RESUMEN
Ciliary outer-arm dynein (OAD) consists of heavy chains (HCs), intermediate chains (ICs), and light chains (LCs), of which HCs are the motor proteins that produce force. Studies using the green alga Chlamydomonas have revealed that ICs and LCs form a complex (IC/LC tower) at the base of the OAD tail and play a crucial role in anchoring OAD to specific sites on the microtubule. In this study, we isolated a novel slow-swimming Chlamydomonas mutant deficient in the IC2 protein. This mutation, E279K, is in the third of the seven WD repeat domains. No apparent abnormality was observed in electron microscope observations of axonemes or in SDS-PAGE analyses of dynein subunits. To explore the reason for the lowered motility in this mutant, in vitro microtubule sliding experiments were performed, which revealed that the motor activity of the mutant OAD was lowered. In particular, a large difference was observed between wild type (WT) and the mutant in the microtubule sliding velocity in microtubule bundles formed with the addition of OAD: ~35.3 µm/sec (WT) and ~4.3 µm/sec (mutant). From this and other results, we propose that IC2 in an OAD interacts with the ß HC of the adjacent OAD, and that an OAD-OAD interaction is important for efficient beating of cilia and flagella.Key words: cilia, axoneme, dynein heavy chain, cooperativity.
Asunto(s)
Chlamydomonas , Dineínas , Dineínas/genética , Dineínas/metabolismo , Microtúbulos/metabolismo , Axonema/metabolismo , Cilios/metabolismo , Flagelos/metabolismo , Chlamydomonas/genética , Chlamydomonas/metabolismo , MutaciónRESUMEN
Chlamydomonas flagella display surface motility such that small polystyrene beads (microspheres) attached to the flagellar membrane move bidirectionally along the flagellum. This surface motility enables cells to glide on a solid substrate to which they are attached by the flagellar surface. Previous studies suggested that microsphere movement and gliding motility result from the movement of transmembrane glycoprotein(s) within the plane of the plasma membrane, driven by intraflagellar transport (IFT), which utilizes cytoplasmic dynein and kinesin-2. However, it is not well understood how a cell can continuously glide in one direction further than a single flagellar length. Here we show that, during microsphere translocation on the flagella of a non-motile mutant, pf18, some flagellar glycoproteins, including FMG-1B and FAP113, detach from the membrane and attach to the microspheres. We propose that such relocation of surface glycoproteins underlies the ability to glide over a long distance. Surface motility is likely common to cilia/flagella of various organisms, as a similar microsphere movement is observed in the apical ciliary tuft in sea urchin embryos.
Asunto(s)
Membrana Celular/fisiología , Chlamydomonas reinhardtii/fisiología , Flagelos/fisiología , Glicoproteínas/fisiología , Microesferas , Animales , Cilios/fisiología , Locomoción , Erizos de Mar/embriologíaRESUMEN
Outer arm dynein (OAD) in cilia and flagella is bound to the outer doublet microtubules every 24 nm. Periodic binding of OADs at specific sites is important for efficient cilia/flagella beating; however, the molecular mechanism that specifies OAD arrangement remains elusive. Studies using the green alga Chlamydomonas reinhardtii have shown that the OAD-docking complex (ODA-DC), a heterotrimeric complex present at the OAD base, functions as the OAD docking site on the doublet. We find that the ODA-DC has an ellipsoidal shape â¼24 nm in length. In mutant axonemes that lack OAD but retain the ODA-DC, ODA-DC molecules are aligned in an end-to-end manner along the outer doublets. When flagella of a mutant lacking ODA-DCs are supplied with ODA-DCs upon gamete fusion, ODA-DC molecules first bind to the mutant axonemes in the proximal region, and the occupied region gradually extends toward the tip, followed by binding of OADs. This and other results indicate that a cooperative association of the ODA-DC underlies its function as the OAD-docking site and is the determinant of the 24-nm periodicity.
