Sec3 Localizes to Desmosomes and Centrosomes
The localization of several endogenous Exocyst subunits in mammalian epithelial cells has been reported previously (Grindstaff et al., 1998
; Lipschutz et al., 2000
; Folsch et al., 2003
; Prigent et al., 2003
; Rogers et al., 2004
; Oztan et al., 2007
; Zuo et al., 2009
) but that of Sec3 has yet to be examined. Although a Sec3-GFP fusion protein was shown to have a cytosolic distribution, it is unclear whether this reflects the localization of endogenous Sec3 (Matern et al., 2001
). We approached this question by preparing polyclonal antibodies against fusion proteins representing either N- or C-terminal regions of human Sec3 (A). Both antibodies were specific, as determined by a variety of criteria (, B–G, and Supplemental Figure S1). Antibodies to both N- and C-terminal domains labeled pools of Sec3 distributed in a punctate, discontinuous pattern along the plasma membrane and in the cytoplasm of MDCK cells (, E and F). Preimmune serum did not label either pool (D), and immune serum depleted of Sec3-specific antibodies showed decreased levels of plasma membrane and cytoplasmic labeling (data not shown). Antibodies against the N terminus of Sec3 additionally labeled a perinuclear compartment in subconfluent MDCK cultures (F). Therefore, in contrast to ectopic GFP-Sec3, endogenous Sec3 was, at least in part, associated with the plasma membrane and cytoplasmic organelles.
Figure 1. Antibodies to Sec3 are specific. (A) Schematic representation of human Sec3. Three coiled-coil motifs (amino acids 152-176, 205-257, and 742-764) are predicted by the COILS program. Lines indicate regions of the protein that were used to generate antibodies. (more ...)
To determine whether Sec3 colocalized with other Exocyst subunits on lateral plasma membranes, we colabeled MDCK cells with anti-Sec3 C-terminal antibodies and monoclonal Sec6 and Sec8 antibodies. Specific Sec3 immunolabeling was coincident with that of Sec6 when one specific mAb (8A5), but not another (9H5), was used (, A and B). In polarized MDCK cells, structures labeled by anti-Sec3 and anti-Sec6(8A5) were concentrated at two distinct sites along lateral membranes (B). In contrast, anti-Sec3 antibodies did not label structures recognized by anti-Sec6(9H5) (A), in spite of the fact that this antibody was previously shown to label Exocyst pools associated with the AJC (Yeaman et al., 2004
). Sec3 localized to lateral membrane sites that were more basal than those detected by anti-Sec6(9H5). In addition, although the labeling pattern of anti-Sec6(9H5) was continuous and even, that of anti-Sec3 and anti-Sec6(8A5) was discontinuous and punctate. Therefore, although localization of Sec3 to lateral membranes was similar to that of other Exocyst subunits reported previously, Sec3 did not colocalize with previously characterized Sec6/8 complexes there.
Figure 2. Exocyst localizes to desmosomes. (A) Polarized MDCK cells were colabeled with anti-Sec3CT and anti-Sec6(9H5) antibodies. Both en face (xy) and vertical (xz) confocal sections are shown, and the arrow denotes the position at which xy images were collected. (more ...)
To confirm that the novel punctate labeling pattern observed with anti-Sec3 and anti-Sec6(8A5) antibodies represented bona fide Exocyst localization, the distribution of several other Exocyst subunits was examined. Because prior studies of Sec8 localization in MDCK cells used a mAb (8F12) that did not colabel structures decorated by anti-Sec3 antibodies, we screened several fixation conditions and a panel of 10 mAbs to Sec8 to determine whether these proteins were colocalized in cells. We found that a cocktail of anti-Sec8 mAbs (2E9, 5C3, 7E8, and 17A10) applied to methanol-fixed cells labeled a pool of Sec8 that colocalized with Sec3 at the lateral plasma membrane (C). Permeabilization of cells with Triton X-100 before fixation revealed that this Sec8 pool was, like Sec3, distributed in a punctate and discontinuous manner on the membrane (Supplemental Figure S2).
Polyclonal anti-Sec15 antibodies also labeled plasma membrane structures that were indistinguishable from those labeled by anti-Sec6(8A5) (D). This labeling was enriched at sites of cell–cell contact and absent from free cell borders, consistent with Sec3 labeling at the plasma membrane. Furthermore, antibodies to Sec15 and two other Exocyst subunits, namely, Exo70 and Exo84, produced identical labeling patterns in A431 cells (Supplemental Figure S3). Thus, at least six different Exocyst subunits localized to similar punctate, lateral plasma membrane structures that were distinct from the sites at which previously identified AJC-associated Sec6/8 complexes localized.
