Sec10 and the Exocyst are Necessary for Renal Epithelial Monolayer Homeostasis and Ciliogenesis In Vitro and In Vivo

Abstract

The eight‐protein exocyst complex regulates targeted delivery of exocytic vesicles. A previous report demonstrated that shRNA‐knockdown of Sec10 (Sec10‐KD), a central subunit of the exocyst, in Madin‐Darby canine kidney (MDCK) cells disrupted primary cilia assembly and 3D cyst formation. To further study the mechanisms by which Sec10 and the exocyst regulate epithelial polarity, morphogenesis, and homeostasis, we used three dimensional collagen cultures of MDCK cells. Sec10‐KD cysts initially showed undisturbed lumen formation, even though subsequently displayed significantly fewer and shorter primary cilia than controls. Later in cystogenesis, while Sec10‐KD cysts showed numerous apoptotic cells extruded basally into the collagen matrix, control cells maintained normal homeostasis. Sec10‐KD MDCK cells were also more sensitive to apoptotic triggers than controls. Apico‐basal polarity appeared normal in Sec10‐KD cysts, while a planar cell polarity defect was suggested by the differences in mitotic spindle angles between knockdowns and controls. Furthermore, analysis of renal tubules in our recently generated kidney‐specific Sec10 knockout mouse model revealed significant defects in primary cilia assembly and in the targeted renal tubules, abnormal epithelial cell extrusion was also observed, supporting our in vitro findings. We hypothesize that in Sec10‐KD cells, the disrupted exocyst activity leads to an increased apoptotic sensitivity through disrupted primary cilia signaling, and in combination with an increased basal cell extrusion rate, it affects epithelial barrier integrity and homeostasis.Supported by: the NIH, March of Dimes, UH RCMI‐BRIDGES and HRFDCC at University of Alabama.