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We have shown previously thatRab6, a small, trans-Golgi-localizedGTPase, acts upstream of the conserved oligomericGolgi complex (COG) andZW10/RINT1 retrograde tether complexes to maintainGolgi homeostasis. In this article, we present evidence from the unbiased and high-resolution approach of electron microscopy and electron tomography thatRab6 is essential to the trans-Golgi trafficking of two morphological classes of coated vesicles; the larger corresponds to clathrin-coated vesicles and the smaller to coat proteinI(COPI)-coated vesicles. On the basis of the site of coated vesicle accumulation, cisternal dilation and the normal kinetics of cargo transport from the endoplasmic reticulum (ER) toGolgi followed by delayedGolgi to cell surface transport, we suggest thatGolgi function in cargo transport is preferentially inhibited at the trans-Golgi/trans-Golgi network (TGN). The >50% increase inGolgi cisternae number inRab6-depletedHeLa cells that we observed may well be coupled to the trans-Golgi accumulation ofCOPI-coated vesicles; depletion of the individualRab6 effector, myosinIIA, produced an accumulation of uncoated vesicles with if anything a decrease in cisternal number. These results are the first evidence for aRab6-dependent protein machine affectingGolgi-proximal, coated vesicle accumulation and probably transport at the trans-Golgi and the first example of concomitant cisternal proliferation and increasedGolgi stack organization under inhibited transport conditions.
Of the 12 different Golgi-associated Rab proteins (reviewed in ), small GTPases of the Rab family, Rab6, is the most abundant . It consists of four different isoforms, Rab6a, Rab6a′, Rab6b and Rab6c (reviewed in  and also see ). Of these, Rab6a and Rab6a′ which are equally abundant and differ in three amino acids are the only family members expressed in all cell types of the mammalian body. Rab6b is neuronal specific , while Rab6c is expressed in a limited number of human tissues and is involved in cell cycle progression . Rab6a and Rab6a′ are preferentially localized to the trans-Golgi cisterna/trans-Golgi network (TGN), while other Golgi-associated Rabs exhibit different localizations with Rab1 and Rab2, e.g. preferentially localized to the cis-Golgi apparatus/cis-Golgi network (CGN).
Like all Rab proteins, Rab6a and Rab6a′ function as molecular switches that regulate membrane trafficking and organelle organization in diverse ways. In the GTP-bound state, they are both associated with trans-Golgi membranes and active in effector recruitment. To date, more than 15 individual Rab6a/a′ effectors have been identified (reviewed in ). These include a series of motor proteins and/or motor protein regulators such as myosin II (MyoII), KIF1C, KIF5B and at least four different members of the golgin family of coiled coil proteins, GCC185, golgin 97, TMF and OCRL. With the exception of Rabkinesin 6, a mitotic kinesin (aka Rab6-KIFL/MKlp2/KIF20A), little, if any, evidence exists to suggest a differential interaction of any of the other effectors with Rab6a versus Rab6a′ and, in fact, considerable, but not all evidence suggests that the two closely related isoforms are functionally redundant. How Rab6a/a′ can produce the contextual sensitive recruitment of these effectors or balance the relative importance of individual effectors to maintain Golgi homeostasis remain open questions. For the sake of simplicity, we shall refer to Rab6a and Rab6a′ collectively as Rab6. This convention is consistent with the original naming of Rab6a as Rab6 as this was the first discovered member of this Rab subfamily.
In previous work, we found that Rab6 knockdown suppressed Golgi fragmentation and vesicle dispersal in HeLa cells depleted of the retrograde Golgi tether proteins, ZW10/RINT1 and conserved oligomeric Golgi complex (COG), in particular, the COG3 subunit . In a formal genetic sense, this outcome strongly indicates that Rab6 is acting upstream of both the ZW10/RINT1 and COG complexes. One plausible common mechanism follows from the fact that these tether systems are important to vesicular trafficking, albeit at different stages. If Rab6 acted to recruit motor proteins important to the Golgi-proximal movement of either set of vesicles, then in the absence of normal vesicle movement and consumption there might well be a feedback inhibition of vesicle budding and hence Golgi fragmentation. Any unbalancing of membrane trafficking under these conditions might well have profound effects on Golgi cisternal homeostasis.
