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. 2020 Oct 6;10(1):16604.
doi: 10.1038/s41598-020-73276-w.

Rab6 is required for rapid, cisternal-specific, intra-Golgi cargo transport

Lindsey James Dickson #  1 , Shijie Liu #  1 , Brian Storrie  2
Affiliations

Rab6 is required for rapid, cisternal-specific, intra-Golgi cargo transport

Lindsey James Dickson et al. Sci Rep. .

Abstract

Rab6, the most abundant Golgi associated small GTPase, consists of 2 equally common isoforms, Rab6A and Rab6A', that differ in 3 amino acids and localize to trans Golgi cisternae. The two isoforms are largely redundant in function and hence are often referred to generically as Rab6. Rab6 loss-of-function inhibits retrograde Golgi trafficking, induces an increase in Golgi cisternal number in HeLa cells and delays the cell surface appearance of the anterograde cargo protein, VSVG. We hypothesized that these effects are linked and might be explained by a cisternal-specific delay in cargo transport. In pulse chase experiments using a deconvolved, confocal line scanning approach to score the distribution of the tsO45 mutant of VSVG protein in Rab6 depleted cells, we found that anterograde transport at 32 °C, permissive conditions, through the Golgi apparatus was locally delayed, almost tenfold, between medial and trans Golgi cisterna. Cis to medial transport was nearly normal as was trans Golgi to TGN transport. TGN exit was unaffected by Rab6 depletion. These effects were the same with either of two siRNAs. Similar intra-Golgi transport delays were seen at 37 °C with RUSH VSVG or a RUSH GPI-anchored construct using a biotin pulse to release the marker proteins from the ER. Using 3D-SIM, a super resolution approach, we found that RUSH VSVG transport was delayed pre-trans Golgi. These visual approaches suggest a selective slowing of anterograde transport relative to 3 different marker proteins downstream of the trans Golgi. Using a biochemical approach, we found that the onset of VSVG endoglycosidase H resistance in Rab6 depleted cells was delayed. Depletion of neither Rab6A or Rab6A' isoforms alone had any effect on anterograde transport through the Golgi suggesting that Rab6A and Rab6A' act coordinately. Delayed cargo transport conditions correlate strongly with a proliferation of Golgi cisternae observed in earlier electron microscopy. Our results strongly indicate that Rab6 is selectively required for rapid anterograde transport from the medial to trans Golgi. We suggest that the observed correlation with localized cisternal proliferation fits best with a cisternal progression model of Golgi function.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Rab6 knockdown strongly inhibits intra-Golgi cargo transport. Confocal immunofluorescence, orthogonal views of tsO45G-GFP distribution in the Golgi apparatus of HeLa cells stained for p115 (cis-Golgi, red) and TGN46 (TGN, blue) at various times. (A) 0 time, 20 min chase and 40 min chase. At 0 time, there is little, if any correspondence between tsO45G-GFP distribution and a Golgi marker while at 20 min there is extensive correspondence in either Control or Rab6 knockdown cells, and at 40 min chase, Control cells exhibit little to no correspondence in TGN46 distribution and tsO45-GFP. In Rab6 knockdown cells, most of the tsO45G-GFP corresponded in distribution to TGN46 (blue). (B) Immunoblot demonstrating the high level of Rab6 protein knockdown with siRNA treatment. (C) Immunofluorescence evidence for high level Ra6 protein knockdown across the HeLa cell population. Software used includes iVision 4.5 (https://www.biovis.com/), Huygens Professional 4.3.0 p3 (https://www.svi.nl) and Photoshop 2019 (https://adobe.com).
Figure 2
Figure 2
