© 2020. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs239814. doi:10.1242/jcs.239814

RESEARCH ARTICLE Vesicular and uncoated Rab1-dependent cargo carriers facilitate ER to Golgi transport Laura M. Westrate1,*, Melissa J. Hoyer2,3,*, Michael J. Nash2 and Gia K. Voeltz2,3,‡

ABSTRACT next recruits the heterodimer ‘inner coat’ complex Sec23/Sec24, Secretory cargo is recognized, concentrated and trafficked from which stabilizes membrane curvature and recruits cargo through the endoplasmic reticulum (ER) exit sites (ERES) to the Golgi. Cargo cargo binding sites on Sec24 (Bi et al., 2002; Kuehn et al., 1998; export from the ER begins when a series of highly conserved COPII Miller et al., 2002, 2003). The inner coat recruits the heterotetramer ‘ ’ coat proteins accumulate at the ER and regulate the formation of outer coat complex composed of Sec13/Sec31, resulting in the cargo-loaded COPII vesicles. In animal cells, capturing live de novo formation of a fully formed COPII-coated transport vesicle that cargo trafficking past this point is challenging; it has been difficult to delivers incorporated cargo to the Golgi complex via anterograde discriminate whether cargo is trafficked to the Golgi in a COPII-coated trafficking from the ER (Kirk and Ward, 2007; Lederkremer et al., vesicle. Here, we describe a recently developed live-cell cargo export 2001; Lee et al., 2004; Rossanese et al., 1999; Stagg et al., 2006). system that can be synchronously released from ERES to illustrate de Many of these studies have been performed in yeast cells because of novo trafficking in animal cells. We found that components of the the advantages of yeast genetics in identifying COPII components, COPII coat remain associated with the ERES while cargo is extruded combined with temperature-sensitive mutants that can stall cargo as it into COPII-uncoated, non-ER associated, Rab1 (herein referring to is trafficked. Rab1a or Rab1b)-dependent carriers. Our data suggest that, in Animal cells may have adapted alternative methods to ensure rapid animal cells, COPII coat components remain stably associated with cytoplasmic trafficking of cargo from ERES to the Golgi in a vast the ER at exit sites to generate a specialized compartment, but once cytoplasm. Early reports on mammalian cells suggested that COPII cargo is sorted and organized, Rab1 labels these export carriers and vesicles rapidly lose their coat after scission from the ER (Antonny facilitates efficient forward trafficking. et al., 2001; Aridor et al., 1995; Scales et al., 1997; Presley et al., 1997; Stephens et al., 2000) and immuno-electron microscopy evidence – This article has an associated First Person interview with the first identified the existence of free COPII-coated vesicles at the ER Golgi De novo author of the paper. interface (Zeuschner et al., 2006). ER to Golgi cargo trafficking is difficult to resolve in live animal cells because secretory KEY WORDS: COPII, Rab1, ERES, TNF-α, MANII, RUSH proteins are continuously being synthesized and move rapidly through the secretory pathway. Additionally, temperature-sensitive mutants of INTRODUCTION the COPII coat are not available in animal cells. Instead, although The endoplasmic reticulum (ER) serves as the entry point for the various techniques have been used to study secretory cargo trafficking secretory pathway. Following successful translation and translocation, in animal cells, the majority of studies have utilized a single secretory proteins synthesized in the ER are concentrated by COPII temperature-sensitive fluorescently tagged cargo, ts-VSVG (Bonfanti proteins at ER exit sites (ERES) and trafficked to the Golgi within et al., 1998; Gallione and Rose, 1983; Kreitzer et al., 2000; Shomron vesicular carriers (D’Arcangelo et al., 2013). The order of assembly of et al., 2019 preprint). At non-permissive temperature, GFP-ts-VSVG is the COPII coat and its role in generating a vesicle that contains secretory stuck in the ER as an unfolded protein. A shift to permissive cargo has been dissected in molecular detail by elegant in vivo and temperature allows VSVG to fold and be exported. This cargo can be in vitro studies. The major components include a guanine exchange visualized live as it leaves the ER in animal cells, and previous factor, Sec12, that recruits and activates the small GTPase Sar1 to experiments with this cargo have suggested that although the cargo the ER (Nakano and Muramatsu, 1989; Nakano et al., 1988). Upon initially co-localizes with a component of the COPII coat at ERES, it GTP activation, Sar1 inserts an amphipathic helix into the ER does not exit the ERES with Sec24 (Presley et al., 1997; Stephens membrane to initiate membrane deformation (Lee et al., 2005). Sar1 et al., 2000). These data first suggested that some aspects of the transition from COPII-coated vesicle formation to a vesicular structure 1Department of Chemistry and Biochemistry, Calvin University, Grand Rapids, MI trafficking towards the Golgi might not require COPII. 49546, USA. 2Howard Hughes Medical Institute, Department of Molecular, Cellular, The current model for mammalian ER to Golgi trafficking remains and Developmental Biology, University of Colorado-Boulder, Boulder, CO 80309, USA. 3Department of Molecular, Cellular, and Developmental Biology, University of somewhat controversial. Many have cited that ER to Golgi trafficking Colorado-Boulder, Boulder, CO 80309, USA. relies on both vesicular and tubular intermediate compartments (Saraste *Co-first authors and Svensson, 1991; Stinchcombe et al., 1995; Watson and Stephens, ‡Author for correspondence ([email protected]) 2005; Xu and Hay, 2004). The proposed function of these structures is to sort the ER exit site cargo to the Golgi (Bannykh et al., 1996). Many G.K.V., 0000-0003-3199-5402 of these structures are not static and can make long-range movements

This is an Open Access article distributed under the terms of the Creative Commons Attribution (Presley et al., 1997; Scales et al., 1997). Studies monitoring the VSVG License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, cargo system have shown that when the temperature is reduced, distribution and reproduction in any medium provided that the original work is properly attributed. intermediate structures become enlarged, accumulate secretory cargo Handling Editor: Jennifer Lippincott-Schwartz and form in the vicinity of COPII-labeled ERES (Mironov et al., 2003;

Received 29 September 2019; Accepted 19 June 2020 Presley et al., 1997; Stephens, 2003). These structures acquire the Journal of Cell Science

