RESUMO
Ciliary dyneins are preassembled in the cytoplasm before being transported into cilia, and a family of proteins containing the PIH1 domain, PIH proteins, are involved in the assembly process. However, the functional differences and relationships between members of this family of proteins remain largely unknown. Using Chlamydomonas reinhardtii as a model, we isolated and characterized two novel Chlamydomonas PIH preassembly mutants, mot48-2 and twi1-1. A new allele of mot48 (ida10), mot48-2, shows large defects in ciliary dynein assembly in the axoneme and altered motility. A second mutant, twi1-1, shows comparatively smaller defects in motility and dynein assembly. A double mutant mot48-2; twi1-1 displays greater reduction in motility and in dynein assembly compared to each single mutant. Similarly, a double mutant twi1-1; pf13 also shows a significantly greater defect in motility and dynein assembly than either parent mutant. Thus, MOT48 (IDA10), TWI1 and PF13 may define different steps, and have partially overlapping functions, in a pathway required for ciliary dynein preassembly. Together, our data suggest the three PIH proteins function in preassembly steps that are both common and unique for different ciliary dyneins.
Assuntos
Dineínas do Axonema/metabolismo , Movimento Celular/genética , Cílios/metabolismo , Transtornos da Motilidade Ciliar/genética , Proteínas de Plantas/genética , Chlamydomonas reinhardtii , Humanos , Mutação , Proteínas de Plantas/metabolismoRESUMO
Cytoplasmic assembly of ciliary dyneins, a process known as preassembly, requires numerous non-dynein proteins, but the identities and functions of these proteins are not fully elucidated. Here, we show that the classical Chlamydomonas motility mutant pf23 is defective in the Chlamydomonas homolog of DYX1C1. The pf23 mutant has a 494 bp deletion in the DYX1C1 gene and expresses a shorter DYX1C1 protein in the cytoplasm. Structural analyses, using cryo-ET, reveal that pf23 axonemes lack most of the inner dynein arms. Spectral counting confirms that DYX1C1 is essential for the assembly of the majority of ciliary inner dynein arms (IDA) as well as a fraction of the outer dynein arms (ODA). A C-terminal truncation of DYX1C1 shows a reduction in a subset of these ciliary IDAs. Sucrose gradients of cytoplasmic extracts show that preassembled ciliary dyneins are reduced compared to wild-type, which suggests an important role in dynein complex stability. The role of PF23/DYX1C1 remains unknown, but we suggest that DYX1C1 could provide a scaffold for macromolecular assembly.
Assuntos
Proteínas de Algas/genética , Axonema/genética , Chlamydomonas reinhardtii/genética , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Animais , Axonema/química , Cílios/química , Cílios/genética , Citoplasma/genética , Citoplasma/metabolismo , Proteínas do Citoesqueleto , Dineínas/química , Dineínas/genética , Flagelos/genética , Humanos , Mutação , Proteínas do Tecido Nervoso/química , Proteínas Nucleares/química , Domínios Proteicos/genéticaRESUMO
Alcohol abuse results in an increased incidence of pulmonary infection, in part attributable to impaired mucociliary clearance. Analysis of motility in mammalian airway cilia has revealed that alcohol impacts the ciliary dynein motors by a mechanism involving altered axonemal protein phosphorylation. Given the highly conserved nature of cilia, it is likely that the mechanisms for alcohol-induced ciliary dysfunction (AICD) are conserved. Thus we utilized the experimental advantages offered by the model organism, Chlamydomonas, to determine the precise effects of alcohol on ciliary dynein activity and identify axonemal phosphoproteins that are altered by alcohol exposure. Analysis of live cells or reactivated cell models showed that alcohol significantly inhibits ciliary motility in Chlamydomonas via a mechanism that is part of the axonemal structure. Taking advantage of informative mutant cells, we found that alcohol impacts the activity of the outer dynein arm. Consistent with this finding, alcohol exposure results in a significant reduction in ciliary beat frequency, a parameter of ciliary movement that requires normal outer dynein arm function. Using mutants that lack specific heavy-chain motor domains, we have determined that alcohol impacts the ß- and γ-heavy chains of the outer dynein arm. Furthermore, using a phospho-threonine-specific antibody, we determined that the phosphorylation state of DCC1 of the outer dynein arm-docking complex is altered in the presence of alcohol, and its phosphorylation correlates with AICD. These results demonstrate that alcohol targets specific outer dynein arm components and suggest that DCC1 is part of an alcohol-sensitive mechanism that controls outer dynein arm activity.