Confocal line scanning measurement of the distance between VSV-G and Golgi markers. (A) Schematic depiction of confocal line scanning method. In brief, after acquiring confocal image stacks, maximum intensity projections (MIPs) or the best single plane image was used for line scanning. Using ImageJ software, a perpendicular line is drawn through the Golgi ribbon in areas of maximal separation of known Golgi markers. This allows for consistent comparison amongst all data derived from this project. Pixel data is converted to distance in microns, which gives the distance in microns between your cargo marker of interest and known Golgi markers, in this instance trans marker (red) and TGN marker (blue). (B) An example image of an immunofluorescently labeled HeLa cell, Golgi apparatus taken with a 63X/1.40 numerical aperture objective with a CARVII spinning disk accessory and deconvolved using Huygens Professional software. The inset in the upper right-hand corner is a ×3 image blowup. (C,D) Line scan graphs as an illustrative example of the outcomes of the approach. Data are averaged for 30 or more individual line scans please see Methods for full detail. Sets of 3 different cis (p115), medial (NAGT1), trans (GalT and SialylT), and TGN (TGN46) Golgi markers are shown. (E) Dot plot representation of resolved Golgi markers from C and D plus and minus standard error of the mean. Distances are similar to those reported by Dejgaard et al. (2008), the originators of the line scan approach. Note that these distances are about twice that seen by electron microscopy. By electron microscopy, the Golgi cisternae in these experiments are dilated (data not shown). This is due to the weak preparative technique (formaldehyde, etc.) used to preserve antigenicity for immunofluorescence. Software used includes iVision 4.5, Huygens Professional 4.3.0 p3, KaleidaGraph 4.5.2, Excel for Mac 16.16.21 and Photoshop 2019. URLs are given in the legends for Table 1 and Fig. 1.
Figure 3
Figure 3
VSV-G transport kinetics from the ER to the cis-Golgi are unaffected by Rab6 depletion. (A) Confocal images of tsO45G-GFP at 0 min in both Control and Rab6 KD cells. At this time point, VSV-G (green) is located in the ER, as evidenced by the web-like distribution of fluorescence across the cytoplasm. There is also no correlation of VSV-G with any known Golgi markers (cis marker p115 in red, TGN marker TGN46 in blue). (B) Upon shifting the temperature to 32 °C, permissive temperature for transport, tsO45G reaches the Golgi at the same time in Control and Rab6 KD cells, co-localizing with cis marker p115. (C,D) Corresponding line scan graphs for 0 and 20 min time chase of VSV-G. Yellow lines trace typical line-scan-area choices. Note: The authors chose in this figure, Part A to show all 3 colors, i.e., all three markers, to illustrate visually their separation. The authors chose in this figure, Part B to show only 2 markers, 2 colors, in order to illustrate visually that with the 20 min chase the high degree of overlap between VSV-GFP distribution and that of p115. Software used includes iVision 4.5, Huygens Professional 4.3.0 p3, KaleidaGraph 4.5.2, Excel for Mac 16.16.21 and Photoshop 2019. URLs are given in the legends for Table 1 and Fig. 1.
Figure 4
Figure 4
tsO45G-GFP accumulated in the medial Golgi (NAGT1 marker, 30 min chase) is slow to reach the TGN (TGN46 marker) in Rab6 KD cells. (A–D) Confocal line scan graphs depicting the medial exit kinetics of VSV-G in Control cells over time. (E–H) Confocal line scan graphs depicting the medial exit kinetics of tsO45G in Rab6 KD cells. There was a significant delay in tsO45G reaching the TGN in Rab6 KD cells. I) Quantification of medial exit kinetics in Control and Rab6 KD cells. Overall, the transport of tsO45G from the ER to the cis-Golgi or medial-Golgi was the same with Rab6 depletion. However, transport of tsO45G was slowed significantly during exit from the medial-Golgi compartment and in its transport to the TGN compartment in Rab6 KD cells, an indication that Rab6 is essential for proper intra-Golgi cargo transport at the medial-to-trans Golgi interface. Software used includes iVision 4.5, Huygens Professional 4.3.0 p3, KaleidaGraph 4.5.2, and Excel for Mac 16.16.21. URLs are given in the legends for Table 1 and Fig. 1.
Figure 5
Figure 5