1 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs239814. doi:10.1242/jcs.239814 vesicular coat complex COPI (Aridor et al., 1995; Lee et al., 2004; cargo trafficking from ERES relative to the COPII coat components Shomron et al., 2019 preprint; Stephens et al., 2000) and, upon and the Rab1 GTPase (herein referring to Rab1a or Rab1b). We have temperature release, the structures move towards the Golgi complex in used three different mammalian cell lines, four fluorescently tagged a microtubule-dependent manner (Presley et al., 1997; Shima et al., COPII coat proteins, two different cargoes and a Rab membrane 1999; Stephens et al., 2000). More recent studies have keyed in on how marker to demonstrate that COPII coat components predominately these intermediate compartment structures rearrange to facilitate remain at stable ERES as cargo leaves in a Rab1-dependent carrier. trafficking of large cargoes such as collagen (McCaughey et al., 2019; Saito et al., 2009). With the help of Tango1, intermediate RESULTS structures could potentially form tunnels from the ER to Golgi (Raote COPII components localize to stable domains on peripheral and Malhotra, 2019). It is currently unclear whether the creation of ER tubules larger tubular clusters is due to large cargoes or, potentially, an We used live confocal fluorescence microscopy to visualize the abundance of cargo in cells. However, with the advent of faster and distribution and dynamics of four major COPII components at more sensitive imaging techniques and the development of newly ERES in three different animal cell lines: COS-7, HeLa and U2OS. trackable trafficking cargoes, we can now combine these advanced First, we assessed the location and dynamics of fluorescently tagged methods to visualize this vital cargo trafficking step in live cells. and exogenously expressed GFP-Sec16s, a resident ER protein that Here, we follow trafficking of COPII ERES markers and ER export serves as a scaffold for COPII assembly at ERES (Watson et al., cargoes, including the classically used ts-VSVG and two cargoes 2006). GFP-Sec16s localized to punctate structures along ER with varying size that release from the ER upon addition of biotin tubules in all three cell types (Fig. 1A; Fig. S1, Movie 1). We using the ‘retention using selective hooks’ (RUSH) system collected 2 min movies (with 5 s intervals) of GFP-Sec16s puncta to (Boncompain et al., 2012; Presley et al., 1997). We have optimized track their location relative to the tubular ER network [labeled with the RUSH system to measure the rapid release of de novo secretory mCherry (mCh)-KDEL] over time. As expected for an ERES

Fig. 1. COPII components localize to stable domains on peripheral ER tubules. (A) Representative merged images of COS-7 cells reveal the distribution of the ER membrane network relative to COPII components by live cell confocal fluorescence microscopy (ER labeled with mCh-KDEL in red; GFP-Sec16s, GFP-Sec23A, GFP-Sec24D, or YFP-Sec31A in green). Magnification of boxed areas shown below. (B) COPII outer coat component YFP-Sec31A (green) is tracked over time for 2 min to quantify YFP-Sec31A puncta association with the ER (mCh-KDEL, red) over time. (C) COPII-labeled puncta (as in B) were tracked live over time to determine whether they remain tightly associated with the ER network for the duration of the 2 min movie in COS-7, HeLa, or

U2OS cells. 3 replicates; 15 cells for Sec16s, 15 cells for Sec23A, 19 cells for Sec24D and 18 cells for Sec31A. Scale bars: 5 µm (A), 2 µm (B). Journal of Cell Science

2 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs239814. doi:10.1242/jcs.239814 scaffold, the vast majority of Sec16s puncta (98.2%) remained COS-7 and HeLa cells, fluorescence recovery occurred at the ROI associated with the ER network throughout the length of the movie marking the original COPII puncta nearly 90% of the time (87.9± (puncta were scored as ‘associated’ if they were visible and tracked 6.53% in COS7, 88.8±4.25% in HeLa cells; Fig. S3D) suggesting with an ER tubule for the entire length of the movie). that COPII components are recruited to stable ERES domains. We performed a similar analysis on fluorescently tagged COPII inner and outer coat components Sec23A, Sec24D and Sec31A Dynamic COPII domains are tethered to sliding ER tubules (Fig. 1A). Although most of the COPII mammalian isoforms are COPII- and ERES-labeled structures have been shown to be functionally redundant, the four mammalian Sec24 isoforms have relatively stable in yeast and plants (DaSilva et al., 2004; Hanton distinct and overlapping specificity in the cargo export signals et al., 2009; Kirk and Ward, 2007; Shindiapina and Barlowe, 2010). recognized (Khoriaty et al., 2018; Stankewich, 2006; Wendeler To characterize the dynamics of COPII coat components in et al., 2007). Sec24D was chosen because it binds strongly to four mammalian cells, we tracked COPII-labeled domains over time. out of six common ER export signals, whereas the other three Sec24 We found that COPII coat components remain tightly associated isoforms strongly bind only two out of six (Wendeler et al., 2007). with the ER. Consistent with previous experiments performed on Similar to GFP-Sec16s, puncta of GFP-Sec23A, GFP-Sec24D and Sec24D, COPII sites were relatively stable in the peripheral ER YFP-Sec31A were all distributed throughout the cytoplasm and (Gupta et al., 2008; Stephens et al., 2000). However, we were all tightly associated with the tubular ER network during 2 min occasionally observed a COPII-labeled domain traveling a long movies and in multiple cell types (Fig. 1B,C; Fig. S1, Movies 2-4). distance (over 2 µm; Fig. 2A,B) while maintaining contact with ER Nearly 100% of all COPII components remained associated with the tubules. The total length of movement was highly variable, with the ER throughout the length of the movie (98.2±4.8% for Sec16s, 99.7± average distance traveled being about 5 µm (4.67±0.4 µm for 1.2% for Sec23A, 98.0±8.6% for Sec24D and 99.0±3.9% for Sec16s and 5.51±0.5 µm for Sec31A; Fig. 2C). This type of coupled Sec31A in COS-7 cells; Fig. 1C). COPII components were scored as movement was reminiscent of ER sliding, whereby ER tubules remaining associated with the ER if they remained tethered to the ER extend along the side of an existing microtubule at a velocity of throughout the 2 min movie. The small percentage of COPII ∼0.5 µm/s (Friedman et al., 2010). To confirm this mechanism of components that were marked as not being associated with the ER movement, we measured the maximum velocity of COPII puncta often represented puncta that appeared to become dissociated from during long-range trajectories. Consistent with known velocities for the ER as a result of the ER network or a COPII punctum moving out ER sliding (Friedman et al., 2010), dynamic ER-associated COPII of the focal plane. Thus, despite being associated with the ER for the domains traveling more than 2 µm had a maximum velocity of majority of the movie, if there were at least two sequential frames ∼0.5 µm/s along microtubules (0.41±0.03 µm/s for Sec16s and 0.51± (10 s) where the ER and COPII punctum could not be tracked 0.04 µm/s for Sec31A; Fig. 2D). Additionally, when we observed together, the COPII component was marked as ‘not associated.’ HeLa these dynamic events in conjunction with fluorescently labeled and U2OS cells, which are thicker than COS7 cells, showed a higher microtubules, we could track COPII puncta traveling along existing propensity for non-association and therefore demonstrated slightly microtubules (Fig. 2F), thus providing additional evidence that these reduced percentages of COPII components remaining associated with long-range movements are trafficking via ER sliding. By tracking the the ER through the 2 min movie (Fig. 1C). Notably, we never direction of these long-range COPII movements, we found that captured a COPII-coated vesicle budding off or trafficking away from the majority of events (73% for Sec16s and 74% for Sec31A) move in the ER in live cell images. These data suggest that COPII coat the retrograde direction towards the Golgi (Fig. 2E). Despite the components form stable structures at ERES in animal cells. distance traveled, these dynamic COPII-labeled domains remain To challenge the strength of the association between the ER and tethered to the ER during long-range movements, suggesting that they COPII-coated domains, we treated cells with ionomycin, an ionophore are not released from the ER membrane. that triggers increased intracellular calcium and subsequent fragmentation of the ER (Fig. S2A) (Koch et al., 1988). COS-7 cells Using the RUSH system to monitor cargo trafficking from ER were treated with 2 µM ionomycin (5 min) and fixed before acquiring to Golgi confocal z-slices through the entire cell. Even upon fragmentation of Next, we analyzed whether COPII-labeled puncta bud off from the the ER, we still observed that COPII-labeled domains remained ER only once they are loaded with cargo to be trafficked. co-localized with ER vesicles: 86.7±13.6% for Sec16s, 91.3±10.2% Conflicting results from the mammalian field have questioned the for Sec23A, 94.1±6.8% for Sec24D and 89.3±15.3% for Sec31A fate of COPII components following cargo packaging at the ER. (Fig. S2B-D). These data further support the notion that COPII Specifically, work by Stephens et al. (2000), Scales et al. (1997) and domains are integral components of ERES in animal cells. Presley et al. (1997) using a temperature block cargo release system Further evidence that COPII components form stable structures at (VSVG) showed that cargo can be transported to the Golgi in the ERES in animal cells came from fluorescent recovery assays used to absence of COPII components, whereas immuno-electron measure the dynamics of the COPII outer coat component Sec31A tomography work by Zeuschner et al. (2006) identified the in COS7 and HeLa cells (Fig. S3A,B, respectively). A 10×10 µm existence of COPII-coated transport vesicles at the ER–Golgi region of interest (ROI) was photobleached in the peripheral ER of interface. We therefore vetted potential systems for visualizing cells expressing outer coat component YFP-Sec31A and a general cargo release from the ERES relative to COPII coat components and ER marker (mCh-KDEL). The YFP signal within the ROI was hit ER that would provide a systematic analysis of secretory cargo with a high-power 488 nm laser to diminish YFP signal selectively. export dynamics from the ER. We first optimized the RUSH system We intentionally picked a laser exposure that did not result in 100% to synchronize and track the release of cargoes exiting the ER in real loss of signal so that we could confidently track whether time (Fig. 3A) (Boncompain et al., 2012). The RUSH system fluorescence recovered to the ROI that marked the original COPII utilizes an ER ‘hook’ protein fused to streptavidin and a secretory site on the ER (Fig. S3C). Fluorescence intensity was calculated at cargo protein fused to streptavidin-binding protein (SBP), which the frame immediately before photobleaching (pre), the frame under normal conditions traps the secretory cargo in the ER as a immediately after bleaching (post) and 30 s after bleaching. In both result of the streptavidin–SBP interaction. Biotin addition Journal of Cell Science

