A Screen for Modifiers of Cilia Phenotypes Reveals Novel MKS Alleles and Uncovers a Specific Genetic Interaction between osm-3 and nphp-4

PLoS Genet. 2016 Feb 10;12(2):e1005841. doi: 10.1371/journal.pgen.1005841. eCollection 2016 Feb.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Alleles*
  • Animals
  • Caenorhabditis elegans / genetics*
  • Caenorhabditis elegans Proteins / genetics*
  • Cilia / metabolism*
  • Ciliary Motility Disorders / genetics*
  • Encephalocele / genetics*
  • Epistasis, Genetic*
  • Genetic Complementation Test
  • Genetic Testing*
  • Kinesins / genetics*
  • Mutagenesis / genetics
  • Mutation / genetics
  • Phenotype
  • Phosphorylation
  • Polycystic Kidney Diseases / genetics*
  • Protein Structure, Tertiary
  • Protein Transport
  • Retinitis Pigmentosa

Substances

  • Caenorhabditis elegans Proteins
  • OSM-3 protein, C elegans
  • nephrocystin 4, C elegans
  • Kinesins

Supplementary concepts

  • Meckel syndrome type 1