ABSTRACT: Secretory cargo is recognized, concentrated and trafficked from 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 utilized a recently developed live cell, cargo export system that can be synchronously released
from ERES to illustrate de novo trafficking in animal cells. We find that components of the COPII
coat remain associated with the ERES, while cargo is extruded into COPII uncoated, non-ER
associated, Rab1-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.
INTRODUCTION:
The ER serves as the entry point for the secretory pathway. Following successful translation and
translocation, secretory proteins synthesized in the ER are concentrated by COPII proteins at ER
exit sites (ERES) and trafficked to the Golgi within vesicular carriers (D’Arcangelo et al., 2013).
The order of assembly of the COPII coat and its role in generating a vesicle that contains secretory
cargo has been dissected in molecular detail by elegant in vivo and in vitro studies. The major
components include a guanine exchange factor (GEF) Sec12 that recruits and activates a small
GTPase Sar1 to the ER (Nakano and Muramatsu, 1989; Nakano et al., 1988). Upon GTP
activation, Sar1 inserts an amphipathic helix into the ER membrane to initiate membrane
deformation (Lee et al., 2005). Sar1 next recruits the heterodimer “inner coat” complex
Sec23/Sec24, which stabilizes membrane curvature and recruits cargo through the cargo binding
sites on Sec24 (Bi et al., 2002; Kuehn et al., 1998; Miller et al., 2002; Miller et al., 2003). The
inner coat recruits the heterotetramer “outer coat” complex composed of Sec13/Sec31 resulting
in the formation of a fully formed COPII-coated transport vesicle that will deliver incorporated
cargo to the Golgi complex via anterograde trafficking from the ER (Kirk and Ward, 2007;
Lederkremer et al., 2001; Lee et al., 2004; Rossanese et al., 1999; Stagg et al., 2006). Many of
these studies have been performed in yeast cells because of the advantages of yeast genetics to
identify COPII components combined with temperature sensitive (ts) mutants that can stall cargo
as it is trafficked.
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Animal cells may have adapted alternative methods to ensure rapid cytoplasmic trafficking of
cargo from ERES to the Golgi in a vast cytoplasm. Early reports in mammalian cells suggest that
COPII vesicles rapidly lose their coat after scission from the ER (Antonny et al., 2001; Aridor et
al., 1995; Scales et al., 1997; Presley et al 1997; Stephens et al., 2000) and immuno-EM evidence
has identified the existence of free COPII coated vesicles at the ER:Golgi interface (Zeuschner et
al., 2006). De novo ER to Golgi cargo trafficking is difficult to resolve in live animal cells because
secretory proteins are continuously being synthesized and move rapidly through the secretory
pathway. Additionally, ts mutants of the COPII coat are not available in animal cells. Instead, while
various techniques have been used to study secretory cargo trafficking in animal cells, the
majority of them have utilized a single temperature sensitive fluorescently tagged cargo, ts-VSVG
(Bonfanti et al., 1998; Gallione and Rose, 1983; Kreitzer et al., 2000; Shomron et al., 2019). At
non-permissive temperature, GFP-ts-VSVG is stuck in the ER as an unfolded protein. A shift to
permissive temperature allows VSVG to fold and be exported. This cargo can be visualized live
as it leaves the ER in animal cells and previous experiments with this cargo have suggested that
although the cargo initially co-localizes with a component of the COPII coat at the ERES, it does
not exit the ERES with Sec24 (Presley et al., 1997; Stephens et al., 2000). These data first
suggested that some aspects of the transition from COPII-coated vesicle formation to a vesicular
structure trafficking towards the Golgi may not require COPII.
The current model for mammalian ER to Golgi trafficking remains somewhat controversial. Many
have cited that ER to Golgi trafficking relies on both vesicular and tubular intermediate
compartments (Saraste and Svensson, 1991; Stinchcombe et al., 1995; Watson and Stephens,
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 of these structures are not static and can make
long range movements (Presley et al., 1997; Scales et al., 1997). In studies monitoring the VSVG
cargo system, when temperature is reduced, intermediate structures become enlarged,
accumulate secretory cargo, and form in the vicinity of COPII-labeled ERES (Mironov et al., 2003;
Presley et al., 1997; Stephens, 2003). These structures acquire the vesicular coat complex COPI
(Aridor et al., 1995; Lee et al., 2004; Shomron et al., 2019; Stephens et al., 2000) and upon
temperature release, these structures move towards the Golgi complex in a microtubule-
dependent manner (Presley et al., 1997; Shima et al., 1999; Stephens et al., 2000). More recent
studies have keyed in on how these intermediate compartment structures rearrange to facilitate
trafficking of large cargoes like collagen (McCaughey et al., 2019; Saito et al., 2009). With the
help of Tango1, intermediate structures could potentially form tunnels from the ER to Golgi (Raote
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and Malhotra, 2019). It is currently unclear if the creation of larger tubular clusters is due to large
cargoes or potentially an abundance of cargo in cells. However, with the advent of faster and
more sensitive imaging techniques and the development of newly trackable trafficking cargoes,
we can now combine these advanced methods to visualize this vital cargo trafficking step in live
cells.
Here we follow trafficking of COPII ERES markers and ER export cargoes including the
classically-used temperature sensitive VSVG as well two cargoes with varying size that release
from the ER upon addition of Biotin using the Retention Using Selective Hooks (RUSH) system
(Boncompain et al., 2012; Presley et al., 1997). We have optimized the RUSH system to measure
the rapid release of de novo secretory cargo trafficking from ERES relative to the COPII coat
components and the Rab1 GTPase. We have used three different mammalian cell lines, four
fluorescently tagged COPII coat proteins, two different cargoes, and a Rab membrane marker to
demonstrate that COPII coat components predominately remain at stable ERES as cargo leaves
in a Rab1-dependent carrier.
RESULTS
COPII components localize to stable domains on peripheral ER tubules
We used live confocal fluorescence microscopy to visualize the distribution and dynamics of four
major COPII components at ERES in three different animal cell lines: COS-7, HeLa and U2OS.
First, we assessed the location and dynamics of fluorescently tagged and exogenously expressed
GFP-Sec16S, a resident ER protein that serves as a scaffold for COPII assembly at ERES
(Watson et al., 2006). GFP-Sec16S localized to punctate structures along ER tubules in all three
cell types (Fig. 1A, Fig. S1, Movie S1). We collected two-minute movies (with 5 sec intervals) of
GFP-Sec16s puncta to track their location relative to the tubular ER network (labelled with mCh-
KDEL) over time. As expected for an ERES scaffold, the vast majority of Sec16s puncta (98.2%)
remained associated with the ER network throughout the length of the movie (puncta were scored
as “associated” if they were visible and tracked with an ER tubule for the entire length of the
movie).
We performed a similar analysis on fluorescently tagged COPII inner and outer coat components;
Sec23A, Sec24D and Sec31A (Fig. 1A). While most of the COPII mammalian isoforms are
functionally redundant, the four mammalian Sec24 isoforms have been previously shown to have
distinct and overlapping specificity in the cargo export signals recognized (Khoriaty et al., 2018;
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Stankewich, 2006; Wendeler et al., 2007). Sec24D was chosen because it binds strongly to 4 out
of 6 common ER export signals, while the other three Sec24 isoforms strongly bind only 2 out of
6 (Wendeler et al., 2007). Similar to GFP-Sec16s, puncta of GFP-Sec23A, GFP-Sec24D or YFP-
Sec31A were all observed to be distributed throughout the cytoplasm and all were tightly
associated with the tubular ER network during two-minute movies and in multiple cell types (Fig.
