RESUMO
Intraflagellar transport (IFT), which is essential for the formation and function of cilia in most organisms, is the trafficking of IFT trains (i.e. assemblies of IFT particles) that carry cargo within the cilium. Defects in IFT cause several human diseases. IFT trains contain the complexes IFT-A and IFT-B. To dissect the functions of these complexes, we studied a Chlamydomonas mutant that is null for the IFT-A protein IFT140. The mutation had no effect on IFT-B but destabilized IFT-A, preventing flagella assembly. Therefore, IFT-A assembly requires IFT140. Truncated IFT140, which lacks the N-terminal WD repeats of the protein, partially rescued IFT and supported formation of half-length flagella that contained normal levels of IFT-B but greatly reduced amounts of IFT-A. The axonemes of these flagella had normal ultrastructure and, as investigated by SDS-PAGE, normal composition. However, composition of the flagellar 'membrane+matrix' was abnormal. Analysis of the latter fraction by mass spectrometry revealed decreases in small GTPases, lipid-anchored proteins and cell signaling proteins. Thus, IFT-A is specialized for the import of membrane-associated proteins. Abnormal levels of the latter are likely to account for the multiple phenotypes of patients with defects in IFT140.This article has an associated First Person interview with the first author of the paper.
Assuntos
Proteínas de Algas/genética , Membrana Celular/metabolismo , Chlamydomonas reinhardtii/genética , Cílios/metabolismo , Flagelos/metabolismo , Proteínas Ligadas a Lipídeos/genética , Proteínas de Algas/química , Proteínas de Algas/metabolismo , Axonema/metabolismo , Axonema/ultraestrutura , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Membrana Celular/ultraestrutura , Ataxia Cerebelar/genética , Ataxia Cerebelar/metabolismo , Ataxia Cerebelar/patologia , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/ultraestrutura , Cílios/ultraestrutura , Síndrome de Ellis-Van Creveld/genética , Síndrome de Ellis-Van Creveld/metabolismo , Síndrome de Ellis-Van Creveld/patologia , Flagelos/ultraestrutura , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Ligadas a Lipídeos/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação , Organismos Geneticamente Modificados , Transporte Proteico , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologia , Transdução de Sinais , Proteína Vermelha FluorescenteRESUMO
Motility of cilia/flagella is generated by a coordinated activity of thousands of dyneins. Inner dynein arms (IDAs) are particularly important for the formation of ciliary/flagellar waveforms, but the molecular mechanism of IDA regulation is poorly understood. Here we show using cryoelectron tomography and biochemical analyses of Chlamydomonas flagella that a conserved protein FAP44 forms a complex that tethers IDA f (I1 dynein) head domains to the A-tubule of the axonemal outer doublet microtubule. In wild-type flagella, IDA f showed little nucleotide-dependent movement except for a tilt in the f ß head perpendicular to the microtubule-sliding direction. In the absence of the tether complex, however, addition of ATP and vanadate caused a large conformational change in the IDA f head domains, suggesting that the movement of IDA f is mechanically restricted by the tether complex. Motility defects in flagella missing the tether demonstrates the importance of the IDA f-tether interaction in the regulation of ciliary/flagellar beating.
Assuntos
Dineínas do Axonema/metabolismo , Dineínas do Axonema/fisiologia , Cílios/metabolismo , Animais , Axonema/metabolismo , Movimento Celular , Chlamydomonas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Cílios/fisiologia , Citoesqueleto/metabolismo , Dineínas/metabolismo , Flagelos/metabolismo , Microtúbulos/metabolismo , Transdução de Sinais , Tetrahymena/genética , Tetrahymena/metabolismoRESUMO
Intraflagellar transport (IFT) trains, multimegadalton assemblies of IFT proteins and motors, traffic proteins in cilia. To study how trains assemble, we employed fluorescence protein-tagged IFT proteins in Chlamydomonas reinhardtii. IFT-A and motor proteins are recruited from the cell body to the basal body pool, assembled into trains, move through the cilium, and disperse back into the cell body. In contrast to this 'open' system, IFT-B proteins from retrograde trains reenter the pool and a portion is reused directly in anterograde trains indicating a 'semi-open' system. Similar IFT systems were also observed in Tetrahymena thermophila and IMCD3 cells. FRAP analysis indicated that IFT proteins and motors of a given train are sequentially recruited to the basal bodies. IFT dynein and tubulin cargoes are loaded briefly before the trains depart. We conclude that the pool contains IFT trains in multiple stages of assembly queuing for successive release into the cilium upon completion.