Asunto(s)
Axonema/metabolismo , Dineínas/metabolismo , Sustancias Macromoleculares/metabolismo , Microtúbulos/metabolismo , Modelos Biológicos , Western Blotting , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Electroporación , Técnica del Anticuerpo Fluorescente , Microscopía Electrónica , Microscopía Fluorescente , Unión Proteica , Colorantes de Rosanilina , UltracentrifugaciónRESUMEN
Cilia and flagella are highly conserved organelles that have diverse roles in cell motility and sensing extracellular signals. Motility defects in cilia and flagella often result in primary ciliary dyskinesia. However, the mechanisms underlying cilia formation and function, and in particular the cytoplasmic assembly of dyneins that power ciliary motility, are only poorly understood. Here we report a new gene, kintoun (ktu), involved in this cytoplasmic process. This gene was first identified in a medaka mutant, and found to be mutated in primary ciliary dyskinesia patients from two affected families as well as in the pf13 mutant of Chlamydomonas. In the absence of Ktu/PF13, both outer and inner dynein arms are missing or defective in the axoneme, leading to a loss of motility. Biochemical and immunohistochemical studies show that Ktu/PF13 is one of the long-sought proteins involved in pre-assembly of dynein arm complexes in the cytoplasm before intraflagellar transport loads them for the ciliary compartment.
Asunto(s)
Axonema/metabolismo , Cilios/metabolismo , Dineínas/metabolismo , Proteínas de Peces/metabolismo , Oryzias , Proteínas/metabolismo , Animales , Axonema/química , Axonema/genética , Axonema/patología , Chlamydomonas/genética , Chlamydomonas/metabolismo , Cilios/química , Cilios/genética , Cilios/patología , Clonación Molecular , Células Epiteliales/citología , Proteínas de Peces/genética , Genes Recesivos/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Síndrome de Kartagener/genética , Síndrome de Kartagener/patología , Masculino , Ratones , Datos de Secuencia Molecular , Mutación/genética , Oryzias/embriología , Oryzias/genética , Oryzias/metabolismo , Unión Proteica , Proteínas/genética , Homología de Secuencia de Aminoácido , Motilidad Espermática , Testículo/citologíaRESUMEN
This review outlines the current knowledge of the functional diversity of axonemal dyneins, as revealed by studies with the model organism Chlamydomonas. Axonemal dyneins, which comprise outer and inner dynein arms, power cilia and flagella beating by producing sliding movements between adjacent outer-doublet microtubules. Outer- and inner-arm dyneins have traditionally been considered similar in structure and function. However, recent evidence suggests that they differ rather strikingly in subunit composition, axonemal arrangement, and molecular motor properties. We posit that these arms make up two largely independent motile systems; whereas outer-arm dynein can generate axonemal beating by itself under certain conditions, inner-arm dynein can generate beating only in cooperation with the central pair/radial spokes. This conclusion is supported by genome analyses of various organisms. Outer-arm dynein appears to be particularly important for nodal cilia of mammalian embryos that function for determination of left-right body asymmetry.
Asunto(s)
Dineínas Axonemales/metabolismo , Chlamydomonas/metabolismo , Regulación de la Expresión Génica/fisiología , Animales , Dineínas Axonemales/genética , Chlamydomonas/genética , Flagelos/metabolismo , Movimiento , MutaciónRESUMEN
In many phototrophic microorganisms and plants, chloroplasts change their positions relative to the incident light to achieve optimal photosynthesis. In the case of motile green algae, cells change their swimming direction by switching between positive and negative phototaxis, i.e., swimming toward or away from the light source, depending on environmental and internal conditions. However, little is known about the molecular signals that determine the phototactic direction. Using the green alga Chlamydomonas reinhardtii, we found that cellular reduction-oxidation (redox) poise plays a key role: Cells always exhibited positive phototaxis after treatment with reactive oxygen species (ROS) and always displayed negative phototaxis after treatment with ROS quenchers. The redox-dependent switching of the sign of phototaxis may contribute in turn to the maintenance of cellular redox homeostasis.
