Functional genomic screen for modulators of ciliogenesis and cilium length

Department of Neurosciences, Institute for Genomic Medicine, Howard Hughes Medical Institute, University of California San Diego, La Jolla, California 92093, USA.
Nature (Impact Factor: 41.46). 04/2010; 464(7291):1048-51. DOI: 10.1038/nature08895
Source: PubMed


Primary cilia are evolutionarily conserved cellular organelles that organize diverse signalling pathways. Defects in the formation or function of primary cilia are associated with a spectrum of human diseases and developmental abnormalities. Genetic screens in model organisms have discovered core machineries of cilium assembly and maintenance. However, regulatory molecules that coordinate the biogenesis of primary cilia with other cellular processes, including cytoskeletal organization, vesicle trafficking and cell-cell adhesion, remain to be identified. Here we report the results of a functional genomic screen using RNA interference (RNAi) to identify human genes involved in ciliogenesis control. The screen identified 36 positive and 13 negative ciliogenesis modulators, which include molecules involved in actin dynamics and vesicle trafficking. Further investigation demonstrated that blocking actin assembly facilitates ciliogenesis by stabilizing the pericentrosomal preciliary compartment (PPC), a previously uncharacterized compact vesiculotubular structure storing transmembrane proteins destined for cilia during the early phase of ciliogenesis. The PPC was labelled by recycling endosome markers. Moreover, knockdown of modulators that are involved in the endocytic recycling pathway affected the formation of the PPC as well as ciliogenesis. Our results uncover a critical regulatory step that couples actin dynamics and endocytic recycling with ciliogenesis, and also provides potential target molecules for future study.

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    • "The characterization of the function of this ion channel in primary cilia, with an additional role in ciliogenesis, provides novel insights into disease pathogenesis in these patients. In contrast to our findings, a functional genomic screen for modulators of ciliogenesis and cilium length in retinal pigment epithelial cells did not identify these four ion channels (Kim et al., 2010). However, we have ascertained that these same ion channels are not expressed in this cell type (RNA sequence data deposited, BioProject ID PRJNA298886, "
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    ABSTRACT: To investigate the contribution of ion channels to ciliogenesis we carried out an siRNA-based reverse genetics screen of all ion channels in the mouse genome in murine inner medullary collecting duct kidney cells. This screen revealed four candidate ion channel genes: Kcnq1, Kcnj10, Kcnf1 and Clcn4. We show that these four ion channels localize to renal tubules, specifically to the base of primary cilia. We report that human KCNQ1 Long QT syndrome disease alleles, regulate renal ciliogenesis; KCNQ1-p.R518X, -p.A178T and -p.K362R could not rescue ciliogenesis after Kcnq1 siRNA-mediated depletion in contrast to wild-type KCNQ1 and benign KCNQ1-p.R518Q, suggesting that the ion channel function of KCNQ1 regulates ciliogenesis. In contrast, we demonstrate that the ion channel function of KCNJ10 is independent of its effect on ciliogenesis. Our data suggest that these four ion channels possibly regulate renal ciliogenesis through the periciliary diffusion barrier or the ciliary pocket, with potential implication as genetic contributors to ciliopathy pathophysiology. The new functional roles of a subset of ion channels provide new insights into the disease pathogenesis of channelopathies and may suggest future therapeutic approaches.
    No preview · Article · Nov 2015 · Journal of Cell Science
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    • "This was confirmed by using SEM to examine mock and RSVinfected cells at 24 hpi, which showed that in both cases the cilia were similar in appearance (Fig. 1C). Although F-actin is not present within the cilia body, certain types of F-actin structure have also been shown to exert an inhibitory effect on the formation of cilia (ciliagenesis) (Kim et al., 2010; Bershteyn et al., 2010; Cao et al., 2012; Bhogaraju et al., 2014; Yan and Zhu, 2013). An association of F-actin and virus filaments has been demonstrated, and virus-induced F-actin remodeling has been proposed to play a role in RSV filament formation (Jeffree et al., 2007; Ravi et al., 2013; Ulloa et al., 1998; Kallewaard et al., 2005). "
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    ABSTRACT: The distribution of cilia and the respiratory syncytial virus (RSV) nucleocapsid (N) protein, fusion (F) protein, attachment (G) protein, and M2-1 protein in human ciliated nasal epithelial cells was examined at between 1 and 5 days post-infection (dpi). All virus structural proteins were localized at cell surface projections that were distinct from cilia. The F protein was also trafficked into the cilia, and while its presence increased as the infection proceeded, the N protein was not detected in the cilia at any time of infection. The presence of the F protein in the cilia correlated with cellular changes in the cilia and reduced cilia function. At 5dpi extensive cilia loss and further reduced cilia function was noted. These data suggested that although RSV morphogenesis occurs at non-cilia locations on ciliated nasal epithelial cells, RSV infection induces changes in the cilia body that leads to extensive cilia loss. Copyright © 2015 Elsevier Inc. All rights reserved.
    Preview · Article · Jul 2015 · Virology
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    • "In contrast, in cancer cells that aberrantly express the lipogenic transcription factor SREBP1c, PLA2G3 gets overexpressed through direct transcriptional regulation (Figure 8B). On secretion, PLA2G3 may act on extracellular phospholipids of the plasma membrane, and/or on intracellular phospholipids during the secretion process or upon endocytosis, and/or upon sequestration at the centrosome/centriole pair (Kim et al., 2010). The increase in LPCs resulting from the hydrolysis of phosphatidtylcholines (PCs) increases the positive membrane curvature in the plasma membrane and intracellular (vesicle) membranes showed that SREBP1c overexpression affected transferrin transport and led to a mislocalization of the recycling marker Rab11, which normally localizes to the perinuclear recycling compartment in proximity of the ciliary base and regulates vesicle trafficking during ciliogenesis (Knodler et al., 2010; Westlake et al., 2011). "
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    ABSTRACT: Distortion of primary cilia formation is increasingly recognized as a key event in many human pathologies. One of the underlying mechanisms involves aberrant activation of the lipogenic transcription factor Sterol Regulatory Element-binding Protein 1c (SREBP1c), as observed in cancer cells. To gain more insight into the molecular pathways by which SREBP1c suppresses primary ciliogenesis, we searched for overlap between known ciliogenesis regulators and targets of SREBP1. One of the candidate genes that was consistently upregulated in cellular models of SREBP1c-induced cilium repression was Phospholipase A2 group III (PLA2G3), a phospholipase that hydrolyses the sn-2 position of glycerophospholipids. Use of RNA interference and a chemical inhibitor of PLA2G3 rescued SREBP1c-induced cilium repression. Cilium repression by SREBP1c and PLA2G3 involved alterations in endosomal recycling and vesicular transport toward the cilium as revealed by aberrant transferrin and Rab11 localization and was largely mediated by an increase in lysophosphatidylcholine and lysophosphatidylethanolamine levels. Together, these findings indicate that aberrant activation of SREBP1c suppresses primary ciliogenesis by PLA2G3-mediated distortion of vesicular trafficking and suggest that PLA2G3 is a novel potential target to normalize ciliogenesis in SREBP1c-overexpressing cells, including cancer cells. © 2015 by The American Society for Cell Biology.
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