SUPPLEMENTARY INFORMATION

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Flora Chambers
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DOI: 10.1038/ncb2647 Figure S1 Other Rab GTPases do not co-localize with the ER.

1 DOI: /ncb2647 Figure S1 Other Rab GTPases do not co-localize with the ER. a, Cos-7 cells cotransfected with an ER luminal marker (either KDEL-venus or mch-kdel) and mch-tagged human Rab5 (mch-rab5, top panel), GFP-tagged human Rab7 (GFP-Rab7, middle panel), or mch-tagged human Rab11 (mch-rab11, bottom panel). These Rabs all localized to distinct vesicular compartments and did not co-localize with the luminal ER marker (Rab, red; KDEL, green). The third and fourth image in each panel shows merged images (ER in green, Rab in red) and zoomed images of boxed regions in merge, respectively. Scale bars = 10 µm. 1

2 Figure S2 Rab10 GTPase localizes to the ER and Golgi. a, Sequence alignment of Xenopus laevis Rab8/10 with Homo sapiens paralogs Rab8 and Rab10. b, Cos-7 cells were co-transfected with an ER luminal marker (KDEL-venus) and low amounts of mch-rab10. c, Cos-7 cells were cotransfected with mch-rab10 and a Golgi marker (GPP130-eGFP), mch- Rab10 and a mitochondrial marker (GFP-mito), BFP-tagged human Rab10 (BFP-Rab10) and an early endosome marker (mch-rab5), or BFP-Rab10 and a recycling endosome marker (mch-rab11), as indicated. Right panels in b and c, show merged images and zoomed images of boxed region (Rab10 in green, organelle markers in red). For b and c, scale bars = 10 µm. 2

3 Figure S3 Rab10 has a similar localization and function in HeLa cells. a, HeLa cells co-expressing mch-rab10 WT or mch-rab10 T23N 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 (Rab in green, ER in red). Scale bars = 10 µm. b, Immuno-blot analysis with antibodies against Rab10 reveals expression levels of endogenous and expressed Rab10 constructs under various conditions: no transfection, mch-rab10 WT or mch-rab10 T23N. GAPDH protein levels were measured as a loading control. When measured by densitometry, fusion proteins were found to be expressed 2.5 and 2.8- fold over endogenous for WT and T23N, respectively. Arrowhead indicates expressed mch-rab10 constructs. Asterisk indicates endogenous Rab

4 Figure S4 Rab10 sirna does not affect Golgi morphology. a, Cos-7 cells transfected with control sirna (top panel) or Rab10 sirna (bottom panel) and expressing Golgi marker GPP130-eGFP and mch-kdel (GPP130 in green, KDEL in red). b, Cos-7 cell transfected with GDP-locked mcherry tagged human Rab5 (mch-rab5 S34N) and KDEL-venus; note that the ER morphology remains highly tubular, (Rab5 S34N in red, KDEL in green). c, Cos-7 cell transfected with KDEL-venus and mcherry ER integral membrane protein, Climp63 (mch-climp63) and KDEL-venus. Note that expression of mch-climp63 results in cisternal ER, (Climp63 in red, KDEL in green). For a, b and c: the third image in each panel shows merged images. The fourth image in each panel shows zoomed, merged images. Boxes highlight the zoomed region. Scale bars = 10 µm. d, Percentage of the ER comprised of cisternae for cells transfected with KDEL-venus and mch-rab5 WT, mch-rab5 S34N or mch- Climp63. As additional controls for our cisternal ER analysis, we expressed both WT and GDP-locked mch-rab5 (S34N); expression of these constructs did not alter the percentage of ER that was cisternal when compared to other controls, 20% for both mch-rab5 WT and mch-rab5 S34N. Means ± standard errors were calculated from each condition, n = 20 cells, 3 boxes per cell. 4

5 Figure S5 Fusion events per area of ER (µm 2 ) a, Quantification of successful ER fusion events/area of ER (µm 2 ) over a time course of 5 min for KDEL-venus alone or KDEL-venus with mch-rab10 WT, mch-rab10 T23N. b, Quantification of successful ER fusion events/area of ER (µm 2 ) over a time course of 5 min for KDEL-venus with control or Rab10 sirna. Means ± standard errors were calculated for a and b, n = 30 cells for each condition. *** P <

