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. 2020 Jul 24;133(14):jcs239814.
doi: 10.1242/jcs.239814.

Vesicular and uncoated Rab1-dependent cargo carriers facilitate ER to Golgi transport

Laura M Westrate  1 , Melissa J Hoyer  2   3 , Michael J Nash  2 , Gia K Voeltz  4   3
Affiliations

Vesicular and uncoated Rab1-dependent cargo carriers facilitate ER to Golgi transport

Laura M Westrate et al. J Cell Sci. .

Abstract

Secretory cargo is recognized, concentrated and trafficked from endoplasmic reticulum (ER) exit sites (ERES) to the Golgi. Cargo export from the ER begins when a series of highly conserved COPII coat proteins accumulate at the ER and regulate the formation of cargo-loaded COPII vesicles. In animal cells, capturing live de novo cargo trafficking past this point is challenging; it has been difficult to discriminate whether cargo is trafficked to the Golgi in a COPII-coated vesicle. Here, we describe a recently developed live-cell cargo export system that can be synchronously released from ERES to illustrate de novo trafficking in animal cells. We found that components of the COPII coat remain associated with the ERES while cargo is extruded into COPII-uncoated, non-ER associated, Rab1 (herein referring to Rab1a or Rab1b)-dependent carriers. Our data suggest that, in animal cells, COPII coat components remain stably associated with the ER at exit sites to generate a specialized compartment, but once cargo is sorted and organized, Rab1 labels these export carriers and facilitates efficient forward trafficking.This article has an associated First Person interview with the first author of the paper.

Keywords: COPII; ERES; MANII; RUSH; Rab1; TNF-α.

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Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

Figures

Fig. 1.
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).
Fig. 2.
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).
Fig. 3.
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).
Fig. 4.
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).
Fig. 5.
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 signal of 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.
Fig. 6.
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.
Fig. 7.
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).
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