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First published online 29 July 2008
doi: 10.1242/jcs.021808

Journal of Cell Science 121, 2768-2781 (2008)
Published by The Company of Biologists 2008
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Rab18 and Rab43 have key roles in ER-Golgi trafficking

Selma Y. Dejgaard1, Ayesha Murshid1, Aysegül Erman1, Özge Kizilay1, David Verbich1, Robert Lodge2, Kurt Dejgaard3, Thi Bach Nga Ly-Hartig4, Rainer Pepperkok4, Jeremy C. Simpson4,* and John F. Presley1,{ddagger}

1 Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada, H3A 2B2
2 Laboratoire d'Immunoretrovirologie, Centre de Recherche d'Infectiologie – CHUL, Quebec, Canada, G1V 4G2
3 Department of Biochemistry, McGill University, Montreal, Quebec, Canada, H3G 1Y6
4 Cell Biology and Biophysics Unit, EMBL, 69117 Heidelberg, Germany


Figure 1
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  		Fig. 1. Localization of GFP constructs. Vero cells on coverslips were transfected with the indicated GFP constructs and fixed 24 hours post transfection. Cells were subsequently stained with DAPI to visualize nuclei and antibodies against GM130 to visualize the Golgi complex. Scale bar: 5 µm.

 

Figure 2
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  		Fig. 2. Disruption of the Golgi complex by overexpression of dominant-negative or wild-type Rab proteins. Cells on coverslips were transfected with a panel of wild-type or dominant-negative GFP constructs. Coverslips were fixed 24 hours post transfection. GM130 staining was visualized by immunofluorescence using a Cy3-tagged secondary antibody. Nuclei were visualized using DAPI. (A) Dispersal of GM130 staining in Vero cells (top) and COS7 cells (bottom) expressing wild-type GFP-Rab18. (B) Dispersal of GM130 staining in multiple COS7 cells expressing dominant-negative Rab43. Scale bars: 5 µm. (C,D) Quantification of Golgi dispersal in Vero cells expressing the indicated wild-type Arl/Rab GFP constructs (C) or corresponding dominant-negative constructs (D). (E,F) Quantification of Golgi dispersal in COS7 cells expressing the indicated wild-type Arl/Rab GFP constructs (E) or corresponding dominant-negative constructs (F). Golgi dispersal was quantified and normalized to Golgi dispersal in untransfected cells on the same coverslip. Error bars indicate s.e.m.

 

Figure 3
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  		Fig. 3. Light microscopic localization of Rab proteins to Golgi subdomains. NRK cells were transfected with the indicated GFP constructs. 24 hours post transfection, cells were fixed and stained for p115 and TGN38. The cis-Golgi marker p115 was visualized using a Cy5-tagged secondary antibody (blue) and the trans-Golgi marker TGN38 was visualized using a Cy3-tagged secondary antibody (red). GFP constructs were scored for localization within the Golgi as described (Dejgaard et al., 2007Go). (A) Schematic illustrating choice of location for linescans. (B) Examples of linescans analyzed. (C) Mean location values determined for the indicated constructs. Mean values for 20 cells/sample are shown. Error bars indicate s.e.m.

 

Figure 4
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  		Fig. 4. Dynamics of Golgi association of GFP constructs. NRK cells in glass-bottomed dishes were transfected with the indicated GFP constructs and time-lapse sequences taken 24 hours post transfection. (A) Frames from a typical time-lapse sequence from a NRK cell expressing GFP-Rab43 showing a prebleach image with typical Golgi and cytoplasmic labeling, ablation of Golgi fluorescence by photobleach and points in the recovery time sequence. (B) Recovery curve from cell shown in A. Golgi fluorescence as proportion of total cell fluorescence is plotted. (C) Mean half-times (seconds) for the indicated proteins. Error bars indicate s.e.m.

 

Figure 5
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  		Fig. 5. The Golgi complex is functional but relocated to ER exit sites in COS7 cells expressing Rab43-T32N. (A) Colocalization of GM130 (Cy3; red) with COPII (Cy5; blue) in cell expressing GFP-Rab43-T32N (24 hours post transfection). (B) VSVG-CFP transport to the cell surface in cells expressing GFP-Rab43-T32N (top, middle) or GFP-Rab43 (bottom). Cells were stained without permeabilization to visualize surface VSVG-CFP (Cy3; red) or with permeabilization to visualize total VSVG-CFP as indicated. (C) Total Golgi fragments in cells expressing the indicated GFP-Rab construct. Cells were transfected with the indicated GFP-Rab constructs and were fixed and stained 24 hours post transfection. Transfected cells were divided into three expression levels based on GFP fluorescence and Golgi fragments were counted. Data are mean ± s.e.m. (D) Dispersal of GFP-p150Glued from microtubules in cells expressing CFP-Rab43 (bottom). (E) Scoring of GFP-p150Glued localization to microtubules in singly transfected cells or cells transfected with CFP-Rab43, CFP-Rab43-T32N, Rab18 or Clontech CFP N1 vector. 100 cells were scored for each condition.

