Abstract
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 remain largely unknown. In the present study, we 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. We then attempted to identify a specific region(s) of Rab34 that is essential for ciliogenesis by performing deletion and mutation analyses of Rab34. Unexpectedly, 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 of Rab34 before the conserved GTPase domain was found to be essential. These findings suggest that Rab34 is an atypical Rab that regulates serum starvation-induced ciliogenesis through its unique N-terminal region.
Highlights
Primary cilia are membrane projections from the cell surface and are thought to function as sensors of chemical and mechanical signals in the extracellular environment [1]
Rab34 is required for primary ciliogenesis siRNAs against 62 human Rabs that we have developed previously [20], we performed a comprehensive knockdown screening in hTERT-RPE1 cells
We performed a comprehensive knockdown screening for human Rabs that regulate ciliogenesis (Fig. 1), and, based on the results together with the results of KO analyses, we succeeded in identifying Rab34 as an essential Rab in serum starvation-induced ciliogenesis in hTERT-RPE1 cells (Fig. 1–3)
Summary
Comprehensive screening for Rabs whose knockdown inhibits ciliogenesis in hTERT-RPE1 cells. The lack of a rescue effect by Rab (DN) is unlikely to be attributable to its protein expression level, because similar amounts of Rab34(DN) and endogenous Rab were observed in hTERT-RPE1 cells (Fig. 6F). We knocked down endogenous FLCN in hTERT-RPE1 cells, and the results showed that FLCN-KD had no effect on ciliogenesis, even though the FLCN immunoreactive bands almost completely disappeared after treatment with two independent siRNAs (Fig. 7B). These findings taken together indicate that the known Rab34-interacting proteins, including RILP and FLCN, are not required for Rab34-regulated serum starvation-induced ciliogenesis in hTERT-RPE1 cells
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