In this study, we tested in HeLa cells several predictions of this hypothesis. First, we predicted that with Rab6 depletion that trans-Golgi proximal vesicles would accumulate and because of possible feedback effects there would a concomitant accumulation of arrested trans-Golgi vesicle budding structures. Second, we predicted that decreased trafficking might well lead to increased cisternal continuity. Vesicle budding is normally thought to limit cisternal continuity. Third, we predicted that the depletion of Rab6 as a trans-Golgi protein might well unbalance Golgi membrane trafficking such that there would be little, if any, inhibition of endoplasmic reticulum (ER) to Golgi trafficking, but a pronounced inhibition of Golgi to plasma membrane trafficking. If so, in accordance with a cisternal maturation model, the number of cisternae per Golgi stack might well increase. We used vesicular stomatitis virus membrane glycoprotein (VSV-G) transport from the ER to plasma membrane as an independent assay for cargo transport. Fourth, we tested the prediction that the depletion of a single Rab6 effector, MyoII, recently shown to be involved in vesicle formation at the TGN, would mimic the central elements of this hypothesis. Because vesicle accumulation should be Golgi proximal, we took as the major experimental readout in our experiments electron microscopy and, in particular, electron tomography where a full three-dimensional (3D) imaging of vesicle structure and size could be established.
The net outcome of these experiments was to place Rab6 as a trans-Golgi specific factor in coated vesicle budding/vesicle consumption. The finding that the vesicles were coated was both novel and unexpected. All previous evidence had indicated a role for Rab6 in coat-free vesicle trafficking [10, 11]. Coated vesicle accumulation in Rab6-depleted cells was accompanied by increased cisternal continuity and cisternal number with effects on cargo transport from the ER to plasma membrane being specific to the Golgi apparatus/TGN. Contrary to expectation, MyoII-depletion did not mimic the Rab6 knockdown phenotype as revealed by electron microscopy. In fact, the MyoII-depletion phenotype displayed in electron micrographs was not a noticeable aspect of the Golgi structure in Rab6 knockdown cells. In sum, we conclude that Rab effectors are not created equal in phenotypic importance to Golgi organization and suggest that the most phenotypically critical Rab6-dependent step(s) affects the transport of Golgi-coated vesicles of which at least a portion morphologically identified as coat protein I (COPI)-coated vesicles are likely intra-Golgi retrograde carriers.
- Top of page
- Materials and Methods
- Supporting Information
The results presented here indicate that Rab6 – the most abundant Golgi-associated Rab protein  – is essential to the normal transport and budding and/or consumption of multiple classes of coated vesicles. Moreover, they suggest that coated vesicle trafficking is crucial to the role of Rab6 in Golgi homeostasis. These results are a novel and unexpected outcome; considerable evidence to date has implicated Rab6 in non-coated, Golgi-derived vesicle trafficking [10, 11, 45]. The experiments themselves and the electron microscope approach essential to this outcome were prompted by our previous findings that in double knockdown, epistasis experiments, that Rab6 was required for the dispersal of Golgi-derived vesicles to the cytosol . We reasoned that as Rab6 effectors include a number of motor proteins and/or motor interacting polypeptides, e.g. MyoIIA, KIF5B, KIF1C, KIF20 and subunits of the dynactin complex that the epistatic suppression observed with Rab6 knockdown of both vesicle dispersal and Golgi fragmentation could be due to a failure to transport Golgi-derived vesicles. If so, Rab6 depletion might well produce vesicle accumulation in the immediate proximity of the Golgi apparatus. Because these vesicles would be Golgi proximal, they would be undetectable at the 200 nm or so resolution of the light microscope. Therefore, we used electron microscopy and, in particular, electron tomography of thick sections with its much higher resolution of approximately 4 nm as our primary experimental readout. In sum, the net outcome of our electron microscopy has been to reveal that Rab6 knockdown produces a diverse set of Golgi phenotypic changes in which the pronounced increase in cisternal number and continuity is accompanied by the accumulation of two classes of vesicles, both coated and concentrated on the trans side of the organelle. We propose that the observed accumulation of coated vesicles is primary to the dilation of the trans-Golgi/TGN and the proliferation of Golgi cisternae in response to Rab6 depletion.
