perinuclear compartment (arrows). With increased incubation times at the permissive temperature (60, 120 minutes) cells expressing wild-type Dyn 2 (c,d) transported most, if not all, of the nascent VSVG-ts045-GFP out of the perinuclear region to the cell surface. During this same time period cells expressing the mutant dynamin (g,h) retained the nascent viral protein in a perinuclear region even after 120 minutes (h). Bar, 10 um.
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