scholarly journals Mechanosignaling between central apparatus and radial spokes controls axonemal dynein activity

2014 ◽  
Vol 204 (5) ◽  
pp. 807-819 ◽  
Author(s):  
Toshiyuki Oda ◽  
Haruaki Yanagisawa ◽  
Toshiki Yagi ◽  
Masahide Kikkawa
Keyword(s):  
Regulatory Mechanisms ◽  
Flagellar Motility ◽  
Downstream Effectors ◽  
Concerted Activity ◽  
Radial Spokes ◽  
Axonemal Dynein ◽  
Central Apparatus ◽  
Dynein Arms ◽  
Protein Tags ◽  
Intermolecular Collision

Cilia/flagella are conserved organelles that generate fluid flow in eukaryotes. The bending motion of flagella requires concerted activity of dynein motors. Although it has been reported that the central pair apparatus (CP) and radial spokes (RSs) are important for flagellar motility, the molecular mechanism underlying CP- and RS-mediated dynein regulation has not been identified. In this paper, we identified nonspecific intermolecular collision between CP and RS as one of the regulatory mechanisms for flagellar motility. By combining cryoelectron tomography and motility analyses of Chlamydomonas reinhardtii flagella, we show that binding of streptavidin to RS heads paralyzed flagella. Moreover, the motility defect in a CP projection mutant could be rescued by the addition of exogenous protein tags on RS heads. Genetic experiments demonstrated that outer dynein arms are the major downstream effectors of CP- and RS-mediated regulation of flagellar motility. These results suggest that mechanosignaling between CP and RS regulates dynein activity in eukaryotic flagella.

scholarly journals Chlamydomonas WDR92 in association with R2TP-like complex and multiple DNAAFs to regulate ciliary dynein preassembly

2018 ◽  
Vol 11 (9) ◽  
pp. 770-780 ◽  
Author(s):  
Guang Liu ◽  
Limei Wang ◽  
Junmin Pan
Keyword(s):  
Mass Spectrometry ◽  
Insertional Mutagenesis ◽  
The Other ◽  
Cytoplasmic Protein ◽  
Flagellar Motility ◽  
Heavy Chains ◽  
The Third ◽  
Axonemal Dynein ◽  
Dynein Arms ◽  
Dna Insertional Mutagenesis

Abstract The motility of cilia or eukaryotic flagella is powered by the axonemal dyneins, which are preassembled in the cytoplasm by proteins termed dynein arm assembly factors (DNAAFs) before being transported to and assembled on the ciliary axoneme. Here, we characterize the function of WDR92 in Chlamydomonas. Loss of WDR92, a cytoplasmic protein, in a mutant wdr92 generated by DNA insertional mutagenesis resulted in aflagellate cells or cells with stumpy or short flagella, disappearance of axonemal dynein arms, and diminishment of dynein arm heavy chains in the cytoplasm, suggesting that WDR92 is a DNAAF. Immunoprecipitation of WDR92 followed by mass spectrometry identified inner dynein arm heavy chains and multiple DNAAFs including RuvBL1, RPAP3, MOT48, ODA7, and DYX1C. The PIH1 domain-containing protein MOT48 formed a R2TP-like complex with RuvBL1/2 and RPAP3, while PF13, another PIH1 domain-containing protein with function in dynein preassembly, did not. Interestingly, the third PIH1 domain-containing protein TWI1 was not related to flagellar motility. WDR92 physically interacted with the R2TP-like complex and the other identified DNNAFs. Our data suggest that WDR92 functions in association with the HSP90 co-chaperone R2TP-like complex as well as linking other DNAAFs in dynein preassembly.

scholarly journals Regulation of flagellar motility by the conserved flagellar protein CG34110/Ccdc135/FAP50

2011 ◽  
Vol 22 (7) ◽  
pp. 976-987 ◽  
Author(s):  
Yong Yang ◽  
Deborah A. Cochran ◽  
Mary D. Gargano ◽  
Iryna King ◽  
Nayef K. Samhat ◽  
...  
Keyword(s):  
Flagellar Motility ◽  
Genetic Screens ◽  
Sperm Flagellum ◽  
Respiratory Epithelia ◽  
Downstream Effectors ◽  
Radial Spokes ◽  
Flagellar Proteins ◽  
Flagellar Protein ◽  
Cilia And Flagella ◽  
Lost Boys

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.

scholarly journals Three-dimensional structure of the radial spokes reveals heterogeneity and interactions with dyneins in Chlamydomonas flagella

2012 ◽  
Vol 23 (1) ◽  
pp. 111-120 ◽  
Author(s):  
Cynthia F. Barber ◽  
Thomas Heuser ◽  
Blanca I. Carbajal-González ◽  
Vladimir V. Botchkarev ◽  
Daniela Nicastro
Keyword(s):  
Electron Tomography ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Flagellar Motility ◽  
Three Dimensional Structure ◽  
Radial Spoke ◽  
Cryo Electron Tomography ◽  
Radial Spokes ◽  
Axonemal Dynein ◽  
Subtomogram Averaging

