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Figure 2. Rab10 localizes to the ER and regulates tubular ER morphologya, Cos-7 cells co-expressing mCh-tagged human Rab8 (mCh-Rab8) or Rab10 (mCh-Rab10) and a luminal ER protein (KDEL-venus) were localized by confocal FM (top and bottom panels, respectively). Third and fourth panels show merged image and zoom of boxed region, respectively (Rab in green, ER in red). Note that Rab10 localizes throughout the ER, and Rab8 does not. b, As in a for cells expressing KDEL-venus with mCh-Rab10 T23N or mCh-Rab10 Q68L, as indicated. Note the expansive cisternae in Rab10 T23N expressing cells. c, Method for quantitative analysis of ER shape. Three identical 28 pixel wide line segments were drawn on the images beginning at the NE away from the MT organizing center (yellow boxes), the Reni Entropy Threshold setting was used to select only ER cisternae (middle panel image) or the total ER (third panel, green + blue). Dividing the number of ER cisternae pixels (green) by the number of total ER pixels (green + blue) gives the percentage of ER cisternae in each line segment. The remaining ER was defined as ER tubules. For a, b and c, scale bars = 10 μm. d, Percentage of the ER comprised of cisternae (using analysis described in c) for cells expressing KDEL-venus alone or together with mCh-Rab10 WT, mCh-Rab10 Q68L, or mCh-Rab10 T23N. Means ± standard errors were calculated from each condition in d, n = 50 cells for each condition, 3 regions per cell. *** P < 0.001..
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Figure 3. Depletion of endogenous Rab10 reduces tubular ER morphologya, Cos-7 cells were co-transfected with a luminal ER marker (KDEL-venus) and either control or Rab10 siRNA, and were then visualized by confocal FM to detect changes in ER morphology (note the expansive cisternae in the Rab10 siRNA depleted sample). Scale bars = 10 μm. b, Immuno-blot analysis with antibodies against Rab10 reveals efficient depletion of Rab10 by siRNA. GAPDH protein levels were measured as a loading control. c, Percentage of the ER comprised of cisternae for cells transfected as in a with control or Rab10 siRNA (analysis performed as described in Fig. 2c and 2d). Means ± standard errors were calculated from each condition, n = 50 cells, 3 regions per cell. *** P < 0.001.
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Figure 4. Rab10 regulates ER tubule dynamics and fusiona, Cos-7 cells expressing KDEL-venus and mCh-Rab10 WT or mCh-Rab10 T23N (top and bottom panel, respectively) were imaged live to visualize ER tubule extension and fusion events at time points indicated (in seconds). Arrowheads mark the sites of successful (top panels) or unsuccessful fusion (bottom panels). b, Cells described in a were imaged live to measure the number of tubular ER extension events that occurred in a 10 μm2 box over a 5 min time course. These tubular extensions were scored as either resulting in a successful or unsuccessful fusion event. c, The successful and unsuccessful fusion events measured in b were alternatively graphed as a percentage of events leading to fusion. d, Cos-7 cells expressing KDEL-venus to visualize ER extension and fusion were co-transfected with control siRNA (top panel) or Rab10 siRNA (bottom panel) and were imaged at the indicated time points (in seconds). Arrowheads mark sites of successful fusion (top panel) or unsuccessful fusion (bottom panel). Images in a and d, scale bars = 2 μm.. e, As in b for d. f, As in c for d. Means ± standard errors were calculated for b, c, e and f, n = 30 cells for each condition, 2 boxes for each cell. *** P < 0.001.
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Figure 5. Rab10 and PIS co-localize to the leading edge of ER dynamicsa, A Cos-7 cell co-expressing luminal ER marker (KDEL-venus) and mCh-Rab10 imaged by live confocal FM to visualize ER dynamics; top panel shows overlay, bottom panel shows KDEL-venus alone to illustrate the absence of the ER from the Rab10 domain. Arrowheads mark the position of a Rab10 dynamic domain (in red, white arrowhead) not initially labeled with the luminal ER marker (in green, yellow arrowhead follows movement of KDEL marker behind the Rab10 domain). b, As in a for cells expressing KDEL-venus, mCh-tagged human PIS (mCh-PIS) and either BFP-Rab10 WT (top panel) or BFP-Rab10 T23N (bottom panel) (PIS in red, Rab10 in blue, KDEL in green). Note that Rab10 and PIS co-localize at dynamic domains not initially labeled with KDEL. Arrowheads mark positions of successful and unsuccessful fusion events (top and bottom panel, respectively) that follow the BFP-Rab10/mCh-PIS dynamic domain structures. Images in a and b, scale bars = 2 μm.. c, Percentage of ER attached puncta that contain both BFP-Rab10 and mCh-PIS, BFP-Rab10 alone, or mCh-PIS alone (n = 444 puncta for BFP-Rab10 WT and 492 puncta for BFP-Rab10 T23N). d, Cos-7 cells co-transfected as in b and imaged live by confocal FM. All ER extension events occurring in a 10 μm2 box over a 5 min time course were observed. We calculated the percentage of total events clearly led by either a PIS/Rab10 WT or PIS/Rab10 T23N dynamic domain (621 and 437 total events, respectively). e, For each of the events in d led by a PIS/Rab10 dynamic domain, we scored if the event led to a successful fusion reaction with adjacent ER domains. The efficiency of fusion was calculated for puncta containing WT or T23N Rab10. Note that PIS/Rab10 T23N puncta had a highly reduced ability to successfully fuse. All times indicated are in seconds. Means ± standard errors were calculated for each graph; for c, n = 10 cells; for d and e, n = 30 cells, 2 boxes per cell. *** P < 0.001.
