Exocyst is involved in cystogenesis and tubulogenesis and acts by modulating synthesis and delivery of basolateral plasma membrane and secretory proteins - PubMed (original) (raw)

Exocyst is involved in cystogenesis and tubulogenesis and acts by modulating synthesis and delivery of basolateral plasma membrane and secretory proteins

J H Lipschutz et al. Mol Biol Cell. 2000 Dec.

Free PMC article

Abstract

Epithelial cyst and tubule formation are critical processes that involve transient, highly choreographed changes in cell polarity. Factors controlling these changes in polarity are largely unknown. One candidate factor is the highly conserved eight-member protein complex called the exocyst. We show that during tubulogenesis in an in vitro model system the exocyst relocalized along growing tubules consistent with changes in cell polarity. In yeast, the exocyst subunit Sec10p is a crucial component linking polarized exocytic vesicles with the rest of the exocyst complex and, ultimately, the plasma membrane. When the exocyst subunit human Sec10 was exogenously expressed in epithelial Madin-Darby canine kidney cells, there was a selective increase in the synthesis and delivery of apical and basolateral secretory proteins and a basolateral plasma membrane protein, but not an apical plasma membrane protein. Overexpression of human Sec10 resulted in more efficient and rapid cyst formation and increased tubule formation upon stimulation with hepatocyte growth factor. We conclude that the exocyst plays a central role in the development of epithelial cysts and tubules.

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Figures

Figure 1

Figure 1

Sec8p partially localizes at the tight junction in cysts. This figure is a confocal section through the middle of a mature, fully polarized cyst formed by MDCK cells grown for 10 d in a collagen matrix. Antibody against Sec8p (a) and antibody against the tight junction protein ZO-1 (b) show colocalization as demonstrated by the yellow color in the merged panel (c). Sec 8, green; ZO-1, red. Bar, 10 μm.

Figure 2

Figure 2

Sec8p relocalizes during tubulogenesis. (a and b) Fluid-filled cyst formed by MDCK cells grown for 10 d in a collagen gel. Staining is seen at the area of the tight junction (arrowhead) by using anti-Sec8 antibody (a). Sec8p colocalized with the tight junction protein ZO-1 (Figure 1). Concurrent staining of actin with phalloidin was performed (b). (c) Merge of a and b. (d–l) Fluid-filled cysts formed by MDCK cells grown for 10 d in collagen and stimulated for 24 h with conditioned medium containing HGF. The exocyst can be seen relocalizing along the growing tubules in a pattern consistent with the changes in polarity that occur as tubules form. (d–f) Extension stage of tubulogenesis. Sec8p is seen relocalizing into the extension (d), in association with actin staining (e). (g–i) Cord stage of tubulogenesis. Arrow in g indicates staining at the region of cell–cell contact in the cord. This region may become the boundary of a new lumen, as shown by the intense actin staining in this region (h). (j–l) Nascent tubule in the final stage of tubulogenesis. Arrow in j shows two vertical lines of Sec8p staining outlining the boundary of the lumen. Intense actin staining in a broad band in k underlies the apical surface surrounding this lumen. (f, i, and l) Merge of d and e, g and h, and j and k shows that the relocalizing exocyst closely surrounds, but does not precisely overlap the actin in the projections of the nascent tubules as indicated by lack of yellow in the merged panels. Sec 8, green; actin, red. Bar, 10 μm.

Figure 3

Figure 3

Sec10p partially localizes at the tight junction. Antibody against Sec10p and antibody against the tight junction marker ZO-1 demonstrated colocalization (yellow in merged panels) at the tight junction in MDCK cell monolayers grown for 7 d on filters. Ap, apical; TJ, tight junction; Bl, basolateral. Sec 10, green; ZO-1, red. Bar, 10 μm.

Figure 4

Figure 4

hSec10-myc partially localizes at the tight junction and the levels of Sec6p and Sec8p are unchanged in hSec10 overexpressing cells. (a) Western blot by using antibody against the myc epitope tag demonstrated three hSec10 expressing clones. C, control. (b) Immunofluorescent staining by using antibody against the myc epitope tag in hSec10 overexpressing cells shows transfected hSec10p localized to the plasma membrane at the level of the tight junction (the level of the tight junction was determined by costaining with ZO-1; Lipschutz and Mostov, unpublished observations). No myc staining is evident in the control cells. (c) Western blot demonstrates equal amounts of Sec6p and Sec8p protein per microgram of total protein in Sec10 overexpressing and control cells. Ap, apical; TJ, tight junction; Bl, basolateral. Bar, 10 μm.