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Fig. 2. Dynamic COPII domains are tethered to sliding ER tubules. (A) Representative image of a COS-7 cell expressing an ER marker (mCh-KDEL in red) and GFP-Sec16s puncta (in green). In the zoomed insets, the Sec16s puncta (arrowheads) tracks with an attached ER tubule over time. (B) As in A for YFP-Sec31A puncta dynamics. (C) Total distance traveled by individual COPII-labeled domains displays long range movement within the cell (9 cells and 23 exit sites for Sec16s; 11 cells and 35 events for Sec31A). (D) Maximum velocity of indicated COPII puncta dynamics during long range movements. Maximum velocity was calculated by determining the maximum distance traveled between sequential frames acquired 5 s apart (9 cells and 17 events for Sec16s; 11 cells and 26 events for Sec31A). Error bars represent s.e.m. (E) Percentage of dynamic Sec16s or Sec31 puncta from C that moved in the retrograde versus anterograde direction. (F) Representative example of Sec31A punctum moving along an established microtubule while still associated with the ER. COS-7 cells expressed mCh-tubulin (gray), YFP-Sec31A (green) and BFP-KDEL (red). Scale bars: 5 µm (A,B), 2 µm (insets in A,B; F). Journal of Cell Science

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Fig. 3. Using the RUSH system to monitor cargo trafficking from ER to Golgi. (A) Cartoon of the RUSH system for cargo release from ERES upon biotin addition. (B) Representative image of cargo distribution in a COS-7 cell expressing mCh-TNF RUSH (cargo, red), GFP-Sec24D (COPII, green) and BFP-Sec61β (ER, gray) prior to biotin addition (0 min) and after biotin addition (20 min). Note that at 0 min, the ER network and ERES and TNF cargo are spread throughout the cytoplasm, with ERES and TNF cargo localized to the ER network. By comparison, 20 min after biotin addition, a dramatic increase in TNF cargo accumulation was observed in the perinuclear box (yellow). (C) Quantification of TNF cargo and Sec24D fluorescence in perinuclear box (representative of Golgi region) compared with peripheral signal before (0 min) or after biotin addition (20 min). The 5×5 μm2 regions selected in the periphery and perinuclear region were used to calculate fluorescence intensity in those cellular regions. The ratio of perinuclear to peripheral fluorescence signal was used to track any re-distribution of cargo or Sec24D-marked COPII exit sites following biotin addition (*P=0.003). (D,E) As in B and C for ER (BFP-Sec61B), mCh-TNF cargo and the outer coat component YFP-Sec31 (*P=0.024). (F,G) As in B and C for ER (BFP-Sec61B), mCh-Sec24D and GFP-ManII cargo (*P=0.019). Error bars represent s.e.m. Scale bars: 5 µm (B,D,F); 2 µm (insets in B,D,F). outcompetes the streptavidin–SBP interaction, resulting in Sec24D and YFP-Sec31A, respectively) with the cargo revealed synchronous release of secretory cargo from the ER that can be that these cargo-enriched puncta are often co-labeled with COPII tracked over time. SBP was linked to two different fluorescently proteins (Fig. 3B,D,F; Fig. 4). This implies that, in the absence of tagged cargoes for comparison: TNFα-SBP-mCh and ManII-SBP- biotin, the RUSH cargo is still capable of being loaded into COPII- GFP. The expression of TNFα-SBP-mCh or ManlI-SBP-GFP marked exit sites that are stalled for export as a result of the resulted in a striking localization pattern where the cargo, while streptavidin–SBP interaction occurring between the cargo and ER localized to the ER (0 min in Fig. 3), was observed to be hook protein. significantly enriched in small puncta reminiscent of what we see Biotin addition released forward trafficking of these SBP-tagged when we label for COPII proteins. Cargo concentration at ERES has secretory cargoes and TNFα-SBP-mCh became enriched at the been demonstrated to be an early step in the secretory pathway, with Golgi within 20 min of biotin treatment (Fig. 3B). By comparison, subsequent quality control checkpoints at the ER to ensure that only GFP-Sec24D (Fig. 3B,C) or YFP-Sec31A (Fig. 3D,E) coat properly folded proteins are allowed to traffic to vesicular-tubular components remained at puncta associated with the ER network clusters and the trans-Golgi network (Dukhovny et al., 2008, 2009; and did not accumulate with cargo at the Golgi. Similar results were Mezzacasa and Helenius, 2002). We therefore predicted that these seen with a different cargo, ManII-SBP-GFP, which also small puncta represent concentrated cargo at COPII sites that was accumulated at the Golgi within 20 min of biotin addition, unable to undergo forward transport because of its association with whereas again the mCh-Sec24D coat component, even though it the streptavidin ER hook protein. Dual labeling of both directly binds cargo during initial cargo sorting, also remained in fluorescently tagged inner and outer COPII coat proteins (GFP- ER-associated puncta (Fig. 3F,G). Significant redistribution of Journal of Cell Science

