Biolistic bombardment and fluorescence microscopy

Tobacco [Nicotiana tabacum cv Bright-Yellow-2 (BY-2)] suspension cell cultures were maintained and prepared for biolistic bombardment as described previously (Lingard et al., 2008). Transient (co-)transformations were performed using 0.5–6μg of plasmid DNA [determined empirically based on the relative strength of the (immuno)fluorescence signal due to expression levels of each plasmid-encoded protein construct] with a biolistic particle delivery system-1000/HE (Bio-Rad Laboratories). Bombarded cells were incubated for ~4–8h to allow for expression and sorting of the introduced gene product(s) and to reduce potential negative effects due to protein over-expression. Cells were either viewed immediately or fixed in 4% (w/v) formaldehyde, followed by permeabilization with 0.01% (w/v) pectolyase Y-23 (Kyowa Chemical Products) and 0.3% (v/v) Triton X-100 (Sigma-Aldrich). Antibodies used for subsequent immunofluorescence staining of cells were as follows: mouse α-Myc antibodies; rabbit α-Myc; rabbit α-HA; goat α-mouse and goat α-rabbit Alexa Fluor 488 IgGs, goat α-mouse and goat α-rabbit Cy5 (Cedar Lane Laboratories); and goat α-rabbit rhodamine red-X IgGs (Jackson ImmunoResearch Laboratories).

Epifluorescent images of cells were acquired using an Axioscope 2 MOT epifluorescence microscope (Carl Zeiss Inc.) with a 63× Plan Apochromat oil-immersion objective. Image capture was performed using a Retiga 1300 CCD camera (Qimaging) and Openlab software (Improvision). Confocal laser-scanning microscopy (CLSM) was carried out using a Leica DM RBE microscope with a 63× Plan Apochromat oil-immersion objective, anTCS SP2 scanning head, and the TCS NT software. Fluorophore emissions were collected sequentially in double- and triple-labeling experiments; single-labeling experiments showed no detectable crossover at the settings used for data collection. Other negative controls including omission of primary or secondary antibodies and mock transformations with vector (pRTL2) alone also yielded no detectable fluorescence. Confocal images were acquired as a z-series or single optical sections of representative cells were saved as 512×512-pixel digital images. Figure compositions and merged images were generated using Northern Eclipse software (Empix Imaging Inc.) and Adobe Photoshop CS or Illustrator CS2 (Adobe Systems). The co-localization of proteins in selected CLSM optical sections was quantified using the ImageJ plugin “Co-localization Finder” and methods based on those described previously by us (Gidda et al., 2011).

In all experiments, at least 50 independently (co-)transformed cells were evaluated to determine subcellular localization(s) of transiently (co)expressed proteins, and all micrographs shown in the figures are representative images. Each biolistic experiment was replicated at least two times.

Subcellular localization of Arabidopsis ESCRT proteins

We next characterized the Arabidopsis ESCRT machinery by examining the subcellular localization of some of the known and putative ESCRT proteins in tobacco BY-2 suspension-cultured cells, which serve as a well-known model plant cell system for studying protein targeting and organelle biogenesis (Brandizzi et al., 2003; Miao and Jiang, 2007). Specifically, epitope- or fluorescent protein-tagged versions of selected ESCRT proteins from Table ​Table1,1, including one or more proteins from each of the various ESCRT subcomplexes, as well as Vps4, or modified versions thereof, were expressed transiently (via biolistic bombardment) either individually or in combination in BY-2 cells. Their resulting subcellular localizations were then assessed, along with that of the co-expressed MVB marker protein, GFP-Syp21, consisting of GFP appended to the N terminus of the Arabidopsis membrane-bound Qa-SNARE Syp21 (Uemura et al., 2004), by standard epifluorescence microscopy, and/or by CLSM, when greater spatial resolution was necessary in order to confirm protein co-localization on a single optical plane or to view a novel MVB-related structure(s) in a 3D distribution. We also chose to examine the subcellular localization of selected plant ESCRT proteins in order to complement the protein–protein interaction results from our yeast two-hybrid assays (Figure ​(Figure1),1), as well as the wealth of information already available on the ESCRT-related functions and/or MVB targeting mechanisms of their homologs in yeasts and mammals. In doing so, this allowed us to discuss these (Arabidopsis) proteins in terms of the current, generalized working models for ESCRT.

