title: A Screen for Modifiers of Cilia Phenotypes Reveals Novel MKS Alleles and Uncovers a Specific Genetic Interaction between osm-3 and nphp-4.
abstract: Nephronophthisis (NPHP) is a ciliopathy in which genetic modifiers may underlie the variable penetrance of clinical features. To identify modifiers, a screen was conducted on C. elegans nphp-4(tm925) mutants. Mutations in ten loci exacerbating nphp-4(tm925) ciliary defects were obtained. Four loci have been identified, three of which are established ciliopathy genes mks-1, mks-2, and mks-5. The fourth allele (yhw66) is a missense mutation (S316F) in OSM-3, a kinesin required for cilia distal segment assembly. While osm-3(yhw66) mutants alone have no overt cilia phenotype, nphp-4(tm925);osm-3(yhw66) double mutants lack distal segments and are dye-filling (Dyf) and osmotic avoidance (Osm) defective, similar to osm-3(mn357) null mutants. In osm-3(yhw66) mutants anterograde intraflagellar transport (IFT) velocity is reduced. Furthermore, expression of OSM-3(S316F)::GFP reduced IFT velocities in nphp-4(tm925) mutants, but not in wild type animals. In silico analysis indicates the S316F mutation may affect a phosphorylation site. Putative phospho-null OSM-3(S316F) and phospho-mimetic OSM-3(S316D) proteins accumulate at the cilia base and tip respectively. FRAP analysis indicates that the cilia entry rate of OSM-3(S316F) is slower than OSM-3 and that in the presence of OSM-3(S316F), OSM-3 and OSM-3(S316D) rates decrease. In the presence OSM-3::GFP or OSM-3(S316D)::GFP, OSM-3(S316F)::tdTomato redistributes along the cilium and accumulates in the cilia tip. OSM-3(S316F) and OSM-3(S316D) are functional as they restore cilia distal segment formation in osm-3(mn357) null mutants; however, only OSM-3(S316F) rescues the osm-3(mn357) null Dyf phenotype. Despite rescue of cilia length in osm-3(mn357) null mutants, neither OSM-3(S316F) nor OSM-3(S316D) restores ciliary defects in nphp-4(tm925);osm-3(yhw66) double mutants. Thus, these OSM-3 mutations cause NPHP-4 dependent and independent phenotypes. These data indicate that in addition to regulating cilia protein entry or exit, NPHP-4 influences localization and function of a distal ciliary kinesin. Moreover, data suggest human OSM-3 homolog (Kif17) could act as a modifying locus affecting disease penetrance or expressivity in NPHP patients.
authors: Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham Medical School, Birmingham, Alabama, United States of America.
authors: Svetlana V
authors: SV
authors: Masyukova
authors: Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham Medical School, Birmingham, Alabama, United States of America.
authors: Dawn E
authors: DE
authors: Landis
authors: Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham Medical School, Birmingham, Alabama, United States of America.
authors: Scott J
authors: SJ
authors: Henke
authors: Department of Pharmacology and Therapeutics, College of Medicine, University of Florida, Gainesville, Florida, United States of America.
authors: Corey L
authors: CL
authors: Williams
authors: Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham Medical School, Birmingham, Alabama, United States of America.
authors: Jay N
authors: JN
authors: Pieczynski
authors: Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham Medical School, Birmingham, Alabama, United States of America.
authors: Kelly N
authors: KN
authors: Roszczynialski
authors: Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham Medical School, Birmingham, Alabama, United States of America.
authors: Jannese E
authors: JE
authors: Covington
authors: Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham Medical School, Birmingham, Alabama, United States of America.
authors: Erik B
authors: EB
authors: Malarkey
authors: Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham Medical School, Birmingham, Alabama, United States of America.
authors: Bradley K
authors: BK
authors: Yoder
mesh_terms: D000483:Alleles; D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics*; D002923:Cilia / Q000378:metabolism*; D002925:Ciliary Motility Disorders / Q000235:genetics*; D004677:Encephalocele / Q000235:genetics*; D004843:Epistasis, Genetic; D005816:Genetic Complementation Test; D005820:Genetic Testing; D016547:Kinesins / Q000235:genetics*; D016296:Mutagenesis / Q000235:genetics; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D010766:Phosphorylation; D007690:Polycystic Kidney Diseases / Q000235:genetics*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012174:Retinitis Pigmentosa
chemical_list: D029742:Caenorhabditis elegans Proteins; C093002:OSM-3 protein, C elegans; C501205:nephrocystin 4, C elegans; D016547:Kinesins