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Fig. 4. COPII components remain associated with the ER as cargo is exported. (A) COS-7 cell expressing mCh-TNF RUSH (red), GFP-Sec24D (green) and BFP-Sec61β (gray). Right: Time-lapse image of two events (from within yellow box) where fluorescently marked cargo (red) is observed leaving COPII fluorescent puncta (green) on the ER (gray) at the indicated time points following biotin addition. Arrows mark the first trafficking event and the arrowheads mark a second event. COPII is marked with a yellow arrow/head in each frame and the location of cargo as it traffics is marked with white. (B) Sec24D, TNF cargo and ER fluorescence was monitored over 2 min within an ROI (yellow circle) that marks the original site of cargo release. Graphs plot the relative fluorescence intensity and reveal that the ER and Sec24D fluorescence levels remain in the circle even after the cargo leaves. (C) As in A, for several events, the coat and cargo fluorescence was measured in pre and and post cargo leaving frames. Circles mark where fluorescence measurements were made. ROI1 is the site from which cargo is released and ROI2 is the site where cargo traffics to. Fluorescence at each region for each marker was background subtracted and the percentage fluorescence at each ROI calculated pre and post cargo leaving (****P<0.0001). (D) As in C for TNF cargo and YFP-Sec31A (****P<0.0001). (E) As in C for GFP- ManII cargo (****P<0.0001) and mCh-Sec24D (*P=0.03). (F) As in C for GFP-VSVG (****P<0.0001) and mCh-Sec24D (***P=0.0004 in ROI2). Error bars represent s.e.m. Scale bars: 5 µm (A); 1 µm (insets in A; B-F). cargo to the perinuclear region of the cell following biotin addition with the movement of the cargo (Fig. 4A, compare yellow and white was noted for mCh-TNFα and GPF-ManII cargo (P=0.003, 0.024 arrows; Movie 5). Cargo carriers dissociated from the tubular ER and 0.019 in Fig. 3C, E and G, respectively). These data show that it network as they trafficked (Fig. 4B). To quantify the amount of TNF is possible to stall and then release cargo in order to visualize how cargo relative to the amount of COPII coat protein that traffics away cargo traffics to the Golgi relative to COPII coat components. from the ER, we measured the fluorescence intensity of the ER, TNFα-SBP-mCh cargo and Sec24D in a region of interest (ROI) COPII components remain linked to the ER while cargo is (Fig. 4B, yellow circle) surrounding the ERES where cargo is exported away released from the ER. Fluorescence intensity at this site was plotted Next, we used the RUSH system to visualize cargo release from over a 2 min time course (Fig. 4B). We measured a drop in relative ERES and further investigate the properties of cargoes upon ER fluorescence for TNFα-SBP-mCh signal in the ERES region when release and trafficking to the Golgi. First, we stalled TNFα-SBP- cargo left the site. By comparison, relative fluorescent intensity for mCh at Sec24D-labeled ERES and labeled the ER with BFP- the COPII coat marker GFP-Sec24D and the ER signal BFP-Sec61β Sec61β (Fig. 4A,B). Following biotin addition, we visualized both remained constant before and after cargo release (Fig. 4B, line TNFα-SBP-mCh cargo as it trafficked away from the ER in punctate scan analysis depicting fluorescence at the site at each time point). carriers. Strikingly, fluorescence associated with the GFP-Sec24D These data suggest that cargo separates from COPII coat puncta stayed associated with the peripheral ER and did not track components as it exits the ER to traffic to the Golgi. Journal of Cell Science

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To obtain a more quantitative picture of cargo trafficking from rate we could capture a small amount of Sec31A following the cargo COPII components at ERES, we captured further events where signal (Fig. S5B). This fluorescence signal was very dim and cargo is exported from the ER. For each event, we analyzed the asymmetrically distributed, marking only a small percentage of the cargo leaving frames just before (pre) and just after (post) the event. entire cargo-containing puncta. In addition, it was lost within 5 s of In these frames, we defined equal area ROIs encircling where cargo cargo leaving (see images at t=8 s in event 1 and t=31 s in event 2, initiated from in the pre frame (ROI1) and encircled where cargo Fig. S5B). The rapid loss of the Sec31A fluorescence signal along trafficked to in the post frame (ROI2) (Fig. 4C). A dramatic drop in with the absence of detectable Sec24D signal suggests that cargo is percentage fluorescence was measured from pre to post frames for largely being exported from the ER in a carrier domain independent the ROI1 location for TNFα-SBP-mCh signal but not for the GFP- of COPII components. Sec24D signal in these events (P<0.0001; Fig. 4C). To further To confirm that these events leave the ER and were not confirm that we saw cargo leaving from the ER without the COPII potentially ER exit sites dividing into two separate sites, we coat, we performed the same analyses done for the TNFα-SBP-mCh repeated the cargo release experiments, imaging the ER (not cargo with the Sec24D COPII coat system, but varied the COPII previously done by Stephens et al., 2000) in relation to cargo release coat marker (Sec31A) (Fig. 4D) and the type of RUSH cargo events (Fig. 5). Even with the addition of the third channel to (ManII-SBP-GFP) (Fig. 4E). Additionally, we monitored the ts- capture the ER, we were still able to image cargo release events at a VSVG system with the Sec24D COPII coat (Fig. 4F). As with the high-speed interval (1 s). We sought to dissect further the steps that TNFα-SBP-mCh/Sec24D system, the TNFα-SBP-mCh cargo left occurred before and after cargo release in order to measure the Sec31A-marked ERES, ManII-SBP-GFP cargo left the Sec24D- accurately Sec24D and Sec31A coverage on the cargo puncta that marked ERES and VSVG cargo left the Sec24D-marked ERES trafficked away from the ER towards the Golgi. For consistency and (Fig. 4D-F; P<0.0001 for each condition except for pre/post analysis to directly compare and contrast the behavior of these two COPII of Sec24D in Fig. 4E where P=0.03, and the pre/post analysis of components, both Sec24D and Sec31A were tagged with the Sec24D in Fig. 4F where P=0.0004, calculated by comparing cargo mNeon fluorophore (Fig. 5). fluorescence pre and post event; Fig. S4). In all examples where We first characterized ERES before cargo release. We were able cargo left ROI1 to traffic to ROI2, the COPII coat components to capture events where the ER, cargo and components moved remained at ROI1 and did not travel with cargo to ROI2 (Fig. 4C-F; together as the ER network rearranged pre cargo release (Fig. 5A,B). Fig. S4). Together, these data support a model whereby the COPII These events demonstrated that cargo was still linked to the ER just coat proteins mark stable sites of protein export where cargo traffics prior to cargo release. All three components (ER, COPII and cargo) from the ER towards the Golgi in an uncoated carrier. Interestingly, first moved together pre cargo release (0-2 s, white arrow, Fig. 5B). we observed a statistically significant but minimal redistribution of Cargo release from ERES was observed at 3 s and was not affiliated Sec24 into ROI2 following ManII and VSVG cargo release with COPII coat components (yellow arrowhead, Fig. 5B). The co- (P=0.03; Fig. 4E). This could be the result of combined COPII/ movement of ER, COPII and cargo pre cargo release (0-2 s before cargo movement being captured in ROI2 before complete cargo release) further emphasized that both COPII and cargo were linked export had occurred or it could represent dynamic uncoating of the to the ER and not just closely associated prior to the moment of COPII coat being captured following cargo export (Fig. S4). cargo export. The cargo that we visualized trafficking from the ER membrane in To visualize the steps of cargo release (Fig. 5C,D; Movies 6 and 7), most cases did not have a tubular structure and usually maintained a we analyzed each event prior to cargo moving (0 s), when cargo circular nature more indicative of a vesicle (see examples in Fig. 4 and began to move from the ER but was still connected to ERES signal Fig. S4). However, due to the resolution of fluorescence microscopy, (1 s) and when cargo had moved from the ERES (2 s). To detect any we could not determine whether these structures were vesicles or a potential Sec signal over background, a threshold was used to convert cluster of vesicles in living cells. If these structures were rapidly Sec24D and Sec31A fluorescent images to binary images (Yen et al., uncoating, however, one would predict that the fluorescence intensity 1995). The cargo images were also converted to binary and this of COPII components at puncta would be significantly reduced as the detectible cargo signal set the cargo ROI (yellow outlines, Fig. 5E,F). cargo was exported from the ER. Instead, we observed negligible loss We then calculated the percentage of Sec24D or Sec31A pixels that of COPII fluorescence from the point of cargo export (Fig. 4), were within the cargo ROI at 0, 1 and 2 s for each event (Fig. 5G,H). suggesting instead that COPII forms stable sites on the ER that do not In the 1 s time points captured, cargo had not yet separated from traffic with cargo as it exports from the ER. the ERES, and both Sec24D and Sec31A signals were detected (Fig. 5G,H). To compare Sec24D and Sec31A distribution just as Rapid time-lapse imaging reveals cargo exports from ER in a cargo was leaving the ER, we scored the time point at which the cargo carrier uncoated by COPII ROI was fully separated from the ER and from any residual faint To better ascertain the dynamics of COPII components on cargo cargo still at the ERES (2 s time point, Fig. 5G-I). At this time point, domains being exported from the ER, we decided to track COPII Sec24D fluorescent signal had little overlap with the cargo ROI and cargo export at more rapid time points (Fig. 5; Fig. S5). First, we (1.2%), whereas Sec31A had a small, yet significantly different imaged TNF cargo export relative to Sec24 or Sec31 coat (P=0.003), percentage overlap with the cargo ROI (12.3%) (Fig. 5I). components at a higher frame rate (every 500 ms) to improve Thus, a small portion of the outer coat Sec31A protein can remain visualization of COPII coat and cargo dynamics (Fig. S5A,B). In associated, albeit briefly, with the cargo carrier, but the cargo is previous studies, time-lapse images tracking temperature-sensitive released from the ERES in a predominately Sec24D/Sec31A-free cargoes had been performed using 1 s intervals (Stephens and cargo carrier. The lack of Sec24D, an inner coat protein, staining Pepperkok, 2002). As before, the majority of COPII coat fluorescent suggests that Sec31A plays another role in facilitating cargo export in signal remained behind for both Sec24D (inner coat, cargo binding addition to its role as an outer coat protein. Sec31A has been shown to component) and Sec31A (outer coat) following cargo extrusion interact directly with both Sec23 and Sar1, facilitating the GTP (Fig. S5). In all Sec24D events, no coat signal could be captured hydrolysis of Sar1 and possible subsequent fission of COPII vesicles leaving with the cargo (Fig. S5A). Interestingly, at this high frame (Bacia et al., 2011; Bi et al., 2007). Thus, the short lived and Journal of Cell Science

