The Exocyst Protein Sec10 Is Necessary for Primary Ciliogenesis and Cystogenesis In Vitro

Abstract

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 Small GTPase Cdc42 Is Necessary for Primary Ciliogenesis in Renal Tubular Epithelial Cells

“…We showed previously that the exocyst localized to the primary cilium in MDCK cells (5). In the Cdc42 and Tuba knockdown MDCK cells, the exocyst (detected by monitoring Sec8, which serves as a marker for the holo complex (31)) no longer localized at the primary cilium or ciliary region (p Ͻ 0.001, supplemental Fig.…”

“…These were used for infection of MDCK cells. The p199 vector encodes GFP, which allowed us to identify and separate the infected cells using fluorescence-activated cell sorting (FACS Vantage S.E., BD Biosciences) as we described previously (5).…”

“…We previously showed that the exocyst complex localizes to the primary cilium (4), is essential for ciliogenesis (5), and genetically and biochemically interacts with polycystin-2 (6). The exocyst is a highly conserved 750-kDa complex comprised of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 that was originally identified as regulating polarized exocytosis in Saccharomyces cerevisiae (7).…”

“…Mammalian homologs of all eight exocyst proteins have been identified (8). Sec10 is a central component of the eight-protein exocyst complex, and knockdown of Sec10 but not Sec8 or Exo70 prevented ciliogenesis, whereas overexpression of Sec10 increased ciliogenesis in Madin-Darby canine kidney (MDCK) cells (5). Sec10 overexpression also resulted in an increase in cyst and tubule morphogenesis when MDCK cells were grown in a collagen matrix to the cyst stage and induced to tubulate with hepatocyte growth factor (9).…”

Regulation of epithelial cell polarity by PAR-3 depends on Girdin transcription and Girdin–Gαi3 signaling

“…PAR-3 interacts with the aPKC-PAR-6 complex to form the tripartite PAR-3-aPKC-PAR-6 complex (Izumi et al, 1998;Joberty et al, 2000;Lin et al, 2000;Suzuki et al, 2001), which is required for aPKC recruitment to cell-cell contacts and for apical domain development (Horikoshi et al, 2009). In addition, by binding to PAR-3, the LIMK2 and Tiam1 actin cytoskeleton regulatory proteins and the Sec 8 exocyst protein (also known as EXOC4) have been implicated in epithelial cell polarization (Bryant et al, 2010;Macara, 2005, 2006;Zuo et al, 2009). Moreover, the Lgl tumor suppressor protein (for which there are two isoforms in mammals, LLGL1 and LLGL2) negatively regulates the tripartite PAR-3-aPKC-PAR-6 complex by competing with PAR-3 for binding to the aPKC-PAR-6 complex (Chalmers et al, 2005;Hutterer et al, 2004;Yamanaka et al, 2006Yamanaka et al, , 2003.…”