Assuntos
Axonema/metabolismo , Depressores do Sistema Nervoso Central/farmacologia , Chlamydomonas/metabolismo , Dineínas/metabolismo , Etanol/farmacologia , Axonema/genética , Chlamydomonas/genética , Cílios/genética , Cílios/metabolismo , Dineínas/genética , MutaçãoRESUMO
The motile cilium is a mechanical wonder, a cellular nanomachine that produces a high-speed beat based on a cycle of bends that move along an axoneme made of 9+2 microtubules. The molecular motors, dyneins, power the ciliary beat. The dyneins are compacted into inner and outer dynein arms, whose activity is highly regulated to produce microtubule sliding and axonemal bending. The switch point hypothesis was developed long ago to account for how sliding in the presence of axonemal radial spoke-central pair interactions causes the ciliary beat. Since then, a new genetic, biochemical, and structural complexity has been discovered, in part, with Chlamydomonas mutants, with high-speed, high-resolution analysis of movement and with cryoelectron tomography. We stand poised on the brink of new discoveries relating to the molecular control of motility that extend and refine our understanding of the basic events underlying the switching of arm activity and of bend formation and propagation.
RESUMO
To identify proteins specific to the proximal ciliary axoneme, we used iTRAQ to compare short (~2 µm) and full-length (~11 µm) axonemes of Chlamydomonas. Known compoents of the proximal axoneme such as minor dynein heavy chains and LF5 kinase as well as the ciliary tip proteins FAP256 (CEP104) and EB1 were enriched in short axonemes whereas proteins present along the length of the axoneme were of similar abundance in both samples. The iTRAQ analysis revealed that FAP93, a protein of unknown function, and protein phosphatase 2A (PP2A) are enriched in the short axonemes. Consistently, immunoblots show enrichment of FAP93 and PP2A in short axonemes and immunofluorescence confirms the localization of FAP93 and enrichment of PP2A at the proximal axoneme. Ciliary regeneration reveals that FAP93 assembles continuously but more slowly than other axonemal structures and terminates at 1.03 µm in steady-state axonemes. The length of FAP93 assembly correlates with ciliary length, demonstrating ciliary length-dependent assembly of FAP93. Dikaryon rescue experiments show that FAP93 can assemble independently of IFT transport. In addition, FRAP analysis of GFP-tagged FAP93 demonstrates that FAP93 is stably anchored in axoneme. FAP93 may function as a scaffold for assembly of other specific proteins at the proximal axoneme.
RESUMO
Recent evidence has revealed that the dynein motors and highly conserved signaling proteins are localized within the ciliary 9+2 axoneme. One key mechanism for regulation of motility is phosphorylation. Here, we review diverse evidence, from multiple experimental organisms, that ciliary motility is regulated by phosphorylation/dephosphorylation of the dynein arms through kinases and phosphatases that are anchored immediately adjacent to their axonemal substrates.