Asunto(s)
Chlamydomonas reinhardtii/fisiología , Chlamydomonas reinhardtii/efectos de la radiación , Procesos Fototróficos/fisiología , Antioxidantes/farmacología , Chlamydomonas reinhardtii/efectos de los fármacos , Óxidos N-Cíclicos/farmacología , Modelos Biológicos , Movimiento/efectos de los fármacos , Movimiento/fisiología , Movimiento/efectos de la radiación , Oxidación-Reducción , Procesos Fototróficos/efectos de los fármacos , Procesos Fototróficos/efectos de la radiación , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/farmacología , Transducción de Señal , Marcadores de SpinRESUMEN
Centriole duplication occurs once per cell cycle through the assembly of daughter centrioles on the side wall of pre-existing centrioles. Little is known about the molecules involved in the assembly of new centrioles. Here, we identify CRC70 as a Chlamydomonas protein with an important role in the accumulation of centriole proteins at the site of assembly. CRC70 contains a highly conserved ~50-amino-acid sequence shared by mammalian Cep70 and preferentially localizes to immature centrioles (the procentrioles). This localization is maintained in the mutant bld10, in which centriole formation is blocked before the assembly of centriolar microtubules. RNA interference (RNAi)-mediated knockdown of CRC70 produces flagella-less cells and inhibits the recruitment of other centriole components, such as SAS-6 and Bld10p to the centriole. Overexpression of CRC70 induces an accumulation of these proteins in discrete spots in the cytoplasm. Overexpression of EGFP-tagged CRC70 in mouse NIH3T3 cells causes the formation of structures apparently related to centrioles. These findings suggest that CRC70 is a member of a conserved protein family and functions as a scaffold for the assembly of the centriole precursor.
Asunto(s)
Centriolos/fisiología , Chlamydomonas/fisiología , Microtúbulos/fisiología , Proteínas de Plantas/fisiología , Secuencia de Aminoácidos , Animales , Centriolos/genética , Centriolos/metabolismo , Chlamydomonas/genética , Chlamydomonas/metabolismo , Chlamydomonas/ultraestructura , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Ratones , Microscopía Electrónica , Microtúbulos/genética , Microtúbulos/metabolismo , Datos de Secuencia Molecular , Células 3T3 NIH , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Interferencia de ARN , TransfecciónRESUMEN
Centrioles/basal bodies have a characteristic cylindrical structure consisting of nine triplet microtubules arranged in a rotational symmetry. How this elaborate structure is formed is a major unanswered question in cell biology [1, 2]. We previously identified a 170 kDa coiled-coil protein essential for the centriole formation in Chlamydomonas. This protein, Bld10p, is the first protein shown to localize to the cartwheel, a 9-fold symmetrical structure possibly functioning as the scaffold for the centriole-microtubule assembly [3]. Here, we report results by using a series of truncated Bld10p constructs introduced into a bld10 null mutant. Remarkably, a transformant (DeltaC2) in which 35% of Bld10p at the C terminus was deleted assembled centrioles with eight symmetrically arranged triplets, in addition to others with the normal nine triplets. The cartwheels in these eight-membered centrioles had spokes approximately 24% shorter than those in the wild-type, suggesting that the eight-triplet centrioles were formed because the cartwheel's smaller diameter. From the morphology of the cartwheel spoke in the DeltaC2 centriole and immunoelectron-microscope localization, we conclude that Bld10p is a major spoke-tip component that extends the cartwheel diameter and attaches triplet microtubules. These results provide the first experimental evidence for the crucial function of the cartwheel in centriolar assembly.
Asunto(s)
Proteínas Algáceas/metabolismo , Centriolos/metabolismo , Centriolos/ultraestructura , Chlamydomonas/metabolismo , Chlamydomonas/ultraestructura , Proteínas Protozoarias/metabolismo , Proteínas Algáceas/genética , Animales , Chlamydomonas/genética , Flagelos/metabolismo , Flagelos/ultraestructura , Genes Protozoarios , Microscopía Inmunoelectrónica , Mutación , Proteínas Protozoarias/genéticaRESUMEN
Centrioles consist of nine-triplet microtubules arranged in rotational symmetry. This structure is highly conserved among various eukaryotic organisms and serves as the base for the ciliary axoneme. Recently, several proteins such as SAS-6 have been identified as essential to the early process of centriole assembly, but the mechanism that produces the 9-fold symmetry is poorly understood. In C. elegans and Drosophila, SAS-6 has been suggested to function in the formation of a centriolar precursor, a central tube that then assembles nine-singlet microtubules on its surface. However, the generality of the central tube is not clear because in many other organisms, the first structure appearing in the centriole assembly is not a tube but a flat amorphous ring or a cartwheel-a structure with a hub and nine radiating spokes. Here we show that in Chlamydomonas the SAS-6 protein localizes to the central part of the cartwheel and that a null mutant of SAS-6, bld12, lacks that part. Intriguingly, this mutant frequently has centrioles composed of 7, 8, 10, or 11 triplets in addition to 9-triplet centrioles. We presume that, in many organisms, SAS-6 is an essential component of the cartwheel, a structure that stabilizes the 9-triplet structure.