Because several Exocyst subunits were distributed in a characteristic spot-like pattern at sites of cell-cell contact, the possibility that these sites represented desmosomes was examined. Anti-Sec6(8A5) and anti-desmoplakin antibodies colabeled identical structures enriched in two regions of the lateral plasma membrane (E). Moreover, Sec3 colocalized with desmoglein-GFP at these lateral membrane puncta (F). These data show that Exocyst complexes containing Sec3 are enriched at desmosomes.
Not only this desmosome-associated Sec3 fraction but also an additional Sec3 pool was observed (A and ). Specifically, Sec3 colocalized with γ-tubulin (A) and kendrin (pericentrin-B) (B) at centrioles. Given that either one or two dots were conspicuous in polarized MDCK cells, Sec3 seems to associate with both maternal and daughter centrioles. In polarized cells, the majority of centrioles are located in the apical cytoplasm in a higher focal plane than desmosomes, so it is not surprising that not all optical sections showed both pools of Sec3 protein (). Antibodies to Sec15, Exo70, and Exo84 also labeled centrosomes in A431 cells, suggesting that a Sec3-containing Exocyst complex is associated with this organelle (Supplemental Fig. S3).
Figure 3. Sec3 localizes to centrioles. (A and B) Polarized MDCK cells were colabeled with anti-Sec3CT and either an anti-γ-tubulin (A) or an anti-kendrin (B) antibody. xy confocal sections are shown. Endogenous Sec3 was localized at kendrin- and γ-tubulin-positive (more ...)
Sec3 Occupies a Subset of Exocyst Complexes in Epithelial Cells
In contrast to published localizations of Sec6 and Sec8 at the AJC, Sec3 colocalized with Sec15, Exo70, and Exo84 and immunologically distinct pools of Sec6 and Sec8 at desmosomes. To determine whether distinct Exocyst complexes are associated with desmosomes and the AJC, we compared the biochemical properties of Sec3 and Sec8. First we examined the detergent solubility of each subunit. Sec3 solubility in buffer containing Triton X-100 was nearly identical to that of every other Exocyst subunit examined, except Exo84 ( and Supplemental S4). Sec3 was, like Sec8, more soluble in deoxycholate-containing RIPA buffer (). However, no significant differences in solubility were observed between Sec3 and Sec8, regardless of the solubilization buffer used.
Figure 4. Sec3 and Sec8 have the same detergent solubility. (A) Insoluble (Insol) and soluble (Sol) MDCK fractions generated using 1% Triton X-100 or RIPA (deoxycholate) were analyzed by SDS-PAGE and immunoblotting with anti-Sec3NT and anti-Sec8(8F12) antibodies. (more ...)
Because the Exocyst associates with membranes, the buoyant densities of Sec3- and Sec8-associated membrane domains were analyzed by isopycnic density gradient centrifugation. Sec8 had been shown previously to cofractionate with proteins associated with the AJC—such as zona occludens (ZO)-1, ZO-2, nectin-2α, and E-cadherin—at δ = 1.16 g/ml (Yeaman et al., 2004
). We found that membrane-associated Sec3 also cofractionated with Sec8 at δ = 1.16 g/ml (A). In addition, the desmosome-associated protein desmoplakin was enriched in these fractions, consistent with immunolocalization of Sec3 to desmosomes. A secondary peak of Sec3 and Sec8 corresponded to cytosolic fractions (δ = 1.20 g/ml).
Figure 5. Sec3 and Sec8 cofractionate in an isopycnic density gradient and coelute on a gel filtration column. (A) MDCK postnuclear supernatants were fractionated on a 10–20-30% iodixanol step gradient. The density of each fraction was determined after (more ...)
To determine whether Sec3 assembles into large, multimeric complexes similar in size to those containing Sec8, we fractionated detergent extracts of MDCK cells by Superose 6 fast-performance liquid chromatography. Most of the Sec3 coeluted with Sec8 in a single peak at fraction 12, corresponding to a protein complex with an apparent molecular size of >3000 kDa, based on the elution of globular protein standards (B). In addition to this major peak, an overlapping peak of Sec8, centered at fraction 14 and corresponding to a protein complex of ~1000 kDa, was observed. Sec3 was not associated with this Sec8 fraction, but a minor pool of Sec3 was observed in fraction 19, as part of a protein complex of ~450 kDa. No Sec3 was recovered in fractions that eluted from the column at later time points, indicating that monomeric Sec3 was not present in detectable quantities in MDCK cells (data not shown). Instead, all of the Sec3 was assembled into high-molecular-weight complexes. In addition, these data hint that Sec8 may exist in at least two distinct complexes that either contain or lack Sec3.