6 Figure S6 Rab10-mediated dynamics occur on microtubules. a, Cos-7 cells expressing BFP-Rab10 WT, mch-tagged α-tubulin (to visualize MTs) and KDELvenus (not shown) were imaged live by confocal fluorescence microscopy. Arrowheads mark dynamic ER domains mediated by BFP-Rab10 punctum (Rab10, green; α-tubulin, red). Times indicated are in seconds. b, Cells as in a were imaged live before and after 5 µm nocodazole (NZ) to depolymerize MTs. Two images, taken 1 minute apart, were collected at 0, 20 and 60 minutes post nocodazole treatment. These images were superimposed in order to visualize the change in ER/Rab10 over a 1 minute time period before and after NZ treatment at each time point. Top panel shows BFP-Rab10 at t = 0 (green) and t = 60 seconds (red), white arrowheads indicate Rab10 rearrangement, blue arrowhead indicates ER fusion event. Bottom panel shows the sensitivity of the MT network to NZ treatment over time. Note that the overlay from samples treated for 60 minutes with NZ, shows very little change in ER/Rab10 positioning, indicating that Rab10 dynamic domains have stopped moving once MTs have depolymerized. Images in a and b, scale bars = 2 µm. 6

Figure S7 CEPT1 localizes to Rab10/PIS dynamic domain. a, A Cos-7 cell coexpressing KDEL-venus and mch-tagged human CEPT1 (mch-cept1) (CEPT1 in red, KDEL in green). Scale bar = 10 µm.

7 Figure S7 CEPT1 localizes to Rab10/PIS dynamic domain. a, A Cos-7 cell coexpressing KDEL-venus and mch-tagged human CEPT1 (mch-cept1) (CEPT1 in red, KDEL in green). Scale bar = 10 µm. b, As in a for cells expressing KDEL-venus, mch-cept1 (CEPT1 in red, KDEL in green). Note that CEPT1 localizes at dynamic domains that do not initially label with KDEL. Arrowheads mark position of ER extension that follows the mch-cept1 dynamic domain structure. Times indicated are in seconds. c, A Cos-7 cell co-expressing KDELvenus, mch-cept1 and BFP-Rab10 WT (CEPT1 in red, Rab10 in blue, KDEL in green); top panel shows overlay, bottom panel shows KDEL-venus alone to illustrate the absence of the ER from the Rab10/CEPT1 domain. Note the positions where Rab10 and CEPT1 co-localize at dynamic domains that do not initially label with KDEL. Arrowheads mark positions of successful fusion events that follow the BFP-Rab10/mCh-CEPT1 dynamic domain structures. Times indicated are in seconds. In b and c, scale bars = 2 µm. d, Percentage of co-localization between Rab10, CEPT1 and PIS; note that the majority of puncta have Rab10, CEPT1 and PIS, n = 10 cells. 7

8 Figure S8 Rab10-mediated ER assembly model. a, Model depicting known ER domains (in green) and domain regulating proteins. Rab10 dynamic domains (in pale green) travel along MTs (in gray) to facilitate homotypic ER fusion reactions. Rab10 (in blue) and the lipid synthesizing proteins PIS (in red) and CEPT1 (in purple) localize to dynamic domains at leading edge of nearly half of all ER tubule dynamics events. Atlastin-mediated ER fusion machinery (in orange) localizes to 3-way junctions. The organization of the peripheral ER cisternae is regulated by Climp63 (in yellow) and large protein complexes such as polyribosomes (not shown). Reticulon proteins (in turquois) oligomerize to maintain the tubular ER and curved edges of the cisternal ER. b, Rab10 and lipid synthesizing enzymes (PIS and CEPT1) localize to punctate structures at the dynamic tips of the ER (in red). GTPbound Rab10 facilitates ER assembly on the MT cytoskeleton (in blue), with the lipid synthesizing enzymes PIS and CEPT1 presumably providing the necessary lipids required for ER growth and dynamics. Depletion of endogenous Rab10 or expression of GDP-locked Rab10 results in the inhibition of ER assembly and an increase in ER cisternae at the expense of ER tubules (ER in green). 8