1B and 1C, Fig. S1, Movies S2-S4). Nearly 100% of all COPII components remain associated
with the ER throughout the length of the movie (98.2 ± 4.8% for Sec16s, 99.7 ± 1.2% for Sec23A,
98.0 ± 8.6% for Sec24D, 99.0 ± 3.9% for Sec31A in COS-7 cells (Fig. 1C). COPII components
were scored as remaining associated with the ER if they remained tethered to the ER throughout
the two-minute movie. The small percentage of COPII components that were marked as not being
associated with the ER often represented puncta that appeared to become dissociated from the
ER due to the ER network or COPII punctum moving out of the focal plane. Thus, despite being
associated with the ER for the majority of the movie, if there were at least 2 sequential frames (10
seconds) where the ER and COPII punctum could not be tracked together, the COPII component
was marked as “not associated.” Hela and U2OS cells, which are thicker than COS7 cells, showed
a higher propensity for this observation and therefore demonstrated slightly reduced percentages
of COPII components remaining associated with the ER through the two-minute movie (Fig 1C).
Notably, we never captured a COPII coated vesicle budding off or trafficking away from the ER in
live cell images. These data suggest that COPII coat components form stable structures at ERES
in animal cells.
To challenge the strength of the association between the ER and COPII coated domains, we
treated cells with ionomycin, an ionophore that triggers increased intracellular calcium and
subsequent fragmentation of the ER (Fig. S2A) (Koch et al., 1988). COS-7 cells were treated
with 2 µM ionomycin (5 min) and fixed before acquiring confocal z-slices through the entire cell.
Even upon fragmentation of the ER, we still observe that COPII labelled domains remained co-
localized with ER vesicles: 86.7 ± 13.6% for Sec16s, 91.3 ± 10.2% for Sec23A, 94.1 ± 6.8% for
Sec24D, 89.3 ± 15.3% for Sec31A (Fig. S2B-D). These data further support the notion that COPII
domains are integral components of ERES in animal cells.
Further evidence that COPII components form stable structures at ERES in animal cells came
from fluorescent recovery assays used to measure the dynamics of COPII outer coat components
Sec31A in COS7 and HeLa cells (Fig. S3A-B, respectively). A 10 µm X 10 µm region of interest
(ROI) was photobleached in the peripheral ER of cells expressing outer coat component YFP-
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Sec31A and a general ER marker (mCh-KDEL). The YFP signal within the ROI was hit with a
high power 488nm laser to selectively diminish YFP signal. We intentionally picked a laser
exposure that did not result in 100% loss of signal so that we could confidently track whether
fluorescence recovered to the ROI that marked the original COPII site on the ER (Fig. S3C).
Fluorescence intensity was calculated at the frame before bleaching (“Pre”), the frame
immediately after photobleaching (“Post”) and 30 seconds post bleaching. In both COS-7 and
HeLa cells, fluorescence recovery occurred at the ROI marking the original COPII puncta nearly
90% of the time (87.9 ± 6.53% in COS7, 88.8 ± 4.25% in HeLa cells Fig. S3D) suggesting that
COPII components are recruited to stable ERES domains.
Dynamic COPII domains are tethered to sliding ER tubules
COPII and ERES labeled structures have been shown to be relatively stable in yeast and plants
(DaSilva et al., 2004; Hanton et al., 2009; Kirk and Ward, 2007; Shindiapina and Barlowe, 2010).
To characterize the dynamics of COPII coat components in mammalian cells, we tracked COPII
labeled domains over time. We found that COPII coat components remain tightly associated with
the ER. Consistent with previous experiments performed on Sec24D, COPII sites were relatively
stable in the peripheral ER (Gupta et al., 2008; Stephens et al., 2000). However, we occasionally
observed a COPII labelled domain traveling a long distance (over 2 microns, Fig. 2A-B) while
maintaining contact with ER tubules. The total length of movement was highly variable, with the
average distance traveled being about 5 µm (Sec16s – 4.67 ± 0.4 µm, Sec31A – 5.51 ± 0.5 µm)
(Fig. 2C). This type of coupled movement was reminiscent of ER sliding, where ER tubules extend
along the side of an existing microtubule (MT) at a velocity of ~0.5 µm/sec (Friedman et al., 2010).
To confirm this mechanism of movement, we measured the max velocity of COPII puncta during
long-range trajectories. Consistent with known velocities for ER sliding (Friedman et al., 2010),
dynamic ER-associated COPII domains traveling more than 2 µm had a max velocity of ~0.5
µm/sec along MTs (0.41 ± 0.03 µm/sec for Sec16s and 0.51 ± 0.04 µm/sec for Sec31A).
Additionally, when we observed these dynamic events in conjunction with fluorescently labeled
microtubules we could track COPII puncta traveling along existing microtubules (Fig. 2F), thus
providing additional evidence that these long-range movements are trafficking via ER sliding.
When tracking the direction of these long-range COPII movements we found that the majority of
events (73% and 74% for Sec16s and Sec31A, respectively) move in the retrograde direction
towards the Golgi (Fig. 2E). Despite the distance traveled, these dynamic COPII labeled domains
remain tethered to the ER during long-range movements suggesting that they are not released
from the ER membrane.
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Using the RUSH system to monitor cargo trafficking from ER to Golgi
Next, we analyzed whether COPII-labelled puncta bud off from the ER only once they are loaded
with cargo to be trafficked. Conflicting results from the mammalian field have questioned the fate
of COPII components following cargo packaging at the ER. Specifically, work by Stephens et al,
Scale et al, and Presley et al, using a temperature block cargo release system (VSVG), showed
that cargo can be transported to the Golgi in the absence of COPII components (Presley et al.,
1997; Scales et al., 1997; Stephens et al., 2000) while EM tomography work by Zeuschner et al
identified the existence of COPII coated transport vesicles at the ER:Golgi interface (Zeuschner
et al., 2006). We therefore vetted potential systems that would allow us to visualize cargo release
from the ERES relative to COPII coat components and ER that would provide a systematic
analysis of secretory cargo export dynamics from the ER. We first optimized the RUSH system
(Retention Using Selective Hooks) to synchronize and track the release of cargoes exiting the ER
in real time (Fig. 3A, (Boncompain et al., 2012)). The RUSH system utilizes an ER “hook” protein
fused to streptavidin and a secretory cargo protein fused to streptavidin-binding protein (SBP),
which under normal conditions traps the secretory cargo in the ER due to the streptavidin:SBP
interaction. Biotin addition outcompetes the streptavidin:SBP interaction resulting in a
synchronous release of secretory cargo from the ER that can be tracked over time. SBP was
linked to two different fluorescently tagged cargoes for comparison: TNF-SBP-mCh and ManII-
SBP-GFP. The expression of TNF-SBP-mCh or ManlI-SBP-GFP resulted in a striking
localization pattern where the cargo, while localized to the ER (“0 min” in Fig. 3). Cargo
concentration at ERES has been demonstrated to be an early step in the secretory pathway with
subsequent quality control checkpoints at the ER to ensure only properly folded proteins are
allowed to traffic to vesicular-tubular clusters and trans-Golgi network (Dukhovny et al., 2008;
Dukhovny et al., 2009; Mezzacasa and Helenius, 2002). We therefore predicted that these small
puncta represented concentrated cargo at COPII sites that were unable to undergo forward
transport due to their association with the streptavidin ER hook protein. Dual labeling of both a
fluorescently tagged inner or outer COPII coat proteins (GFP-Sec24D or YFP-Sec31A,
respectively) with the cargo revealed that these cargo-enriched puncta are often co-labeled with
COPII proteins (Fig. 3B, D and F and Fig. 4). This implies that in the absence of biotin, the RUSH
cargo is still capable of being loaded into COPII marked exit sites which are stalled for export due
to the streptavidin:SBP interaction occurring between the cargo and ER hook protein.
