To have a greater understanding of the nature of transport carriers operating between the ER and Golgi complex, we decided to follow the spatial and temporal dynamics of various classes of cargo molecules that operate between these compartments in living cells. The KDEL receptor (KDEL-R) is one such example of a transmembrane protein that functions to retrieve soluble cargo containing an extreme C-terminal tetra-peptide motif “KDEL” from the Golgi back to the ER (Lewis and Pelham, 1990
). In Vero cells microinjected with a construct encoding a GFP-tagged version of the KDEL-R (GFP-KDEL-R), using time-lapse microscopy it was possible to visualize a large number of punctate and highly motile structures moving between the juxta-nuclear Golgi area and the cell periphery ( and corresponding movie). However, in addition to the punctate carriers, in a number of the cells in the population it was also possible to observe motile structures that were distinctly tubular in appearance (, arrowheads, and corresponding movie). These tubular transport intermediates (TTIs) were variable in length, but most were also highly motile cycling in a manner similar to the punctate carriers. Surprisingly, the appearance of TTIs in cells was strongly dependent on the amount of plasmid DNA injected. Simply increasing the concentration of DNA resulted in an increase from 30 to 50% in the number of cells containing TTIs (). To see if this phenomenon was specific for this construct, we repeated these experiments but this time microinjecting a DNA encoding GFP-tagged ERGIC-53, another recycling protein of the ER-Golgi interface (Schindler et al., 1993
). Again it was possible to see TTIs in some cells of the population, although overall they were not as distinct as those seen with the KDEL-R ( and corresponding movie). However, in a trend similar to that seen with the KDEL-R, microinjection of higher concentrations of the plasmid DNA also resulted in a greater frequency of cells containing TTIs after a fixed time period (unpublished data).
Figure 1. Expression levels of GFP-KDEL-R and GFP-ERGIC-53 influence TTI abundance. (A) Vero cell expressing low amounts GFP-KDEL-R showing the protein localizing to the Golgi and motile punctate structures. (B) Vero cell expressing a higher level of GFP-KDEL-R (more ...)
Because the amount of DNA available for transcription and translation appeared to have an effect on the presence of TTIs within cells, we next considered if expression time might also play a role. To this end we repeated the previous microinjection experiments but this time using a fixed concentration of DNA and adjusting the length of expression time. These experiments revealed a clear correlation between expression time and the frequency of TTIs in the population (). For the GFP-ERGIC-53 construct, although after 1 h of expression no cells containing TTIs could be detected, this proportion reached 20% after 5 h. In contrast, for GFP-KDEL-R, it was already possible to visualize TTIs in 12% of the cells after just 1 h, and this number increased to 35% after 5 h. Incubation times longer than this did not significantly increase the frequency of cells in the population containing TTIs (unpublished data). Together this suggests that either the availability of more DNA or greater expression times are factors contributing to the appearance of tubular ER-Golgi carriers. Both of these situations should result in cells producing a greater amount of protein, which in the case of both ERGIC-53 and the KDEL-R requires transport between the ER and Golgi. We therefore examined the time course experiments described above and quantified the mean fluorescence from individual cells either containing or not containing TTIs. In the case of the cells expressing GFP-KDEL-R, at all of the time points after microinjection those cells with TTIs showed an increased level of fluorescence compared with those cells without (). This was most striking at the longer expression times; for example, after 5 h, the cells without TTIs had on average only 40% of the fluorescence of the cells with TTIs. A similar, albeit less striking effect was also seen in the experiments with the GFP-ERGIC-53 construct. For example after 5 h of expression the cells without TTIs had on average 60% of the fluorescence of the cells with TTIs (). To validate our approach and to be certain that increased fluorescence correlated with an elevated amount of protein, we transfected Vero cells with the GFP-KDEL-R plasmid DNA and incubated them for increasing lengths of time. We were clearly able to identify a number of cells containing tubular structures, which was similar to the microinjection experiments. At each time point we visually determined the percentage of GFP-KDEL-R-expressing cells, quantified their mean fluorescence as before, and subjected equivalent numbers of transfected cells to SDS-PAGE and Western blotting with anti-GFP antibodies (). An increase in the amount of GFP-KDEL-R protein in the cells over time could clearly be detected by both analysis methods. We therefore attempted to correlate our fluorescence measurements with amounts of protein. For example in , the fluorescence measurements of cells containing TTIs indicated an increase in GFP-KDEL-R of ~5.5-fold over the time course. This would correspond to 4.8-fold if measured biochemically. This confirmed that our approach of using fluorescence to determine the relative increase in expressed protein yielded results similar to more conventional biochemical techniques.
