Sec15B regulates ciliogenesis in polarized epithelial cells

GALLO LI; APODACA G

Pittsburgh

Simposio; AHA Fellows Research Day; 2012

American Heart Association

Primary cilia, which project from the surfaces of most cells, consist of a microtubule-based axoneme that assembles on a basal body. Although immotile, the primary cilia act as sensory transducers that coordinate a diversity of physiological and developmental processes. Importantly, defects in their assembly or function are tightly coupled to developmental defects and diseases, including polycystic kidney disease, skeletal abnormalities and defects in early heart development. Cilia biogenesis is dependent on vesicular trafficking. The exocyst is a tethering complex comprised of eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15A/B, Exo70 and Exo84) that is involved in a variety of cellular processes, many of which are coupled to membrane trafficking. Two variants of Sec15 are found in mammals but little is known about Sec15B compared to the well-studied Sec15A isoform. Exocyst subunits, like Sec10 and Sec8, have been described to localize to the primary cilium of epithelial cells and their depletion impairs ciliogenesis. However, it is unknown the mechanism by which the exocyst complex regulates this process. My hypothesis is that Sec15 isoforms form distinct exocyst complexes and thus differently regulate vesicular trafficking pathways to the primary cilium. To study this hypothesis, I used MDCK cells, a well-established polarized epithelial cell model. By immunofluorescence analysis, I found that the two existing Sec15 isoforms show different localization with respect to the centrosome and the cilia. Using shRNA stable cell lines, I observed that when Sec15B is depleted, the cilia is impaired, but slightly affected with Sec15A depletion. Furthermore, emerging evidence from other groups shows that Rab11a and Rab8a regulate vesicular traffic of ciliary proteins and I find that Sec15B knock down affects Rab8 ciliary localization. In conclusion, Sec15B regulates the vesicular trafficking of ciliary proteins, while Sec15A is likely to play a role in the subsequent trafficking along the axoneme.