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Biotin addition released forward trafficking of these SBP tagged secretory cargoes and TNF-
SBP-mCh became enriched at the Golgi within 20 min of biotin treatment (Fig. 3B, right panels).
By comparison, GFP-Sec24D (Fig. 3B-C) or YFP-Sec31A (Fig. 3D-E) coat components remain
at puncta associated with the ER network and do not accumulate with cargo at the Golgi. Similar
results were seen with a different cargo, ManII-SBP-GFP, which also accumulates at the Golgi
within 20min of biotin addition, while again the mCh-Sec24D coat component, even though it
directly binds cargoes during initial cargo sorting, also remains in ER-associated puncta (Fig. 3F-
G). Significant redistribution of cargo to the perinuclear region of the cell following biotin addition
was noted in mCh-TNFα and GPF-ManII cargo (p = 0.003, 0.024 and 0.019 for C, E and G
respectively). These data show that we can stall and then release cargo in order to visualize how
cargo traffics to the Golgi relative to COPII coat components.
COPII components remain linked to the ER while cargo is exported away
Next, we aimed to use the RUSH system to visualize cargo release from ERES and further
investigate the properties of cargoes upon ER release and trafficking to the Golgi. First, we stalled
TNF-SBP-mCh at Sec24D labelled ERES and labelled the ER with BFP-Sec61β (Fig. 4A and
4B). Following biotin addition, we visualized TNF-SBP-mCh cargo as it trafficked away from the
ER in puntate carriers. Strikingly, fluorescence associated with the GFP-Sec24D puncta stayed
associated with the peripheral ER and did not track with the movement of the cargo (Fig. 4A,
compare yellow to white arrows and Movie S5). Cargo carriers dissociated from the tubular ER
network as they trafficked (Fig. 4B). To quantitate the amount of TNF cargo relative to the amount
of COPII coat protein that traffics away from the ER, we measured the fluorescence intensity of
the ER, TNF-SBP-mCh cargo, and Sec24D in a region of interest (ROI) (yellow circle)
surrounding the ERES where cargo is released from the ER. Fluorescence intensity at this site
was plotted over a 2min time course (Fig. 4B, images and graphs). We measured a drop in relative
fluorescence for TNF-SBP-mCh signal in the ERES region when cargo left the site. By
comparison, relative fluorescent intensity for the COPII coat marker GFP-Sec24D and the ER
signal BFP-Sec61β remained constant pre and post cargo release (Fig. 4B, line scan analysis
depicting fluorescence at the site at each time point). These data suggest that cargo separates
from COPII coat components as it exits the ER to traffic to the Golgi.
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To obtain a more quantitative picture of cargo trafficking from COPII components at ERES, we
captured further events where cargo is exported from the ER. For each event, we analyzed the
“pre” and “post” cargo leaving frames. In these frames, we defined equal area regions of interest
(ROIs) encircling where cargo initiated from in the “pre” frame (ROI1) and encircled where cargo
trafficked to in the “post” frame (ROI2) (Fig. 4C). A dramatic drop in percent fluorescence was
measured from the “pre” to “post” for the ROI1 location for TNF-SBP-mCh signal but not for the
GFP-Sec24D signal in these events (Fig. 4C graphs, p<0.0001). To further confirm that we saw
cargo leaving from the ER without the COPII coat, we performed the same analyses done for the
TNF-SBP-mCh cargo with Sec24D COPII coat system, but varied the COPII coat marker
(Sec31A) (Fig. 4D) and the type of RUSH cargo (ManII-SBP-GFP) (Fig. 4E). Additionally, we
monitored the temperature sensitive VSVG system with the Sec24D COPII coat. (Fig 4F). As with
the TNF-SBP-mCh/Sec24D system, TNF-SBP-mCh cargo left the Sec31A-marked ERES,
ManII-SBP-GFP cargo left the Sec24D-marked ERES and VSVG cargo left the Sec24D-marked
ERES (Fig. S4 and Fig. 4D, 4E, and 4F respectively, p<0.0001 for each condition except for
pre/post analysis of Sec24D in 4E where p=0.03, and the pre/post analysis of Sec24D in panel
4F where p=0.0004, calculated by comparing cargo fluorescence pre and post event). In all
examples where cargo left ROI1 to traffic to ROI2, the COPII coat components remain at ROI1
and do not travel with cargo to ROI2 (Fig 4C-4F, Fig S4). Together, these data support a model
whereby the COPII coat proteins mark stable sites of protein export where cargo traffics from the
ER towards the Golgi in an uncoated carrier. Interestingly, we observed a statistically significant
but minimal redistribution of Sec24 into ROI2 following ManII and VSVG cargo release (Fig. 4E,
p=0.03). This could be due to a combined COPII/cargo movement being captured in ROI2 before
complete cargo export has occurred or it could represent dynamic uncoating of the COPII coat
being captured following cargo export (Fig. S4).
The cargo we visualized trafficking from the ER membrane in most cases did not have a tubular
structure and usually maintained a circular nature more indicative of a vesicle (see examples in
Fig. 4 and Fig. S4). However, due to the resolution of fluorescence microscopy, we cannot
determine if these structures are vesicles or a cluster of vesicles in living cells. If these structures
are rapidly uncoating, however, one would predict that the fluorescence intensity of COPII
components at puncta would be significantly reduced as the cargo was exported from the ER.
Instead, we observe negligible loss of COPII fluorescence from the point of cargo export (in Fig.
4) suggesting instead that COPII is forming stable sites on the ER that do not traffic with cargo as
it exports from the ER.
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Rapid time-lapse imaging reveals cargo exports from ER in a carrier uncoated by COPII
To better ascertain the dynamics of COPII components on cargo domains being exported from
the ER, we decided to track COPII and cargo export at more rapid time points (Fig. 5 and Fig.
S5). First, we imaged TNF cargo export relative to Sec24 or Sec31 coat components at a higher
frame rate (every 500ms) to better visualize COPII coat and cargo dynamics (Fig. S5A and S5B,
respectively). Previously, time-lapse images tracking temperature sensitive cargos had been
performed using 1 second intervals (Stephens and Pepperkok, 2002). As before, the majority of
COPII coat fluorescent signal remained behind for both Sec24D (inner coat, cargo binding
component) and Sec31A (outer coat) following cargo extrusion (Fig. S5). In all Sec24D events,
no coat signal could be captured leaving with the cargo (Fig. S5A). Interestingly, at this high frame
rate, we could capture a small amount of Sec31A following the cargo signal (Fig. S5B). This
fluorescence signal was very dim and asymmetrically distributed, marking only a small percentage
of the entire cargo-containing puncta. In addition, it was lost within 5 seconds of cargo leaving
(see image in t=8s in event 1 and t=31s in event 2 Fig. S5B). The rapid loss of the Sec31A
fluorescence signal along with the absence of detectable Sec24D signal suggests that cargo is
largely being exported from the ER in a carrier domain independent of COPII components.
To be able to confirm that these events leave the ER and were not potentially ER exit sites dividing
into two separate sites, we repeated the cargo release experiments, imaging the ER (not
previously done in Stephens et al, 2002) in relation to cargo release events (Fig. 5). Even with
the addition of the third channel to capture the ER, we were still able to image cargo release
events at a high-speed interval (1 sec). We sought to further dissect the steps that occur pre and
post cargo release in order to accurately measure Sec24D and Sec31A coverage on the cargo
puncta that trafficked away from the ER towards the Golgi. For consistency and to directly
compare and contrast the behavior of these two COPII components, both Sec24D and Sec31A
were tagged with the mNeon fluorophore (Fig. 5).
We first characterized ERES pre cargo release. We were able to capture events where the ER,
cargo, and components moved together as the ER network rearranged pre cargo release (Fig
5A-B). These events demonstrate that cargo is still linked to the ER just prior to cargo release.
