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Electron tomography reveals Rab6 is essential to the trafficking of trans-Golgi clathrin and COPI-coated vesicles and the maintenance of Golgi cisternal number - PubMed

Electron tomography reveals Rab6 is essential to the trafficking of trans-Golgi clathrin and COPI-coated vesicles and the maintenance of Golgi cisternal number

Brian Storrie et al. Traffic. 2012 May.

Abstract

We have shown previously that Rab6, a small, trans-Golgi-localized GTPase, acts upstream of the conserved oligomeric Golgi complex (COG) and ZW10/RINT1 retrograde tether complexes to maintain Golgi homeostasis. In this article, we present evidence from the unbiased and high-resolution approach of electron microscopy and electron tomography that Rab6 is essential to the trans-Golgi trafficking of two morphological classes of coated vesicles; the larger corresponds to clathrin-coated vesicles and the smaller to coat protein I (COPI)-coated vesicles. On the basis of the site of coated vesicle accumulation, cisternal dilation and the normal kinetics of cargo transport from the endoplasmic reticulum (ER) to Golgi followed by delayed Golgi to cell surface transport, we suggest that Golgi function in cargo transport is preferentially inhibited at the trans-Golgi/trans-Golgi network (TGN). The >50% increase in Golgi cisternae number in Rab6-depleted HeLa cells that we observed may well be coupled to the trans-Golgi accumulation of COPI-coated vesicles; depletion of the individual Rab6 effector, myosin IIA, produced an accumulation of uncoated vesicles with if anything a decrease in cisternal number. These results are the first evidence for a Rab6-dependent protein machine affecting Golgi-proximal, coated vesicle accumulation and probably transport at the trans-Golgi and the first example of concomitant cisternal proliferation and increased Golgi stack organization under inhibited transport conditions.

© 2012 John Wiley & Sons A/S.

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Figures

Fig. 1
Fig. 1. Treatment of GalNAcT2-GFP HeLa cells with either of two different siRNAs directed against Rab6 results in a similarly expanded Golgi apparatus by electron microscopy

Cells were chemically fixed and then thin sectioned. A: Appearance of the Golgi apparatus (GA) in chemically fixed cells treated with a Control siRNA, extracellular space (Ex). B: Appearance of the Golgi apparatus in chemically fixed cells treated with siRab6(Sun) for 4 d to deplete Rab6 protein levels (see D, E, F). C: Appearance of the Golgi apparatus in chemically fixed cells treated with siRab6(Young) for 4 d to deplete Rab6 protein levels (see D, E, F). Arrows in B and C point to examples of accumulated coated vesicles of various sizes.

Fig. 2
Fig. 2. Rab6-depletion arrests Golgi vesicle release and results in increased cisternal number as well as increased lateral continuity along the length of the Golgi ribbon

HeLa cells stably expressing GalNAcT2-GFP were incubated with siControl (A) or siRNA sequences specific for both the Rab6a and Rab6a’ isoforms (B) for 72 h, followed by processing for electron microscopy by high-pressure freezing (A and B) or Western blot analysis (C). GA, Golgi apparatus. Asterisks indicate subregions relatively enriched for arrested vesicle profiles within the larger Golgi region per se, consistent with the lateral rims of cisternae/non-compact regions where vesicular traffic is more prevalent (2x magnification, insets). The asterisk in B highlights an example of clathrin-coated profile as denoted by the spiked, electron-dense coat (2x magnification, inset). The arrowhead in B highlights an example of a putative COPI-coated budding profile attached at the level of medial-cisternae (2x magnification, inset). Bar, 0.5 μm (A and B).

Fig. 3
Fig. 3. Golgi stacks in Rab6-depleted cells retain a polarized distribution of resident Golgi proteins along the cis-trans axis

Wild-type HeLa cells incubated with either scrambled (siControl, A) or Rab6 siRNA (siRab6, B) for 96 h were fixed for indirect immunofluorescence localization using antibodies specific for p115 and β-galactosyltransferase (GalT). Images shown are maximum intensity projections of confocal image stacks. (C) HeLa cell cultures were processed by Western blot analysis to confirm the efficiency of Rab6 protein knockdown. Bar, 10 μm (A and B).

Fig. 4
Fig. 4. The Golgi ribbon in control cells is organized as a series of ‘compact regions’ of stacked cisternae

HeLa cells were treated for 96 h with control siRNA and then high-pressure frozen immediately from 37°C. (A) A representative Golgi ribbon is shown; inset - an example of ER membranes in close physical apposition with trans-Golgi cisternae (trans-ER, tER). (B) The 3D s model derived from (A) illustrating Golgi spatial organization. (C) Model clockwise rotated −30° about the vertical axis. (D) Model clockwise rotated −120° about the vertical axis. CGN, cis-Golgi network; MT, microtubule; TGN, trans-Golgi network. Color-coding: cis-cisternae/CGN, blue; clathrin-coated vesicles, white; COPI vesicles, purple; ER, red; medial Golgi cisternae, green; trans-Golgi, yellow; trans-cisternae/TGN, tan. Bar, 0.5 μm.