To investigate whether the KDEL-R and ERGIC-53 TTIs that we had been analyzing with GFP-tagged proteins could also be observed with their endogenous counterparts in nonmanipulated Vero cells, we fixed and immunostained cells for these proteins. A number of ER-Golgi recycling proteins such as GRASP65 and indeed the KDEL-R have been previously reported to be present in tubular structures (Marra et al., 2001
). We were also able to detect a number of cells with this characteristic, which was similar to this earlier study (), although the occurrence of such cells in the population was typically below 2%. Furthermore, unlike the situation seen with the GFP-tagged proteins in , the number of these tubular structures per cell was also very low, with typically only one or two being visible in those cells in which they could be detected. Because we could not exclude the possibility that this low tubule abundance was due to poor preservation of these structures after fixation and the fact that this approach was unable to provide information about the dynamics of TTIs, we reverted to live cell experiments using fluorescently tagged reporters.
Figure 2. Visualization of endogenous TTIs. (A–D) Vero cells growing at 37°C were fixed and immunostained for endogenous KDEL-R (A and B) and ERGIC-53 (C and D). A small population of the cells contained visible tubular structures (arrowheads). (more ...)
Because both KDEL-R and ERGIC-53 are transmembrane proteins, we next considered if TTI formation could also be observed and similarly modulated with a soluble cargo. Lumenal/soluble GFP is a previously described variant of GFP that contains a signal peptide in order to target the protein into the ER lumen, but because it contains no other targeting information, it is rapidly exported from the ER and is expelled from the cell via the classical secretory pathway (Blum et al., 2000
). To follow a wave of ER-to-Golgi transport of soluble GFP it was necessary to first incubate the cells at 15°C, in order to accumulate the newly synthesized cargo in the ERGIC compartment (Saraste and Kuismanen, 1984
). On release of the temperature block, the soluble GFP was observed to travel rapidly toward the juxta-nuclear Golgi area of the cell, in most cases then appearing to fuse with existing structures at this location. Although these transport intermediates were highly pleiomorphic in size, within the population it was possible to find cells where the carriers were only punctate in shape ( and corresponding movie) and other cells where TTIs were more prevalent (, arrowheads, and corresponding movie). We first decided to investigate the effect of holding the cells at 15°C for increasing lengths of time and then after 5 min at 37°C determining the percentage of cells in the population containing TTIs. These experiments revealed that the length of the 15°C block had little effect on the overall frequency of TTI-containing cells within the population, with typically 60% of the cells having discernable tubular carriers (). We next considered the level of cargo expression in these cells compared with those cells lacking TTIs. We consistently found that cells with TTIs had on average 40% more expressed protein than those cells without TTIs, which was similar to the results obtained with the transmembrane proteins, but this was also irrespective of the time at 15°C ().
Figure 3. Soluble GFP cargo influences TTI abundance. (A and B) HeLa cells expressing soluble GFP after accumulation of the cargo at 15°C for 3 h followed by shifting the temperature to 37°C for 5 min. Some cells show transport of the cargo via (more ...)
To understand in more detail the significance of TTIs with respect to transport, we examined the structures on an individual cell basis. Initially we counted the number of distinct punctate and tubular structures in cells that had been incubated at 15°C for either 1 or 5 h. We recorded that after 5 h of incubation, individual cells had 50% more soluble GFP-containing tubular structures than those cells only incubated for 1 h (), and in both cases 90% of these were motile (, black bars). In contrast, only about 40% of the punctate structures showed motility (, gray bars). When we considered only the motile structures in each cell, we found however that irrespective of incubation time, the TTIs only contributed to about 40% of the overall number of transport carriers (). Therefore although the longer incubation time resulted in a greater proportion of tubular structures in each cell, they were not per se contributing to an increase in transport of the cargo. What might therefore be the role of TTIs compared with punctate carriers? Finally we considered the average distance traveled by the two types of carrier. Reproducibly we found that the tubular carriers were moving 60% further than the punctate carriers, on average 13 μm compared with 8 μm in the fixed time intervals used in these experiments (see Materials and Methods for details; ).