As shown in Figure ​Figure2A,2A, N-terminal Myc-epitope-tagged versions of various members of the Arabidopsis Tom1 protein family displayed either (for Tom1A and Tom1D) a diffuse cytosolic and small punctate fluorescence pattern or (for Tom1C and Tom1E) an exclusively cytosolic fluorescence pattern, all of which were distinct from the punctate/globular fluorescence pattern attributable to GFP-Syp21 in the same representative co-transformed BY-2 cells, suggesting that these Tom1 proteins do not localize at steady state to MVBs. Moreover, the punctate structures observed in Myc-Tom1D-expressing cells did not colocalize with other endosomal pathway marker proteins, including those for the trans-Golgi or vacuole (data not shown). We also found that the subcellular localization of each of the selected Tom1 proteins was not affected by their co-expression with GFP-Syp21, since similar localization results were observed when Myc-Tom1A/C/D/E were expressed on their own (Figure ​(Figure2B).2B). Moreover, Tom1A-Myc (consisting of the Myc-epitope appended to the C terminus of Tom1A) and GFP-Tom1A (consisting of GFP appended to the N terminus of Tom1A), similar to Myc-Tom1A, localized to both the cytosol and small punctae in formaldehyde-fixed or living BY-2 cells, which were co-transformed with GFP-Syp21 or RFP-Ara7, respectively (Figures ​(Figures2C,D);2C,D); RFP-Ara7 serving as another well characterized endosomal (MVB) marker fusion protein consisting of RFP fused to the N terminus of the Arabidopsis Rab5-related soluble GTPase, Ara7 (Lee et al., 2004; Ueda et al., 2004). Collectively, these results indicate that the subcellular localization of the Tom1 proteins is not influenced by their appended epitope or fluorescent protein tags, or despite modest changes in endosome morphology due to formaldehyde fixation.

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Figure 2

Subcellular localization of Arabidopsis Tom1 proteins, GFP-Syp21, YFP-Rha1, and RFP-Ara7. BY-2 cells were either transiently (co-)transformed with either (A) various Myc-tagged Tom1 proteins and GFP-Syp21, (B) the same Myc-Tom1 proteins on their own, (C) Tom1A-Myc and GFP-Syp21, (D) GFP-Tom1A and RFP-Ara7, (E) GFP-Syp21 and RFP-Ara7 or YFP-Rha1 and RFP-Ara7, or (F) GFP-Syp21 and RFP-Ara7. Cells were then either formaldehyde-fixed and processed for immuno-epifluorescence microscopy or [in (F)] CLSM. Alternatively, cells [in (D) bottom row) were viewed living (i.e., non-fixed) using epifluorescence microscopy. Each micrograph is labeled at the top left with the name of the (co-)expressed protein construct. Also shown is the corresponding differential interference contrast (DIC) image or [in (F)] merged image for each set of (co-)transformed cell(s). Hatched boxes in [(A) and (C–F)] represent the portion of cells shown at higher magnification and colorized in the panels below. The yellow color in the merged images indicates co-localization between co-expressed proteins; white arrowheads [in (E,F)] also indicate obvious protein co-localization, while the open arrowheads in (E,F) indicate obvious non-co-localization. Bar=10μm.