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Fig. 5. Rapid time-lapse imaging of release suggests cargo is extruded not uncoated. (A) Representative example of COS-7 cell expressing BFP-Sec61β, mNeon-31A and mCh-TNFα. (B) Time-lapse images of zoomed inset (yellow box in A) demonstrating COPII and cargo dynamics with 1 s time frames. White arrows track combined movement of ER associated cargo and COPII components. The yellow arrowhead tracks release of cargo from the COPII component. (C,D) Post biotin cargo release event in a COS-7 cell expressing BFP- Sec61β, mCh-TNF RUSH (red) and either mNeon-Sec31A (green; C) or mNeon-Sec24D (green; D). (E,F) Cargo signal was thresholded and converted to binary to create the cargo ROI (yellow outline). To capture any low signalof COPII/Sec protein fluorescence leaving the ER, the Sec31A or Sec24D images were also thresholded and converted to binary. (G) Percentage of Sec24D coverage in the cargo ROI was calculated at 0, 1 and 2 s. (H) As in G but for Sec31A coverage. (I) For the 2 s time point where cargo has completely moved from the ER, the percentage coverage of the cargo ROI per event was measured and this was further averaged per cell to compare Sec24D with Sec31A (1.2%, 13 cells, 27 events versus 12.3%, 17 cells, 32 events), Student’s t-test (**P=0.003). Error bars represent s.e.m. Scale bars: 1 µm. asymmetrical distribution of Sec31A (Fig. 5; Fig. S5) on exported cargo carriers from ERES. As expected, BFP-Rab1a and BFP- cargo domains could be consistent with a model whereby Sec31 Rab1b exogenously expressed in COS-7 cells localized to the functions at the point of fission. cytosol and to the perinuclear/Golgi area within the cell (Fig. 6A) (Plutner et al., 1991; Saraste and Svensson, 1991). BFP-Rab1a and Rab1 regulates release and trafficking of cargo carriers BFP-Rab1b also strongly co-localized with TNFα-SBP-mCh cargo from ERES enriched in ERES puncta throughout the ER (Fig. 6). We then used We also set out to identify membrane markers that would define and the RUSH system to trap cargo at ERES and asked whether Rab1 traffic with these uncoated cargo carriers. Rab1 is a small GTPase regulates cargo release upon biotin addition. Under conditions with two mammalian isoforms (Rab1a and Rab1b) that has been where we expressed BFP-Rab1, biotin addition resulted in the shown to regulate the transport of membranes and cargo from the translocation of TNFα-SBP-mCh cargo to the Golgi (Fig. 6A,B). ER to the Golgi (Galea et al., 2015; Martinez et al., 2016; Pind et al., We generated dominant-negative mutants of Rab1a and Rab1b to 1994; Plutner et al., 1991; Slavin et al., 2011; Wilson et al., 1994). test whether trafficking of the mCh-TNF cargo in the RUSH system Rab1 (Ypt1p in yeast) plays an essential role in the secretory is regulated by Rab1 family members. Both mutants for Rab1 pathway, regulating cargo transport from the ER to the Golgi (Rab1a N124I and Rab1b N121I) are defective in guanine (Bacon et al., 1989; Yuan et al., 2017). Specifically, Rab1 (Ypt1p) nucleotide binding (Takacs et al., 2017; Wagner et al., 1987; has been implicated in regulating COPII vesicle formation and Wilson et al., 1994). The mutant N124I and N121I Rab1 proteins subsequent tethering and fusion of the COPII vesicle at the Golgi were tolerated at low expression levels in cells, but they did not mark membrane (Cai et al., 2008; Cao and Barlowe, 2000; Cao et al., any structures ( just cytosolic localization) (Fig. 6A,B). In the 1998; Morsomme and Riezman, 2002). We thus asked at what steps presence of dominant-negative forms of Rab1, TNFα-SBP-mCh

Rab1 labels and regulates the release and trafficking of uncoated cargo still accumulated in COPII-marked puncta at 0 min after Journal of Cell Science