Further detailed analysis of soluble GFP transport carriers was difficult because of the short residence time of this cargo within the cell. Therefore we decided to focus on a recycling component of the early secretory pathway. The p24 family of proteins are small single transmembrane molecules that in living cells have been observed to recycle between the ER and Golgi complex (Blum et al., 1999
). Their precise function has not yet been elucidated, and although a role as putative cargo receptors between these compartments is one possibility (Muniz et al., 2000
), in yeast at least their presence is not essential for vesicular transport (Springer et al., 2000
). To work with a more homogeneous population of cells, we constructed a stable HeLa cell line expressing a YFP-tagged version of the p24 protein (see Materials and Methods
for details). SDS-PAGE and Western blotting from these cells revealed the tagged protein to migrate at the expected molecular weight (). After quantifying the intensity of the p24 bands, and taking into account that only 18% of the cells in this population were actually expressing the tagged p24 protein, we determined the level of overexpression to be 3.5-fold over endogenous. Next we analyzed the subcellular localization of the p24-YFP protein using immunostaining with known markers of the endomembrane system. At 37°C the protein was found in a juxta-nuclear Golgi localization and in a number of distinct peripheral structures, overall displaying a similar distribution to that observed with the COPI coat complex (). This finding was in good agreement with previous colocalization studies of these two proteins at the ultrastructural level (Lavoie et al., 1999
). In addition the p24-YFP protein also showed a good overlap in the Golgi area with p27, another member of the p24 family of proteins (), although few peripheral p27 structures were visualized with this antibody. A strong colocalization was also observed with endogenous KDEL-R and ERGIC-53, and a partial colocalization with the COPII subunit sec23 (unpublished data). In contrast there was little overlap of p24-YFP with the trans
-Golgi network (TGN) and endosomal coat complex AP-1 (), altogether indicating a correct distribution of p24-YFP in the early secretory pathway. Finally we assessed the functionality of these cells by performing a secretion assay using the temperature-sensitive variant (ts045G) of the vesicular stomatitis virus glycoprotein (VSV-G) as a cargo marker, and observing its arrival at the plasma membrane (). We were unable to detect any difference in the secretion rate of ts045G in the transfected compared with nontransfected cells in the population, suggesting that the presence of overexpressed p24-YFP was having little effect on normal membrane trafficking events as highlighted by ts045G.
Figure 4. Characterization of a HeLa cell line stably expressing p24-YFP. (A) HeLa cells stably expressing p24-YFP were lysed and subjected to SDS-PAGE and Western blotting with either anti-GFP or anti-p24 antibodies. The blots revealed the presence of endogenous (more ...)
In living cells the punctate p24-YFP structures were seen to be highly motile, continually moving between the cell periphery and the juxta-nuclear area ( and corresponding movie). Again we looked in the population for cells containing tubular rather than vesicular carriers. At 37°C steady-state conditions, TTIs were detectable in only 18% of the cells, suggesting that transport was largely being mediated by carriers of a vesicular nature. Because p24 cycles via the ERGIC compartment we attempted to create an accumulation of p24-YFP cargo in this compartment using temperature blocks, as utilized in the experiments with soluble GFP. Such temperature treatments have previously been shown to cause a progressive accumulation of ERGIC-53 at this location (Klumperman et al., 1998
). We incubated cells for either 30 or 60 min at 15°C, then shifted them back to 37°C, and imaged them after a further 5 min. We observed a striking increase in the frequency of cells in the population now containing TTIs, compared with control conditions (, arrowheads, and corresponding movie). For example a 1-h arrest of transport in the ERGIC compartment, resulted in an increase of almost threefold in the number of cells now containing TTIs (). This increase in the prevalence of tubules was also visible within individual cells. Counting the distinct punctate and tubular structures revealed that under 37°C control conditions TTIs constituted only 10% of the total number of structures, whereas in the time immediately after release from the temperature blocks this proportion rose to 18% (). This increase in TTI abundance within individual cells returned to control levels after 30 min of incubation at 37°C, indicating that this was a transient response to the applied conditions (unpublished data). The actual length of the TTIs after the various temperature treatments remained constant however ().
