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Analysis of Rab small GTPases that regulate primary ciliogenesis

Oguchi Mai 東北大学

2021.03.25

概要

Primary cilia are sensors of chemical and mechanical signals in the extracellular environment. The formation of primary cilia (i.e., ciliogenesis) requires dynamic membrane trafficking events, and several Rab small GTPases, key regulators of membrane trafficking, have recently been reported to participate in ciliogenesis. However, the precise mechanisms of Rab-mediated membrane trafficking during ciliogenesis largely remain unknown. In this thesis, I used a collection of siRNAs against 62 human Rabs to perform a comprehensive knockdown screening for Rabs that regulate serum-starvation-induced ciliogenesis in human telomerase reverse transcriptase retinal pigment epithelium 1 (hTERT-RPE1) cells and succeeded in identifying Rab34 as an essential Rab. Knockout (KO) of Rab34, but not of Rabs previously reported to regulate ciliogenesis (e.g., Rab8 and Rab10), in hTERT-RPE1 cells drastically impaired serum-starvation-induced ciliogenesis. Rab34 was also required for serum-starvation-induced ciliogenesis in NIH/3T3 cells and MCF10A cells, but not for ciliogenesis in Madin-Darby canine kidney (MDCK)-II cysts. I then attempted to identify a specific region(s) of Rab34 that is essential for serum-starvation-induced ciliogenesis in hTERT-RPE1 cells by performing deletion and mutation analyses of Rab34. Instead of a specific sequence in the switch II region, which is generally important for recognizing effector proteins (e.g., Rab interacting lysosomal protein [RILP]), a unique long N-terminal region (amino acids 1–49) of Rab34 before the conserved GTPase domain was found to be essential. Moreover, I performed an in-depth deletion analysis of the N-terminal region of Rab34 together with Ala-based site-directed mutagenesis to identify the essential amino acids required for ciliogenesis. The results showed that a Rab34 mutant lacking an N-terminal 18 amino acids and a Rab34 mutant carrying an LPQ-to-AAA mutation (amino acids 16–18) failed to rescue a Rab34-KO phenotype. I also found that Rab36, the closest paralogue of Rab34, which lacks an LPQ sequence in its N-terminal region failed to restore ciliogenesis. These findings suggest that Rab34 is an atypical Rab that regulates serum-starvation-induced ciliogenesis through the Leu-Pro-Gln sequence of Rab34, which is highly conserved in vertebrates.

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