Fig. 5
Fig. 5. The Golgi ribbon in Rab6-depleted cells exhibits elongated cisternae/lateral continuity, increased cisternal stacking and extensive arrest of vesicular budding

HeLa cells were incubated with siRab6 for 96 h then high pressure frozen immediately from 37°C. (A) Tomogram; inset, 5.6x view of Z slices from area proximal to asterisk more clearly reveals the spiked, clathrin coat-protein associated with trans-Golgi/TGN. (B and C) 3D models of Golgi ribbon organization highlighting the extended continuity of stacked Golgi cisternae. (C) Model clockwise rotated −120° about the vertical axis, and (D) Trapped budding profiles together with ‘free’ vesicles in the region displayed. For abbreviations and color scheme see Fig. 4. (E) Vesicles and arrested budding structures were assigned as clathrin- or COPI-coated profiles on the basis of their electron dense staining and diameters determined from a sphere fitted to the profile. Bar, 0.5 μm (A–D).

Fig. 6
Fig. 6. The Golgi in Rab6-depleted HeLa cells remains sensitive to nocodazole-induced microtubule depolymerization

GalNAcT2-GFP HeLa cells incubated with either scrambled (siControl, A and B) or Rab6 siRNA (siRab6, C and D) for 96 h were either fixed immediately or treated with nocodazole at 10 μM for various time periods to depolymerize the microtubule cytoskeleton. Since we have shown that the fragmentation of the Golgi ribbon in response to microtubule polymerization is slow (54,56), an intermediate (i.e. 2 h) time point is presented to capture the Golgi ribbon prior to complete disassembly. Qualitatively, the punctate Golgi-derived elements induced by nocodazole-treatment appeared somewhat larger in siRab6-treated cells compared to controls. The kinetic incidence of peripheral Golgi elements (E) was computer scored as described in Materials and Methods. Bar, 10 μm (A–D).

Fig. 7
Fig. 7. The formation of multivesicular bodies/autophagic compartments is upregulated in Rab6-depleted cells

(A and B) Tomograms show the formation of extended endo-membrane compartments containing multiple/internal membranes and abundant coated vesicle profiles. (A′ and B′) Higher magnification views neighboring the Golgi clearly demonstrate two predominant types of coated vesicle profiles within the lumen associated with internalized membranes (black arrow versus white arrow). (C and C′) Image slices corresponding to different Z planes in the tomographic volume of a different Golgi region; coated budding profiles arrested in a ‘pre-scission’ state (C, black arrow) are evident, along with multiple ‘free’ coated vesicles (C′, white arrow). MVB, multivesicular body.

Fig. 8
Fig. 8. Depletion of Rab6 inhibits VSV-G transport Golgi to cell surface, but not from the ER to Golgi apparatus

Wild-type HeLa cells were either incubated with Control or Rab6 (Sun) siRNAs for 4 d and then microinjected with plasmids encoding GFP tagged VSV-G protein. At the end of the expression period at 39.5°C, VSV-G protein was located in the ER by wide field light microscopy, 63x/1.40 numerical aperture objective (A, 0 time). Cells were then shifted to permissive conditions, 32°C, and fixed at various chase times (B). At the end of a 20 min chase, there was significant juxtanuclear, i.e., Golgi, accumulation of VSV-G protein in both Control and siRab6 cells (total VSV-G, left hand 2 columns). There was no quantitatively significant cell surface accumulation (Surface VSV-G, right hand 2 columns and Fig. 8C). At later chase times, cell surface accumulation was obvious qualitatively and quantitatively much earlier for Control than siRab6 cells. Rab6 cells were confirmed to be depleted for Rab6 protein by antibody staining. Scale bars in wide field images shown in A and B equal 20 μm.

Fig. 9
Fig. 9. Small uncoated Golgi-associated vesicles and tubules accumulate with selective depletion of the Rab6-effector MyoIIA indicating the specificity of the Rab6-knockdown Golgi phenotype

(A) Thin section electron micrograph of the Golgi region in a MyoIIA-depleted HeLa cell. Arrow points to an example of beads-on-a-string set of vesicles and a membrane-linked, stalked vesicle. (B) Tomogram of the Golgi region in a MyoIIA-depleted cell. Arrow and arrowheads point to examples as above. Asterisk marks an en face structure from which tubular elements appear to be extending. (C and D) Immunofluorescence (4 d) and (E) Western blot analysis for strong MyoIIA depletion with siRNA treatment. (F) Tomogram example showing apparent stages in formation of small arrested budding structures (arrows, rose color circles) in MyoIIA-depleted HeLa cell. Rose and bronze colored structures indicate budding or nearly fully released vesicles that were diameter quantified in Table 2. Bars, 10 μm (A, B, and F); 20 μm (C and D).

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