Primary cilia are found on many epithelial cell types, including renal tubular epithelial cells, in which they are felt to participate in flow sensing and have been linked to the pathogenesis of cystic renal disorders such as autosomal dominant polycystic kidney disease. We previously localized the exocyst, an eight-protein complex involved in membrane trafficking, to the primary cilium of Madin-Darby canine kidney cells and showed that it was involved in cystogenesis. Here, using short hairpin RNA (shRNA) to knockdown exocyst expression and stable transfection to induce exocyst overexpression, we show that the exocyst protein Sec10 regulates primary ciliogenesis. Using immunofluorescence, scanning, and transmission electron microscopy, primary cilia containing only basal bodies are seen in the Sec10 knockdown cells, and increased ciliogenesis is seen in Sec10-overexpressing cells. These phenotypes do not seem to be because of gross changes in cell polarity, as apical, basolateral, and tight junction proteins remain properly localized. Sec10 knockdown prevents normal cyst morphogenesis when the cells are grown in a collagen matrix, whereas Sec10 overexpression results in increased cystogenesis. Transfection with human Sec10 resistant to the canine shRNA rescues the phenotype, demonstrating specificity. Finally, Par3 was recently shown to regulate primary cilia biogenesis. Par3 and the exocyst colocalized by immunofluorescence and coimmunoprecipitation, consistent with a role for the exocyst in targeting and docking vesicles carrying proteins necessary for primary ciliogenesis.

The exocyst is an evolutionarily conserved octameric protein complex that tethers post-Golgi secretory vesicles at the plasma membrane for exocytosis. To elucidate the mechanism of vesicle tethering, it is important to understand how the exocyst physically associates with the plasma membrane (PM). In this study, we report that the mammalian exocyst subunit Exo70 associates with the PM through its direct interaction with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ). Furthermore, we have identified key conserved residues at the C-terminus of Exo70 that are crucial for the interaction of Exo70 with PI(4,5)P 2 . Disrupting Exo70-PI(4,5)P 2 interaction abolished the membrane association of Exo70. We have also found that wild-type Exo70 but not the PI(4,5)P 2 -binding-deficient Exo70 mutant is capable of recruiting other exocyst components to the PM. Using the ts045 vesicular stomatitis virus glycoprotein trafficking assay, we demonstrate that Exo70-PI(4,5)P 2 interaction is critical for the docking and fusion of post-Golgi secretory vesicles, but not for their transport to the PM. INTRODUCTIONExocytosis is important for a variety of cellular functions, ranging from the release of hormones to the incorporation of membrane proteins for cell growth and morphogenesis. The late stage of exocytosis is a multistep process that includes directional transport, tethering, docking, and fusion of postGolgi secretory vesicles with the plasma membrane (PM). The tethering step, defined as the initial contact of secretory vesicles with the PM before SNARE-mediated docking and fusion (Pfeffer, 1999;Guo et al., 2000;Waters and Hughson, 2000;Whyte and Munro, 2002), is mediated by the exocyst, an evolutionarily conserved octameric complex composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 (for review, see Guo et al., 2000;Hsu et al., 2004;Munson and Novick, 2006;Wang and Hsu, 2006). In budding yeast, the exocyst components localize to the growing end of the daughter cell ("bud"), where active exocytosis and membrane addition take place (TerBush and Novick, 1995;Finger et al., 1998, Guo et al., 1999. This localization pattern contrasts that of the membrane fusion machine, the t-SNAREs, which are evenly distributed along both the mother and daughter cell membrane (Brennwald et al., 1994). In mammalian cells, the exocyst components were found in the cytosol, recycling endosomes and trans-Golgi network Fö lsch et al., 2003;Ang et al., 2004;Langevin et al., 2005). However, they are recruited to the PM during a number of cellular processes. For example, in epithelial cells, the exocyst is recruited to the adherens junction region upon cell-cell contact, where it mediates protein and membrane addition at the basolateral domain (Grindstaff et al., 1998;Yeaman et al., 2001); in developing neurons, the exocyst is localized to the growing neurites, where it mediates membrane expansion (Hazuka et al., 1999;Vega and Hsu, 2001); during cell migration, the exocyst is recruited to the leading edges of the PM (Rosse et al., 2006;...