To directly test this possibility, we performed coimmunoprecipitation studies. We performed three consecutive rounds of Sec8 immunoprecipitation in order to deplete this protein it from MDCK RIPA lysates (A). Sec3 was almost completely cleared from the lysates after the first round of Sec8 immunoprecipitation, indicating that all of the Sec3 was associated with Sec8 in MDCK cells (A). In contrast, exhaustive immunoprecipitation of Sec3 from the same lysates left ~30% of the Sec8 in the Sec3-depleted lysate (B). Subsequent immunoprecipitation of this Sec8 pool also recovered Sec6 (C). These results indicate that at least two distinct Sec8-containing Exocyst complexes are present in MDCK cells, and that these are distinguished by the presence or absence of Sec3.
Figure 6. All endogenous Sec3 is bound to Sec8. (A and B) MDCK RIPA extracts were immunoprecipitated several times with anti-Sec8- (A) or anti-Sec3NT (B)-bound protein A-Sepharose. Input lysate (Input) represents 10% of starting material. Depleted lysate (Dep. (more ...)
Assuming that all Sec3 and Sec8 were present in Exocyst complexes at a 1:1 stoichiometry, but additional Sec8-containing complexes lacking Sec3 were also present in cells, Sec8 should be more abundant than Sec3. To test this prediction, we immunoprecipitated Sec3 and Sec8 from radiolabeled cells (D). After correcting for immunoprecipitation efficiencies and differences in the methionine and cysteine contents of the two proteins, we determined that MDCK cells had ~65% more Sec8 than Sec3.
Finally, the spatiotemporal redistribution of Sec3 during early stages of cell–cell contact formation was compared with that of Sec6 and Sec8. The rationale for this experiment was that if distinct Exocyst complexes that either contain or lack Sec3 are associated with desmosomes and AJCs, respectively, it might be possible to observe differences in how they become associated with plasma membranes during polarity development. Contact-naïve MDCK cells were allowed to adhere to a collagen substrate at low plating density in LCM for 1 h, and then either maintained in LCM (A) or switched to high calcium medium (HCM) for 1 h (B). In cells cultured in LCM for 2 h, nascent E-cadherin–containing intercellular contacts formed, and these were labeled with anti-Sec6(9H5) and anti-Sec8(8F12), but not anti-Sec3 antibodies (A). Although Sec3 was not present at these nascent cellular contacts in LCM, it did colocalize with Sec6, Sec8 and desmosomal cadherins in cytoplasmic puncta (, A and C). Only after culturing cells in HCM for 1 h was Sec3 observed to accumulate at sites of intercellular contact (B). Therefore, membrane recruitment of the Sec3 protein, which ultimately becomes associated with desmosomes, was slower than that of the Sec6/8 complexes that associate with the AJC.
Figure 7. Plasma membrane recruitment of Sec3 is temporally distinct from that of Sec6 and Sec8. Low-density cultures of contact-naïve MDCK cells were seeded in LCM on collagen-coated coverslips and incubated for 2 h (A and C) or for 1 h, before shifting (more ...)
Sec3 Is Required for Desmosome Assembly
To study the role of Sec3 in epithelial cells, we reduced its expression in human MCF-10A cells by transduction with lentiviral vectors containing a puromycin selection cassette and shRNAs targeting both splice variants of human Sec3. It was necessary to switch to these cells because multiple efforts to stably reduce Sec3 to levels below ~50% of control levels in MDCK cells were unsuccessful. MCF-10A cells form a polarized, cuboidal epithelium with well-developed desmosomes and adherens junctions (Tait et al., 1990
). We identified two hairpins that reduced Sec3 protein levels by >90%, as assessed by Western blotting (A). These also led to greatly reduced immunofluorescence labeling intensity after selection in puromycin (B).
Figure 8. Sec3 is required for desmosome stability. (A) Lentiviruses producing hairpin 1 (nucleotides 1473-1493) or hairpin 2 (nucleotides 1244-1264), which are common to both splice variants of Sec3, were constructed. MCF-10A cells were transduced with these viruses (more ...)
Because Sec3 accumulated at desmosomes, we determined whether it was required for the structural integrity of these junctions. Pan-specific desmoglein antibodies labeled spot-like desmosomes along plasma membranes of control cells, but this plaque-like labeling was disrupted in Sec3 knockdown cells. In these cells, most of the membrane-proximal desmoglein-positive structures were organized into linear arrays that ran perpendicular to cell–cell contacts (, C and E). Similar results were observed with antibodies specific for Dsg1, Dsg2, and Dsc2/3 (data not shown). Furthermore, anti-plakoglobin antibodies labeled cell–cell contacts in both control and Sec3 knockdown cells, but the morphology of these contacts was abnormal when Sec3 expression was reduced (C). In addition, plasma membrane labeling of desmoplakin and the Exocyst [as defined by Sec15 and Sec6(8A5) labeling] was substantially reduced in Sec3 knockdown cells (C). In contrast, E-cadherin localization was largely unchanged when Sec3 expression was suppressed (, D and E). On reexpression of Sec3 in these cells, desmoplakin and Sec15 were returned to the plasma membrane, and normal desmosomal morphology was restored (C). Therefore, the absence of Sec3 seems to affect the morphology of desmosomes specifically and not that of adherens junctions.
