The Exocyst Protein Sec10 Interacts with Polycystin-2 and Knockdown Causes PKD-Phenotypes

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by formation of renal cysts that destroy the kidney. Mutations in PKD1 and PKD2, encoding polycystins-1 and -2, cause ADPKD. Polycystins are thought to function in primary cilia, but it is not well understood how these and other proteins are targeted to cilia. Here, we provide the first genetic and biochemical link between polycystins and the exocyst, a highly-conserved eight-protein membrane trafficking complex. We show that knockdown of exocyst component Sec10 yields cellular phenotypes associated with ADPKD, including loss of flow-generated calcium increases, hyperproliferation, and abnormal activation of MAPK. Sec10 knockdown in zebrafish phenocopies many aspects of polycystin-2 knockdown—including curly tail up, left-right patterning defects, glomerular expansion, and MAPK activation—suggesting that the exocyst is required for pkd2 function in vivo. We observe a synergistic genetic interaction between zebrafish sec10 and pkd2 for many of these cilia-related phenotypes. Importantly, we demonstrate a biochemical interaction between Sec10 and the ciliary proteins polycystin-2, IFT88, and IFT20 and co-localization of the exocyst and polycystin-2 at the primary cilium. Our work supports a model in which the exocyst is required for the ciliary localization of polycystin-2, thus allowing for polycystin-2 function in cellular processes.

Cdc42 Deficiency Causes Ciliary Abnormalities and Cystic Kidneys

“…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. Other ciliary phenotypes include hydrocephalus and loss of photoreceptor cilia.…”

“…The disorganized cilia were similar to what we observed in sec10 and pkd2 morphants. 29 Also similar to sec10 and pkd2 morphant embryos, 3 cdc42AUG MO embryos showed glomerular expansion in the pronephros by in situ hybridization with the glomerular marker, Wilms tumor 1a (wt1a) at 3 dpf. Wild-type embryos showed a condensed glomerular stain (Figure 2, H and H', 100% condensed; n=30), whereas cdc42AUG MO embryos showed an enlarged stain (Figure 2, I and I'; 18 of 19 embryos with enlarged stain).…”

“…51 It should be noted, however, that we previously showed that polycystin-2, one of the two causative proteins in ADPKD, co-localized with the exocyst at the primary cilium 18 and biochemically interacted with the exocyst. 29 Cell proliferation is also a well known characteristic of ADPKD cells and plays a major role in the formation of the cysts that destroy the kidney, leading some to refer to ADPKD as "neoplasia in disguise." 52 Thus, involvement of Cdc42 and the exocyst in ADPKD is also quite possible.…”

“…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. 18 Here, using two different living organisms, we confirm and extend our in vitro findings.…”

Laterality defects are influenced by timing of treatments and animal model

“…Many treatments affecting LR asymmetry result in 20–40% heterotaxia, a much lower level than the 50–90% expected (depending on the number of organs assessed), an issue that is rarely if ever discussed [see for example (Nagai et al, 2010; Fogelgren et al, 2011; Marszalek et al, 1999; Bajoghli et al, 2007; Amack et al, 2007; Danilchik et al, 2006)]. A considerable number of studies on the mechanisms of LR asymmetry are available and comparable endpoints (i.e.…”