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
BACKGROUND: Centriole duplication is a key step in the cell cycle whose mechanism is completely unknown. Why new centrioles always form next to preexisting ones is a fundamental question. The simplest model is that preexisting centrioles nucleate the assembly of new centrioles, and that although centrioles can in some cases form de novo without this nucleation, the de novo assembly mechanism should be too slow to compete with normal duplication in order to maintain fidelity of centriole duplication. RESULTS: We have measured the rate of de novo centriole assembly in vegetatively dividing cells that normally always contain centrioles. By using mutants of Chlamydomonas that are defective in centriole segregation, we obtained viable centrioleless cells that continue to divide, and find that within a single generation, 50% of these cells reacquire new centrioles by de novo assembly. This suggests that the rate of de novo assembly is approximately half the rate of templated duplication. A mutation in the VFL3 gene causes a complete loss of the templated assembly pathway without eliminating de novo assembly. A mutation in the centrin gene also reduced the rate of templated assembly. CONCLUSIONS: These results suggest that there are two pathways for centriole assembly, namely a templated pathway that requires preexisting centrioles to nucleate new centriole assembly, and a de novo assembly pathway that is normally turned off when centrioles are present.
Assuntos
Centríolos/fisiologia , Animais , Ciclo Celular , Chlamydomonas/genética , Chlamydomonas/fisiologia , CinéticaRESUMO
We present a new Chlamydomonas reinhardtii flagellar mutant in which central pair projections are missing and the central pair microtubules are twisted along the length of the flagellum. We have named this mutant tcp1 for twisted central pair. Immunoblots using an antibody that recognizes the heavy chain of sea urchin kinesin reveal that a 70 kDa protein present in wild-type and pf18 (central pairless) axonemes is absent in tcp1, suggesting the presence of an uncharacterized kinesin associated with the central pair apparatus. We demonstrate that the kinesin-like protein Klp1 is not attached to central pair microtubules in tcp1, but rather is located in, or is part of, a region we have termed the internal axonemal matrix. It is proposed that this matrix acts as a scaffold for axonemal proteins that may also be associated with the central pair apparatus.