 

Figure 6
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  		Fig. 6. Effects of overexpression of Rab18 in COS7 cells. (A) Colocalization of GM130 and COPII in immunofluorescence images of cells expressing moderate levels of GFP-Rab18. GM130 was visualized using a Cy3-tagged secondary antibody (red), COPII using Cy5 (blue). (B) Colocalization of GM130 and COPII staining in cells overexpressing very high levels of GFP-Rab18 staining was as in A. Magnifications of boxed areas are shown below merged images in A and B. (C,D) Trafficking of VSVG in cells strongly expressing Rab18 then incubated at 40°C for 48 hours to accumulate VSVG-YFP in the ER. Cells were then fixed and stained with VG antibody without permeabilization to detect cell-surface VSVG. (C) Singly transfected cells (VSVG-YFP only) were incubated at 32°C for 3 hours to allow exit from the ER. (D) Cells double-transfected with VSVG-YFP and the indicated Rab18 construct after shifting to 32°C for 3 hours. (E) Uptake of Cy3-EGF (100 ng/ml; for 30 minutes following a 2 hour serum starvation) in COS7 cells expressing GFP-Rab18 for 48 hours. Scale bars: 10 µm. (F) Quantification of the VSVG trafficking experiment in D. Each measurement is an average of >100 cells. (G) Quantification of endocytosis of Cy3-Tf (25 µg/ml; for 30 minutes following 2 hours of serum starvation) or EGF in cells expressing the indicated constructs for 48 hours. >100 cells were averaged for each sample. Data are mean ± s.e.m.

 

Figure 7
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  		Fig. 7. Effect of siRNAs against Rab18 in HeLa cells. HeLa cells were incubated for 50 hours with either nonsilencing siRNAs (scrambled) or siRNAs targeting Rab18 or Rab1A, as indicated. (A) RT-qPCR determination of remaining mRNA in cells after treatment with the various siRNAs. (B) Quantification of relative cell surface arrival of VSVG (ts-O45-G) (Simpson et al., 2007Go) in cells treated with various siRNAs. (C) Examination of Golgi morphology, as judged by immunostaining of GM130, in cells incubated with various siRNAs. Quantitative determination of the number of detectable Golgi fragments in these cells as described (Liebel et al., 2003Go) is shown in the lower right graph. Scale bar: 10 µm. Error bars in graphs show the s.d. between three replicate experiments.

 

Figure 8
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  		Fig. 8. Influence of Rab18 and Rab18-S22N on ER-Golgi cycling of Galtase-YFP and p58-YFP in COS7 cells. (A) Frames from representative photobleach sequences acquired with a confocal microscope as described in Materials and Methods. (B) Frames from similar representative photobleach sequences taken from cells expressing p58-YFP. Cells doubly transfected with p58-YFP and CFP-Rab18-S22N or CFP-Rab18 had a similar appearance. (C) Representative recovery curves showing Golgi-associated Galtase-YFP fluorescence as a function of time in cells singly expressing Galtase-YFP or doubly transfected with Galtase-YFP and CFP-Rab18-S22N. (D) Representative recovery curves show Golgi-associated p58-YFP fluorescence as a function of time in cells singly transfected with p58-YFP or double transfected with p58-YFP and CFP-Rab18 or CFP-Rab18-S22N. Experimental design was as in C. (E) A simple model using pseudo-first-order kinetics to describe cycling of resident Golgi proteins between the ER and Golgi in these experiments. (F) Mean Golgi residence times calculated from photobleach recovery curves and ratios of Golgi/total cellular fluorescence (n>20) transfected with the indicated constructs (Galtase or p58-YFP, CFP-Rab18 or CFP-Rab18-S22N). (G) Mean ER residence times determined from the same cells shown in F. Error bars show s.e.m.

 

Figure 9
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  		Fig. 9. Effects of Rab18 mutants and knockdowns on rates of brefeldin-A-induced ER-Golgi fusion. (A) Frames taken from a representative time-lapse imaging sequence showing effects of brefeldin A (5 µg/ml)-induced `blinkout' in COS7 cells expressing Galtase-YFP. (B) Golgis were visualized by Galtase-YFP fluorescence and blinkout after brefeldin A addition and quantified as described (Sciaky et al., 1997Go). (C) Effects of two siRNAs on rate of Golgi blinkout. hsiRNA1 (expressed from pSilencer Neo) was transiently transfected into HeLa cells. Blinkout was measured as in B, 72 hours after transfection. (D) Quantification of brefeldin-A-induced blinkout in COS7 cells treated with nocodazole and brefeldin A. Cells double-transfected with CFP-Rab18-S22N and Galtase-YFP (48 hours post transfection) were placed on ice for 5 minutes in the presence of nocodozole (1 µg/ml), subsequently they were transferred to a heated microscope stage (37°C) in fresh medium containing both nocodazole and brefeldin A (5 µg/ml). Image sequences were taken as described (Sciaky et al., 1997Go). (E,F) Quantification of the experiments in B and C, respectively, showing median time to blinkout in cells transfected with the indicated constructs. 100-150 cells were scored for each condition.

 

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© The Company of Biologists Ltd 2008