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Figure 6. CEPT1 and PIS partition with Rab10 to ER-associated dynamic domainsa, A Cos-7 cell expressing BFP-Rab10 with mCh-CEPT1 (first panel), mCh-PIS (second panel), mCh-Sec61β (third panel) or KDEL-venus (fourth panel). Left image shows mCh-CEPT1, mCh-PIS, mCh-Sec61β or KDEL-venus, respective of panel; middle image shows BFP-Rab10. Right image shows merge for each condition. Scale bars = 2 μm.. b, Percentage of co-localization for cells expressing BFP-Rab10 with mCh-CEPT1, mCh-PIS, mCh-Sec61β or KDEL-venus, as measured by Spearman-Pearsonâs co-localization coefficient in a 10 μm2 box. Note that CEPT1 and PIS are enriched in a domain with Rab10 relative to the other ER markers tested. Means ± standard errors were calculated from each condition, n = 10 cells for each condition. ** P < 0.01.
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Figure 7. Rab10 regulates the formation of Rab10/PIS/CEPT1 dynamic punctaa, A Cos-7 cell co-expressing mCh-CEPT, GFP-PIS and BFP-Rab10 WT (CEPT1 in red, PIS in green, Rab10 WT in blue). Note that CEPT1, PIS and Rab10 co-localize throughout the ER and at punctate structures. b, A Cos-7 cell co-expressing mCh-CEPT, GFP-PIS and BFP-Rab10 T23N (CEPT1 in red, PIS in green, Rab10 T23N in blue). Note that CEPT1 no longer forms punctate structures and PIS puncta are markedly smaller. For a and b, third and fourth panels show merged image and zoom of boxed region, respectively, top panel scale bars = 10 μm, bottom panel scale bars = 2 μm.. c, Cells as in a and b were imaged live, with two images acquired, taken 1 minute apart. These images were superimposed in order to visualize the change in GFP-PIS over a 1 minute time period with BFP-Rab10 WT (left panel) or BFP-Rab10 T23N (right panel) expression. Each image shows GFP-PIS at t = 0 (green) and t = 60 s (red). Immobile puncta will appear yellow. Note that the 60 second overlay for GFP-PIS expressed with BFP-Rab10 T23N shows very little change in PIS dynamics. Scale bars = 2 μm. d, Percentage of co-localization for cells expressing GFP-PIS withBFP-Rab10 WT or BFP-Rab10 T23N, as measured by Spearman-Pearsonâs co-localization coefficient. Note that the higher the percentage co-localization, the less overall dynamics. Means ± standard errors were calculated from each condition, n = 10 cells for each condition. *** P < 0.001.
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Figure 8. Atlastin and Rab10 do not overlap in function or locationa, Atl3 accumulates at 3-way junctions. Confocal images of Cos-7 cells co-expressing GFP-tagged human Atl3 (GFP-Atl3 in green) and an ER luminal marker (mCh-KDEL in red). b, Cos-7 cells expressing GFP-Atl3 (green) and mCh-KDEL (red) with BFP-Rab10 (blue) WT (top panel) or BFP-Rab10 T23N (bottom panel). Note that expansion of cisternal ER induced by Rab10 T23N expression, even in the presence of Atl3. c, Cos-7 cells were co-transfected with GFP-Atl3 (green), mCh-KDEL (red) and either control or Rab10 siRNA, note the expansive cisternae in the Rab10 siRNA depleted sample, even in the presence of Atl3. d, Live images of Cos-7 cells expressing GFP-Atl3 (green), mCh-PIS (red), and BFP-Rab10 WT (blue). Note that Atl3 puncta do not co-localize with Rab10/PIS puncta. For all images: the second to last images shows merged images. The last panels show zoomed images of boxed regions in merge. Images in a, b, c and d, scale bars = 10 μm.
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