RESUMEN
Primary cilia are ubiquitous sensory organelles that mediate diverse signaling pathways. Cilia position on the cell surface is determined by the location of the basal body (BB) that templates the cilium. The mechanisms that regulate BB positioning in the context of ciliogenesis are largely unknown. Here we show that the conserved signaling and scaffolding protein Girdin localizes to the proximal regions of centrioles and regulates BB positioning and ciliogenesis in Caenorhabditis elegans sensory neurons and human RPE-1 cells. Girdin depletion alters localization of the intercentriolar linker and ciliary rootlet component rootletin, and rootletin knockdown in RPE-1 cells mimics Girdin-dependent phenotypes. C. elegans Girdin also regulates localization of the apical junction component AJM-1, suggesting that in nematodes Girdin may position BBs via rootletin- and AJM-1-dependent anchoring to the cytoskeleton and plasma membrane, respectively. Together, our results describe a conserved role for Girdin in BB positioning and ciliogenesis.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Cilios/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas de Microfilamentos/genética , Morfogénesis/genética , Proteínas de Transporte Vesicular/genética , Animales , Cuerpos Basales/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/biosíntesis , Centriolos/genética , Cilios/genética , Citoesqueleto/genética , Humanos , Proteínas de Microfilamentos/biosíntesis , Microtúbulos/genética , Orgánulos/genética , Células Receptoras Sensoriales/metabolismo , Transducción de Señal/genética , Proteínas de Transporte Vesicular/biosíntesisRESUMEN
The structure and function of primary cilia are critically dependent on intracellular trafficking pathways that transport ciliary membrane and protein components. The mechanisms by which these trafficking pathways are regulated are not fully characterized. Here we identify the transmembrane protein OSTA-1 as a new regulator of the trafficking pathways that shape the morphology and protein composition of sensory cilia in C. elegans. osta-1 encodes an organic solute transporter alpha-like protein, mammalian homologs of which have been implicated in membrane trafficking and solute transport, although a role in regulating cilia structure has not previously been demonstrated. We show that mutations in osta-1 result in altered ciliary membrane volume, branch length and complexity, as well as defects in localization of a subset of ciliary transmembrane proteins in different sensory cilia types. OSTA-1 is associated with transport vesicles, localizes to a ciliary compartment shown to house trafficking proteins, and regulates both retrograde and anterograde flux of the endosome-associated RAB-5 small GTPase. Genetic epistasis experiments with sensory signaling, exocytic and endocytic proteins further implicate OSTA-1 as a crucial regulator of ciliary architecture via regulation of cilia-destined trafficking. Our findings suggest that regulation of transport pathways in a cell type-specific manner contributes to diversity in sensory cilia structure and might allow dynamic remodeling of ciliary architecture via multiple inputs.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans , Cilios/genética , Membranas Intracelulares/metabolismo , Proteínas de Transporte de Membrana/fisiología , Animales , Animales Modificados Genéticamente , Transporte Biológico/genética , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cilios/fisiología , Cilios/ultraestructura , Embrión no Mamífero , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Redes y Vías Metabólicas/genética , Modelos Biológicos , Neuronas/metabolismo , Neuronas/fisiología , Neuronas/ultraestructura , Tamaño de los Órganos/genéticaRESUMEN
The membrane of the primary cilium is a highly specialized compartment that organizes proteins to achieve spatially ordered signaling. Disrupting ciliary organization leads to diseases called ciliopathies, with phenotypes ranging from retinal degeneration and cystic kidneys to neural tube defects. How proteins are selectively transported to and organized in the primary cilium remains unclear. Using a proteomic approach, we identified the ARL3 effector UNC119 as a binding partner of the myristoylated ciliopathy protein nephrocystin-3 (NPHP3). We mapped UNC119 binding to the N-terminal 200 residues of NPHP3 and found the interaction requires myristoylation. Creating directed mutants predicted from a structural model of the UNC119-myristate complex, we identified highly conserved phenylalanines within a hydrophobic ß sandwich to be essential for myristate binding. Furthermore, we found that binding of ARL3-GTP serves to release myristoylated cargo from UNC119. Finally, we showed that ARL3, UNC119b (but not UNC119a), and the ARL3 GAP Retinitis Pigmentosa 2 (RP2) are required for NPHP3 ciliary targeting and that targeting requires UNC119b myristoyl-binding activity. Our results uncover a selective, membrane targeting GTPase cycle that delivers myristoylated proteins to the ciliary membrane and suggest that other myristoylated proteins may be similarly targeted to specialized membrane domains.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Caenorhabditis elegans , Cilios/metabolismo , GTP Fosfohidrolasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Cinesinas , Proteínas de Unión al GTP Monoméricas , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Línea Celular , Cilios/enzimología , GTP Fosfohidrolasas/genética , Técnicas de Silenciamiento del Gen , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Cinesinas/genética , Cinesinas/metabolismo , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Mutación , Fenotipo , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Alineación de Secuencia , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
We isolated an Escherichia coli mutant in the iraD gene, sensitive to various forms of DNA damage. Our data are consistent with the function of IraD to promote accumulation of the alternative transcription sigma factor, RpoS, by binding to the adaptor RssB protein that targets RpoS for degradation. Our results demonstrate the physiological importance of this mode of regulation for DNA damage tolerance. Although RpoS is best known for its regulation of genes induced in stationary phase, our work underscores the importance of the RpoS regulon in a DNA damage response in actively growing cells. We show that iraD transcription is induced by DNA damage by a mechanism independent of the SOS response. The IraD and SOS regulatory pathways appear to act synergistically to ensure survival of cells faced with oxidative or DNA damaging stress during cellular growth.