Figure 5. Release from temperature blocks enhances TTI formation. (A) HeLa cells stably expressing p24-YFP at 37°C. (B) HeLa cells stably expressing p24-YFP were incubated at 15°C for 30 min and then incubated at 37°C for 5 min. Tubular (more ...)
Quantification of TTI length in HeLa p24-YFP cells after various treatments
We next examined the importance of the tubular carriers to transport. We noted that regardless of the conditions during the course of the experiment, on average 90% of the TTIs in the cells were moving, which was similar to the results with the soluble GFP cargo (, black bars). However when we calculated the contribution to motility made by all the moving carriers, we observed that after release from the temperature blocks the TTIs now accounted for double (40%) the total number of moving structures compared with control conditions (). Finally we compared the distances moved by the two classes of transport intermediates. Although the various incubation conditions did not greatly affect the distances moved, we observed that some TTI structures often moved very large distances (up to 20 μm); however, because of the spread in this population the final average was not significantly different from that recorded for the vesicular carriers ().
The results above indicate that the enforced accumulation and subsequent release of transport-competent cargo from a donor membrane contributes to the formation of tubular carriers. Because the ultimate destination of these structures is most likely the same membranes as vesicular carriers, we were curious to determine whether similar transport machinery was utilized. Bi-directional transport between the ER and Golgi is known to involve the microtubule cytoskeleton. Disruption of this network using nocodazole has been shown by various studies to dramatically reduce both retrograde and anterograde transport between these membranes (Lippincott-Schwartz et al., 1990
; Scales et al., 1997
). Although these studies have proposed that microtubules only serve to increase the efficiency of transport, inevitably the reduced flow of material between these compartments is likely to result in a gradual accumulation of transport-competent cargo. Again we made use of the p24-YFP expressing cells for these experiments and used control conditions of a 30-min incubation at 15°C followed by a 5-min release at 37°C to reliably induce TTI formation. Initially we fixed the cells after this treatment to confirm that the microtubule cytoskeleton had indeed been depolymerized and that the overexpressed p24-YFP protein was not interfering with this process (). In addition we also immunostained the cells for the cis
-Golgi marker GM130, and the TGN marker TGN46, to verify that the Golgi complex was still intact (). Next we analyzed the motility of the p24-YFP containing structures in living cells. When nocodazole was included during the 30-min incubation at 15°C, and after warming the cells to 37°C, we still observed a number of tubular structures present (, arrowheads, and corresponding movie). However when these experiments were repeated, but the nocodazole washed out, these tubules became more prominent (, arrowheads, and corresponding movie). The average length of these TTIs was also increased compared with control conditions (). Within the population of cells we observed an increase in the frequency of cells containing TTIs from 30 to 45% in the presence of nocodazole and an even more dramatic increase to 75% after nocodazole removal (). Analysis of the cells 30 min after the nocodazole washout revealed control levels of TTIs in the population (unpublished data). Although nocodazole treatment resulted in more cells containing visible tubules, when we looked at the actual number of these in individual cells, we found that not only had the number decreased (from 18 to 8% of the total distinct structures in nocodazole-treated cells; ), but also that very few were motile (). Although it might have been expected that the motility of all the transport structures be abolished by this treatment, our previous immunostainings for the microtubule cytoskeleton had revealed that a small number of very short microtubules were still present in the cells at this time (, inset and arrowheads). In these cells we were also able to observe that p24-YFP TTIs and vesicular elements colocalized with these microtubule remnants, making it likely that the residual motility we detected was taking place on these structures. The removal of nocodazole resulted in an increase in the presence of TTIs in individual cells (25% of all structures; ), and the motility of these was similar to the control (). Similarly, TTI contribution to overall motility in the cells treated with nocodazole was almost not detectable (). Analysis of the distance traveled by TTIs in the presence of nocodazole also showed that on average they were unable to travel more than 2 μm (), consistent with the lengths of microtubule remnants that could be detected ().
