A comprehensive analysis of Rab GTPases reveals a role for Rab34 in serum starvation-induced primary ciliogenesis

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 largely remain 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.

Rab34 GTPase mediates ciliary membrane biogenesis in the intracellular ciliogenesis pathway

“…A key advance in our study comes from the application of volumetric high-resolution FIB-SEM to reconstruct the membrane structures that form (and fail to form) in RAB34 mutant cells, allowing us to unambiguously determine that cells were arrested at the DAV stage ( G). Our studies also extend recent reports that Rab34 can sometimes localize to cilia (Oguchi et al, 2020;Xu et al, 2018) by defining the precise subset and sub-domains of cilia where Rab34 is found ( Figure 5 A-D). Specifically, the In/Out Assay, 3D-SIM, and expansion microscopy showed that Rab34 is present on the ciliary sheath of nascent intracellular cilia.…”

“…However, we found that Rilpl1/2 double-knockout cells neither exhibit ciliogenesis defects nor modulate the Rab34 mutant phenotype (data not shown). Additionally, Rab34 variants that uncouple ciliogenesis from RILP binding have recently been reported (Oguchi et al, 2020). EHD1 is another plausible candidate effector, though we did not detect coimmunoprecipitation of EHD1 with Rab34 (data not shown).…”

“…EHD1) or downstream ciliary membrane growth (Rab8 and ARL13B) are not ( Figure 2). We note that while our work was in progress, Rab34 was suggested to promote early stages of ciliary membrane formation, but the precise step mediated by Rab34 was unclear (Oguchi et al, 2020;Xu et al, 2018). A key advance in our study comes from the application of volumetric high-resolution FIB-SEM to reconstruct the membrane structures that form (and fail to form) in RAB34 mutant cells, allowing us to unambiguously determine that cells were arrested at the DAV stage ( G).…”

“…Recently, we and others conducted genome-wide CRISPR screens that found Rab34 to be required for Hedgehog (Hh) signaling (Breslow et al, 2018;Pusapati et al, 2018), which is strictly dependent on cilia in vertebrates (Bangs and Anderson, 2017). Concordantly, Rab34 mutant mice exhibit ciliopathy features such as polydactyly (Dickinson et al, 2016;Xu et al, 2018), and loss of Rab34 causes ciliary defects (Oguchi et al, 2020;Pusapati et al, 2018;Xu et al, 2018). Despite these recent findings, Rab34 remains one of the least characterized members of the Rab family.…”

Rab34 GTPase mediates ciliary membrane formation in the intracellular ciliogenesis pathway

“…Here, we found that Rab34 is a key mediator of ciliary membrane formation in the intracellular ciliogenesis pathway (Figure 4H). While Rab34 was recently suggested to promote early stages of ciliary membrane formation and to localize to some cilia, 12,13 we now show that Rab34 mediates ciliary vesicle formation and is specifically found on the ciliary sheath of intracellular cilia. Rab34 is also dynamically recruited to the mother centriole during ciliogenesis, and the persistent localization of Rab34-S166N at this site suggests that Rab34 is present on DAVs (Figures 3C-3G).…”