Assuntos
Proteínas de Transporte/metabolismo , Chlamydomonas reinhardtii/metabolismo , Cílios/metabolismo , Substâncias Macromoleculares/metabolismo , Biogênese de Organelas , Multimerização Proteica , Recuperação de Fluorescência Após FotodegradaçãoRESUMO
Intraflagellar transport (IFT) moves IFT trains carrying cargoes from the cell body into the flagellum and from the flagellum back to the cell body. IFT trains are composed of complexes IFT-A and IFT-B and cargo adaptors such as the BBSome. The IFT-B core proteins IFT74 and IFT81 interact directly through central and C-terminal coiled-coil domains, and recently it was shown that the N termini of these proteins form a tubulin-binding module important for ciliogenesis. To investigate the function of IFT74 and its domains in vivo, we have utilized Chlamydomonas reinhardtii ift74 mutants. In a null mutant, lack of IFT74 destabilized IFT-B, leading to flagella assembly failure. In this null background, expression of IFT74 lacking 130 amino acids (aa) of the charged N terminus stabilized IFT-B and promoted slow assembly of nearly full-length flagella. A further truncation (lacking aa 1-196, including part of coiled-coil 1) also stabilized IFT-B, but failure in IFT-A/IFT-B interaction within the pool at the base of the flagellum prevented entry of IFT-A into the flagellum and led to severely decreased IFT injection frequency and flagellar-assembly defects. Decreased IFT-A in these short flagella resulted in aggregates of stalled IFT-B in the flagella. We conclude that IFT74 is required to stabilize IFT-B; aa 197-641 are sufficient for this function in vivo. The N terminus of IFT74 may be involved in, but is not required for, tubulin entry into flagella. It is required for association of IFT-A and IFT-B at the base of the flagellum and flagellar import of IFT-A.
Assuntos
Chlamydomonas reinhardtii/metabolismo , Proteínas do Citoesqueleto/metabolismo , Flagelos/metabolismo , Transporte Biológico , Proteínas do Citoesqueleto/genética , Flagelos/genética , Mutação , Ligação ProteicaRESUMO
Eukaryotic cilia and flagella are vital sensory and motile organelles. The calcium channel PKD2 mediates sensory perception on cilia and flagella, and defects in this can contribute to ciliopathic diseases. Signaling from Pkd2-dependent Ca²+ rise in the cilium to downstream effectors may require intermediary proteins that are largely unknown. To identify these proteins, we carried out genetic screens for mutations affecting Drosophila melanogaster sperm storage, a process mediated by Drosophila Pkd2. Here we show that a new mutation lost boys (lobo) encodes a conserved flagellar protein CG34110, which corresponds to vertebrate Ccdc135 (E = 6e-78) highly expressed in ciliated respiratory epithelia and sperm, and to FAP50 (E = 1e-28) in the Chlamydomonas reinhardtii flagellar proteome. CG34110 localizes along the fly sperm flagellum. FAP50 is tightly associated with the outer doublet microtubules of the axoneme and appears not to be a component of the central pair, radial spokes, dynein arms, or structures defined by the mbo waveform mutants. Phenotypic analyses indicate that both Pkd2 and lobo specifically affect sperm movement into the female storage receptacle. We hypothesize that the CG34110/Ccdc135/FAP50 family of conserved flagellar proteins functions within the axoneme to mediate Pkd2-dependent processes in the sperm flagellum and other motile cilia.
Assuntos
Movimento Celular/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomia & histologia , Flagelos/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Cálcio/metabolismo , Chlamydomonas reinhardtii/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Feminino , Flagelos/ultraestrutura , Masculino , Proteínas Associadas aos Microtúbulos/genética , Mutação , Transdução de Sinais/fisiologia , Espermatozoides/metabolismo , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismoRESUMO
In humans, seven evolutionarily conserved genes that cause the cilia-related disorder Bardet-Biedl syndrome (BBS) encode proteins that form a complex termed the BBSome. The function of the BBSome in the cilium is not well understood. We purified a BBSome-like complex from Chlamydomonas reinhardtii flagella and found that it contains at least BBS1, -4, -5, -7, and -8 and undergoes intraflagellar transport (IFT) in association with a subset of IFT particles. C. reinhardtii insertional mutants defective in BBS1, -4, and -7 assemble motile, full-length flagella but lack the ability to phototax. In the bbs4 mutant, the assembly and transport of IFT particles are unaffected, but the flagella abnormally accumulate several signaling proteins that may disrupt phototaxis. We conclude that the BBSome is carried by IFT but is an adapter rather than an integral component of the IFT machinery. C. reinhardtii BBS4 may be required for the export of signaling proteins from the flagellum via IFT.