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Fig. 6. Rab1 regulates release of cargo carriers from ERES. (A) Representative examples of cargo localization before or 20 min after biotin treatment in COS-7 cells expressing mCh-TNF RUSH (red), BFP-Rab1a or BFP-Rab1a N124I (green) and GFP-Sec61β (gray). (B) As in A, but for BFP-Rab1b or BFP-Rab1b N121I. (C,D) Quantification of TNF cargo recruitment to the Golgi was measured by tracking fluorescence intensity measured within a 10×10 μm2 ROI around the Golgi region (perinuclear area, marked by anti-Giantin) versus peripheral ER at 0 and 20 min following biotin addition in the presence of either BFP-Rab1a, BFP-Rab1a N124I, BFP-Rab1b or BFP-Rab1b-N121I. Redistribution of cargo to the perinuclear/Golgi region was quantified by plotting the ratio of fluorescence intensity of TNF cargo between the perinuclear and peripheral ROIs (***P<0.001). Error bars represent s.e.m. Scale bars: 5 µm. biotin treatment and the distribution of COPII-marked puncta was cells were co-transfected with a cargo marker (TNFα-SBP-mCh), a unaltered, suggesting that Rab1 is not required for cargo COPII marker (GFP-Sec24D) and fluorescently tagged Rab1b concentration into COPII exit sites (Fig. 6A,B). However, the (BFP-Rab1b). Before biotin release, Rab1b signal co-localized with Rab1 mutants caused a potent block in the export of cargo from the sites where TNFα-SBP-mCh cargo was stalled in Sec24D-marked ER, even at 20 min after biotin release (Fig. 6A,B). Cargo ERES (Fig. 7A). Upon biotin release, cargo still accumulated at redistribution to the perinuclear region of the cell was quantified ERES; however, cargo now could be seen in structures marked with by tracking the ratio of cargo fluorescence in equally sized ROIs in Rab1b but not with Sec24D (Fig. 7B,C). the perinuclear and peripheral region of the cell. A more positive We captured events where TNFα-SBP-mCh cargo disassociated perinuclear/peripheral ratio marked cells with a higher proportion of from Sec24D, and in all of these events the carrier was labeled with cargo signal located in the perinuclear region compared to the Rab1b (Fig. 7C-E; Movie 8). Monitoring fluorescence intensity peripheral. Significant redistribution of cargo to the perinuclear area throughout 2 min movies at ERES showed that the Sec24D COPII of the cell was only observed in cells expressing wild-type Rab1a coat fluorescence remained stable, but there was a drop in intensity for and Rab1b (P<0.001; Fig. 6C,D). SiRNA depletion of Rab1a or both the Rab1 and TNFα-SBP-mCh fluorescence as cargo left the Rab1b in HeLa cells similarly impaired trafficking of the Rush ERES (Fig. 7D). For these events, we measured fluorescence at the cargoes to the Golgi following biotin release compared with ER exit site (ROI1) and at the site to which cargo was trafficked controls (P<0.0001 for control, P=0.04 for Rab1b; Fig. S6). These (ROI2). We analyzed both pre and post cargo leaving frames, and data confirm that Rab1 family members localize to ERES/cargo showed that Rab1b followed the cargo signal to ROI2 in the post carriers and regulate trafficking of these cargo carriers to the Golgi. frame (Fig. 7E). No significant differences between the distribution of Rab1b and cargo signals in the post frames were observed, indicating Rab1 labels uncoated cargo carriers that when cargo leaves the ERES, it leaves together with Rab1b. By Finally, we aimed to visualize whether cargo is released from ERES comparison, we saw a significant difference in the distribution of into Rab1 carriers. Given previous reports implicating Rab1b in Sec24D in the post frame compared with those of both Rab1b and the cargo delivery to the Golgi, we focused on the role of Rab1b in cargo (P<0.0001 for both cases; Fig. 7E), indicating that the released regulating cargo export from ER-associated COPII vesicles. Cos-7 cargo leaves in a membrane carrier without Sec24D. Journal of Cell Science

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Fig. 7. Rab1 labels uncoated cargo carriers. (A,B) Representative examples of a COS-7 cell expressing mCh-TNF RUSH (red), GFP-Sec24D (gray) and BFP-Rab1b (green) before (A) or after (B) biotin addition. In A, before biotin addition, the Sec24D, TNF cargo and Rab1b all accumulate in the same place. In B, after biotin is added, Rab1b and cargo localize to structures not marked by Sec24D. (C) Time-lapse image series of an event from B where fluorescently marked cargo (red) and Rab1b (green) are observed leaving the Sec24D-labeled ERES (gray). The original site is marked with a yellow arrow in each frame and the leaving cargo vesicle is marked with a white arrow. (D) For the event in C, BFP-Rab1b, mCh-TNF RUSH cargo and GFP-Sec24D fluorescence intensity was monitored throughout the 2 min movie at the ERES, as indicated by the dotted circle region. Sec24D fluorescence levels do not drop when the cargo leaves. TNF cargo fluorescence and Rab1b fluorescence both drop when cargo leaves (frame defined by the arrow). (E) Quantification of Rab1b, TNF cargo and Sec24D fluorescence immediately before (pre) and immediately after (post) cargo leaves the ERES for multiple export events (16 events from 9 cells). Circles mark where fluorescence measurements were made: ROI1 is the site where cargo leaves from and ROI2 is the site where cargo traffics to. Fluorescence at each region for each marker was background subtracted and the percentage fluorescence at each ROI was calculated pre and post cargo leaving. Rab1b fluorescence follows the cargo fluorescence pre and post cargo leaving (for Rab1 and TNF ****P<0.0001, for Sec24D **P=0.09). Error bars represent s.e.m. Scale bars: 5 µm (A,B), 1 µm (zoomed images in A; C-E).

Additionally, we measured Rab1b fluorescence distribution in between the COPII components and Rab1 at the point of vesicle the pre frame and observed a significant difference from that of both scission from the ER. cargo (P=0.008) and Sec24D (P=0.003), suggesting that Rab1b dramatically accumulates at domains marked by Sec24D-cargo DISCUSSION prior to export. Given the role of COPII components in regulating Due to the dynamic nature of cargo trafficking in animal cells, we the formation and maturation of cargo-loaded vesicles, as well as the have optimized a live cell system to visualize co-trafficking of cargo role of Rab1 role in Golgi delivery, the delayed accumulation of tethered to the ER at exit sites and then subsequent rapid cargo release

Rab1b onto cargo-marked structures suggests a potential hand-off from the ER in real time. Emerging technology developments have Journal of Cell Science