$Figure S9 Uncropped scans of immunoblots. a, Uncropped immunoblot from Figure 1e. Reactions in Figure 1b were alternatively spun down and separated into soluble (S) and membrane (M) fractions.$

9 Figure S9 Uncropped scans of immunoblots. a, Uncropped immunoblot from Figure 1e. Reactions in Figure 1b were alternatively spun down and separated into soluble (S) and membrane (M) fractions. Soluble and membrane proteins were analyzed by immuno-blotting (IB) with antibodies recognizing Xenopus Rab8/10. Note that Rab 8/10 is displaced from the ER vesicles by concentrations of Rab GDI that inhibit tubule formation (as assayed in Figure 1a and 1b). b, Uncropped immuno-blot from Figure 3b. Immuno-blot analysis with antibodies against Rab10 reveals efficient depletion of Rab10 by sirna. GAPDH protein levels were measured as a loading control. c, Uncropped immuno-blot from Supplementary Figure 3b. Immuno-blot analysis with antibodies against Rab10 reveals expression levels of endogenous and expressed Rab10 constructs under various conditions: no transfection, mch-rab10 WT or mch-rab10 T23N. GAPDH protein levels were measured as a loading control. When measured by densitometry, fusion proteins were found to be expressed 2.5 and 2.8-fold over endogenous for WT and T23N, respectively. Arrowhead indicates expressed mch-rab10 constructs. Asterisk indicates endogenous Rab

