Rab34 GTPase mediates ciliary membrane biogenesis in the intracellular ciliogenesis pathway

Abstract

Primary cilia form by two pathways: an extracellular pathway in which the cilium grows out from the cell surface and an intracellular pathway in which the nascent cilium forms inside the cell. Here we identify the GTPase Rab34 as a selective mediator of intracellular ciliogenesis. We find that Rab34 is required for formation of the ciliary vesicle at the mother centriole and that Rab34 marks the ciliary sheath, a unique sub-domain of assembling intracellular cilia. Rab34 activity is modulated by divergent residues within its GTPase domain, and ciliogenesis requires GTP binding and turnover by Rab34. Because Rab34 is found on assembly intermediates that are unique to intracellular ciliogenesis, we tested its role in the extracellular pathway used by MDCK cells. Consistent with Rab34 acting specifically in the intracellular pathway, MDCK cells ciliate independently of Rab34 and paralog Rab36. Together, these findings reveal a new context-specific molecular requirement for ciliary membrane biogenesis.

Rab34 is necessary for early stages of intracellular ciliogenesis

“…Independent analysis of myosin5a and Rab8 indicates that the effects of Rab34 on their local concentration might vary from cell type to cell type, but defects in ciliary vesicle formation are universally observed. 6,17 IFT-B proteins are found at centrosomes, regardless of whether ciliary assembly is occurring or not. 18,19 This localization appears to be independent of Rab34, as we detected no changes to the distribution of the IFT-A component Ift140 and the IFT-B component Ift27 when Rab34 is missing (Figure 4C).…”

“…Report ciliogenesis in hTERT-RPE1, NIH 3T3, and MCF10 cells, but not in MDCK cells. 6,11,17 Ciliary assembly in MCF10 cells has not been explored, but hTERT-RPE1 and NIH 3T3 cells are thought to use the intracellular pathway although MDCK cells are thought to use the extracellular pathway. The relatively subtle phenotype of the Rab34 KO mouse compared to animals completely lacking cilia is consistent with cilia defects being found in only a subset of cells in the animal.…”

A disease-associated PPP2R3C-MAP3K1 phospho-regulatory module controls centrosome function

“…Silent mutations for sgRNA resistance and/or to encode the L193S mutation were introduced by gene synthesis and Gibson assembly. Stable expression of PPP2R3C-GFP was achieved by replacement of Rab34 with PPP2R3C-GFP in pCW-Pgk-Rab34_hPgk-miRFP-670-Centrin2 70 (used in HeLa cells) and by replacement of Rab8 with PPP2R3C-GFP in pCW-Pgk-mScarlet-Rab8_hPgk-NeoR 71 (used in RPE1 cells). For tetracycline-inducible expression of Cas9-BFP, pCW-Tet-Cas9-BFP was prepared by Gibson assembly from pCW-Cas9 (Addgene #50661).…”

“…For CRISPR KO in RPE1 cells, sgRNAs were lentivirally transduced into RPE1 cells stable expressing Cas9-Blast. 71 Following puromycin selection for sgRNA-expressing cells, clonal cell lines were isolated by FACS sorting on a FACS Aria III instrument (BD Scientific). HeLa MAP3K1 KO cells were prepared by lentiviral transduction with pKHH030-sgMAP3K1-A, selected with puromycin, treated with doxycycline to induce Cas9, and FACS sorted to isolate clonal cell lines.…”