The exocyst is a multiprotein complex essential for tethering secretory vesicles to specific domains of the plasma membrane for exocytosis. Here, we report that the exocyst component Exo70 interacts with the Arp2/3 complex, a key regulator of actin polymerization. We further show that the exocyst-Arp2/3 interaction is regulated by epidermal growth factor (EGF) signalling. Inhibition of Exo70 by RNA interference (RNAi) or antibody microinjection blocks the formation of actin-based membrane protrusions and affects various aspects of cell motility. We propose that Exo70, in addition to functioning in exocytosis, also regulates actin at the leading edges of migrating cells, therefore coordinating cytoskeleton and membrane traffic during cell migration.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by formation of renal cysts that destroy the kidney. Mutations in PKD1 and PKD2, encoding polycystins-1 and -2, cause ADPKD. Polycystins are thought to function in primary cilia, but it is not well understood how these and other proteins are targeted to cilia. Here, we provide the first genetic and biochemical link between polycystins and the exocyst, a highly-conserved eight-protein membrane trafficking complex. We show that knockdown of exocyst component Sec10 yields cellular phenotypes associated with ADPKD, including loss of flow-generated calcium increases, hyperproliferation, and abnormal activation of MAPK. Sec10 knockdown in zebrafish phenocopies many aspects of polycystin-2 knockdown—including curly tail up, left-right patterning defects, glomerular expansion, and MAPK activation—suggesting that the exocyst is required for pkd2 function in vivo. We observe a synergistic genetic interaction between zebrafish sec10 and pkd2 for many of these cilia-related phenotypes. Importantly, we demonstrate a biochemical interaction between Sec10 and the ciliary proteins polycystin-2, IFT88, and IFT20 and co-localization of the exocyst and polycystin-2 at the primary cilium. Our work supports a model in which the exocyst is required for the ciliary localization of polycystin-2, thus allowing for polycystin-2 function in cellular processes.

Arl13b belongs to the ADP-ribosylation factor family within the Ras superfamily of regulatory GTPases. Mutations in Arl13b cause Joubert syndrome, which is characterized by congenital cerebellar ataxia, hypotonia, oculomotor apraxia, and mental retardation. Arl13b is highly enriched in cilia and is required for ciliogenesis in multiple organs. Nevertheless, the precise role of Arl13b remains elusive. Here we report that the exocyst subunits Sec8, Exo70, and Sec5 bind preferentially to the GTP-bound form of Arl13b, consistent with the exocyst being an effector of Arl13b. Moreover, we show that Arl13b binds directly to Sec8 and Sec5. In zebrafish, depletion of arl13b or the exocyst subunit sec10 causes phenotypes characteristic of defective cilia, such as curly tail up, edema, and abnormal pronephric kidney development. We explored this further and found a synergistic genetic interaction between arl13b and sec10 morphants in cilia-dependent phenotypes. Through conditional deletion of Arl13b or Sec10 in mice, we found kidney cysts and decreased ciliogenesis in cells surrounding the cysts. Moreover, we observed a decrease in Arl13b expression in the kidneys from Sec10 conditional knockout mice. Taken together, our results indicate that Arl13b and the exocyst function together in the same pathway leading to functional cilia.

Primary cilia are found on many epithelial cell types, including renal tubular epithelial cells, where they participate in flow sensing. Disruption of cilia function has been linked to the pathogenesis of polycystic kidney disease. We demonstrated previously that the exocyst, a highly conserved eight-protein membrane trafficking complex, localizes to primary cilia of renal tubular epithelial cells, is required for ciliogenesis, biochemically and genetically interacts with polycystin-2 (the protein product of the polycystic kidney disease 2 gene), and, when disrupted, results in MAPK pathway activation both in vitro and in vivo. The small GTPase Cdc42 is a candidate for regulation of the exocyst at the primary cilium. Here, we demonstrate that Cdc42 biochemically interacts with Sec10, a crucial component of the exocyst complex, and that Cdc42 colocalizes with Sec10 at the primary cilium. Expression of dominant negative Cdc42 and shRNA-mediated knockdown of both Cdc42 and Tuba, a Cdc42 guanine nucleotide exchange factor, inhibit ciliogenesis in Madin-Darby canine kidney cells. Furthermore, exocyst Sec8 and polycystin-2 no longer localize to primary cilia or the ciliary region following Cdc42 and Tuba knockdown. We also show that Sec10 directly interacts with Par6, a member of the Par complex that itself directly interacts with Cdc42. Finally, we show that Cdc42 knockdown results in activation of the MAPK pathway, something observed in cells with dysfunctional primary cilia. These data support a model in which Cdc42 localizes the exocyst to the primary cilium, whereupon the exocyst then targets and docks vesicles carrying proteins necessary for ciliogenesis.