Assuntos
Axonema/enzimologia , Cílios/enzimologia , Sequência Conservada , Movimento , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Quinases/metabolismo , Animais , Axonema/metabolismo , Cílios/metabolismo , Humanos , Monoéster Fosfórico Hidrolases/química , Proteínas Quinases/químicaRESUMO
Motile cilia (also interchangeably called "flagella") are conserved organelles extending from the surface of many animal cells and play essential functions in eukaryotes, including cell motility and environmental sensing. Large motor complexes, the ciliary dyneins, are present on ciliary outer-doublet microtubules and drive movement of cilia. Ciliary dyneins are classified into two general types: the outer dynein arms (ODAs) and the inner dynein arms (IDAs). While ODAs are important for generation of force and regulation of ciliary beat frequency, IDAs are essential for control of the size and shape of the bend, features collectively referred to as waveform. Also, recent studies have revealed unexpected links between IDA components and human diseases. In spite of their importance, studies on IDAs have been difficult since they are very complex and composed for several types of IDA motors, each unique in composition and location in the axoneme. Thanks in part to genetic, biochemical, and structural analysis of Chlamydomonas reinhardtii, we are beginning to understand the organization and function of the ciliary IDAs. In this review, we summarize the composition of Chlamydomonas IDAs particularly focusing on each subunit, and discuss the assembly, conservation, and functional role(s) of these IDA subunits. Furthermore, we raise several additional questions/challenges regarding IDAs, and discuss future perspectives of IDA studies.
Assuntos
Chlamydomonas reinhardtii , Chlamydomonas , Animais , Axonema/metabolismo , Chlamydomonas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Cílios/metabolismo , Dineínas/metabolismo , Flagelos/metabolismo , Humanos , MutaçãoRESUMO
How ciliary and flagellar motility is regulated is a challenging problem. The flagellar movement in Chlamydomonas reinhardtii is in part regulated by phosphorylation of a 138 kD intermediate chain (IC138) of inner arm dynein f (also called I1). In the present study, we found that the axoneme of mutants lacking dynein f lacks a novel protein having ankyrin repeat motifs, registered as FAP120 in the flagellar proteome database. FAP120 is also missing or decreased in the axonemes of bop5, a mutant that has a mutation in the structural gene of IC138 but assembles the dynein f complex. Intriguingly, the amounts of FAP120 in the axonemes of different alleles of bop5 and several dynein f-lacking mutants roughly parallel their contents of IC138. These results suggest a weak but stoichiometric interaction between FAP120 and IC138. We propose that FAP120 functions in the regulatoryprocess as part of a protein complex involving IC138. Cell Motil. Cytoskeleton 2008. (c) 2008 Wiley-Liss, Inc.
Assuntos
Repetição de Anquirina , Chlamydomonas reinhardtii/metabolismo , Dineínas/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Axonema/metabolismo , Chlamydomonas reinhardtii/fisiologia , Cílios/metabolismo , Eletroforese em Gel de Poliacrilamida , Immunoblotting , Microscopia de Fluorescência , Mutação , Ligação Proteica , Proteínas de Protozoários/genéticaRESUMO
Biochemical studies of Chlamydomonas flagellar axonemes revealed that radial spoke protein (RSP) 3 is an A-kinase anchoring protein (AKAP). To determine the physiological role of PKA anchoring in the axoneme, an RSP3 mutant, pf14, was transformed with an RSP3 gene containing a mutation in the PKA-binding domain. Analysis of several independent transformants revealed that the transformed cells exhibit an unusual phenotype: a fraction of the cells swim normally; the remainder of the cells twitch feebly or are paralyzed. The abnormal/paralyzed motility is not due to an obvious deficiency of radial spoke assembly, and the phenotype cosegregates with the mutant RSP3. We postulated that paralysis was due to failure in targeting and regulation of axonemal cAMP-dependent protein kinase (PKA). To test this, reactivation experiments of demembranated cells were performed in the absence or presence of PKA inhibitors. Importantly, motility in reactivated cell models mimicked the live cell phenotype with nearly equal fractions of motile and paralyzed cells. PKA inhibitors resulted in a twofold increase in the number of motile cells, rescuing paralysis. These results confirm that flagellar RSP3 is an AKAP and reveal that a mutation in the PKA binding domain results in unregulated axonemal PKA activity and inhibition of normal motility.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Chlamydomonas reinhardtii/fisiologia , Flagelos/fisiologia , Proteínas de Protozoários/fisiologia , Sequência de Aminoácidos , Animais , Movimento Celular , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Sequência Conservada , Dados de Sequência Molecular , Proteínas de Plantas , RegeneraçãoRESUMO
Motility of cilia (also known as flagella in some eukaryotes) is based on axonemal doublet microtubule sliding that is driven by the dynein molecular motors. Dyneins are organized into intricately patterned inner and outer rows of arms, whose collective activity is to produce inter-microtubule movement. However, to generate a ciliary bend, not all dyneins can be active simultaneously. The switch point model accounts, in part, for how dynein motors are regulated during ciliary movement. On the basis of this model, supported by key direct experimental observations as well as more recent theoretical and structural studies, we are now poised to understand the mechanics of how ciliary dynein coordination controls axonemal bend formation and propagation.