While the appearance (i.e., number, size, and distribution) of GFP-Syp21- or RFP-Ara7-labeled MVBs in BY-2 cells co-expressing selected Tom1 proteins (or other ESCRT or ESCRT-associated proteins) often varied among cells, this variability has been also reported for other plant cell types and was expected given the highly dynamic nature of the plant endosomal system overall. In addition, over-expression of Syp21 has been previously shown to prevent fusion of MVBs with the vacuole, leading to accumulation of vacuole-destined proteins at MVBs, ultimately resulting in the formation of enlarged MVBs (Foresti et al., 2006). Regardless, in a series of control experiments (Figure ​(Figure2E),2E), we confirmed that GFP-Syp21 partially colocalized with co-expressed RFP-Ara7, while YFP-Rha1, consisting of YFP fused to Rha1, another MVB-localized Arabidopsis Rab5-related GTPase (Lee et al., 2004), partially colocalized with co-expressed RFP-Ara7, and that in both sets of co-transformed cells at least some of the MVBs appeared to be enlarged and/or aggregated (i.e., clustered in certain regions of these cells); the partial co-localization between GFP-Syp21 and RFP-Ara7 also verified by CLSM (Figure ​(Figure2F)2F) and quantified based on the mean Pearson's correlation coefficient r, which revealed a significant amount of fluorescence signal overlap (r=0.67±0.09; see Materials and Methods for details). Overall, these results match those from similar experiments which showed that Ara7/Rha1 and Syp21 are preferentially localized in at least two types of partially overlapping and structurally related endosomal (MVB) compartments in plant cells (Lee et al., 2004; Ueda et al., 2004; Foresti et al., 2010). For the remainder of experiments described in this study, however, we chose to utilize GFP-Syp21 as a marker for MVBs, since, for among other reasons, it represents one of the best characterized MVB marker proteins in terms of its trafficking and biological activities (Shirakawa et al., 2010; Uemura et al., 2010). We also attempted to control for any potential negative effects due to GFP-Syp21 over-expression (Foresti et al., 2006), or transient protein (over)expression in general, by focusing on cells exhibiting relatively low or moderate levels of (immuno)fluorescence, as previously described (e.g., Ueda et al., 2004; Kato et al., 2010). In addition, as we did for with the abovementioned Tom1 proteins, all of the other ESCRT or ESCRT-associated proteins examined in this study in terms of their localization in BY-2 cells were expressed on their own to assess the effects of co-expression with GFP-Syp21 and/or other ESCRT proteins.

Given that both Dictyostelium Tom1 and human Tom1L1 bind Tsg101 (Yanagida-Ishizaki et al., 2008; Blanc et al., 2009) we tested further whether the Arabidopsis Tom1 proteins, using Tom1A and Tom1D as representatives of this group [i.e., both proteins bound Vps23A and/or Vps23B in yeast two-hybrid assays (Figure ​(Figure1)],1)], are redistributed to MVBs when co-expressed with Vps23A, which represents the best characterized component of ESCRT-I in plants (Spitzer et al., 2006). As shown in Figure ​Figure3,3, HA-Vps23A partially colocalized with co-expressed GFP-Syp21 in MVBs in BY-2 cells (r=0.62±0.13), as expected (Spitzer et al., 2006). Figure ​Figure33 shows also that Myc-Tom1A partially localized to HA-Vps23A-containing structures (r=0.59±0.12) that, based on the co-localization of HA-Vps23A and GFP-Syp21, were presumed to be MVBs. By contrast, Myc-Tom1D co-expressed with HA-Vps23A was not redirected to HA-Vps23A-containing MVBs (r=0.22±0.07), but rather remained localized in the cytosol and small punctae in these cells (Figure ​(Figure3),3), similar to when it is either expressed on its own or co-expressed with GFP-Syp21 (Figure ​(Figure2).2). Additionally, we showed that GFP-Tom1A partially colocalized with Myc-Vps23B in co-transformed cells (Figure ​(Figure3;3; r=0.61±0.06), suggesting that Vps23A and Vps23B are equivalent in terms of their ability to recruit Tom1A to MVBs.

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Figure 3

Subellular localization of Vps23A and co-expression of Tom1A and/or Tom1D with Vps23A or Vps23B. BY-2 cells were co-transformed with the indicated gene constructs and then cells were processed, imaged and analyzed by CLSM as described in the legend of Figure ​Figure2.2. The yellow color in the merged images indicates co-localization between co-expressed proteins; white arrowheads also indicate obvious protein co-localization. Bar=10μm.

That Tom1D, unlike Tom1A, is not recruited to MVBs upon co-expression of Vps23A/B was unexpected, given their high degree of sequence similarity and comparable domain organization (Winter and Hauser, 2006), as well as their shared ability to interact with ESCRT-I components in yeast two-hybrid assays (Figure ​(Figure1).1). We speculate that this may be due to stoichiometric differences in the relative amounts of these co-expressed proteins and/or additional endogenous factors. Regardless, the co-localization of Tom1A and Vps23 observed in this study is consistent with the localization of Tom1-related proteins in other organisms (Yanagida-Ishizaki et al., 2008; Blanc et al., 2009). Additional studies of the Arabidopsis Tom1-related protein family, Vps23 homologs, plus the multitude of other uncharacterized ubiquitin-binding proteins encoded in the Arabidopsis genome that may function in MVB protein cargo selection (Leung et al., 2008) are needed in order to better understand the potential role of an “alternate” ESCRT-0 complex in plants. This task will also need to take into consideration that the Tom1 family members display distinct organ/tissue expression patterns and thus the subcellular localization of these proteins (as well as all the other ESCRT proteins examined in this study) should also be studied in planta. There is also a possibility that specific ubiquitinated MVB cargo proteins may serve to initiate the assembly of an ESCRT-0 complex, notably, several candidates have been recently identified as plant ESCRT cargo proteins (Spitzer et al., 2009; Viotti et al., 2010).