Asunto(s)
Proteínas Algáceas/metabolismo , Centriolos/metabolismo , Proteínas Algáceas/genética , Animales , Centriolos/genética , Centriolos/ultraestructura , ChlamydomonasRESUMEN
Flagellar beating in Chlamydomonas was found to be activated by mechanical stimulation. Immediately after a wild-type cell suspension was vortexed, the average swimming velocity of cells increased from 130 mum/second to 150 mum/second, due to an elevation of flagellar beat frequency from approximately 60 Hz to approximately 70 Hz without detectable change in the flagellar waveforms. This response required outer arm dynein. Treatment with EGTA, Ca(2+)-channel blockers, or mechanosensitive-channel blockers inhibited it. In demembranated and reactivated cell models, a modest increase in Ca(2+) concentration elevated the axonemal beat frequency. These data indicate that the mechanical agitation increases beat frequency because it causes Ca(2+) influx into flagella, which then activates outer arm dynein.
Asunto(s)
Calcio/metabolismo , Chlamydomonas reinhardtii/fisiología , Dineínas/metabolismo , Flagelos/fisiología , Mecanotransducción Celular , Animales , Línea Celular , Movimiento Celular , Quelantes/farmacología , Chlamydomonas reinhardtii/efectos de los fármacos , Chlamydomonas reinhardtii/genética , Dineínas/efectos de los fármacos , Dineínas/genética , Ácido Egtácico/farmacología , Flagelos/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacosRESUMEN
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.
Asunto(s)
Repetición de Anquirina , Chlamydomonas reinhardtii/metabolismo , Dineínas/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Axonema/metabolismo , Chlamydomonas reinhardtii/fisiología , Cilios/metabolismo , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Microscopía Fluorescente , Mutación , Unión Proteica , Proteínas Protozoarias/genéticaRESUMEN
The single-cell green alga Chlamydomonas reinhardtii possesses two α-tubulin genes (tua1 and tua2) and two ß-tubulin genes (tub1 and tub2), with the two genes in each pair encoding identical amino acid sequences. Here, we screened an insertional library to establish eight disruptants with defective tua2, tub1, or tub2 expression. Most of the disruptants did not exhibit major defects in cell growth, flagellar length, or flagellar regeneration after amputation. Because few tubulin mutants of C. reinhardtii have been reported to date, we then used our disruptants, together with a tua1 disruptant obtained from the Chlamydomonas Library Project (CLiP), to isolate tubulin-mutants resistant to the anti-tubulin agents propyzamide (pronamide) or oryzalin. As a result of several trials, we obtained 8 strains bearing 7 different α-tubulin mutations and 12 strains bearing 7 different ß-tubulin mutations. One of the mutations is at a residue similar to that of a mutation site known to confer drug resistance in human cancer cells. Some strains had the same amino acid substitutions as those reported previously in C. reinhardtii; however, the mutants with single tubulin genes showed slightly stronger drug-resistance than the previous mutants that express the mutated tubulin in addition to the wild-type tubulin. Such increased drug-resistance may have facilitated sensitive detection of tubulin mutation. Single-tubulin-gene disruptants are thus an efficient background of generating tubulin mutants for the study of the structure-function relationship of tubulin.
Asunto(s)
Chlamydomonas reinhardtii , Genes de Plantas , Mutación , Proteínas de Plantas , Tubulina (Proteína) , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMEN
To investigate the force generation properties of Chlamydomonas axonemal inner-arm dyneins in response to external force, we analyzed microtubule gliding on dynein-coated surfaces under shear flow. When inner-arm dynein c was used, microtubule translocation in the downstream direction accelerated with increasing flow speed in a manner that depended on the dynein density and ATP concentration. In contrast, the microtubule translocation velocity in the upstream direction was unaffected by the flow speed. The number of microtubules on the glass surface was almost constant with and without flow, suggesting that gliding acceleration was not simply caused by weakened dynein-microtubule binding. With other inner-arm dynein species, the microtubule gliding velocity was unaffected by the flow regardless of the flow direction or nucleotide concentration. The flow-generated force acting on a single dynein was estimated to be as small as approximately 0.03 pN/dynein. These results indicate that dynein c possesses a ratchetlike property that allows acceleration only in one direction by a very small external force. This property should be important for slow- and fast-moving dyneins to function simultaneously within the axoneme.