Assuntos
Axonema/fisiologia , Cílios/fisiologia , Dineínas/fisiologia , Animais , Chlamydomonas , Cílios/ultraestrutura , Microscopia Crioeletrônica , Humanos , Modelos Biológicos , MovimentoRESUMO
We determined how the ciliary motor I1 dynein is transported. A specialized adapter, IDA3, facilitates I1 dynein attachment to the ciliary transporter called intraflagellar transport (IFT). Loading of IDA3 and I1 dynein on IFT is regulated by ciliary length.
Assuntos
Axonema/metabolismo , Chlamydomonas/metabolismo , Cílios/metabolismo , Dineínas/metabolismo , Flagelos/metabolismo , Cinesinas/metabolismo , Modelos Biológicos , Mutação , Proteínas de Plantas/metabolismo , Biossíntese de Proteínas , Transporte ProteicoRESUMO
Increased phosphorylation of dynein IC IC138 correlates with decreases in flagellar microtubule sliding and phototaxis defects. To test the hypothesis that regulation of IC138 phosphorylation controls flagellar bending, we cloned the IC138 gene. IC138 encodes a novel protein with a calculated mass of 111 kDa and is predicted to form seven WD-repeats at the C terminus. IC138 maps near the BOP5 locus, and bop5-1 contains a point mutation resulting in a truncated IC138 lacking the C terminus, including the seventh WD-repeat. bop5-1 cells display wild-type flagellar beat frequency but swim slower than wild-type cells, suggesting that bop5-1 is altered in its ability to control flagellar waveform. Swimming speed is rescued in bop5-1 transformants containing the wild-type IC138, confirming that BOP5 encodes IC138. With the exception of the roadblock-related light chain, LC7b, all the other known components of the I1 complex, including the truncated IC138, are assembled in bop5-1 axonemes. Thus, the bop5-1 motility phenotype reveals a role for IC138 and LC7b in the control of flagellar bending. IC138 is hyperphosphorylated in paralyzed flagellar mutants lacking radial spoke and central pair components, further indicating a role for the radial spokes and central pair apparatus in control of IC138 phosphorylation and regulation of flagellar waveform.
Assuntos
Chlamydomonas/enzimologia , Dineínas/química , Dineínas/metabolismo , Flagelos/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Clonagem Molecular , Dineínas/genética , Flagelos/genética , Humanos , Dados de Sequência Molecular , Mutação/genética , Fosforilação , Filogenia , Proteínas de Plantas , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Ciliary motility is crucial for the development and health of many organisms. Motility depends on the coordinated activity of multiple dynein motors arranged in a precise pattern on the outer doublet microtubules. Although significant progress has been made in elucidating the composition and organization of the dyneins, a comprehensive understanding of dynein regulation is lacking. Here, we focus on two conserved signaling complexes located at the base of the radial spokes. These include the I1/f inner dynein arm associated with radial spoke 1 and the calmodulin- and spoke-associated complex and the nexin-dynein regulatory complex associated with radial spoke 2. Current research is focused on understanding how these two axonemal hubs coordinate and regulate the dynein motors and ciliary motility.
Assuntos
Axonema/fisiologia , Cílios/fisiologia , Dineínas/metabolismo , Animais , Humanos , MovimentoRESUMO
Chlamydomonas reinhardtii is an outstanding model genetic organism for study of assembly of cilia. Here, methods are described for synchronization of ciliary regeneration in Chlamydomonas to analyze the sequence in which ciliary proteins assemble. In addition, the methods described allow analysis of the mechanisms involved in regulation of ciliary length, the proteins required for ciliary assembly, and the temporal expression of genes encoding ciliary proteins. Ultimately, these methods can contribute to discovery of conserved genes that when defective lead to abnormal ciliary assembly and human disease.
