The Small GTPase Cdc42 Is Necessary for Primary Ciliogenesis in Renal Tubular Epithelial Cells

Abstract

Primary cilia are found on many epithelial cell types, including renal tubular epithelial cells, where they participate in flow sensing. Disruption of cilia function has been linked to the pathogenesis of polycystic kidney disease. We demonstrated previously that the exocyst, a highly conserved eight-protein membrane trafficking complex, localizes to primary cilia of renal tubular epithelial cells, is required for ciliogenesis, biochemically and genetically interacts with polycystin-2 (the protein product of the polycystic kidney disease 2 gene), and, when disrupted, results in MAPK pathway activation both in vitro and in vivo. The small GTPase Cdc42 is a candidate for regulation of the exocyst at the primary cilium. Here, we demonstrate that Cdc42 biochemically interacts with Sec10, a crucial component of the exocyst complex, and that Cdc42 colocalizes with Sec10 at the primary cilium. Expression of dominant negative Cdc42 and shRNA-mediated knockdown of both Cdc42 and Tuba, a Cdc42 guanine nucleotide exchange factor, inhibit ciliogenesis in Madin-Darby canine kidney cells. Furthermore, exocyst Sec8 and polycystin-2 no longer localize to primary cilia or the ciliary region following Cdc42 and Tuba knockdown. We also show that Sec10 directly interacts with Par6, a member of the Par complex that itself directly interacts with Cdc42. Finally, we show that Cdc42 knockdown results in activation of the MAPK pathway, something observed in cells with dysfunctional primary cilia. These data support a model in which Cdc42 localizes the exocyst to the primary cilium, whereupon the exocyst then targets and docks vesicles carrying proteins necessary for ciliogenesis.

Cdc42 Deficiency Causes Ciliary Abnormalities and Cystic Kidneys

“…27,28 Knockdown of both Sec10 29 and Cdc42 increased mitogen-activated protein kinase (MAPK) activation. 18 Here, using two different living organisms, we confirm and extend our in vitro findings. We show that cdc42 knockdown in zebrafish phenocopies many aspects of sec10 and pkd2 knockdown-including curved tail, glomerular expansion, and MAPK activation-suggesting, in conjunction with our previous data, 12,18,29 that cdc42 may be required for sec10 (and possibly pkd2) function in vivo.…”

“…This difference was highly significant at P,0.0001. sec10 and cdc42 Genetically Interact for Cilia-Related Phenotypes Our in vitro 12,18 and in vivo 29 analyses together support a link between exocyst sec10 and cdc42. The curved tail, hydrocephalus, small eyes, edema, and wt1a expansion phenotypes shared between sec10MO and cdc42MO embryos have also been observed upon knockdown of other ciliary proteins.…”

“…Exocyst Sec8 and polycystin-2 also no longer localized to the primary cilium, or the ciliary region, after Cdc42 and Tuba knockdown. 18 As noted, we showed that Sec10 directly binds to Par6, and others have shown that Cdc42 also directly binds to Par6. 27,28 Knockdown of both Sec10 29 and Cdc42 increased mitogen-activated protein kinase (MAPK) activation.…”

“…Given the ciliogenesis defects observed with cdc42 knockdown in vitro 18 and in the eyes of cdc42AUG MO embryos ( Figure 2, A and B), we predicted that pronephric cilia would be shorter or absent in the kidneys of the cdc42AUG MO embryos. Somewhat surprisingly, pronephric cilia at 27 hours postfertilization appeared of normal length in cdc42AUG MO embryos by immunofluorescence (Figure 2, D-G); however, the cilia were disordered within the medial (and to a lesser degree the posterior) pronephros, where dilations and pronephric cysts have been observed in other zebrafish cilia mutants.…”

“…We previously showed that the exocyst colocalizes with Par3 12 and directly interacts with Par6, 18 both components of the Par complex, which also includes atypical PKC. Cdc42 is associated with the Par complex.…”