All three components (ER, COPII and cargo) first move together pre cargo release (0-2 seconds,
white arrow, Fig 5B). Cargo release from ERES was observed at 3 seconds and was not observed
to be affiliated with COPII coat components (yellow arrow, Fig 5B). The co-movement of ER,
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COPII and cargo pre cargo release (from 0-2 seconds before release) further emphasizes that
both COPII and cargo were linked to the ER and not just closely associated prior to the moment
of cargo export.
To then visualize the steps of cargo release (Fig. 5C-D, Movie S6 Movie S7), we analyzed each
event at pre cargo moving (0 sec), when cargo begins to move from the ER but is still connected
to ERES signal (1 sec), and when cargo has moved from the ERES (2 sec). To detect any
potential Sec signal over background, a threshold was used to convert Sec24D and Sec31A
fluorescent images to binary images (Jui-Cheng Yen et al., 1995). The cargo images were also
converted to binary and this detectible cargo signal set the cargo ROI (yellow outlines, Fig.5 E-
F). We then calculated the percent of Sec24D or Sec31A pixels that were within the cargo ROI
at 0, 1, and 2 sec for each event (average percent coverage, Fig. 5G-H). In the 1 second time
points captured, cargo has not yet separated from the ERES and both Sec24D and Sec31A
signals are detected (Fig. 5 G-H). To compare Sec24D and Sec31A distribution just as cargo is
leaving the ER, we scored the time point at which the cargo ROI was fully separated from the ER
and from any residual faint cargo still at the ERES (2 sec time point, Fig. 5G-I). At this time point,
Sec24D fluorescent signal had little overlap with the cargo ROI (1.2%), while Sec31A had a small,
yet significantly different (p=0.003), percent overlap with the cargo ROI (12.3%) (Fig. 5I). Thus, a
small portion of the outer coat Sec31A protein can remain associated albeit briefly with the cargo
carrier, but the cargo is released from the ERES in a predominately Sec24D/Sec31A free cargo
carrier. The lack of Sec24D staining, an inner coat protein, suggests that Sec31A may play
another role in facilitating cargo export in addition to its role as an outer coat protein. Sec31A has
been previously shown to directly interact with both Sec23 and Sar1, facilitating the GTP
hydrolysis of Sar1 and possible subsequent fission of COPII vesicles (Bacia et al., 2011; Bi et al.,
2007). Thus the short lived, and asymmetrical distribution of Sec31A (Fig. 5 and S5) on exported
cargo domains could be consistent with a model whereby Sec31 is functioning at the point of
fission.
Rab1 regulates release and trafficking of cargo carriers from ERES
Finally, we set out to identify membrane markers that would define and traffic with these uncoated
cargo carriers. Rab1 is a small GTPase with two mammalian isoforms (Rab1a and Rab1b) that
has been previously shown to regulate the transport of membranes and cargo from the ER to the
Golgi (Galea et al., 2015; Martinez et al., 2016; Pind et al., 1994; Plutner et al., 1991; Slavin et
al., 2011; Wilson et al., 1994). Rab1(Ypt1p in yeast) has been shown to play an essential role in
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the secretory pathway, regulating cargo transport from the ER to the Golgi (Bacon et al., 1989;
Yuan et al., 2017). Specifically, Rab1 (Ypt1p) has been implicated in regulating COPII vesicle
formation and subsequent tethering and fusion of the COPII vesicle at the Golgi membrane as
(Cai et al., 2008; Cao and Barlowe, 2000; Cao et al., 1998; Morsomme and Riezman, 2002). We
thus asked at what steps Rab1 labels and regulates the release and trafficking of uncoated cargo
carriers from ERES. As expected, BFP-Rab1a and BFP-Rab1b exogenously expressed in COS-
7 cells localized to the cytosol and to the perinuclear/Golgi area within the cell (Fig. 6A) (Plutner
et al., 1991; Saraste and Svensson, 1991). BFP-Rab1a and BFP-Rab1b also strongly co-localized
with TNF-SBP-mCh cargo enriched in ERES puncta throughout the ER (Fig. 6). We then used
the RUSH system to trap cargo at ERES and asked whether Rab1 regulates cargo release upon
biotin addition. Under conditions where we expressed BFP-Rab1, biotin addition resulted in the
translocation of TNF-SBP-mCh cargo to the Golgi (Fig. 6A-B). We generated a dominant
negative mutant of Rab1a and Rab1b to test whether trafficking of the mCh-TNF cargo in the
RUSH system is regulated by Rab1 family members. Both mutants for Rab1 (Rab1a N124I and
Rab1b N121I) are reported to be defective in guanine nucleotide binding (Takacs et al., 2017;
Wagner et al., 1987; Wilson et al., 1994). The mutant N124I and N121I Rab1 proteins were
tolerated at low expression levels in cells, but they did not mark any structures (just cytosolic
localization) (Fig. 6A-B). In the presence of dominant negative forms of Rab1, TNF-SBP-mCh
cargo still accumulated in COPII marked puncta at 0min biotin and the distribution of COPII
marked puncta was unaltered, suggesting that Rab1 is not required for cargo concentration into
COPII exit sites (Fig. 6A-B). However, the Rab1 mutants caused a potent block in the export of
cargo from the ER even at 20 minutes post biotin release (Fig. 6A-B). Cargo redistribution to the
perinuclear region of the cell was quantified by tracking the ratio of cargo fluorescence in equally
sized ROIs in the perinuclear and peripheral region of the cell. A perinuclear/peripheral ratio that
was more positive would mark cells with a higher proportion of cargo signal located in the
perinuclear region compared to the peripheral. Significant redistribution of cargo to the perinuclear
area of the cell was only observed in cells expressing wildtype Rab1a and Rab1b (Fig. 6C-D, p-
value <0.001). SiRNA depletion of Rab1a or Rab1b in HeLa cells similarly impaired trafficking of
the Rush cargoes to the Golgi following biotin release compared to controls (Fig. S6, p-value for
Control <0.0001, for Rab1b p =0.04). These data confirm that Rab1 family members localizes to
ERES/cargo carriers and regulate trafficking of these cargo carriers to the Golgi.
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Rab1 labels uncoated cargo carriers
Finally, we aimed to visualize whether cargo is released from the ERES into Rab1 carriers. Given
previous reports implicating Rab1b in cargo delivery to the Golgi, we focused on Rab1b’s role in
regulating cargo export from ER-associated COPII vesicles. Cos-7 cells were co-transfected with
a cargo marker (TNF-SBP-mCh), a COPII marker (GFP-Sec24D) and fluorescently tagged
Rab1b (BFP-Rab1b). Before biotin release, Rab1b signal co-localized with sites where TNF-
SBP-mCh cargo was stalled in Sec24D-marked ERES (Fig. 7A). Upon biotin release, cargo still
accumulated at ERES; however, cargo now could be seen in structures marked with Rab1b but
not with Sec24D (Fig. 7B-C).
We captured events where TNF-SBP-mCh cargo disassociated from Sec24D, and in all of these
events the carrier was labelled with Rab1b (Fig. 7C-E Movie S8). When we monitored
fluorescence intensity throughout 2-min movies at the ERES, the Sec24D COPII coat
fluorescence remained stable, but there was a drop in intensity for both the Rab1 and TNF-SBP-
mCh fluorescence as cargo left the ERES (Fig. 7D). For these events, we measured fluorescence
at the ER exit site (ROI1) and at the site where cargo trafficked to (ROI2). We analyzed both “pre”
and “post” cargo leaving frames, and score that Rab1b followed the cargo signal to ROI2 in the
“post” frame (Fig. 7E). No significant difference between the distribution of Rab1b and cargo
signals in the “post” frames were observed indicating that when cargo leaves the ERES, it leaves
together with Rab1b. By comparison, we see a significant difference in the distribution of Sec24D
in the “post” frame compared to that of both Rab1b and the cargo (Fig. 7E, p-value <0.0001 for
both cases) indicating that the released cargo is leaving in a membrane carrier without Sec24D.