In addition to Vps23A and Vps23B, the putative ESCRT-I complex in Arabidopsis also includes Vps28A/B and Vps37A/B (Table ​(Table1)1) and while we showed via yeast two-hybrid assays (Figure ​(Figure1)1) that Vps23A/B interacts with Vps28A/B and Vps37A, only Vps23A, Vps28B, and Vps37A have been shown elsewhere to form a complex in plant cells (Spitzer et al., 2006). Thus, to further investigate the interactions detected between ESCRT-I proteins (Figure ​(Figure1),1), we examined the subcellular localization of the putative Vps28A and Vps28B proteins when expressed with or without Vps23A. As shown in Figure ​Figure4A,4A, both Myc-tagged Vps28A and Vps28B localized exclusively to the cytosol in BY-2 cells co-expressing GFP-Syp21, similar to the cytosolic localization of ectopically expressed Vps28 proteins in other organisms (Bishop and Woodman, 2001). By contrast, co-expression of Myc-Vps28A or Myc-Vps28B with HA-Vps23A resulted in both sets of proteins being colocalized partially to the cytosol and also to the periphery of large, ring-like structures (Figure ​(Figure4B);4B); shown also in Figure ​Figure4B4B (bottom row) are three related CLSM 3D projection images illustrating the localization of Myc-Vps28A in the large, ring-like structures found in an Myc-Vps28A and HA-Vps23A co-transformed cell. While similar structures were not observed in cells co-expressing any of these proteins with GFP-Syp21 alone (Figures ​(Figures3A3A and ​and4A),4A), these structures were readily observed in cells triple-transformed with Myc-Vps28A (or Myc-Vps28B), HA-Vps23A, and GFP-Syp21, wherein all three proteins colocalized (Figure ​(Figure4C;4C; results presented only for co-expressed Myc-Vps28A-HA-Vps23-GFP-Syp21), indicating they are derived from MVBs.

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Figure 4

Subcellular localization of Vps28A and Vps28B and co-expression of Vps28A or Vps28B with Vps23A. BY-2 cells were co-transformed the indicated gene constructs and then cells were processed, imaged, and analyzed either by (A,B) epifluorescence microscopy or [(B) bottom row and (C)] CLSM as described in the legend of Figure ​Figure2.2. The yellow color in the corresponding merged images indicates co-localization between co-expressed proteins; white arrowheads also indicate obvious protein co-localization in the large, ring-like MVB-related structures. Note that the CLSM images presented in [(B) bottom row] are three different rotations (i.e., left ~−15°, center, and right ~+15°) obtained from the same 3D projection (full z-series) of immunostained Myc-Vps28A in an Myc-Vps28A and HA-Vps23A co-transformed cell; refer also to Figure S4 in Supplementary Material for the corresponding movie. The CLSM images of the triple-transformed cell in (C) are single z-sections. Bar=10μm.

The relocalization of Vps28 to MVB-derived structures upon co-expression with Vps23A is consistent with its classification as a core ESCRT-I component. Furthermore, the formation of these structures is reminiscent of the aberrant MVB-related structures or so-called “class E compartments” formed in yeast or mammalian cells upon over-expression or deletion of certain ESCRT components (or mutants versions thereof) that act in a dominant-negative fashion to disrupt normal ESCRT function (Roxrud et al., 2010). While we do not know the ultrastructure of the class E-like compartments in Vps23 and Vps28-co-transformed BY-2 cells (Figure ​(Figure4)4) or the molecular mechanism underlying their formation, the appearance of these unique structures, combined with the observation that Arabidopsis Vps23 and Vps28 interact directly (Figure ​(Figure1;1; Spitzer et al., 2006) provides further evidence that these proteins function together in MVB biogenesis. On the other hand, until Vps23 and Vps28 have been studied in more detail, we also urge caution in interpretation of results obtained from studies of the subcellular localization and/or MVB morphology changes associated with expression of these proteins (or any other plant ESCRT protein described in this study), and their extrapolation to cellular function(s).