10 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs239814. doi:10.1242/jcs.239814 allowed us to use a high frame rate and high-resolution multicolor proteins. Importantly, Rab1 localized to the cargo prior to export imaging system with limited phototoxicity to monitor both ERES from the ER and tracked with cargo as it trafficked away. Due to the dynamics on the ER and cargo release from the ER in multiple dynamic nature of cargo trafficking, a live cell system was needed to mammalian cell lines. COPII puncta remain linked to the ER over understand how ER to Golgi trafficking occurs in real time. This time and when the ER rearranged along microtubules, these COPII study monitored the role of several major ER cargo trafficking puncta co-trafficked. Interestingly, when cargo left the ER, trafficking players and visually constructed the stepwise hand-offs required for away from the ERES, components of the COPII coat remained cargo movement from the ERES to the microtubule trafficking predominately with the ERES. The COPII pool remained stable on the roadways. This visual framework opens up future studies for ER at the ERES, even when cargo was released. These results are probing how each of these pathway components is regulated and consistent with previous work suggesting that ERES are relatively how this stepwise process can be altered or potentially rerouted long-lived on the ER (Hammond and Glick, 2000; Shomron et al., following various perturbations to cellular homeostasis. 2019 preprint; Stephens et al., 2000). This could indicate an adaptive mechanism by which mammalian cells maximize secretory efficiency MATERIALS AND METHODS by retaining COPII coat proteins at stable ERES. In doing so, it Tissue culture ensures that COPII fulfills the primary role of partitioning secretory COS-7 (ATCC-CRL-1651), HeLa (ATCC-CCL-2) and U2OS (ATCC- cargo, providing a quality control checkpoint that selectively advances HTB-96) cells were purchased from ATCC (Table S1). COS-7 and HeLa the forward transport of properly folded cargo (Mezzacasa and cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) Helenius, 2002). Given the expansive network of the ER in supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/ mammalian cells and the sheer volume of the cytoplasm it might be streptomycin. U2OS cells were grown in McCoy’s 5A medium advantageous to maintain COPII organization on the ER to provide supplemented with 10% FBS and 1% penicillin/streptomycin. stable sites where proteins can accumulate. Taken together with our data, it is intriguing to postulate that the stable organization of COPII DNA plasmids proteins on the ER provides specificity and regulatory control over the Steptavidin (Str)-KDEL-TNFα-SBP-mCh, Str-KDEL-ManII-SBP-EGFP, way cargo is compartmentalized and organized prior to export. GFP-Sec16s, GFP-Sec23A, GFP-Sec24D, GFP-Sec31A and EGFP-VSVG We observed that cargo and COPII markers moved together with were acquired from Addgene (Table S1) (Bhattacharyya and Glick, 2007; Boncompain et al., 2012; Presley et al., 1997; Richards et al., 2011; the ER on the microtubules but when released, cargo left the ER in Stephens et al., 2000). The mCh-KDEL, BFP-KDEL and mCh-tubulin were COPII-free puncta. This suggests that the cargo is being trafficked previously described (Friedman et al., 2010, 2011; Zurek et al., 2011). Ras- away from the ER in a vesicle or cluster of vesicles, largely related protein Rab1b (NM_030981.3) was cloned from HeLa cDNA and unmarked by COPII. We asked, if not COPII, what could be inserted into Xho1/Kpn1 sites of the pAcGFP-C1 (Clontech, Mountain regulating this trafficking? We specifically looked for other View, CA) to make GFP-Rab1b. Site-directed mutagenesis was used to membrane components thought to regulate ER to Golgi generate the dominant-negative GFP-Rab1b N121I, which is unable to bind trafficking. In a report by Slavin et al. (2011), inhibition of Rab1b nucleotides (Takacs et al., 2017). BFP-Rab1b wild-type (WT) and BFP- activity was sufficient to delay cargo sorting at the ER. We found Rab1b N121I were generated by subcloning Rab1b WT and Rab1b N121I Xho Kpn that both Rab1a and Rab1b localized to cargo-containing COPII- and inserting them into 1/ 1 sites of mTag-BFP (Evrogen, Russia). labeled sites still attached to the ER before tracking with the COPII coat-free cargo carriers upon export from the ER. This, together Transfection and imaging with our data, suggests that Rab1 is a necessary regulator of the Prior to imaging experiments, all three cell types were seeded in six-well, plastic-bottom dishes at 1×105 cells/ml about 18 h prior to transfection. unmarked cargo vesicle that releases from COPII-marked sites Plasmid transfections were performed as described previously (Hoyer et al., before export to the Golgi. 2018). The following standard amounts of DNA were transfected per ml: Our observation that Rab1b is recruited to COPII-marked sites 0.1 µg GFP-Sec24D, 0.1 µg YFP-Sec31A, 0.1 µg GFP-Sec23A, 0.1 µg prior to cargo export suggests that Rab1b recruitment is an early GFP-Sec16s, 0.2 µg BFP-KDEL, 0.2 µg mCh-KDEL, 0.075 µg mCh- event in secretory trafficking that promotes cargo export from tubulin, 0.4 µg Str-KDEL-TNFα-SBP-mCh, and Str-KDEL-ManII-SBP- COPII-coated domains on the ER. We additionally report that when EGFP, 0.2 µg EGFP-VSVG and 0.075 µg Rab1a or Rab1b (WT or mutant) a dominant-negative Rab1 is introduced or when Rab1 is depleted, fluorescent constructs. cargo cannot accumulate at the Golgi region and remains at ERES All images (except those used for FRAP analysis) were acquired on an puncta. Wild-type Rab1a and Rab1b both localize to ERES, but the inverted fluorescence microscope (TE2000-U; Nikon) equipped with a dominant-negative forms of Rab1 localize diffusely in the Yokogawa spinning-disk confocal system (CSU-Xm2; Nikon or Yokogawa CSU X1). Images were taken with a 100× NA 1.4 oil objective on an cytoplasm and do not localize to the cargo puncta in the ER that electron-multiplying charge-coupled device (CCD) camera 50×50 (Cascade is stuck at ERES. These findings further emphasize the importance II; Photometrics), 50×50 (Andor) or 1TB (Andor). Lasers were aligned to of Rab1 at the early steps of cargo release from ERES. However, release at least 12 Mw out of the optic cable prior to experiments. Images Rab1 could be playing this upstream role at ERES and still play a were acquired with Nikon Elements or with Micromanager and then downstream role to aid in later fusion with the Golgi. In yeast, Rab1 analyzed, merged and contrasted using Fiji (ImageJ), as well as converted to regulates the tethering of COPII vesicles to Golgi acceptor 400 dpi using Photoshop (Adobe, San Jose, CA). Scale bars were generated membranes and may therefore play a similar role in mammalian using Fiji (Schindelin et al., 2012, https://fiji.sc/). Movies 1-8 were cells (Barlowe, 1997; Nakajima et al., 1991). Rab1b continues to generated using ImageJ, Adobe Photoshop and Quicktime. Live cells were track with uncoated carriers and may therefore function to facilitate imaged at 37°C in prewarmed Fluorobrite supplemented with 10% FBS. In eventual fusion with the Golgi, potentially through the subsequent ionomycin experiments, COS-7 cells were imaged just prior and 5 min after ionomycin addition (2 µM). For all immunofluorescence experiments, cells recruitment of COPI machinery. were first fixed at room temperature with 4% paraformaldehyde plus 0.5% Emerging technological developments have allowed us to use glutaraldehyde in PBS, solubilized in 0.1% Triton-X in PBS and then higher frame rate imaging and a more exhaustive examination of the immunostained. Anti-Giantin was used at 1:500 and AlexaFluor 647- dynamics of cargo export from the ER in mammalian cells. We conjugated antibody (Thermofisher) was used at 1:300. Images of fixed observed cargo leaving ERES in a carrier unmarked by COPII cells were captured at room temperature. Journal of Cell Science

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Fluorescence recovery assays (FRAP) were performed at the University thresholded using Yen threshold in Fiji image analysis software to convert of Colorado BioFrontiers Advanced Light Microscopy Core on a Nikon the fluorescent images to binary (Yen et al., 1995). With the cargo binary A1R Laser Scanning Confocal equipped with an Andor Ixon 3 EMCCD images, the analyzed particles function was used to create the cargo ROI. camera (DU897E-C50). ROIs of 10×10 µm were bleached with a 488 nm Then, with the SEc24D or Sec31A binary images, the percentage of positive laser to selectively target the GFP-marked exit sites in the peripheral ER. pixels in the cargo ROI was calculated and this was termed percentage Images were captured with a 100× NA 1.45 oil objective. Cells were tracked coverage. The number of cells analyzed is indicated and P was calculated for 2 min after photobleaching to monitor fluorescence recovery. using Student’s t-tests assuming unequal variance.