Additionally, when we measure Rab1b fluorescence distribution in the “pre” frame, we measured
a significant difference from that of both cargo (p-value =0.008) and Sec24D (p-value = 0.003)
suggesting that Rab1b is dramatically accumulating at Sec24D:cargo marked domains prior to
export. Given the role of COPII components in regulating the formation and maturation of cargo
loaded vesicles as well as Rab1’s role in Golgi delivery, the delayed accumulation of Rab1b onto
cargo marked structures suggests a potential hand-off between the COPII components and Rab1
at the point of vesicle scission from the ER.
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DISCUSSION
Due to the dynamic nature of cargo trafficking in animal cells, we have optimized a live cell system
to visualize co-trafficking of cargo tethered to the ER at exit sites and then subsequent rapid cargo
release from the ER in real time. Emerging technology developments have allowed us to use a
high frame rate and high-resolution multi-color imaging system with limited photo-toxicity to
monitor both ERES dynamics on the ER and cargo release from the ER in multiple mammalian
cell lines. COPII puncta remain linked to the ER over time and when the ER rearranges along
MTs, these COPII puncta cotraffic. Interestingly, when cargo left the ER, trafficking away from the
ERES, components of the COPII coat remained predominately with the ERES. The COPII pool
remains stable on the ER at the ERES even when cargo is released. These results are consistent
with previous work suggesting that ERES are relatively long-lived on the ER (Hammond and Glick,
2000; Shomron et al., 2019; Stephens et al., 2000). This may indicate an adaptive mechanism by
which mammalian cells maximize secretory efficiency by retaining COPII coat proteins at stable
ERES. In doing so, this ensures COPII fulfills the primary role of partitioning secretory cargo,
providing a quality control checkpoint that selectively advances the forward transport of properly
folded cargo (Mezzacasa and Helenius, 2002). Given the expansive network of the ER in
mammalian cells and the sheer volume of the cytoplasm it may be advantageous to maintain
COPII organization on the ER to provide stable sites where proteins can accumulate. Taken
together with our data, it is intriguing to postulate that the stable organization of COPII proteins
on the ER provide specificity and regulatory control over the way cargo is compartmentalized and
organized prior to export.
We observed that cargo and COPII markers moved together with the ER on the microtubules but
when released, cargo left the ER in a COPII-free puncta. This suggests that the cargo is being
trafficked away from the ER in a vesicle or cluster of vesicles, largely unmarked by COPII. We
asked, if not COPII, what could be regulating this trafficking? We specifically looked for other
membrane components thought to regulate ER to Golgi trafficking. In a report by Slavin et al,
inhibition of Rab1b activity was sufficient to delay cargo sorting at the ER (Slavin et al., 2011). We
found that both Rab1a and Rab1b localized to cargo-containing COPII labeled sites still attached
to the ER before tracking with the COPII coat-free cargo carriers upon export from the ER. Taken
together with our data, it would suggest that Rab1 is a necessary regulator of the unmarked cargo
vesicle that releases from COPII marked sites before export to the Golgi.
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Our observation that Rab1b is recruited to COPII marked sites prior to cargo export suggests that
Rab1b recruitment may be an early event in secretory trafficking that promotes cargo export from
COPII coated domains on the ER. We additionally report that when a dominant negative Rab1 is
introduced or when Rab1 is depleted, cargo cannot accumulate at the Golgi region and remains
at the ERES puncta. Wildtype Rab1a and Rab1b both localize to ERES, but the dominant negative
forms of Rab1 localize diffusely in the cytoplasm and do not localize to the cargo puncta in the
ER that is stuck at ERESs. These findings further emphasize the importance of Rab1 at the early
steps of cargo release from the ERES. However, Rab1 could be playing this upstream role at the
ERES and still play a downstream role to aid in later fusion with the Golgi. In yeast, Rab1 regulates
the tethering of COPII vesicles to Golgi acceptor membranes and may therefore be playing a
similar role in mammalian cells (Barlowe, 1997; Nakajima et al., 1991). Rab1b continues to track
with uncoated carriers and may therefore function to facilitate eventual fusion with the Golgi,
potentially through the subsequent recruitment of COPI machinery.
Emerging technological developments have allowed us to use higher frame rate imaging and a
more exhaustive examination in the dynamics of cargo export from the ER in mammalian cells.
We observed cargo leaving ERESs in a carrier unmarked by COPII proteins. Importantly, Rab1
localized to the cargo prior export from the ER and tracked with cargo as it trafficked away. Due
to the dynamic nature of cargo trafficking, a live cell system was needed to understand how ER
to Golgi trafficking occurs in real time. This study monitored the role of several major ER cargo
trafficking players and visually constructed the stepwise handoffs required for cargo movement
from the ERES to the microtubule trafficking roadways. This visual framework opens up future
studies to further probe how each of these pathway components are regulated and how this
stepwise process can be altered or potentially rerouted following various perturbations to cellular
homeostasis.
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MATERIAL AND METHODS Tissue Culture COS-7 cells (ATCC-CRL-1651), HeLa cells (ATCC-CCL-2) and U2OS (ATCC-HTB-96) were purchased from ATCC (Table S1). COS-7 and HeLa cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). U2OS cells were grown in McCoy’s 5A medium supplemented with 10% FBS and 1% P/S. DNA plasmids Str_KDEL_TNFalpha_SBP_mcherry, Str_KDEL_ManII_SBP_EGFP, GFP_Sec16s, GFP_Sec23A, GFP_Sec24D, GFP_Sec31A, and EGFP_VSVG were acquired from addgene (Table S1) (Bhattacharyya and Glick, 2007; Boncompain et al., 2012; Presley et al., 1997; Richards et al., 2011; Stephens et al., 2000) Mcherry_KDEL, BFP_KDEL, mcherry_tubulin were previously described (Friedman et al., 2010; Friedman et al., 2011; Zurek et al., 2011). Ras-related protein Rab1b (NM_030981.3) was cloned from HeLa cDNA and inserted into Xho1/Kpn1 sites of the pAcGFP_C1 (Clontech, Mountain View, CA) to make GFP_Rab1b. Site directed mutagenesis was used to generate the dominant negative GFP_Rab1b_N121I which is unable to bind nucleotides (Takacs et al., 2017). BFP_Rab1b_WT and BFP_Rab1b_N121I were generated by subcloning Rab1b_WT and Rab1b_N121I and inserting them into Xho1/Kpn1 sites of mTag_BFP (Evrogen, Russia). Transfection and Imaging Prior to imaging experiments, all three cell types were seeded in 6-well, plastic bottom dishes at 1 x 105 cells/mL about 18 hours prior to transfection. Plasmid transfections were performed as described previously (Hoyer et al., 2018). The following standard amounts of DNA were transfected per mL: 0.1 µg GFP_Sec24D, 0.1 µg YFP_Sec31A, 0.1 µg GFP_Sec23A, 0.1 µg GFP_Sec16s, 0.2 µg BFP_KDEL, 0.2 µg mcherry_KDEL, 0.075 µg mcherry_tubulin, 0.4 µg Str_KDEL_TNFalpha_SBP_mcherry, and Str_KDEL_ManII_SBP_EGFP, 0.2 µg EGFP_VSVG, 0.075 µg Rab1a or Rab1b (WT or mutant) fluorescent constructs. All images (except those used for FRAP analysis), were acquired on an inverted fluorescence microscope (TE2000-U; Nikon) equipped with a 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 electron-multiplying charge-coupled device (CCD) camera 50X50 (Cascade II; Photometrics), 50x50 (Andor) or 1TB (Andor). Lasers were aligned to release at least 12 Mw out of the optic cable prior to experiments. Images were acquired with Nikon Elements or with Micromanager and then analyzed, merged, and contrasted using Fiji (ImageJ), as well as converted to 400dpi using Photoshop (Adobe, San Jose, CA). Scale bars were generated using Fiji (Schindelin et al., 2012, https://fiji.sc/). Supplemental videos were generated using ImageJ, Adobe Photoshop, and Quicktime. Live cells were imaged at 37°C in pre-warmed Fluorobrite supplemented with 10% FBS. In ionomycin experiments, COS7 cells were imaged just prior and 5 minutes after ionomycin addition (2 µM). For all immunofluorescence experiments, cells were first fixed at room temperature with 4% Paraformaldehyde + 0.5% glutaraldehyde in PBS, solubilized in 0.1% Triton-X in PBS and then immunostained. Anti-Giantin was used at 1:500 and Alexa-Fluor 647 conjugated antibody (thermofisher) was used at 1:300. Images of fixed cells were captured at room temperature.