Because the Exocyst has been implicated in vesicle tethering to target membranes, we investigated the possibility that Sec3 was required for trafficking of desmoglein-containing vesicles to the plasma membrane. The delivery of Dsg2 to the plasma membrane was assayed by biotin accessibility in a surface repopulation assay. Cells were suspended in trypsin to dissociate them and remove preexisting surface proteins, and then replated in LCM at confluent density. At various time points after transferring cells to HCM, Dsg2 expression at the plasma membrane was quantified by cell surface biotinylation, followed by streptavidin precipitation of biotinylated proteins. In control cells, Dsg2 gradually accumulated at the cell surface over a 72-h time course (, A and B). Surprisingly, in Sec3-knockdown cells the rate of Dsg2 accumulation at the plasma membrane was increased relative to that in control cells, reaching steady state within 15–30 h of the calcium switch (, A and B). However, the Dsg2 that was delivered to the plasma membrane did not seem to assemble into desmosomes (, C and E). In contrast, E-cadherin transport was not altered after the suppression of Sec3 expression (C). Therefore, loss of Sec3 expression is associated with changes in transport or incorporation of Dsg2 into desmosomes but not with trafficking of E-cadherin to developing adherens junctions.
Figure 9. Sec3 is required for normal Dsg2 trafficking. A surface repopulation assay was performed in MCF-10A control and shSec3 cells, as described in Materials and Methods. At various time points after the calcium switch, surface proteins were biotinylated and (more ...)
To gain further insight into how a reduction in Sec3 expression affected desmosome structure and function, we quantified overall expression levels of several desmosome- and adherens junction-associated proteins in two independent clones of MCF-10A cells expressing hairpin 2. These clones exhibited an ~80–90% reduction of Sec3 expression (A). In both clones, as well as in pools of cells expressing either hairpin 1 or hairpin 2, the levels of all desmosomal cadherins examined were elevated relative to those in control cells (B and Supplemental Figure S5). Dsg1expression was 1.4- and 3.2-fold higher in clones 1 and 2, respectively. Likewise, Dsg2 expression was 1.9- and 4.2-fold higher, and Dsc2/3 expression was 13- and 23-fold higher, in the Sec3 knockdown clones (B). In addition, plakoglobin protein levels were increased, but desmoplakin expression was not substantially altered (B). Expression of Sec3hr in these cells partially reversed these effects, leading to a substantial reduction in Dsc2/3 expression and a more modest reduction in plakoglobin expression (E). Adherens junction components, such as E-cadherin and α-catenin, were not consistently affected, indicating that effects of Sec3 knockdown were specific to desmosomes (, C and D, and Supplemental Figure S5).
Figure 10. Suppression of Sec3 expression is associated with increases in overall expression levels of desmosomal cadherins and plakoglobin. (A) Two clonal populations of MCF-10A cells stably transduced with shSec3 (hairpin 2) were isolated and characterized. Sec3 (more ...)
To determine whether observed changes in desmosome composition and morphology in Sec3-knockdown cells had functional consequences, we evaluated intercellular adhesion strength by hanging drop assay (Kim et al., 2000
; Huen et al., 2002
; Lorch et al., 2004
). After 20 h in suspension, both control and Sec3-knockdown cells had formed cellular aggregates of similar size and distribution (). However, application of an external force revealed a clear difference in relative adhesive strengths of the two cultures. Although the frequency of large aggregates (>50 cells) was reduced after trituration of either population, the extent to which each culture was affected was not the same. Whereas essentially all of the large cell aggregates were disrupted in Sec3-knockdown cultures, nearly 50% of such clusters were resistant to trituration in the control cultures (). Consistent with the disruption of large aggregates, the number of single cells and doublets in Sec3-knockdown cultures was higher than that of larger aggregates (). Collectively, these findings indicate that a Sec3-containing Exocyst localizes to desmosomes and is required for the assembly of functioning intercellular junctions between epithelial cells.
Figure 11. Sec3 is important for cell–cell adhesion. (A) MCF-10A parental and shSec3 cells before and after trituration. Representative images are shown. (B) Percentages of clusters with indicated numbers of cells were calculated, for control and shSec3 (more ...)