Cdc42 Deficiency Causes Ciliary Abnormalities and Cystic Kidneys

Ciliogenesis and cystogenesis require the exocyst, a conserved eight-protein trafficking complex that traffics ciliary proteins. In culture, the small GTPase Cdc42 co-localizes with the exocyst at primary cilia and interacts with the exocyst component Sec10. The role of Cdc42 in vivo, however, is not well understood. Here, knockdown of cdc42 in zebrafish produced a phenotype similar to sec10 knockdown, including tail curvature, glomerular expansion, and mitogen-activated protein kinase (MAPK) activation, suggesting that cdc42 and sec10 cooperate in ciliogenesis. In addition, cdc42 knockdown led to hydrocephalus and loss of photoreceptor cilia. Furthermore, there was a synergistic genetic interaction between zebrafish cdc42 and sec10, suggesting that cdc42 and sec10 function in the same pathway. Mice lacking Cdc42 specifically in kidney tubular epithelial cells died of renal failure within weeks of birth. Histology revealed cystogenesis in distal tubules and collecting ducts, decreased ciliogenesis in cyst cells, increased tubular cell proliferation, increased apoptosis, increased fibrosis, and led to MAPK activation, all of which are features of polycystic kidney disease, especially nephronophthisis. Taken together, these results suggest that Cdc42 localizes the exocyst to primary cilia, whereupon the exocyst targets and docks vesicles carrying ciliary proteins. Abnormalities in this pathway result in deranged ciliogenesis and polycystic kidney disease.Cilia are thin rod-like organelles, found on the surface of many eukaryotic cells, with complex functions in signaling, cell differentiation, and growth control. Cilia extend outward from the basal body, a cellular organelle related to the centriole. In kidney cells, a single primary cilium projects from the basal body, is nonmotile, and exhibits an axoneme microtubule pattern of 9+0. In the mammalian kidney, primary cilia have been observed on renal tubule cells in the parietal layer of the Bowman capsule, the proximal tubule, the distal tubule, and in the principal, but not intercalated, cells of the collecting duct.¹Multiple proteins that, when mutated, result in the development of polycystic kidney disease (PKD) have been localized to renal primary cilia. These include polycystin-1 and -2, the causal proteins in autosomal dominant PKD (ADPKD) (reviewed by Smyth et al.²). Research into pkd2 function in zebrafish has further strengthened the idea that polycystin-2 functions in cilia. Knockdown of pkd2 by morpholino (MO)^3–5 or in mutants^5,6 produces phenotypes that are consistent with a role in cilia function, such as curved tails, pronephric cysts, and edema.Although we are beginning to identify the roles ciliary proteins play in diverse biologic processes, relatively little is known about how these proteins are transported to the cilium.⁷ The exocyst, originally identified in Saccharomycescerevisiae,⁸ is a highly conserved 750-kD eight-protein complex known for the targeting and docking of vesicles carrying membrane proteins.⁹ It is composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 (also known as EXOC1–8).¹⁰ Notably, in addition to being found near the tight junction, exocyst proteins were localized to the primary cilium in kidney cells.^11,12 Sec10 and Sec15 are the most vesicle-proximal of the exocyst components. Sec10 directly binds to Sec15, which, in turn, directly binds Sec4/Rab8, a Rab GTPase found on the surface of transport vesicles. Sec10 then acts as a “linker” by binding the other exocyst components through Sec5.¹³ Our previous studies suggested that the exocyst would no longer be able to bind Sec15, and thereby target/dock transport vesicles, without Sec10, and would, instead, disintegrate and be degraded. Importantly, we showed that knockdown of Sec10 in Madin-Darby canine kidney (MDCK) cells abrogated ciliogenesis, while Sec10 overexpression enhanced ciliogenesis. Furthermore, Sec10 knockdown caused abnormal cystogenesis when the cells were grown in a collagen matrix and decreased the levels of other exocyst components and the intraflagellar transport protein 88. This was in contrast to knockdown of exocyst components Sec8 and Exo70, which had no effect on ciliogenesis, cystogenesis, or levels of other exocyst components.¹² On the basis of these data, and its known role in trafficking proteins to the plasma membrane,^14–17 we proposed that Sec10 and the exocyst are required to build primary cilia by targeting and docking vesicles carrying ciliary proteins.A possible mechanism to target the exocyst to nascent primary cilia, so it can participate in ciliogenesis, is through the Par complex. We previously showed that the exocyst co-localizes with Par3¹² and directly interacts with Par6,¹⁸ both components of the Par complex, which also includes atypical PKC. Cdc42 is associated with the Par complex.^19,20 In addition to their well studied function at cell-cell contacts, the Par complex has been immunolocalized to primary cilia and is necessary for ciliogenesis.^21,22 The exocyst is regulated by multiple Rho and Rab family GTPases (reviewed by Lipschutz and Mostov⁹), including Cdc42, which regulates polarized exocytosis via interactions with the exocyst in yeast.²³ Using inducible MDCK cell lines that express constitutively active or dominant negative forms of Cdc42,^24,25 we established that Cdc42 is centrally involved in three-dimensional collagen gel cystogenesis and tubulogenesis.²⁶ Whether and how Cdc42 might participate in ciliogenesis and cooperate with the exocyst in ciliary membrane trafficking are open questions.Toward this end, we showed, in cell culture, that Cdc42 co-immunoprecipitated and co-localized with Sec10 and that Cdc42 was necessary for ciliogenesis in renal tubule cells, in that Cdc42-dominant negative expression, small hairpin RNA knockdown of Cdc42, and small hairpin RNA knockdown of Tuba, a guanine nucleotide exchange factor (GEF) for Cdc42, all inhibited ciliogenesis. 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 Sec10²⁹ and Cdc42 increased mitogen-activated protein kinase (MAPK) activation.¹⁸Here, 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. Other ciliary phenotypes include hydrocephalus and loss of photoreceptor cilia. We also demonstrate a synergistic genetic interaction between zebrafish cdc42 and sec10 for these cilia-related phenotypes, indicating that cdc42 and sec10 function in the same pathway. Demonstrating that the phenotypes were not due to off-target effects from the cdc42 MOs, we rescued the phenotypes with mouse Cdc42 mRNA. Cdc42 kidney-specific knockout mice died of kidney failure within weeks of birth; histologic examination revealed cystogenesis in distal tubules and collecting ducts and decreased ciliogenesis in cyst cells. Cdc42 conditional knockout kidneys showed increased tubular epithelial cell proliferation, increased apoptosis, increased interstitial fibrosis, and MAPK pathway activation, all features of the nephronophthisis form of PKD. These data, along with our previously published results, 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; if this does not occur, the result is abnormal ciliogenesis and PKD.