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Fluorescence Recovery Assays (FRAP) was performed at the University of Colorado BioFrontiers Advanced Light Microscopy Core on a Nikon A1R Laser Scanning Confocal equipped with an Andor Ixon 3 EMCCD Camera (DU897E-C50). Region of interests (ROIs) of 10 µm x 10 µm were bleached with a 488 nm laser to selectively target the GFP marked exit sites in the peripheral ER. Images were captured with a 100x NA 1.45 oil objective. Cells were tracked for 2 minutes after photobleaching to monitor fluorescence recovery. Image Analysis All image processing was performed using Fiji (Schindelin et al., 2012). To track association of exit sites with the peripheral ER, 10 µm ROIs were used to follow the movement of exit sites throughout a two-minute movie, frames taken every 5 seconds. Only exit sites that remained associated with the ER throughout the extent of the entire movie were marked as having remained associated with the ER. Exit sites that moved out of the focal plane or became detached for more than 2 consecutive frames were marked as not associated. To track exit site dynamics, exit sites trajectories were tracked by drawing a segmented line connecting the location of a particular exit site throughout a two-minute movie. Total distance (in microns) was calculated by summing the entire distance traveled while max velocity represented the speed calculated by dividing the max distance traveled between adjacent images in the time lapse by 5 seconds (images acquired every 5 seconds). Fluorescence Recovery was measured by calculating the change in fluorescence post photobleaching and 30 seconds later compared to the measured fluorescence of the exit site immediately prior (Pre) to photobleaching. All t-tests performed assumed unequal variance. Cargo Export Analysis The following protocol was modified from that first described in Boncompain et al (Boncompain et al., 2012) to track the export dynamics of RUSH cargo. Briefly, COS-7 cells were seeded in 6 well dishes and transfected with 3x fluorescently tagged constructs: (1) ER marker, (2) exit site marker, and (3) secretory RUSH cargo marker according to the protocol described above. Twenty-four hours after transfection, media was replaced with pre-warmed imaging media (Fluorobrite + 10% FBS). COS7 cells were marked for imaging and z-stacks that went throughout the entire cell were acquired just prior to biotin addition to initiate cargo release from the ER. Biotin was added to a final concentration of 40 µM and z-stacks of marked cells were taken at time points indicated in Results. To quantify cargo recruitment to the ER, one 5 x 5 μm2 area was selected in the peripheral ER and one 5 x 5 μm2 area was drawn around the Golgi (marked by Giantin). The ratio of cargo fluorescence for each cell at 0 minutes and 20 minutes post cargo release following addition of biotin was used as a proxy to measure cargo accumulation at the Golgi. Live cell images tracking cells every 5 seconds post biotin addition were also acquired to track the dynamics of cargo over time following release from the ER. When cargo moved away from the site marked with coat towards perinuclear area of the cell, the frames pre and post cargo movement were analyzed. Fluorescent measurements at both ROI1 (where the cargo initially rested) and ROI2 (where the cargo moves to) in the pre and post frames. The ROIs were the same size and each ROI measurement was background subtracted. Percent fluorescence at each ROI1 was calculated by comparing fluorescence in one ROI to total fluorescence in both ROI1 and 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 BFP-Rab1 to monitor Rab1 moving away with the cargo signal.
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VSVG cargo export dynamics were monitored in real time following the modified protocol from Presley et al [Bresley]. COS-7 cells were seeded in a 6 well dish and transfected with the 3x fluorescently tagged constructs: (1) ER marker, (2) exit site marker, and (3) VSVG cargo marker according to the protocol described above. Five hours after transfection, media was replaced with
pre-warmed imaging media (Fluorobrite + 10% FBS) and cells were stored at 40°C overnight.
The following morning (~18 hours post transfection), COS-7 cells were moved to a live cell
chamber equilibrated at 32°C. Individual cells were allowed to equilibrate for 5 minutes at the
new temperature before being tracked every 2 seconds for 60 seconds. Cargo dynamics were analyzed as described above. For the Sec24D and Sec31A signal tracking with cargo at high frame rate experiment (Figure 6), 5x5 micron cropped image of the ERES with cargo release event were threshold using Yen threshold in Fiji image analysis software to convert the fluorescent images to binary (Yen et al 1996). With the cargo binary images, the analyze particles function was used to create the cargo ROI. Then with the SEc24D or Sec31A binary images, the percent positive pixels in the cargo ROI was calculated and this was termed percent coverage. N=number of cells and p-value was calculated using student’s t tests assuming unequal variance. Rab1a/Rab1b knockdown RNAi knockdown of Rab1a and Rab1b was performed using ON-TARGETplus Human siRNASMARTpools (Horizon Discovery). HeLa cells were seeded at 150K cells per well of a 6 well dish, ~16 hrs prior to transfection. Cells were first transfected ~48 hours before fixation with 5 µL Dharmafect (Horizon Discovery) in optimum with 25nM of the siRNA SMARTpool or 25nM Silencer Negative Control #1 siRNA (ambion AM4635). After 5 hours of transfection, cells were washed and replenished with DMEM media supplemented with 10% FBS and 1% penicillin/streptomycin. After 24 hrs, cells were transfected again with plasmid DNA as described above (0.4 ug Str_KDEL_TNFalpha_SBP_mcherry, 0.2 ug BFP_KDEL, 0.1 ug GFP_Sec24D) with the addition of 12.5 nM siRNA SMARTpool or Silencer Negative Control. Cells were seeded in 35mm imaging dishes (CellVis) and analysis of cargo export was performed at 48 hours post knockdown as described above. HeLa cell lysates were collected at 48 hours post knockdown and protein knockdown was confirmed by SDS-Page as described previously [Hoyer et al]. Blots were incubated overnight with primary antibodies against Rab1a (CST #13075) and Rab1b (Thermo #PA5-68302) at 1:1000 dilution. Alpha tubulin (sigma T3562) was used at 1:5000 as a loading control. HRP-conjugated goat anti-rabbit antibody (Sigma-Aldrich) was used at 1:1000 as described previously (Hoyer et al., 2018). Signal was detected with SuperSignal Femto Chemiluminescent Substrate (Thermo). ACKNOWLEDGEMENTS The imaging work was performed at the BioFrontiers Institute Advanced Light Microscopy Core.
Laser scanning confocal microscopy was performed on a Nikon A1R microscope supported by
NIST-CU Cooperative Agreement award number 70NANB15H226. This work was supported by
grants from the National Institutes of Health to G.K.V. (GM083977), L.M.W. (F32GM116371), and
to M.J.N. (T32GM008497). G.K.V. is an Investigator of the Howard Hughes Medical Institute.
