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

ABSTRACT 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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    • "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.
    Molecular biology of the cell 04/2015; 26(12). DOI:10.1091/mbc.E14-10-1472 · 4.47 Impact Factor
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    • "FIP3 is involved in the regulation of the actin cytoskeleton both in late telophase, which is essential for the severing of the connecting bridge between daughter cells during cytokinesis (Schiel et al., 2012), and in breast cancer cell migration (Jing et al., 2009). Because blocking actin assembly facilitates ciliogenesis (Kim et al., 2010), the role of FIP3 and ASAP1 might include the control of actin polymerization in the periciliary region to facilitate ciliary trafficking. Activated Arf4 significantly increases direct ASAP1–rhodopsin interaction (Wang et al., 2012). "
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    ABSTRACT: Primary cilia have gained considerable importance in biology and disease now that their involvement in a wide range of human ciliopathies has been abundantly documented. However, detailed molecular mechanisms for specific targeting of sensory receptors to primary cilia are still unknown. Here, we show that the Arf and Rab11 effector FIP3 (also known as RAB11FIP3) promotes the activity of Rab11a and the Arf GTPase-activating protein (GAP) ASAP1 in the Arf4-dependent ciliary transport of the sensory receptor rhodopsin. During its passage out of the photoreceptor Golgi and trans-Golgi network (TGN), rhodopsin indirectly interacts with FIP3 through Rab11a and ASAP1. FIP3 competes with rhodopsin for binding to ASAP1 and displaces it from the ternary complex with Arf4–GTP and ASAP1. Resembling the phenotype resulting from lack of ASAP1, ablation of FIP3 abolishes ciliary targeting and causes rhodopsin mislocalization. FIP3 coordinates the interactions of ASAP1 and Rab11a with the Rab8 guanine nucleotide exchange factor Rabin8 (also known as RAB3IP). Our study implies that FIP3 functions as a crucial targeting regulator, which impinges on rhodopsin–ASAP1 interactions and shapes the binding pocket for Rabin8 within the ASAP1–Rab11a–FIP3 targeting complex, thus facilitating the orderly assembly and activation of the Rab11–Rabin8–Rab8 cascade during ciliary receptor trafficking.
    Journal of Cell Science 02/2015; DOI:10.1242/jcs.162925 · 5.43 Impact Factor
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    • "Alternatively, and perhaps more importantly, proteins that accumulate in the cilium as a result of active transport by microtubule-associated motors may be selectively removed when they reach the transition zone by active endocytosis . Endocytic recycling is critically important for ciliogenesis (Kim et al., 2010), and the preciliary membrane at the base of the cilium is enriched in clathrin-coated pits (Molla-Herman et al., 2010). These observations suggest that, as in yeast, coupling of targeted exocytosis, in tandem with active transport mechanisms and focal endocytosis, could contribute significantly to retention of specific membrane proteins in cilia. "
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    ABSTRACT: Biological membranes segregate into specialized functional domains of distinct composition, which can persist for the entire life of the cell. How separation of their lipid and (glyco)protein components is generated and maintained is not well understood, but the existence of diffusional barriers has been proposed. Remarkably, the physical nature of such barriers and the manner whereby they impede the free diffusion of molecules in the plane of the membrane has rarely been studied in depth. Moreover, alternative mechanisms capable of generating membrane inhomogeneity are often disregarded. Here we describe prototypical biological systems where membrane segregation has been amply documented and discuss the role of diffusional barriers and other processes in the generation and maintenance of their structural and functional compartmentalization. © 2015 Trimble and Grinstein.
    The Journal of Cell Biology 02/2015; 208(3):259-271. DOI:10.1083/jcb.201410071 · 9.83 Impact Factor
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