Figure 6. TTI motility is dependent on an intact microtubule cytoskeleton. (A and B) HeLa p24-YFP cells were incubated at 15°C for 30 min and then at 37°C for 5 min in the presence of 10 μM nocodazole before fixing and immunostaining for (more ...)
Membrane traffic is known to depend on a variety of other factors that associate with the cytoplasmic face of transport carriers. These include the Rab family of small GTPases (reviewed in Deneka et al., 2003
), and the COPI and COPII coat complexes (reviewed in Barlowe, 2000
). In addition the p24 proteins themselves are also implicated in the regulation of transport between the ER and Golgi (Lavoie et al., 1999
; Belden and Barlowe, 2001
). To test the importance of these factors with respect to TTI formation and motility, we microinjected various agents that selectively interfere with these components in turn and then incubated the cells at 15°C and then 37°C as before in order to promote TTI formation. Initially we injected synthetic peptides representing the cytoplasmic tails of the p24 and p23 proteins. Their presence had a dramatic effect on both the abundance and motility of all the carriers in the cell (, and corresponding movies). TTIs were hardly detectable in these cells (), and the few that were identified showed no motility (, black bars). In addition the motility of the vesicular carriers was also dramatically reduced in these cells; for example, when the p24 tail peptides were injected, <5% of these structures showed any significant movement (). To confirm that this striking result was not a nonspecific effect of peptide microinjection into the cells, we designed a third peptide representing a portion of the lumenal domain of p24, but having a pI and length similar to the p24 tail peptide. In living cells microinjected with this peptide and treated with the same temperature regime as before, the motility of p24-YFP structures (both tubular and vesicular) was observed to be similar to control noninjected cells (). Detailed quantification of p24-YFP motility from these cells also revealed a profile comparable to control noninjected cells ().
Figure 7. Regulation of TTIs involves other known membrane traffic components. (A–E) HeLa cells stably expressing p24-YFP after microinjection with various factors that may perturb membrane traffic. Example cells microinjected with 1 mg/ml p24 tail peptide (more ...)
To test the importance of Rab proteins for TTI activity, we next injected purified Rab-GDI (GTPase dissociation inhibitor) protein. Rab-GDI normally functions in the cytoplasm and holds Rab proteins in a soluble, inactive state, before their delivery to the membrane. Cells injected with Rab-GDI contained only a very small number of tubular structures ( and corresponding movie, and ), and we were unable to detect any motile TTIs in these cells (, black bars), which was similar to the experiments with the p23 and p24 peptides. Finally we injected cells with the poorly hydrolysable analogue of GTP, GTPγS. GTPγS has the effect of locking coat complexes on to transport-competent structures. On inspection of cells injected with GTPγS, we observed a large reticular network of tubular structures (, arrowheads, and corresponding movie). The frequency of these tubules within cells was much greater than in control cells (), and they were still contributing to the overall transport observed in the cells (, black bars). However despite this treatment appearing to have the smallest effect with respect to motility, the tubules generated in these cells were the most dramatic, with their mean length increasing from 2.7 to 5.6 μm ().
From all the reagents used to try and modulate the formation of TTIs, the peptide tails of the p23 and p24 proteins proved to be most effective. Because transport between the ER and Golgi complex is highly dependent on the activity of the COPI and COPII coat complexes, we decided to visualize these coats in cells injected with these peptides. To our surprise we observed that despite the lack of both vesicular and tubular motility in these cells, both coats appeared to have a relatively normal appearance, being largely present on distinct membrane structures. Similar observations were made in cells injected with both the p23 () and p24 tail peptides ().
Figure 8. Peptide tails of p23 and p24 proteins do not affect COPI and COPII coat distribution. (A and B) Appearance of coat proteins in HeLa cells after microinjection with p23 or p24 peptide tails. HeLa cells were microinjected with 1 mg/ml p23 or p24 tail peptides (more ...)