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Ciliogenesis is a highly ordered and complex process involving membrane trafficking, fusion, and reorganization. Myosin-Va (Myo-Va) had been demonstrated that it could facilitate the transport of preciliary vesicles (PCVs) to the distal appendages of the mother centriole, thereby initiating ciliogenesis. However, the mechanisms underlying ciliary membrane assembly during ciliogenesis remain poorly understood. The exocyst complex is known to play a critical role in vesicle trafficking across various cellular processes. In this study, I investigated the role of EXOC6A, a component of the exocyst complex, in ciliogenesis. Utilizing advanced microscopy techniques and biochemical analysis, I found that EXOC6A interacts and colocalizes with Myo-Va during multiple stages of ciliogenesis, including preciliary vesicle (PCV), ciliary vesicle (CV), and ciliary sheath membrane, spanning early to later stages of the process. Deletion of EXOC6A resulted in defects in the assembly of the intact transition zone complex and impaired the recruitment of several ciliary proteins, including TCTN2, AHI1, MKS3, MKS1, NPHP8, GPR161, and BBS9. Additionally, deletion of EXOC6A also exhibited alterations in INPP5E localization and phospholipid composition in ciliary membrane. Notably, EXOC6A deletion significantly inhibited cilia assembly and disrupted ciliary membrane homeostasis. In addition, our data revealed that Myo-Va transports EXOC6A vesicles to the mother centriole in the early stages of ciliogenesis via a dynein-, microtubule-, and Arp2/3-dependent mechanism. Furthermore, EXOC6A vesicles were continuously recruited and fused into the CV at early stages or the ciliary sheath membrane at later stages of ciliogenesis. Interestingly, we observed that the Rab8a-Rab11-Rabin8 cascade operates in parallel with the EXOC6A-mediated pathway during ciliogenesis. Depletion of either EXOC6A or the components of Rab8a-Rab11-Rabin8 cascade individually did not affect the other pathway, but depletion of both exacerbated cilia defects. Furthermore, inhibition of Rab11 recruitment to the centrosomal region using pitstop2 mimicked the effects of double knockout (EXOC6A-/Rab11-) cells, suggesting that multiple vesicle sources contribute to ciliogenesis. Collectively, these findings suggest the involvement of both Golgi-derived and recycling endosome-derived vesicles. Collectively, the interaction of EXOC6A with Myo-Va on Golgi-derived vesicles plays a crucial role in ciliary membrane assembly during the early stages of ciliogenesis.

Taiwan International Graduate Program in Molecular Medicine, National Yang-Ming University and Academia Sinica Doctoral Dissertation