Objective:To estimate birth population-based perinatal-neonatal mortality and preterm rate in China from a regional survey in 2010.Study design:Data of total births in 2010 obtained from 151 level I–III hospitals in Huai’an, Jiangsu, were prospectively collected and analyzed.Results:From 61 227 birth registries (including 60 986 live births and 241 stillbirths), we derive a birth rate of 11.3‰ (of 5.4 million regional population), a male-to-female ratio of 116:100 and valid data from 60 615 newborns. Mean birth weight (BW) was 3441 ± 491 g with 13.6% macrosomia. Low BW was 2.8% (1691/60 372) with 8.83% mortality. Preterm rate was 3.72% (2239/60 264) with 7.61% mortality. Cesarean section rate was 52.9% (31 964/60 445), multiple pregnancy 1.8% (1088/60 567) and birth defects 6.7‰ (411/61 227). There were 97.4% healthy newborns and 2.2% (1298) requiring hospitalized after birth. The perinatal mortality was 7.7‰ (471/61 227, including 241 stillbirths, 230 early neonatal deaths). The neonatal mortality was 4.4‰ (269/60 986). The main causes of neonatal death were birth asphyxia (24.5%), respiratory diseases (21.5%), prematurity related organ dysfunction (18.5%) and congenital anomalies (7.7%), whereas incidence of congenital heart disease and respiratory distress syndrome was 8.6‰ and 6.1‰, respectively.Conclusions:This regional birth population-based data file contains low perinatal-neonatal mortality rates, associated with low proportion of LBW and preterm births, and incidences of major neonatal disease, by which we estimate, in a nationwide perspective, in 16 million annual births, preterm births should be around 800 000, perinatal and neonatal mortality may be 128 000–144 000 and 80 000–96 000, respectively, along with 100 000 respiratory distress syndrome.

Most cases of congenital obstructive nephropathy are the result of ureteropelvic junction obstructions, and despite their high prevalence, we have a poor understanding of their etiology and scarcity of genetic models. The eight-protein exocyst complex regulates polarized exocytosis of intracellular vesicles in a large variety of cell types. Here we report generation of a conditional knockout mouse for Sec10, a central component of the exocyst, which is the first conditional allele for any exocyst gene. Inactivation of Sec10 in ureteric bud-derived cells using Ksp1.3-Cre mice resulted in severe bilateral hydronephrosis and complete anuria in newborns, with death occurring 6–14 hours after birth. Sec10FL/FL;Ksp-Cre embryos developed ureteropelvic junction obstructions between E17.5 and E18.5 as a result of degeneration of the urothelium and subsequent overgrowth by surrounding mesenchymal cells. The urothelial cell layer that lines the urinary tract must maintain a hydrophobic luminal barrier again urine while remaining highly stretchable. This barrier is largely established by production of uroplakin proteins that are transported to the apical surface to establish large plaques. By E16.5, Sec10FL/FL;Ksp-Cre ureter and pelvic urothelium showed decreased uroplakin-3 protein at the luminal surface, and complete absence of uroplakin-3 by E17.5. Affected urothelium at the UPJ showed irregular barriers that exposed the smooth muscle layer to urine, suggesting this may trigger the surrounding mesenchymal cells to overgrow the lumen. Findings from this novel mouse model show Sec10 is critical for the development of the urothelium in ureters, and provides experimental evidence that failure of this urothelial barrier may contribute to human congenital urinary tract obstructions.