We have taken the top down approach of knocking out Rab6 (Rab6a/a′) to study the role of Rab6 regulated protein machines in the regulation of Golgi dynamics and homeostasis. This approach has the virtue of revealing what are likely the dominant or most important phenotypic consequences of Rab6 for Golgi organization and dynamics. In our initial studies, we found that two different siRNAs directed against Rab6 produced the same morphological phenotype in electron micrographs of chemically fixed, thin-sectioned HeLa cells. In both cases, there appeared to be considerable cisternal proliferation, dilation of several cisternae and some accumulation of coated vesicles and cisternal-associated, coated-swellings that we term Ω figures. As both siRNAs produced the same morphological phenotype, we chose in all subsequent experiments to use the siRab6 (Sun) previously described by this laboratory . In additional validation of this choice, we had shown previously that the Rab6 phenotype suppression is mimicked by overexpression of GDP-restricted Rab6 and in some cases by the overexpression of a mutant Rab6 effector . A final methodological outcome from our initial electron microscopy was the decision to use high-pressure freezing as the first step in all subsequent sample preparation for electron microscopy. We found that high-pressure freezing followed by freeze substitution gave a much better preservation of Golgi structure in Rab6-depleted cells. Under these conditions, cisternal dilation was clearly restricted to the trans-Golgi/TGN, the site of coated vesicle accumulation.
To the best of our knowledge, our observations provide the first example in which depletion of a Golgi-associated regulatory molecule resulted in significantly increased Golgi organization, increased cisternal stacking and lateral continuity. In striking contrast, we found that knockdown of a single Rab6 effector, MyoIIA , resulted in the accumulation of small, uncoated structures that often remained linked to Golgi membranes but was not accompanied by a concomitant accumulation of multiple classes of coated vesicles or Ω figures as observed for Rab6. In siMyoIIA-treated cells, the general architecture of the Golgi ribbon was disrupted and cisternae within individual Golgi stacks were slightly shorter and less numerous than in control cells. At the electron microscope level, we did observe evidence for MyoIIA regulated Golgi tubule extension as reported by others . Rab6 has also been reported to regulate Golgi tubule extension in response to hypotonic shock . As MyoIIA is a known motor protein in Golgi to plasma membrane trafficking [11, 45], the failure to observe any increase in Golgi cisternal number with MyoIIA knockdown strongly indicates that inhibition of Golgi to plasma membrane trafficking per se is not tightly coupled to cisternal homeostasis. Our experiments indicate that Rab6-dependent recruitment of a cisternal adherence factor, likely a golgin, is of limited structural consequence to the overall organization of the Golgi apparatus. Although the disruption or depletion of individual golgins can lead to fragmentation of the Golgi ribbon (reviewed in ), we found that Rab6 depletion did not fragment the Golgi ribbon rather the Golgi ribbon was, if anything, more continuous. We did observe some de-adherence of TGN from trans-Golgi cisternae and correspondingly trans-Golgi cisternae from medial cisternae. Together these observations suggest that a limited set of Rab6 effecters are most important to establishing the morphological organization of the mammalian Golgi apparatus. A major goal of our current research is defining what that crucial subset might be.
The work presented in this article provides an explanation for the apparent dominant role of Rab6 in Golgi organization that is entirely consistent with our previous double knockdown experiments . In epistatic knockdown experiments, Rab6 loss-of-function suppressed Golgi fragmentation following knockdown of either the multimeric COG retrograde tether complex or the putative Golgi retrograde tether proteins ZW10 or RINT1 (reviewed in ). Importantly, co-depletion of Rab6 together with COG suppressed the cytoplasmic accumulation of Golgi-derived vesicles , suggesting that Rab6 regulates a key early step in either vesicle formation or transport. Based on the present results, we suggest that a major effect of Rab6 is at the level of coated vesicle formation/transport. Whether this is the crucial function of Rab6 in Golgi organization remains an open question. An inhibition of coated vesicle transport, especially that of COPI-coated vesicles, presumed intra-Golgi retrograde carriers, could well have feedback/knock-on effects through delayed recycling on coated vesicle formation and cargo transport through the Golgi apparatus. A primary defect in transport could then have secondary consequences on cisternal progress/maturation and the proliferation of Golgi cisternae. Considering the number of motor proteins known to be Rab6 effectors, we favor the interpretation that the failure to recruit a motor(s) may produce a primary defect in coated vesicle transport that then produces the potential secondary consequences cited previously. In addition, Rab6 may have a hitherto unsuspected role in autophagy and/or non-conventional secretion. We observed the accumulation of multivesicular/autophagic compartments in Rab6 knockdown cells. Whether this is a direct Golgi-associated consequence of Rab6-depletion, or mediated via a second Rab, e.g. Rab33b, a functionally overlapping Golgi Rab  known also to be involved in autophagy [51, 52] is unknown.
