Par3 functions in the biogenesis of the primary cilium in polarized epithelial cells

Sfakianos, Jeff; Togawa, Akashi; Maday, Sandra; Hull, Mike; Pypaert, Marc; Cantley, Lloyd G.; Toomre, Derek; Mellman, Ira

doi:10.1083/jcb.200709111

Cited by 88 publications

(102 citation statements)

References 30 publications

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“…Recent findings have also localized Par3, Par6, and atypical protein kinase C to primary cilia. These proteins seem to be important in ciliogenesis, perhaps through their interactions with critical ciliary proteins such as KIF3A and Crumbs3a (13,14). This is the first report to show that Cdc42 and Tuba, a Cdc42 GEF, also contribute to ciliogenesis in epithelial cells.…”

Section: Discussionmentioning

confidence: 75%

“…We previously found that the exocyst coimmunoprecipitated and colocalized with Par3 (5), a main component of the Par complex that consists of Par3, Par6, atypical protein kinase C, and Cdc42 (11,12). In addition to their studied function at cell-cell contacts, the Par complex has been immunolocalized to primary cilia and has been shown to be necessary for ciliogenesis (13,14). It is known that the exocyst is regulated by multiple Rho and Rab family GTPases (reviewed in 15), which, like the exocyst, play central roles in cell polarization, morphogenesis, membrane trafficking, cell growth, and development (16,17).…”

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confidence: 99%

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

Zuo

Fogelgren

Lipschutz

2011

Journal of Biological Chemistry

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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.

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Section: Discussionmentioning

confidence: 75%

mentioning

confidence: 99%

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

Zuo

Fogelgren

Lipschutz

2011

Journal of Biological Chemistry

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“…Par3, as discussed, was (Sfakianos et al, 2007). To test for a possible exocyst-Par3 interaction, we performed double labeling immunofluorescence microscopy by using antibodies against Par3 and the exocyst component Sec8.…”

Section: Discussionmentioning

confidence: 99%

“…It was recently shown that the PDZ protein Par3 localizes to the tight junction and primary cilium and knockdown of Par3 inhibits primary ciliogenesis (Sfakianos et al, 2007). Given a similar defect in ciliogenesis in the Sec10 knock- Figure 5.…”

Section: The Exocyst and Par3 Colocalize And Coimmunoprecipitatementioning

confidence: 99%

“…Several gene products, which when mutated result in the development of PKD, including polycystin-1 and polycystin-2, have been localized to and are crucial for the function of renal primary cilia (for review, see Smyth et al, 2003). It was recently shown that the postsynaptic density 95/disc-large/ zona occludens (PDZ) protein Par3 is necessary for the biogenesis of primary renal cilia (Fan et al, 2004;Sfakianos et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

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The Exocyst Protein Sec10 Is Necessary for Primary Ciliogenesis and Cystogenesis In Vitro

Zuo

Guo

Lipschutz

2009

MBoC

162

259

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Primary cilia are found on many epithelial cell types, including renal tubular epithelial cells, in which they are felt to participate in flow sensing and have been linked to the pathogenesis of cystic renal disorders such as autosomal dominant polycystic kidney disease. We previously localized the exocyst, an eight-protein complex involved in membrane trafficking, to the primary cilium of Madin-Darby canine kidney cells and showed that it was involved in cystogenesis. Here, using short hairpin RNA (shRNA) to knockdown exocyst expression and stable transfection to induce exocyst overexpression, we show that the exocyst protein Sec10 regulates primary ciliogenesis. Using immunofluorescence, scanning, and transmission electron microscopy, primary cilia containing only basal bodies are seen in the Sec10 knockdown cells, and increased ciliogenesis is seen in Sec10-overexpressing cells. These phenotypes do not seem to be because of gross changes in cell polarity, as apical, basolateral, and tight junction proteins remain properly localized. Sec10 knockdown prevents normal cyst morphogenesis when the cells are grown in a collagen matrix, whereas Sec10 overexpression results in increased cystogenesis. Transfection with human Sec10 resistant to the canine shRNA rescues the phenotype, demonstrating specificity. Finally, Par3 was recently shown to regulate primary cilia biogenesis. Par3 and the exocyst colocalized by immunofluorescence and coimmunoprecipitation, consistent with a role for the exocyst in targeting and docking vesicles carrying proteins necessary for primary ciliogenesis.

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Visualization of Live Primary Cilia Dynamics Using Fluorescence Microscopy

Ott

2012

CP Cell Biology

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Methods useful for exploring the formation and functions of primary cilia in living cells are described here. First, multiple protocols for visualizing solitary cilia that extend away from the cell body are described. Primary cilia collect, synthesize, and transmit information about the extracellular space into the cell body to promote critical cellular responses. Problems with cilia formation or function can lead to dramatic changes in cell physiology. These methods can be used to assess cilia formation and length, the location of the cilium relative to other cellular structures, and localization of specific proteins to the cilium. The subsequent protocols describe how to quantify movement of fluorescent molecules within the cilium using kymographs, photobleaching, and photoconversion. The microtubules that form the structural scaffold of the cilium are also critical avenues for kinesin and dynein‐mediated movement of proteins within the cilium. Assessing intraflagellar dynamics can provide insight into mechanisms of ciliary‐mediated signal perception and transmission. Curr. Protoc. Cell Biol. 57:4.26.1‐4.26.22. © 2012 by John Wiley & Sons, Inc.

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Par3 functions in the biogenesis of the primary cilium in polarized epithelial cells

Cited by 88 publications

References 30 publications

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

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

The Exocyst Protein Sec10 Is Necessary for Primary Ciliogenesis and Cystogenesis In Vitro

Visualization of Live Primary Cilia Dynamics Using Fluorescence Microscopy

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