10 Supplementary Videos Supplementary Video 1 Relative localization over time of Rab10 WT and an ER luminal protein. Cos-7 cells expressing mch-rab10 WT and KDEL-venus (Rab10 WT, red; KDEL, green) were imaged live by confocal fluorescence microscopy. Scale bar = 10 µm. Supplementary Video 2 Relative localization of PIS, Rab10 WT and an ER luminal protein. Cos-7 cells co-expressing mch-pis, BFP-Rab10 WT and KDELvenus (PIS, red; Rab10 WT, blue; KDEL, green) were imaged live by confocal fluorescence microscopy. Scale bar = 10 µm. Supplementary Video 3 Relative localization of Rab10 WT, CEPT1 and PIS. Cos-7 cells co-expressing BFP-Rab10 WT, mch-cept1 and GFP-PIS (Rab10 WT, blue; CEPT1, red, GFP, green) were imaged live by confocal fluorescence microscopy. Scale bar = 10 µm. Supplementary Video 4 Relative localization and structure of the ER in cells expressing Rab10 T23N, CEPT1 and PIS. Cos-7 cells co-expressing BFP-Rab10 T23N, mch-cept1 and GFP-PIS (Rab10 T23N, blue; CEPT1, red, GFP, green) were imaged live by confocal fluorescence microscopy. Scale bar = 10 µm. Supplementary Video 5 Relative localization of PIS, Rab10 WT and Atl3 puncta. Cos-7 cells expressing mch-pis, BFP-Rab10 WT and GFP-Atl3 (mch-pis, red; BFP-Rab10 WT, blue; GFP-Atl3, green) were imaged live by confocal fluorescence microscopy. Scale bar = 10 µm. Table S1 Rab10 Isoforms PER Counts. Raw data presented in Figure 2d. Percentage of the ER comprised of cisternae for cells expressing KDEL-venus alone or together with mch-rab10 WT, mch-rab10 Q68L, or mch-rab10 T23N. To determine ER morphology composition, three 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 or the total ER. 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. Means ± standard errors were calculated from each condition, n = 50 cells for each condition, 3 regions per cell. *** P < Table S2 Controls for PER Counts. Raw data presented in Supplementary Figure 4d. Percentage of the ER comprised of cisternae for cells transfected with KDEL-venus and mch-rab5 WT, mch-rab5 S34N or mch-climp63 (analysis performed as described in Table S1). Means ± standard errors were calculated from each condition, n = 20 cells, 3 boxes per cell. Table S3 Control vs Rab10 sirna PER Counts. Raw data presented in Figure 3c. Percentage of the ER comprised of cisternae for cells transfected with control or Rab10 sirna (analysis performed as described in Table S1). Means ± standard errors were calculated from each condition, n = 50 cells, 3 regions per cell. *** P < Table S4 Rab10 Isoforms Fusion Counts. Raw data presented in Figure 4b, 4c and Supplementary Figure 5a. Cos-7 cells expressing KDEL-venus and mch-rab10 WT or mch-rab10 T23N were imaged live to visualize ER tubule extension and fusion events that occurred in a 10 µm 2 box over a 5 min time course. These tubular extensions were scored as either resulting in a successful or unsuccessful fusion event. The successful and unsuccessful fusion events measured were alternatively assayed as a percentage of events leading to fusion and ER fusion events/area of ER (µm 2 ). Means ± standard errors were calculated from each condition, n = 30 cells, 2 regions per cell. *** P < Table S5 Control vs Rab10 sirna Fusion Counts. Raw data presented in Figure 4e, 4f and Supplementary Figure 5b. Cos-7 cells expressing KDEL-venus to visualize ER extension and fusion were co-transfected with control sirna or Rab10 sirna and imaged live to visualize ER tubule extension and fusion events that occurred in a 10 µm 2 box over a 5 min time course. These tubular extensions were scored as either resulting in a successful or unsuccessful fusion event. The successful and unsuccessful fusion events measured were alternatively assayed as a percentage of events leading to fusion and ER fusion events/ area of ER (µm 2 ). Means ± standard errors were calculated from each condition, n = 30 cells, 2 regions per cell. *** P < Table S6 Puncta Counts WT vs T23N BFP-Rab10 + mch-pis. Raw data presented in Figure 5c. Cos7 cells expressing KDEL-venus, mch-pis and either BFP- Rab10 WT or BFP-Rab10 T23N. Percentage of ER attached puncta that contain both BFP-Rab10 and mch-pis, BFP-Rab10 alone, or mch-pis alone (n = 444 puncta from 10 cells for BFP-Rab10 WT and 492 puncta from 10 cells for BFP-Rab10 T23N). *** P < Table S7 ER Dynamics WT vs T23N BFP-Rab10 + mch-pis. Raw data presented in Figure 5d, 5e. Cos7 cells expressing KDEL-venus, mch-pis and either BFP-Rab10 WT or BFP-Rab10 T23N were imaged live by confocal FM. All ER extension events occurring in a 10 µm 2 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). For each of the events 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. Means ± standard errors were calculated for each graph; n = 30 cells, 2 boxes per cell. *** P < Table S8. Rab10 Co-Localization. Raw data presented in Figure 6b. Cos-7 cell expressing BFP-Rab10 with mch-cept1, mch-pis, mch-sec61β or KDELvenus. Percentage of co-localization for cells expressing BFP-Rab10 with mch-cept1, mch-pis, mch-sec61β or KDEL-venus, as measured by Spearman- Pearson s co-localization coefficient in a 10 µm 2 box. Means ± standard errors were calculated from each condition, n = 10 cells for each condition. ** P < Table S9 Puncta Profile. Raw data presented in Supplementary Figure 7d. We determined the presence of BFP-Rab10, mch-cept1 and GFP-PIS at all of the puncta present in the imaged cells; n = 10 cells/condition. Table S10 WT vs T23N BFP-Rab10 T0-T60. Raw data presented in Figure 7d. Percentage of co-localization for cells expressing GFP-PIS withbfp-rab10 WT or BFP-Rab10 T23N, as measured by Spearman-Pearson s 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 <

13-3. Synthesis-Secretory pathway: Sort lumenal proteins, Secrete proteins, Sort membrane proteins

13-3. Synthesis-Secretory pathway: Sort lumenal proteins, Secrete proteins, Sort membrane proteins Molecular sorting: specific budding, vesicular transport, fusion 1. Why is this important? A. Form and