Image analysis Rab1a/Rab1b knockdown All image processing was performed using Fiji (Schindelin et al., 2012). To RNAi knockdown of Rab1a and Rab1b was performed using ON- track association of exit sites with the peripheral ER, 10 µm ROIs were used to TARGETplus Human siRNASMARTpools (Horizon Discovery). HeLa follow the movement of exit sites throughout a 2 min movie; frames were taken cells were seeded at 150,000 cells per well of a six-well dish, ∼16 h prior to every 5 s. Only exit sites that remained associated with the ER throughout the transfection. Cells were first transfected ∼48 h before fixation with 5 µl extent of the entire movie were marked as having remained associated with the Dharmafect (Horizon Discovery) in Opti-MEM medium with 25 nM of ER. Exit sites that moved out of the focal plane or became detached for more siRNA SMARTpool or 25 nM Silencer Negative Control #1 siRNA than two consecutive frames were marked as not associated. To track exit site (Ambion AM4635). After 5 h of transfection, cells were washed and dynamics, exit site trajectories were tracked by drawing a segmented line replenished with DMEM medium supplemented with 10% FBS and 1% connecting the location of a particular exit site throughout a 2 min movie. Total penicillin/streptomycin. After 24 h, cells were transfected again with distance (in microns) was calculated by summing the entire distance traveled. plasmid DNA as described above (0.4 µg Str-KDEL-TNFα-SBP-mCh, Maximum velocity was the speed calculated by dividing the maximum 0.2 µg BFP-KDEL, 0.1 µg GFP-Sec24D) with the addition of 12.5 nM distance traveled between adjacent images in the time lapse by 5 s (images siRNA SMARTpool or Silencer Negative Control. Cells were seeded in acquired every 5 s). Fluorescence recovery was measured by calculating the 35 mm imaging dishes (CellVis) and analysis of cargo export was change in fluorescence immediately after photobleaching and 30 s later, performed at 48 h after knockdown as described above. compared with the measured fluorescence of the exit site immediately prior to HeLa cell lysates were collected at 48 h after knockdown and protein photobleaching. All t-tests performed assumed unequal variance. knockdown was confirmed by SDS-Page as described previously (Hoyer et al., 2018). Blots were incubated overnight with primary antibodies against Cargo export analysis Rab1a (CST #13075) and Rab1b (Thermo #PA5-68302) at 1:1000 dilution. The following protocol was modified from that first described by α-Tubulin (Sigma T3562) was used at 1:5000 as loading control. HRP- Boncompain et al. (2012) to track the export dynamics of RUSH cargo. conjugated goat anti-rabbit antibody (Sigma-Aldrich) was used at 1:1000 as Briefly, COS-7 cells were seeded in six-well dishes and transfected with the described previously (Hoyer et al., 2018). Signal was detected with following 3× fluorescently tagged constructs: (1) ER marker, (2) exit site SuperSignal Femto Chemiluminescent Substrate (Thermo). marker and (3) secretory RUSH cargo marker according to the protocol described above. At 24 h after transfection, medium was replaced with prewarmed imaging medium (Fluorobrite plus 10% FBS). COS-7 cells were Acknowledgements The imaging work was performed at the BioFrontiers Institute Advanced Light marked for imaging and z-stacks that went throughout the entire cell were Microscopy Core. Laser scanning confocal microscopy was performed on a Nikon acquired just prior to biotin addition to initiate cargo release from the ER. A1R microscope supported by NIST-CU Cooperative Agreement award number Biotin was added to a final concentration of 40 µM and z-stacks of marked 70NANB15H226. cells were taken at the time points indicated. 2 To quantify cargo recruitment to the ER, one 5×5 μm area was selected Competing interests in the peripheral ER and one 5×5 μm2 area was drawn around the Golgi The authors declare no competing or financial interests. (marked by Giantin). The ratio of cargo fluorescence for each cell at 0 min and 20 min after cargo release following addition of biotin was used as a Author contributions proxy to measure cargo accumulation at the Golgi. Conceptualization: G.K.V., L.M.W., M.J.H.; Methodology: G.K.V., L.M.W., M.J.H.; Live cell images tracking cells every 5 s after biotin addition were also Validation: M.J.H.; Formal analysis: G.K.V., L.M.W., M.J.H.; Investigation: L.M.W., acquired to track the dynamics of cargo over time following release from the M.J.H.; Data curation: L.M.W., M.J.N.; Writing - original draft: L.M.W., M.J.H.; ER. When cargo moved away from the site marked with coat towards the Writing - review & editing: G.K.V., M.J.N.; Supervision: G.K.V.; Funding acquisition: G.K.V., L.M.W., M.J.N. perinuclear area of the cell, the frames pre and post cargo movement were analyzed. Fluorescent measurements were made at both ROI1 (where the Funding cargo initially rested) and ROI2 (where the cargo moves to) in the pre and post This work was supported by grants from the National Institutes of Health to G.K.V. frames. The ROIs were the same size and each ROI measurement was (GM083977), L.M.W. (F32GM116371) and M.J.N. (T32GM008497). G.K.V. is an background subtracted. Percentage fluorescence at each ROI1 was calculated Investigator of the Howard Hughes Medical Institute. Deposited in PMC for by comparing fluorescence in one ROI to total fluorescence in both ROI1 and immediate release. ROI2 in either the pre or post frames [ROI#/ (ROI1+ROI2)] The same analysis was performed with cells transfected with exit site marker, cargo and Supplementary information BFP-Rab1 to monitor Rab1 moving away with the cargo signal. Supplementary information available online at VSVG cargo export dynamics were monitored in real time following the https://jcs.biologists.org/lookup/doi/10.1242/jcs.239814.supplemental modified protocol from Presley et al. (1997). COS-7 cells were seeded in a six-well dish and transfected with the following 3× fluorescently tagged Peer review history constructs: (1) ER marker, (2) exit site marker and (3) VSVG cargo marker The peer review history is available online at according to the protocol described above. At 5 h after transfection, medium https://jcs.biologists.org/lookup/doi/10.1242/jcs.239814.reviewer-comments.pdf was replaced with prewarmed imaging medium (Fluorobrite plus 10% FBS) and cells were stored at 40°C overnight. The following morning (∼18 h after transfection), COS-7 cells were moved to a live cell chamber References equilibrated at 32°C. Individual cells were allowed to equilibrate for 5 min Antonny, B., Madden, D., Hamamoto, S., Orci, L. and Schekman, R. (2001). Dynamics of the COPII coat with GTP and stable analogues. Nat. Cell Biol. 3, at the new temperature before being tracked every 2 s for 60 s. Cargo 531-537. doi:10.1038/35078500 dynamics were analyzed as described above. Aridor, M., Bannykh, S. I., Rowe, T. and Balch, W. E. (1995). Sequential coupling For Sec24D and Sec31A signal tracking with cargo at a high frame rate between COPII and COPI vesicle coats in endoplasmic reticulum to Golgi

(Fig. 6), 5×5 µm cropped images of the ERES with cargo release event were transport. J. Cell Biol. 131, 875-893. doi:10.1083/jcb.131.4.875 Journal of Cell Science

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