Figure 5

Figure 5

Partial association of Sec8p and hSec10p. (a) Glycerol gradient from homogenized cells overexpressing hSec10, with Western blot performed on each fraction by using antibody against Sec8p and the myc epitope tag of hSec10p. Size markers (arrows) are bovine serum albumin (4.3S), β-amylase (11.2S), and thyroglobulin (19.2S). (b) Four sequential immunoprecipitations by using antibody against Sec8p followed by an immunoprecipitation with antibody against the myc epitope tag on the transfected hSec10p demonstrated that 65% of the transfected hSec10p is not in a complex with endogenous Sec8p. Blotting was performed by using antibody against the myc epitope tag (blotting was also done with antibody against Sec8p to confirm depletion). Note that there is a doublet of ∼116 and 120 kDa in the fifth immunoprecipitation. The upper band (120 kDa) of the doublet is a nonspecific contaminant seen in immunoprecipitations with antibody to myc (Lipschutz and Mostov, unpublished observations). The lower band (116 kDa) is a small amount of Sec8p that has escaped the first four rounds of immunoprecipitation, but is assumed to be associated with hSec10p as it was coimmunoprecipitated with hSec10p in the fifth round. (c) Four sequential immunoprecipitations by using antibody against the myc epitope tag followed by an immunoprecipitation with anti-Sec8 antibody demonstrated that 35% of the endogenous Sec8p was not complexed with the transfected hSec10p. Blotting was performed by using antibody against Sec8p (blotting was also done with antibody against myc to confirm depletion).

Figure 6

Figure 6

Morphological changes in hSec10 overexpressing cells, as shown by EM. (a and b) hSec10 overexpressing cells were vertically elongated (a) compared with control cells (b). (c and d) Increased numbers of vesicles were seen at the apical plasma membrane in hSec10 overexpressing cells (c) compared with controls (d). Increased numbers of vesicles were also seen basolaterally (Lipschutz and Mostov, unpublished observations). Arrows point to examples of 50–100-nm vesicles found within 500 nm of the plasma membrane. a and b and c and d were taken at equal magnifications. Bar, 1 μm.

Figure 7

Figure 7

Synthesis and delivery of secretory proteins in hSec10 overexpressing cells. (a) Western blot by using antibody against gp80 demonstrates increased steady-state levels of gp80 in three hSec10 transfected clones (1, 2, 3) compared with control cells (C). (b–g) Synthesis and secretion assays were performed by pulse and pulse-chase with [35S]methionine. (b) hSec10 overexpressing and control cells were metabolically pulse labeled for 20 min with [35S]methionine and the amount of newly synthesized gp80 was measured. (c) Other secretory proteins were also increased in hSec10 overexpressing versus control cells. Cells were pulse labeled for 20 min with [35S]methionine, and chased for 1 h. Then the apical (A) and basolateral (B) media were collected and aliquots run on SDS-PAGE and analyzed by phosphorimaging. The entire gel is shown and the position of gp80 indicated by the arrow. We show this experiment in triplicate to demonstrate that even though there is some variability, there is a general consistency with respect to the increase in apical and basolateral protein secretion across a broad spectrum of secretory proteins. (d and f) hSec10 overexpressing and control cells were metabolically pulse labeled for 20 min. The apical and basolateral media were collected for the indicated time intervals during the subsequent chase. Aliquots were analyzed by SDS-10% PAGE and radioactivity associated with gp80 was determined by using a phosphorimager. Total secretion of gp80 was increased both apically (d) and basolaterally (f). (e and g) Data in d and f were replotted in e and g, respectively, to emphasize the kinetics of secretion, i.e., the fraction of the total gp80 secreted as a function of time. Data for each condition were normalized, so that the total gp80 secreted into the apical (e) or basolateral (g) media by the end of the 120-min time course was taken as 100%. The cumulative secretion at each time point was then plotted. These results indicate that although the amounts of gp80 secreted apically and basolaterally were increased by hSec10 transfection, the kinetics of secretion, measured as cumulative percent of total over time, was unchanged.

Figure 8

Figure 8

Synthesis and delivery of plasma membrane proteins in hSec10 overexpressing cells. In contrast to gp135, E-Cadherin synthesis and delivery were increased in hSec10 overexpressing versus control cells. The p values were the result of at least three experiments and were determined by the Mann-Whitney nonparametric test. Units are arbitrary phosphorimager units. NS, nonsignificant. Sec10 (▪), control (□).

Figure 9

Figure 9

Effect of hSec10 overexpression on cystogenesis. (a and b) After 7 d of growth in a collagen matrix, cysts composed of hSec10 transfected cells (a) were mature, while cysts composed of control cells (b) were incompletely formed (confocal microscopy, cysts were stained for actin with phalloidin). (c) Quantitation demonstrated the increased rate and efficiency of mature cyst formation, as noted in a and b, in hSec10 overexpressing compared with control cell cysts. a and b were taken at equal magnification. Bar, 30 μm.

Figure 10

Figure 10

Effect of hSec10 overexpression on tubulogenesis. (a and b, insets) Nomarski imaging of mature hSec10 overexpressing (a, inset) and control (b, inset) cell cysts grown for 10 d in collagen again showed that the cysts composed of hSec10 overexpressing cells were larger than cysts composed of control cells. (a and b) HGF-induced stimulation, for 48 h, of mature cysts revealed greater numbers of tubules per cyst in the cysts composed of hSec10 overexpressing (a) versus control cells (b). (c) Quantitation of the number of tubules formed per cyst, as measured at the level of greatest cyst diameter, revealed increased tubulogenesis in all the clones of hSec10 overexpressing versus control cell cysts. Similar effects were seen as early as 6 and 12 h after addition of HGF (Lipschutz and Mostov, unpublished observations), indicating that the increase in tubule number was not simply a result of enhanced tubular lumen formation, which is a later event (Pollack et al., 1998). a and b were taken at equal magnification. Bar, 30 μm.

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