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Figures
Figure 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 labelled with mch-KDEL in red; GFP-Sec16s, GFP-Sec23A, GFP-Sec24D, or YFP-Sec31A in green). A zoom in of the boxes shown below. B. A COPII outer coat component YFP-Sec31A (in green) is tracked over time for 2 minutes to quantify YFP-Sec31A puncta association with the ER (mch-KDEL, red) over time. C. COPII labelled 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 minute movie in COS-7, HeLa, or U2OS cells. N=3 replicates; 15 cells for Sec16s, 15 cells for Sec23A, 19 cells for Sec24D, and 18 cells for Sec31A. Scale Bars: A = 5 µm, B = 2 µm.
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Figure 2: Dynamic COPII domains are tethered to sliding ER tubules. A. 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 (marked by a cyan arrow head) tracks with an attached ER tubule over time. B. As in (A) for YFP-Sec31A puncta dynamics. C. Quantification representing the total distance traveled by individual COPII labelled domains displaying long range movement within the cell (N = 9 cells and 23 exit sites for Sec16s, 11 cells and 35 events for Sec31A). D. Quantification of the max velocity of indicated COPII puncta dynamics during long range movements. Max velocity was calculated by determining the max distance traveled between sequential frames acquired 5 seconds apart (N = 9 cells and 17 events for Sec16s, and 11 cells and 26 events for Sec31A). Error bars represent standard error about the mean. E. Percent 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 A,B = 5 µm for whole cell inset and 2 µm for the zoomed inset and (F).
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Figure 3: Using the RUSH system to monitor cargo trafficking from ER to Golgi. A. Cartoon description of the RUSH system for cargo release from ERES upon biotin addition. B. Representative image of cargo distribution in a COS7 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 at 0 min, the ER network and ERES and TNF cargo 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 to peripheral signal before (0 min) or after biotin addition (20 min). 5 x 5 μm2 regions selected in the periphery and perinuclear region were used to calculate fluorescence intensity in those cellular regions. The ratio of Perinuclear/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-C for ER (BFP-Sec61B), mCh-TNF cargo and the outer coat component YFP-Sec31(p=0.024). F-G. As in B-C for ER (BFP-Sec61B), mCh-Sec24D and GFP-ManII cargo (p=0.019). Error bars represent s.e.m in C, E, and G. Scale bars = 5 µm in whole cell; 2 µm in zoomed insets.
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Figure 4: COPII components remain associated with the ER as cargo is exported. A. A COS-7 cell expressing mCh-TNF RUSH (red), GFP-Sec24D (green), and BFP-Sec61β (gray).
Right panels show time-lapse image of two events (from within yellow box, bottom left panel)
where fluorescently marked cargo (red) is observed leaving COPII fluorescent puncta (green) on
the ER (gray) at timepoints following biotin addition. The arrow marks the first trafficking event
and the arrowhead marks a second event. COPII is marked with yellow arrow/head in each frame
and the location of cargo as it traffics is marked with white arrow/head. B. Sec24D, TNF cargo,
and ER fluorescence was monitored over 2 minutes within an ROI (yellow circle) that marks the
original site of cargo release. Graph plots the relative fluorescence intensity (RFI) and reveals
that the ER and Sec24D fluorescence levels remain in the circle even after the cargo leaves. C.
As in A, for several events, coat and cargo fluorescence was measured pre and post cargo
leaving. 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 then percent fluorescence at each ROI was
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 and mCh-Sec24D. (p<0.0001 for ManII, p=0.03 for
Sec24D) F. As in C for GFP-VSVG and mCh-Sec24D. (p<0.0001 for VSVG, p=0.0004 for Sec24D
in ROI2) Error bars represent s.e.m. Scale bars: A = 5 µm in whole cell; 1 µm for zoom panels in
A, and B-E.
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Figure 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, mCh-TNFα. B. Time-lapse images of zoomed inset (yellow box in A) demonstrating COPII and cargo dynamics with 1 s time frames. C. A post biotin cargo release event in a COS-7 cell expressing BFP- Sec61β, mCh-TNF RUSH (red) and either mNeon-Sec31A (green) or D. mNeon-Sec24D (green). E-F. Cargo signal was thresholded and coverted 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. The percent of Sec24D coverage in the cargo ROI was calculated at 0, 1, or 2 seconds. H. As in G but for Sec31A coverage I. For the 2 second time point where cargo has completely moved from the ER, the percent coverage of the cargo ROI per event was measured and this was further averaged per cell to compare Sec24D to Sec31A (1.2% n=17cells (32 events) vs. 12.3% n=13 cells (27 events), students t test, (p-value=0.003). Error bars represent s.e.m. Scale bar = 1 µm.
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Figure 6: Rab1 regulates release of cargo carriers from ERES. A. Representative examples of cargo localization before or 20min after biotin treatment in COS-7 cells expressing mCh-TNF RUSH (red), BFP-Rab1a or Rab1a N124I (green) and GFP-Sec61β (grey). B. As in A but for BFP-Rab1b or Rab1b N121I. C and D. Quantification of TNF cargo recruitment to the Golgi was measured by tracking fluorescence intensity measured within a 10 x 10 μm2 ROI around the Golgi region (perinuclear area, marked by anti-Giantin) versus peripheral ER at 0 min 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 ROI’s (p<0.001). Error bars represent s.e.m. Scale bars=5 µm.
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Figure 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 post (B) biotin addition. In A, before biotin addition, the Sec24D, TNF cargo and the Rab1b all accumulate in the same place, but 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) is observed leaving the Sec24D-labelled ERES (grey). 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-minute 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 pre and post cargo leaving 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 then percent fluorescence at each ROI was calculated pre and post cargo leaving. The Rab1b fluorescence follows the cargo fluorescence pre and post cargo leaving. (p-values for Rab1 and TNF <0.0001, p-vale for Sec24D = 0.09) Error bars represent s.e.m. Scale bars: 5 µm or 1 µm for whole cell or zoom image, respectively.
pEGFP-VSVG was a gift from Jennifer Lippincott-Schwartz
Plasmid mcherry_tubulin Friedman et al, JCB 2010
Plasmid GFP_Rab1b WT HeLa cDNA
Plasmid GFP_Rab1b_N121I HeLa cDNA
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Plasmid mNeon-Sec24D mNeon subcloned in to pEGFP-Sec24D to replace EGFP
Plasmid mNeon-Sec31A mNeon subcloned in to pEYFP-Sec21A to replace YFP
Chemical Biotin Sigma Sigma: B4501
Re-suspended in PBS
Chemical Ionomycin Invitrogen I24222 2 µM
J. Cell Sci.: doi:10.1242/jcs.239814: Supplementary information
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Movie 1, related to Figure 1. Sec16s puncta localize to stable domains on peripheral ER tubules. 10x10 micron region of a COS-7 cell expressing mch-KDEL (ER in red) and GFP-Sec16s (COPII component in green), imaged for 2 minutes with 5 second intervals.
Movie 2, related to Figure 1. Sec23A puncta localize to stable domains on peripheral ER tubules. 10x10 micron region of a COS-7 cell expressing mch-KDEL (ER in red) and GFP-Sec23A (COPII component in green), imaged for 2 minutes with 5 second intervals.
J. Cell Sci.: doi:10.1242/jcs.239814: Supplementary information
Movie 3, related to Figure 1. Sec24D puncta localize to stable domains on peripheral ER tubules. 10x10 micron region of a COS-7 cell expressing mch-KDEL (ER in red) and GFP-Sec24D (COPII component in green), imaged for 2 minutes with 5 second intervals.