Asunto(s)
Chlamydomonas/citología , Chlamydomonas/enzimología , Chlamydomonas/metabolismo , Dineínas/metabolismo , Microtúbulos/metabolismo , Animales , Transporte Biológico , CinéticaRESUMEN
How centrioles and basal bodies assemble is a long-standing puzzle in cell biology. To address this problem, we analyzed a novel basal body-defective Chlamydomonas reinhardtii mutant isolated from a collection of flagella-less mutants. This mutant, bld10, displayed disorganized mitotic spindles and cytoplasmic microtubules, resulting in abnormal cell division and slow growth. Electron microscopic observation suggested that bld10 cells totally lack basal bodies. The product of the BLD10 gene (Bld10p) was found to be a novel coiled-coil protein of 170 kD. Immunoelectron microscopy localizes Bld10p to the cartwheel, a structure with ninefold rotational symmetry positioned near the proximal end of the basal bodies. Because the cartwheel forms the base from which the triplet microtubules elongate, we suggest that Bld10p plays an essential role in an early stage of basal body assembly. A viable mutant having such a severe basal body defect emphasizes the usefulness of Chlamydomonas in studying the mechanism of basal body/centriole assembly by using a variety of mutants.
Asunto(s)
Proteínas Algáceas/metabolismo , Centriolos/metabolismo , Chlamydomonas reinhardtii/metabolismo , Flagelos/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Algáceas/genética , Proteínas Algáceas/aislamiento & purificación , Secuencia de Aminoácidos/genética , Animales , Secuencia de Bases/genética , Células Cultivadas , Centriolos/genética , Centriolos/ultraestructura , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/ultraestructura , ADN Complementario/análisis , ADN Complementario/genética , Flagelos/genética , Flagelos/ultraestructura , Inmunohistoquímica , Microscopía Electrónica , Microtúbulos/genética , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Modelos Animales , Datos de Secuencia Molecular , Orgánulos/genética , Orgánulos/metabolismo , Orgánulos/ultraestructura , Proteínas Protozoarias/genética , Proteínas Protozoarias/aislamiento & purificación , Huso Acromático/genética , Huso Acromático/metabolismo , Huso Acromático/ultraestructuraRESUMEN
Cilia and flagella have multiple dyneins in their inner and outer arms. Chlamydomonas inner-arm dynein contains at least seven major subspecies (dynein a to dynein g), of which all but dynein f (also called dynein I1) are the single-headed type that are composed of a single heavy chain, actin, and either centrin or a 28-kDa protein (p28). Dynein d was found to associate with two additional proteins of 38 kDa (p38) and 44 kDa (p44). Following the characterization of the p38 protein (R. Yamamoto, H. A. Yanagisawa, T. Yagi, and R. Kamiya, FEBS Lett. 580:6357-6360, 2006), we have identified p44 as a novel component of dynein d by using an immunoprecipitation approach. p44 is present along the length of the axonemes and is diminished, but not absent, in the ida4 and ida5 mutants, both lacking this dynein. In the ida5 axoneme, p44 and p38 appear to form a complex, suggesting that they constitute the docking site of dynein d on the outer doublet. p44 has potential homologues in other ciliated organisms. For example, the mouse homologue of p44, NYD-SP14, was found to be strongly expressed in tissues with motile cilia and flagella. These results suggest that inner-arm dynein d and its subunit organization are widely conserved.