As mentioned previously, in yeast and mammals, ESCRT-II is recruited to the endosomal membrane by ESCRT-I, where it plays an essential role in the concentration of ubiquitinated cargo proteins and the recruitment of ESCRT-III (reviewed in Hurley and Hanson, 2010; Peel et al., 2010). As shown in Figure ​Figure5A,5A, HA-Vps36 localized in BY-2 cells to the plasma membrane and to numerous small punctae/globular or ring-like structures that occasionally colocalized with GFP-Syp21. Myc-Vps25, a second component of ESCRT-II that we showed interacts directly with Vps36 (Figure ​(Figure1;1; Figure S3 in Supplementary Material) localized exclusively to the cytosol when co-expressed with GFP-Syp21 (Figure ​(Figure5A),5A), but was localized primarily to the plasma membrane when co-expressed with HA-Vps36 (Figure ​(Figure5B);5B); refer also to CLSM images in Figure ​Figure5B5B (bottom row) confirming the co-localization of HA-Vps36 and Myc-Vps25 at the plasma membrane (r=0.90±0.10). Together, these data are consistent with the premise that these two proteins function together as part of the ESCRT-II complex in Arabidopsis, just as they do in yeasts and mammals (Hurley and Hanson, 2010; Peel et al., 2010). However, the functional significance of the plasma membrane localization of Vps36 (and co-expressed Vps25) is still unknown and needs to be confirmed in planta, and further investigation of ESCRT-II and the functional relationship between Vps36 and Vps25 in Arabidopsis may reveal additional and/or unique roles for ESCRT-II in plants.

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Figure 5

Subcellular localization of Vps25 and Vps36. BY-2 cells were co-transformed with the indicated gene constructs and then cells were processed, imaged, and analyzed either by epifluorescence microscopy [(A,B) top row] or [(B) bottom row] CLSM as described in the legend of Figure ​Figure2.2. The yellow color in the corresponding merged images indicates co-localization between co-expressed proteins; white arrowheads also indicate obvious protein co-localization, while open arrowheads in (A) indicate the localization of HA-Vps36 (but not co-expressed GFP-Syp21) at the plasma membrane. Bar=10μm.

We examined next the subcellular localization of several putative Arabidopsis ESCRT-III components, including Vps20A, Vps20B, and Snf7A. Based on the working models for ESCRT in yeast and mammals (Hurley and Hanson, 2010; Peel et al., 2010), each of the four core components of ESCRT-III (e.g., yeast Vps2, Vps20, Vps24, and Snf7; Table ​Table1)1) contain a similar domain organization, including an N-terminal basic domain required for membrane interaction and (homo- and/or hetero-) oligomerization, and a C-terminal acidic autoinhibitory domain that prevents their oligomerization and recruitment from the cytosol to endosomes. By a mechanism that is not fully understood, autoinhibition is relieved, allowing the ESCRT-III proteins to be sequentially assembled on the endosomal membrane as two subcomplexes; the Vps20-Snf7 subcomplex that binds to the endosomal membrane through the N-terminal myristoylation of Vps20 and a direct interaction with Vps25 of ESCRT-II, and the Vps2-Vps24 subcomplex that binds via an interaction between Vps24 and Snf7A. Consistent with this model, our yeast two-hybrid assays (Figure ​(Figure1)1) revealed that both of the putative Arabidopsis Vps20 isoforms (Vps20A and Vps20B) interact with Vps25, as well as with Snf7A and Snf7B. Moreover, both Snf7A/B and all of the other putative Arabidopsis ESCRT-III proteins share a relatively high degree of primary sequence and secondary structure similarity with their yeast and mammalian counterparts (Winter and Hauser, 2006; Leung et al., 2008), including conserved N-terminal basic and C-terminal acidic regions. In addition, both Arabidopsis Vps20A/B, like Vps20 proteins in other organisms, possess a predicted N-terminal myristoylation signal (Leung et al., 2008).