Movie 4, related to Figure 1. Sec31A puncta localize to stable domains on peripheral ER tubules. 10x10 micron region of a COS-7 cell expressing mch-KDEL (ER in red) and GFP-Sec31A (COPII component in green), imaged for 2 minutes with 5 second intervals.
J. Cell Sci.: doi:10.1242/jcs.239814: Supplementary information
Movie 5, related to Figure 5. COPII components remain linked to the ER as cargo is exported. 5x5 micron region of a COS-7 cell expressing BFP-Sec61β (ER in gray), mCh-TNF RUSH (cargo in red), and GFP-Sec24D (COPII component in green), imaged for 2 minutes with 5 second intervals. This movie shows a 50 sec time period (10 frames of the image sequence) highlighting when cargo moves from two ER-linked COPII sites. The yellow arrow marks the first cargo export event and the yellow arrowhead marks the second cargo export event.
Movie 6, related to Figure 6. Rapid time-lapse imaging of release from Sec24D labelled ERES. 5x5 micron region of a COS-7 cell expressing BFP-Sec61β (ER in gray), mCh-TNF RUSH (cargo in red), and GFP-Sec24D (COPII component in green), imaged for 2 minutes with 1 second intervals. This movie shows a 5 sec time period (5 frames of the image sequence) highlighting when cargo moves from the ER-linked Sec24D site.
J. Cell Sci.: doi:10.1242/jcs.239814: Supplementary information
Movie 7, related to Figure 6. Rapid time-lapse imaging of release from Sec31A labelled ERES. 5x5 micron region of a COS-7 cell expressing BFP-Sec61β (ER in gray), mCh-TNF RUSH (cargo in red), and GFP-Sec31A (COPII component in green), imaged for 2 minutes with 1 second intervals. This movie shows a 10 sec time period (10 frames of the image sequence) highlighting when cargo moves from the ER-linked Sec31A site and when Sec31A signal diminishes.
Movie 8, related to Figure 8. Figure 8: Rab1 labels uncoated cargo carriers. 5x5 micron region of a COS-7 cell expressing BFP-Rab1b (Rab1b in green), mCh-TNF RUSH (cargo in red), and GFP-Sec24D (COPII component in gray), imaged for 2 minutes with 5 second intervals. This movie shows a 50 sec time period (10 frames of the image sequence) highlighting when cargo moves from the ER-linked Sec24D site in a Rab1b labelled vesicle.
J. Cell Sci.: doi:10.1242/jcs.239814: Supplementary information
Figure S1: COPII Exit Sites are Tightly Associated with the Peripheral ER in HeLa and U2OS
A and C. Distribution of ERES within the peripheral ER (mch-KDEL, red) of HeLa (A) or U2OS (C) cells
expressing fluorescently tagged markers of COPII exit sites (GFP-Sec16s, GFP-Sec23A, GFP-Sec24D,
and YFP-Sec31A, green). Scale bars 5 µm. B and D. Exit Sites (YFP-Sec31A, green) were tracked over
time for 2 minutes to quantify whether sites were associated with ER (mch-KDEL, red) throughout the 2
minutes. Scale bars 2 µm.
J. Cell Sci.: doi:10.1242/jcs.239814: Supplementary information
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D0 min ionomycin
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Figure S2: COPII components remain ER-associated upon ionomycin-induced 1 fragmentation. A. Representative images of COS-7 cells expressing fluorescently tagged 2 markers of the ER (mch-KDEL, red) and COPII exit sites (YFP-Sec31A, green) before and after 3 (2 µM) ionomycin treatment. Zoomed insets (from white square in whole cell view) show exit site 4 association with ER following ionomycin fragmentation. B. Quantification of the percentage of ER 5 exit sites associated with the ER following ionomycin treatment. N = 3 replicates; 20 cells for 6 Sec16s, 20 cells for Sec23A, 11 cells for Sec24D, and 23 cells for Sec31A. Scale Bars: 5 µm for 7 both whole cell and zoomed insets. 8
J. Cell Sci.: doi:10.1242/jcs.239814: Supplementary information
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Figure S3
COS-7 cellA
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Figure S3: COPII coat proteins cycle at long lived ERES. 1 A. A COS-7 cell expressing fluorescent markers to label the ER network (mCh-KDEL) and YFP-2 Sec31A (in green). Zoomed insets (from white square in whole cell view) show the temporal 3 dynamics of Sec31A fluorescence before and after targeted photobleaching within the ROI 4 indicated. White circles track the fluorescence of an individual COPII puncta during recovery. B. 5 As described in A but for a HeLa cell. C. Representative images demonstrating the quantification 6 protocol used to measure fluorescence recovery by measuring fluorescence intensity within the 7 dashed circles at pre, post and 30 seconds after photobleaching. D. Quantification of fluorescence 8 recovery (at 30 sec relative to fluorescence just prior to photobleaching) for COS-7 and HeLa 9 cells (from 60 and 78 events in 13 COS7 and 14 HeLa cells, respectively). Error bars represent 10 standard error about the mean. A-C scale bars = 5 µm for the whole cell and 2µm for zoom insets. 11
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Figure S4: COPII ERES remain linked to the ER with various coat or cargo markers 1 A. Example time-lapse image series of events in COS-7 cells where fluorescently marked cargo 2 (mCh-TNF,red) is observed leaving the COPII fluorescent puncta (YFP-Sec31A, green) on the 3 ER (BFP- Sec61β, gray) after biotin addition. B As in A for a different cargo (GFP-Manll RUSH, 4 red), a different coat (mCh-Sec24D, ER (BFP- Sec61β, gray). C. As in B but for a different cargo 5 (GFP-VSVG), ER (BFP-KDEL). Arrow marks the first event and if there is a second event, the 6 arrowhead marks the second event. The ERES is marked with yellow in each frame and the 7 leaving cargo vesicle is marked in white. Scale bars 1 µm. 8
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Figure S5
Figure S5. Rapid time-lapse imaging of release suggests cargo is extruded not uncoated. 1 A. A COS-7 cell expressing mCh-TNF RUSH (red) and GFP-Sec24D (green) with marked insets 2 labeling two different events where fluorescently marked cargo (red) is observed leaving COPII 3 fluorescent puncta (green) on the ER and tracked through time. In the zoomed insets, white 4 circles mark the beginning position of the dual labeled Sec24D and TNFalpha marked site and 5 white arrows track the dynamics of the TNFalpha cargo (red) over time. GFP-Sec24D only 6 panels demonstrate dynamics of COPII coat which does not track with cargo (arrows). B. As in 7 A for mCh-TNF RUSH cargo (red) and YFP-Sec31A (green). The Golgi region is highlighted in 8 grey. Scale bar 5 µm 9
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Figure S6
Figure S6: Loss of Rab1 impairs cargo export from the ER. 1 A. Representative examples of cargo localization before or 20min after biotin treatment in HeLa 2 cells expressing mCh-TNF RUSH (red), GFP-Sec24D (green) and BFP-KDEL (ER, white). Cells 3 belonged in1 of 3 groups, Control, Rab1a or Rab1b siRNA treated cells. B.. Quantification of 4 TNF cargo recruitment to the Golgi was measured by tracking fluorescence intensity measured 5 within a 10 x 10 μm2 ROI in the perinuclear region versus peripheral ER at 0 min and 20 min 6 following biotin addition in Control, Rab1a or Rab1b siRNA treated cells. Redistribution of cargo 7 to the perinuclear/Golgi region was quantified by plotting the ratio of fluorescence intensity of 8 TNF cargo between the perinuclear and peripheral ROI’s. C. Western blot confirmation of 9 protein knockdown for Rab1a and Rab1b. Error bars represent standard error about the mean. 10 Scale bars=5 µm. 11
J. Cell Sci.: doi:10.1242/jcs.239814: Supplementary information
