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      Functional genomic screen for modulators of ciliogenesis and cilium length

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          Abstract

          Primary cilia are evolutionarily conserved cellular organelles that organize diverse signaling pathways 1, 2. Defects in the formation or function of primary cilia are associated with a spectrum of human diseases and developmental abnormalities 3. Genetic screens in model organisms have discovered core machineries of cilium assembly and maintenance 4. 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. PPC was labeled by recycling endosome markers. Moreover, knockdown of modulators that are involved in the endocytic recycling pathway affected the formation of PPC as well as ciliogenesis. Our results uncover a critical regulatory step that couples actin dynamics and endocytic recycling with ciliogenesis, and also provide potential target molecules for future study.

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          Most cited references17

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          The vertebrate primary cilium in development, homeostasis, and disease.

          Cilia are complex structures that have garnered interest because of their roles in vertebrate development and their involvement in human genetic disorders. In contrast to multicellular invertebrates in which cilia are restricted to specific cell types, these organelles are found almost ubiquitously in vertebrate cells, where they serve a diverse set of signaling functions. Here, we highlight properties of vertebrate cilia, with particular emphasis on their relationship with other subcellular structures, and explore the physiological consequences of ciliary dysfunction.
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            Cilia and developmental signaling.

            Recent studies have revealed unexpected connections between the mammalian Hedgehog (Hh) signal transduction pathway and the primary cilium, a microtubule-based organelle that protrudes from the surface of most vertebrate cells. Intraflagellar transport proteins, which are required for the construction of cilia, are essential for all responses to mammalian Hh proteins, and proteins required for Hh signal transduction are enriched in primary cilia. The phenotypes of different mouse mutants that affect ciliary proteins suggest that cilia may act as processive machines that organize sequential steps in the Hh signal transduction pathway. Cilia on vertebrate cells are likely to be important in additional developmental signaling pathways and are required for PDGF receptor alpha signaling in cultured fibroblasts. Cilia are not essential for either canonical or noncanonical Wnt signaling, although cell-type-specific modulation of cilia components may link cilia and Wnt signaling in some tissues. Because ciliogenesis in invertebrates is limited to a very small number of specialized cell types, the role of cilia in developmental signaling pathways is likely a uniquely vertebrate phenomenon.
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              Functional dissection of Rab GTPases involved in primary cilium formation

              Primary cilia are sensory structures involved in morphogen signalling during development, liquid flow in the kidney, mechanosensation, sight, and smell (Badano, J.L., N. Mitsuma, P.L. Beales, and N. Katsanis. 2006. Annu. Rev. Genomics Hum. Genet. 7:125–148; Singla, V., and J.F. Reiter. 2006. Science. 313:629–633.). Mutations that affect primary cilia are responsible for several diseases, including neural tube defects, polycystic kidney disease, retinal degeneration, and cancers (Badano et al., 2006; Singla and Reiter, 2006). Primary cilia formation and function requires tight integration of the microtubule cytoskeleton with membrane trafficking (Singla and Reiter, 2006), and this is poorly understood. We show that the Rab GTPase membrane trafficking regulators Rab8a, -17, and -23, and their cognate GTPase-activating proteins (GAPs), XM_037557, TBC1D7, and EVI5like, are involved in primary cilia formation. However, other human Rabs and GAPs are not. Additionally, Rab8a specifically interacts with cenexin/ODF2, a basal body and microtubule binding protein required for cilium biogenesis (Ishikawa, H., A. Kubo, S. Tsukita, and S. Tsukita. 2005. Nat. Cell Biol. 7:517–524), and is the sole Rab enriched at primary cilia. These findings provide a basis for understanding how specific membrane trafficking pathways cooperate with the microtubule cytoskeleton to give rise to the primary cilia.
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                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                0028-0836
                1476-4687
                19 August 2010
                15 April 2010
                1 October 2010
                : 464
                : 7291
                : 1048-1051
                Affiliations
                [1 ]Departments of Neurosciences and Pediatrics, Institute for Genomic Medicine, Howard Hughes Medical Institute, University of California San Diego, La Jolla, CA 92093, USA
                [2 ]High Content Screening Systems, Burnham Institute, La Jolla, CA 92037, USA
                [3 ]Functional Genomics Core, Burnham Institute, La Jolla, CA 92037, USA
                [4 ]Departments of Medicine and Bioengineering, University of California San Diego, La Jolla, CA 92093, USA
                [5 ]Department of Biomedical Informatics, Ajou University School of Medicine, Suwon 443-749, Korea
                Author notes
                Correspondence and requests for materials should be addressed to Joseph G. Gleeson: 9500 Gilman Drive, M/C 0665, Leichtag Biomedical Research Building R482, University of California San Diego, La Jolla, CA 92093, Tel: 858-822-3535, Fax: 858-822-1021, jogleeson@ 123456ucsd.edu

                Author Information Reprints and permissions information is available at www.nature.com/reprints.

                Article
                nihpa178187
                10.1038/nature08895
                2929961
                20393563
                5328060e-d147-4e14-a75e-3efc92d9b6f9

                Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

                History
                Funding
                Funded by: National Institute of Neurological Disorders and Stroke : NINDS
                Award ID: R01 NS052455-05 ||NS
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