As shown in Figure ​Figure6A,6A, both Vps20A-Myc and Vps20B-Myc, consisting of full-length Arabidopsis Vps20A and Vps20B appended to the Myc-epitope tag at their C terminus in order to preserve their putative N-terminal myristoylation signal, localized exclusively to the cytosol in BY-2 cells. By contrast, C-terminal-mutant versions of both proteins, namely Vps20A_1–170-Myc and Vps20B_1–170-Myc (consisting of their N-terminal 1–170 out of ~220 amino acid residues), localized to the cytosol and to punctate/globular structures, at least some of which also contained co-expressed GFP-Syp21 (Figure ​(Figure6B).6B). Combined, these results are similar to those for the localization of human (full-length) Chmp6 (the mammalian homolog of Arabidopsis Vps20) in the cytosol and the partial localization of a C-terminally truncated mutant version of Chmp6 to endosomes (Peck et al., 2004), suggesting that a C-terminal autoinhibitory mechanism may be conserved between Chmp6 and Arabidopsis Vps20.

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Figure 6

Subcellular localization of full-length and C-terminal-truncated versions of Vps20A and Vps20B, and co-expression of Snf7A and Vps4 or Vps4^E232Q. (A–D) BY-2 cells were co-transformed with the indicated gene constructs and then cells were processed, imaged and analyzed by epifluorescence microscopy as described in the legend of Figure ​Figure2.2. The yellow color in the corresponding merged images [in (B–D)] indicates co-localization, between co-expressed proteins; white arrowheads indicate obvious protein co-localization including in the enlarged globular-like structures in (C). Bar=10μm.

As shown in Figure ​Figure6C,6C, the putative Arabidopsis Snf7A (i.e., HA-Snf7A) localized in BY-2 cells to punctae and large, globular-like structures that mostly colocalized with co-expressed GFP-Syp21. Given the ability of Snf7 and Chmp4 yeast and mammalian cells, respectively, to form large, higher-ordered polymers on the endosomal surface (Teis et al., 2010) and induce the formation of aberrant MVB-related structures upon their over-expression in cells (Peck et al., 2004; Boysen and Mitchell, 2006), it is possible that the large globular structures observed in BY-2 cells may be also the result of the assembly of abnormal Snf7A oligomers. This premise is supported, albeit indirectly, by the ability of Arabidopsis Snf7A/B to interact in yeast two-hybrid assays (Figure ​(Figure1),1), just as their yeast and mammalian counterparts do (Nikko and André, 2007).

Following membrane scission, ESCRT-III subunits (mainly Snf7) are disassembled and recycled to the cytosol for further rounds of vesicle formation, a process that is mediated by Vps4 and its regulators in yeasts and mammals (Hurley and Hanson, 2010; Peel et al., 2010), and, based on available data, also in plants (Jou et al., 2006; Haas et al., 2007; Shahriari et al., 2010). Unexpectedly, however, Vps4 and Snf7A/B did not interact in the yeast two-hybrid assay (Figure ​(Figure1),1), which contrasts the interaction data reported elsewhere (Shahriari et al., 2010), and Myc-Vps4 was localized exclusively to the cytosol in BY-2 cells co-expressing HA-Snf7A (Figure ​(Figure6D).6D). We therefore took advantage of a catalytically inactive version of Vps4 that is well-known in yeasts and mammals to have a potent dominant-negative effect on MVB biogenesis by binding irreversibly to MVBs and “trapping” the ESCRT(-III) machinery on the MVB surface (Babst et al., 1998). The equivalent mutation in the Arabidopsis protein (Vps4^E232Q) has also been shown to be catalytically inactive and disrupt MVB biogenesis in plant cells (Haas et al., 2007; Shahriari et al., 2010). As shown also in Figure ​Figure6D,6D, when HA-Snf7A was co-expressed with Myc-Vps4^E232Q, both proteins mostly colocalized in irregular-shaped structures that appeared to be distinct from punctate and large, globular-like structures observed in cells expressing HA-Snf7A and GFP-Syp21, further supporting the classification of Snf7A (and Snf7B) as a component of ESCRT-III, and suggesting a possible functional relationship between Snf7A and Vps4, just as in other organisms (Hurley and Hanson, 2010; Peel et al., 2010).