Assuntos
Chlamydomonas/fisiologia , Cílios/fisiologia , Regeneração , Transporte Biológico , Células Cultivadas , HumanosRESUMO
We developed quantitative assays to test the hypothesis that the N-DRC is required for integrity of the ciliary axoneme. We examined reactivated motility of demembranated drc cells, commonly termed "reactivated cell models." ATP-induced reactivation of wild-type cells resulted in the forward swimming of â¼90% of cell models. ATP-induced reactivation failed in a subset of drc cell models, despite forward motility in live drc cells. Dark-field light microscopic observations of drc cell models revealed various degrees of axonemal splaying. In contrast, >98% of axonemes from wild-type reactivated cell models remained intact. The sup-pf4 and drc3 mutants, unlike other drc mutants, retain most of the N-DRC linker that interconnects outer doublet microtubules. Reactivated sup-pf4 and drc3 cell models displayed nearly wild-type levels of forward motility. Thus, the N-DRC linker is required for axonemal integrity. We also examined reactivated motility and axoneme integrity in mutants defective in tubulin polyglutamylation. ATP-induced reactivation resulted in forward swimming of >75% of tpg cell models. Analysis of double mutants defective in tubulin polyglutamylation and different regions of the N-DRC indicate B-tubule polyglutamylation and the distal lobe of the linker region are both important for axonemal integrity and normal N-DRC function. © 2016 Wiley Periodicals, Inc.
Assuntos
Axonema/metabolismo , Chlamydomonas reinhardtii/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Plantas/metabolismo , Axonema/genética , Chlamydomonas reinhardtii/genética , Cílios/genética , Cílios/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Plantas/genéticaRESUMO
Kinesin-13, an end depolymerizer of cytoplasmic and spindle microtubules, also affects the length of cilia. However, in different models, depletion of kinesin-13 either lengthens or shortens cilia, and therefore the exact function of kinesin-13 in cilia remains unclear. We generated null mutations of all kinesin-13 paralogues in the ciliate Tetrahymena. One of the paralogues, Kin13Ap, localizes to the nuclei and is essential for nuclear divisions. The remaining two paralogues, Kin13Bp and Kin13Cp, localize to the cell body and inside assembling cilia. Loss of both Kin13Bp and Kin13Cp resulted in slow cell multiplication and motility, overgrowth of cell body microtubules, shortening of cilia, and synthetic lethality with either paclitaxel or a deletion of MEC-17/ATAT1, the α-tubulin acetyltransferase. The mutant cilia assembled slowly and contained abnormal tubulin, characterized by altered posttranslational modifications and hypersensitivity to paclitaxel. The mutant cilia beat slowly and axonemes showed reduced velocity of microtubule sliding. Thus kinesin-13 positively regulates the axoneme length, influences the properties of ciliary tubulin, and likely indirectly, through its effects on the axonemal microtubules, affects the ciliary dynein-dependent motility.
Assuntos
Axonema/fisiologia , Cílios/fisiologia , Cinesinas/genética , Tubulina (Proteína)/metabolismo , Acetiltransferases/genética , Axonema/genética , Cílios/genética , Técnicas de Inativação de Genes , Morfogênese/efeitos dos fármacos , Morfogênese/genética , Mutação , Paclitaxel/farmacologia , Processamento de Proteína Pós-Traducional , Tetrahymena thermophila/genética , Tetrahymena thermophila/fisiologiaRESUMO
Radial spokes are conserved macromolecular complexes that are essential for ciliary motility. A triplet of three radial spokes, RS1, RS2, and RS3, repeats every 96 nm along the doublet microtubules. Each spoke has a distinct base that docks to the doublet and is linked to different inner dynein arms. Little is known about the assembly and functions of individual radial spokes. A knockout of the conserved ciliary protein FAP206 in the ciliate Tetrahymena resulted in slow cell motility. Cryo-electron tomography showed that in the absence of FAP206, the 96-nm repeats lacked RS2 and dynein c. Occasionally, RS2 assembled but lacked both the front prong of its microtubule base and dynein c, whose tail is attached to the front prong. Overexpressed GFP-FAP206 decorated nonciliary microtubules in vivo. Thus FAP206 is likely part of the front prong and docks RS2 and dynein c to the microtubule.