We found that the Rab6-depleted, Golgi apparatus still supported secretory cargo transport, albeit at a slower rate, a result consistent with the previous evidence of Grigoriev et al.  and Miserey-Lenkei et al.  that Rab6 – facilitated VSV-G transport from the Golgi to plasma membrane. Rab6 has long been known to have diverse effects on Golgi transport kinetics and organization. Depletion of Rab6 is known to affect both anterograde and retrograde Golgi trafficking [11, 12, 45]. Furthermore, Rab6 has been implicated in the regulation of endosome to TGN trafficking [53, 54]. Overexpression of GTP-restricted Rab6 causes the redistribution of Golgi proteins to the ER [47, 55] while, on the other hand, overexpression of GDP-restricted Rab6 delays secretory cargo transport from the TGN to the plasma membrane and inhibits Golgi to ER retrograde trafficking of Golgi enzymes [10, 56]. In this study, we found using quantitative fluorescence microscopy that transport of the model cargo protein, VSV-G, from the ER to Golgi was unaffected by Rab6-depletion while transport of VSV-G from the Golgi apparatus to the plasma membrane was substantially inhibited. Likely, this inhibition occurs at the trans-Golgi/TGN as that is the site of cisternal dilation and coated vesicle accumulation.
We suggest that the two morphological classes of coated vesicles/Ω figures that accumulated in response to Rab6 depletion correspond to clathrin- and COPI-coated structures. This reasoning is based on the morphology of the coats in electron tomograms where the coat can be observed in different planes and orientation and on the size difference between the two vesicle classes. The one coat appeared to be spikey and the other smooth. The spikey coat was associated with vesicles/Ω structures with an average diameter of approximately 75 nm, while the smooth coat was associated with vesicles/Ω figures of an average diameter of approximately 45 nm. These properties are consistent with the larger structures being clathrin coated and the smaller COPI coated. Other possible approaches to identifying the coats are immunolabeling and biochemistry/cell fractionation. Biochemically, our previous report that the lysosomal membrane protein LAMP2 is shifted to a higher molecular weight, i.e. further processed, in Rab6 knockdown cells  is consistent with the delayed transport implicit in the accumulation of putative clathrin-coated vesicles, known carriers in Golgi to endosome/lysosome cargo transport. Our light microscope immunolabeling experiments with antibody directed against COPI indicate an increase in the level of Golgi-associated COPI coat (Storrie, unpublished observations). Our initial efforts at a high-resolution immunogold labeling approach failed to give sufficient labeling to permit any conclusions to be drawn (Micaroni and Marsh, unpublished observations). To date, no direct effector link has been made between Rab6 and COPI or clathrin. However, the linkage to vesicles may well not be to the coat but rather to a motor protein. Several motor proteins are known Rab6 effectors. Whether specific effects due to Rab6a or Rab6a′ are evident in our data set remains for now an open question. In our experiments, we have used proven siRNAs directed against both Rab6 isoforms [9, 12] and hence the total level of Rab6 protein is knocked down 80 to >95%. The maximal level of total Rab6 protein reduction expected for the knockdown of the individual Rab6a or Rab6a′ isoforms is approximately 50%.
In conclusion, we present evidence from the unbiased and high-resolution approach of electron microscopy and electron tomography that Rab6 regulates, be it directly or indirectly, the trans-Golgi proximal accumulation of two morphological classes of coated vesicles. The morphological properties of these vesicles are suggestive that one class corresponds to clathrin-coated vesicles and the other to COPI-coated vesicles. On the basis of the site of vesicle accumulation, cisternal dilation and the kinetics of cargo transport from the ER to the plasma membrane, we suggest that Golgi function in transport is preferentially inhibited at the trans-Golgi/TGN. The observed proliferation of Golgi cisternae in Rab6-depleted HeLa cells may then be a secondary consequence of slowed Golgi export and/or inhibited intra-Golgi retrograde trafficking mediated by COPI-coated vesicles. Such an outcome is consistent with a cisternal maturation/progression model of Golgi function in secretion and the accumulated putative COPI vesicles are likely intermediates in cisternal maturation. These results are the first evidence for a Rab6-dependent coated vesicle transport at the trans-Golgi and the first example of concomitant cisternal proliferation and increased Golgi stack organization under such conditions.