Asunto(s)
Proteínas Algáceas/genética , Axonema/enzimología , Chlamydomonas reinhardtii/enzimología , Dineínas/química , Proteínas Protozoarias/química , Proteínas Algáceas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Movimiento Celular , Chlamydomonas reinhardtii/genética , Cilios/metabolismo , Secuencia Conservada , Dineínas/genética , Dineínas/metabolismo , Flagelos/genética , Flagelos/metabolismo , Técnica del Anticuerpo Fluorescente , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Immunoblotting , Ratones , Datos de Secuencia Molecular , Subunidades de Proteína , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Conejos , Homología de Secuencia de AminoácidoRESUMEN
The outer dynein arm of Chlamydomonas flagella contains three heavy chains (alpha, beta, and gamma), each of which exhibits motor activity. How they assemble and cooperate is of considerable interest. Here we report the isolation of a novel mutant, oda2-t, whose gamma heavy chain is truncated at about 30% of the sequence. While the previously isolated gamma chain mutant oda2 lacks the entire outer arm, oda2-t retains outer arms that contain alpha and beta heavy chains, suggesting that the N-terminal sequence (corresponding to the tail region) is necessary and sufficient for stable outer-arm assembly. Thin-section electron microscopy and image analysis localize the gamma heavy chain to a basal region of the outer-arm image in the axonemal cross section. The motility of oda2-t is lower than that of the wild type and oda11 (lacking the alpha heavy chain) but higher than that of oda2 and oda4-s7 (lacking the motor domain of the beta heavy chain). Thus, the outer-arm dynein lacking the gamma heavy-chain motor domain is partially functional. The availability of mutants lacking individual heavy chains should greatly facilitate studies on the structure and function of the outer-arm dynein.
Asunto(s)
Chlamydomonas/enzimología , Dineínas/metabolismo , Flagelos/enzimología , Mutación , Proteínas Protozoarias/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Western Blotting , Chlamydomonas/química , Chlamydomonas/genética , Chlamydomonas/fisiología , Dineínas/química , Dineínas/genética , Dineínas/ultraestructura , Flagelos/química , Flagelos/genética , Flagelos/fisiología , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/ultraestructuraRESUMEN
HSP40s are regarded as cochaperones, perpetually shuttling client polypeptides to HSP70s for refolding. However, many HSP40s that are central for disparate processes diverge from this paradigm. To elucidate the noncanonical mechanisms, we investigated HSP40 in the radial spoke (RS) complex in flagella. Disruption of the gene by the MRC1 transposon in Chlamydomonas resulted in jerky flagella. Traditional electron microscopy, cryo-electron tomography, and sub-tomogram analysis revealed RSs of various altered morphologies that, unexpectedly, differed between the two RS species. This indicates that HSP40 locks the RS into a functionally rigid conformation, facilitating its interactions with the adjacent central pair apparatus for transducing locally varied mechanical feedback, which permits rhythmic beating. Missing HSP40, like missing RSs, could be restored in a tip-to-base direction when HSP40 mutants fused with a HSP40 donor cell. However, without concomitant de novo RS assembly, the repair was exceedingly slow, suggesting HSP40/RS-coupled intraflagellar trafficking and assembly. Biochemical analysis and modeling uncovered spoke HSP40's cochaperone traits. On the basis of our data, we propose that HSP40 accompanies its client RS precursor when traveling to the flagellar tip. Upon arrival, both refold in concert to assemble into the mature configuration. HSP40's roles in chaperoning and structural maintenance shed new light on its versatility and flagellar biology.
Asunto(s)
Flagelos/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Axonema/metabolismo , Axonema/ultraestructura , Proteínas Bacterianas/metabolismo , Chlamydomonas , Elementos Transponibles de ADN/genética , Tomografía con Microscopio Electrónico , Flagelos/ultraestructura , Modelos Moleculares , Mutagénesis Insercional/genética , Mutación/genética , Unión ProteicaRESUMEN
Cilia and flagella are equipped with multiple species of dyneins that have diverse motor properties. To assess the properties of various axonemal dyneins of Chlamydomonas, in vitro microtubule translocation by isolated dyneins was examined with and without flow of the medium. With one inner-arm dynein species, dynein c, most microtubules became aligned parallel to the flow and translocated downstream after the onset of flow. When the flow was stopped, the gliding direction was gradually randomized. In contrast, with inner-arm dyneins d and g, microtubules tended to translocate at a shallow right angle to the flow. When the flow was stopped, each microtubule turned to the right, making a curved track. The clockwise translocation was not accompanied by lateral displacement, indicating that these dyneins generate torque that bends the microtubule. The torque generated by these dyneins in the axoneme may modulate the relative orientation between adjacent doublet microtubules and lead to more efficient functioning of total dyneins.
Asunto(s)
Dineínas/química , Dineínas/ultraestructura , Flagelos/química , Microtúbulos/química , Microtúbulos/ultraestructura , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/ultraestructura , Simulación por Computador , Modelos Químicos , Modelos Moleculares , Movimiento (Física) , Unión Proteica , Estrés Mecánico , TorqueRESUMEN
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.