Assuntos
Axonema/metabolismo , Dineínas/metabolismo , Microtúbulos/metabolismo , Proteínas de Protozoários/fisiologia , Tetrahymena/metabolismo , Cílios/metabolismo , Cílios/fisiologia , Tomografia com Microscopia Eletrônica , Técnicas de Inativação de Genes , Microtúbulos/ultraestrutura , Modelos Moleculares , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Tetrahymena/genética , Tetrahymena/ultraestruturaRESUMO
To determine mechanisms of assembly of ciliary dyneins, we focused on the Chlamydomonas inner dynein arm, I1 dynein, also known as dynein f. I1 dynein assembles in the cytoplasm as a 20S complex similar to the 20S I1 dynein complex isolated from the axoneme. The intermediate chain subunit, IC140 (IDA7), and heavy chains (IDA1, IDA2) are required for 20S I1 dynein preassembly in the cytoplasm. Unlike I1 dynein derived from the axoneme, the cytoplasmic 20S I1 complex will not rebind I1-deficient axonemes in vitro. To test the hypothesis that I1 dynein is transported to the distal tip of the cilia for assembly in the axoneme, we performed cytoplasmic complementation in dikaryons formed between wild-type and I1 dynein mutant cells. Rescue of I1 dynein assembly in mutant cilia occurred first at the distal tip and then proceeded toward the proximal axoneme. Notably, in contrast to other combinations, I1 dynein assembly was significantly delayed in dikaryons formed between ida7 and ida3. Furthermore, rescue of I1 dynein assembly required new protein synthesis in the ida7 × ida3 dikaryons. On the basis of the additional observations, we postulate that IDA3 is required for 20S I1 dynein transport. Cytoplasmic complementation in dikaryons using the conditional kinesin-2 mutant, fla10-1 revealed that transport of I1 dynein is dependent on kinesin-2 activity. Thus, I1 dynein complex assembly depends upon IFT for transport to the ciliary distal tip prior to docking in the axoneme.
Assuntos
Axonema/metabolismo , Chlamydomonas/metabolismo , Cílios/metabolismo , Dineínas/metabolismo , Flagelos/metabolismo , Transporte Biológico , Cinesinas/metabolismo , Modelos Biológicos , Mutação , Proteínas de Plantas/metabolismo , Biossíntese de ProteínasRESUMO
Assembly of cilia and flagella requires intraflagellar transport (IFT), a highly regulated kinesin-based transport system that moves cargo from the basal body to the tip of flagella [1]. The recruitment of IFT components to basal bodies is a function of flagellar length, with increased recruitment in rapidly growing short flagella [2]. The molecular pathways regulating IFT are largely a mystery. Because actin network disruption leads to changes in ciliary length and number, actin has been proposed to have a role in ciliary assembly. However, the mechanisms involved are unknown. In Chlamydomonas reinhardtii, conventional actin is found in both the cell body and the inner dynein arm complexes within flagella [3, 4]. Previous work showed that treating Chlamydomonas cells with the actin-depolymerizing compound cytochalasin D resulted in reversible flagellar shortening [5], but how actin is related to flagellar length or assembly remains unknown. Here we utilize small-molecule inhibitors and genetic mutants to analyze the role of actin dynamics in flagellar assembly in Chlamydomonas reinhardtii. We demonstrate that actin plays a role in IFT recruitment to basal bodies during flagellar elongation and that when actin is perturbed, the normal dependence of IFT recruitment on flagellar length is lost. We also find that actin is required for sufficient entry of IFT material into flagella during assembly. These same effects are recapitulated with a myosin inhibitor, suggesting that actin may act via myosin in a pathway by which flagellar assembly is regulated by flagellar length.