The TGN exit kinetics for tsO45G were the same in both Control and Rab6 KD cells. (A–C) Confocal line scan graphs depicting the TGN exit kinetics of tsO45G-GFP in Control cells at indicated time points. (D–F) Deconvolved, confocal line scan graphs depicting the TGN exit kinetics of tsO45G-GFP in Rab6 KD cells. While the entry into the TGN compartment was significantly delayed by 20 min in Rab6 KD, the rate of exit from the TGN was the same in Control and Rab6 KD cells. (G) Quantification of TGN exit kinetics in Control and Rab6 KD cells. (H) Overall transport kinetics of tsO45G in Control and Rab6 KD cells. Software used included iVision 4.5, Huygens Professional 4.3.0 p3, KaleidaGraph 4.5.2, and Excel for Mac 16.16. URLs are given in the legends for Table 1 and Fig. 1.
Figure 6
Figure 6
Rab6 depletion results in slow and incomplete tsO45G-GFP endoglycosidase H resistance. Control and Rab6 depleted HeLa cells were lysed at various chase times and samples incubated with and without EndoH. Following gel electrophoresis, samples were immunoblotted for GFP. (A,B) Immunoblot patterns. Note that ihe onset of EndoH resistance appeared to be the same for both Control and Rab6 depleted samples. (C) Rab6 protein knockdown levels by immunoblotting. Preliminary, near full length gel patterns are shown in Supplemental Fig. S-7. Software used includes iVision 4.5, Excel for Mac 16.16.21 and Photoshop 2019. URLs are given in the legends for Table 1 and Fig. 1.
Figure 7
Figure 7
Rab6 depletion significantly delayed 37 °C cargo transport from Golgi apparatus to plasma membrane. Control and Rab6 depleted HeLa cells transfected with RUSH plasmids encoding either VSVG-GFP (A) or GFP-GPI (B). Following the addition of biotin to release the VSVG-GFP from the ER, cells were incubated at 37 °C for various chase times. Cells were then fixed and visualized by confocal light microscopy. (A) At 0 min, VSVG-GFP was distributed uniformly in the ER. With Rab6 depletion, the transport of VSVG-GFP from ER to Golgi apparatus showed little to no change, while the cargo transport within the Golgi apparatus was significantly delayed at 37 °C. Images shown are maximum intensity projections. (B) Control and Rab6 depleted HeLa cells transfected with RUSH plasmids encoding GFP-GPI were treated with biotin to release the expressed protein from the ER and then incubated at 37 °C for various chase. Cells were fixed and visualized by confocal light microscopy. At 0 min, GFP-GPI was accumulated in the ER. At the end of a 40- or 60-min chase, transport of GFP-GPI within the Golgi apparatus was slower in Rab6 depleted cells than Control cells. Images shown are maximum intensity projections. Software used included iVision 4.5 and Photoshop 2019. URLs are given in the legends for Table 1 and Fig. 1.
Figure 8
Figure 8
Super-resolution, 3D-SIM microscopy indicates that transport of RUSH VSVG-GFP in Rab6 depleted cells was stalled before trans Golgi. Control and Rab6 depleted HeLa cells transfected with RUSH plasmids encoding VSVG-GFP were incubated at 37 °C for various chase. Cells were then fixed and stained for GalT (red), and visualized by super-resolution, 3D-SIM microscopy. (A,B) At 0 min, RUSH VSVG-GFP was accumulated in the ER in both Control cells and Rab6 depleted cells. (C,D) At 40 min, in Control cells, VSVG-GFP was transported to the cells surface, while in Rab6 depleted cells, VSVG-GFP was still distributed in the Golgi apparatus and had little to no overlap with GalT distribution. Images shown are maximum intensity projections. Software used included. Software used includes Zeiss Zen 2.0 (https://www.zeiss.com/microscopy/int/products/microscope-software/zen.html) and Photoshop 2019 (https://adobe.com).
Figure 9
Figure 9
Neither siRNAs directed against Rab6A nor siRab6A′ individually inhibit anterograde cargo transport. (A) WT Hela were treated with siRNAs directed selectively against Rab6A or Rab6A′. Biotin was used to release at 37 °C ER accumulated Neither had any obvious effect on the transport of VSVG RUSH construct as a marker for anterograde cargo relative to Control. (B) Co-transfection of WT HeLa cells with siRab6A and siRab6A′, GFP-GPI RUSH cargo, strong inhibition of cargo transport. Software used includes iVision 4.5 (https://biovis.com) and Photoshop 2019 (https://adobe.com).
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