RESUMO
Intraflagellar transport (IFT) is a rapid movement of multi-subunit protein particles along flagellar microtubules and is required for assembly and maintenance of eukaryotic flagella. We cloned and sequenced a Chlamydomonas cDNA encoding the IFT88 subunit of the IFT particle and identified a Chlamydomonas insertional mutant that is missing this gene. The phenotype of this mutant is normal except for the complete absence of flagella. IFT88 is homologous to mouse and human genes called Tg737. Mice with defects in Tg737 die shortly after birth from polycystic kidney disease. We show that the primary cilia in the kidney of Tg737 mutant mice are shorter than normal. This indicates that IFT is important for primary cilia assembly in mammals. It is likely that primary cilia have an important function in the kidney and that defects in their assembly can lead to polycystic kidney disease.
Assuntos
Chlamydomonas/genética , Cílios/metabolismo , Flagelos/metabolismo , Rim Policístico Autossômico Recessivo/genética , Proteínas/química , Proteínas de Protozoários/metabolismo , Proteínas Supressoras de Tumor , Sequência de Aminoácidos , Animais , Chlamydomonas/citologia , Cílios/genética , Cílios/patologia , Cílios/ultraestrutura , Clonagem Molecular , Sequência Conservada , Flagelos/genética , Flagelos/patologia , Flagelos/ultraestrutura , Humanos , Rim/metabolismo , Rim/patologia , Meiose , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/metabolismo , Proteínas Motores Moleculares/patologia , Proteínas Motores Moleculares/ultraestrutura , Dados de Sequência Molecular , Mutação/genética , Fenótipo , Proteínas de Plantas , Rim Policístico Autossômico Recessivo/patologia , Rim Policístico Autossômico Recessivo/fisiopatologia , Ligação Proteica , Subunidades Proteicas , Proteínas/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Sequências Repetitivas de Aminoácidos/genética , Sequências Repetitivas de Aminoácidos/fisiologia , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Tctex2 is thought to be one of the distorter genes of the mouse t haplotype. This complex greatly biases the segregation of the chromosome that carries it such that in heterozygous +/t males, the t haplotype is transmitted to >95% of the offspring, a phenomenon known as transmission ratio distortion. The LC2 outer dynein arm light chain of Chlamydomonas reinhardtii is a homologue of the mouse protein Tctex2. We have identified Chlamydomonas insertional mutants with deletions in the gene encoding LC2 and demonstrate that the LC2 gene is the same as the ODA12 gene, the product of which had not been identified previously. Complete deletion of the LC2/ODA12 gene causes loss of all outer arms and a slow jerky swimming phenotype. Transformation of the deletion mutant with the cloned LC2/ODA12 gene restores the outer arms and rescues the motility phenotype. Therefore, LC2 is required for outer arm assembly. The fact that LC2 is an essential subunit of flagellar outer dynein arms allows us to propose a detailed mechanism whereby transmission ratio distortion is explained by the differential binding of mutant (t haplotype encoded) and wild-type dyneins to the axonemal microtubules of t-bearing or wild-type sperm, with resulting differences in their motility.
Assuntos
Chlamydomonas reinhardtii/genética , Dineínas/genética , Genes de Protozoários , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Associadas aos Microtúbulos , Proteínas de Protozoários/genética , Animais , Movimento Celular , Clonagem Molecular , Flagelos/genética , Flagelos/ultraestrutura , Camundongos , Microscopia Eletrônica , Microtúbulos/ultraestrutura , Mutação , Proteínas Nucleares/genética , Fenótipo , Proteínas de Protozoários/metabolismo , Homologia de Sequência , Transformação Genética , Ubiquitina-Proteína Ligases , Região do Complexo-t do GenomaRESUMO
Dyneins are microtubule-based molecular motors involved in many different types of cell movement. Most dynein heavy chains (DHCs) clearly group into cytoplasmic or axonemal isoforms. However, DHC1b has been enigmatic. To learn more about this isoform, we isolated Chlamydomonas cDNA clones encoding a portion of DHC1b, and used these clones to identify a Chlamydomonas cell line with a deletion mutation in DHC1b. The mutant grows normally and appears to have a normal Golgi apparatus, but has very short flagella. The deletion also results in a massive redistribution of raft subunits from a peri-basal body pool (Cole, D.G., D.R. Diener, A.L. Himelblau, P.L. Beech, J.C. Fuster, and J.L. Rosenbaum. 1998. J. Cell Biol. 141:993-1008) to the flagella. Rafts are particles that normally move up and down the flagella in a process known as intraflagellar transport (IFT) (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. USA. 90:5519-5523), which is essential for assembly and maintenance of flagella. The redistribution of raft subunits apparently occurs due to a defect in the retrograde component of IFT, suggesting that DHC1b is the motor for retrograde IFT. Consistent with this, Western blots indicate that DHC1b is present in the flagellum, predominantly in the detergent- and ATP-soluble fractions. These results indicate that DHC1b is a cytoplasmic dynein essential for flagellar assembly, probably because it is the motor for retrograde IFT.