Radial spokes (RSs) play an essential role in the regulation of axonemal dynein activity and thus of ciliary and flagellar motility. However, few details are known about the complexes involved. Using cryo–electron tomography and subtomogram averaging, we visualized the three-dimensional structure of the radial spokes in Chlamydomonas flagella in unprecedented detail. Unlike many other species, Chlamydomonas has only two spokes per axonemal repeat, RS1 and RS2. Our data revealed previously uncharacterized features, including two-pronged spoke bases that facilitate docking to the doublet microtubules, and that inner dyneins connect directly to the spokes. Structures of wild type and the headless spoke mutant pf17 were compared to define the morphology and boundaries of the head, including a direct RS1-to-RS2 interaction. Although the overall structures of the spokes are very similar, we also observed some differences, corroborating recent findings about heterogeneity in the docking of RS1 and RS2. In place of a third radial spoke we found an uncharacterized, shorter electron density named “radial spoke 3 stand-in,” which structurally bears no resemblance to RS1 and RS2 and is unaltered in the pf17 mutant. These findings demonstrate that radial spokes are heterogeneous in structure and may play functionally distinct roles in axoneme regulation.

scholarly journals Oscillatory movement of a dynein-microtubule complex crosslinked with DNA origami

2021 ◽  
Author(s):  
Shimaa A. Abdellatef ◽  
Hisashi Tadakuma ◽  
Kangmin Yan ◽  
Takashi Fujiwara ◽  
Kodai Fukumoto ◽  
...  
Keyword(s):  
Basic Mechanism ◽  
Dna Origami ◽  
Mt Dna ◽  
Oscillatory Movement ◽  
Radial Spokes ◽  
Simple Model System ◽  
Regulatory Complex ◽  
Axonemal Dynein ◽  
Central Apparatus ◽  
Cilia And Flagella

AbstractDuring repetitive bending of cilia and flagella, axonemal dynein molecules move in an oscillatory manner along a microtubule (MT), but how the minus-end-directed motor dynein can oscillate back and forth is unknown. There are various factors that may regulate the dynein activities, e.g., the nexin-dynein regulatory complex, radial spokes, and central apparatus. In order to understand the basic mechanism of the oscillatory movement, we constructed a simple model system composed of MTs, outer-arm dyneins, and DNA origami that crosslinks the MTs. Electron microscopy (EM) showed patches of dynein molecules crossbridging two MTs in two opposite orientations; the oppositely oriented dyneins are expected to produce opposing forces. The optical trapping experiments showed that the dynein-MT-DNA-origami complex actually oscillate back and forth after photolysis of caged ATP. Intriguingly, the complex, when held at one end, showed repetitive bending motions. The results show that a simple system composed of ensembles of oppositely oriented dyneins, MTs, and inter-MT crosslinkers, without the additional regulatory structures, has an intrinsic ability to cause oscillation and repetitive bending motions.

scholarly journals Phosphoregulation of an Inner Dynein Arm Complex in Chlamydomonas reinhardtii Is Altered in Phototactic Mutant Strains

1997 ◽  
Vol 136 (1) ◽  
pp. 177-191 ◽  
Author(s):  
Stephen J. King ◽  
Susan K. Dutcher
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Kinase Inhibitor ◽  
Flagellar Motility ◽  
Biochemical Phenotype ◽  
Phototactic Behavior ◽  
Mutant Strains ◽  
Axonemal Dynein ◽  
Dynein Arms ◽  
Intermediate Chain

To gain a further understanding of axonemal dynein regulation, mutant strains of Chlamydomonas reinhardtii that had defects in both phototactic behavior and flagellar motility were identified and characterized. ptm1, ptm2, and ptm3 mutant strains exhibited motility phenotypes that resembled those of known inner dynein arm region mutant strains, but did not have biochemical or genetic phenotypes characteristic of other inner dynein arm mutations. Three other mutant strains had defects in the f class of inner dynein arms. Dynein extracts from the pf9-4 strain were missing the entire f complex. Strains with mutations in pf9/ida1, ida2, or ida3 failed to assemble the f dynein complex and did not exhibit phototactic behavior. Fractionated dynein from mia1-1 and mia2-1 axonemes exhibited a novel f class inner dynein arm biochemical phenotype; the 138-kD f intermediate chain was present in altered phosphorylation forms. In vitro axonemal dynein activity was reduced by the mia1-1 and mia2-1 mutations. The addition of kinase inhibitor restored axonemal dynein activity concomitant with the dephosphorylation of the 138-kD f intermediate chain. Dynein extracts from uni1-1 axonemes, which specifically assemble only one of the two flagella, contained relatively high levels of the altered phosphorylation forms of the 138-kD intermediate chain. We suggest that the f dynein complex may be phosphoregulated asymmetrically between the two flagella to achieve phototactic turning.

Mechanosignaling between central apparatus and radial spokes controls axonemal dynein activity

2014 ◽  
Vol 143 (4) ◽  
pp. 1434OIA11
Author(s):  
Toshiyuki Oda ◽  
Haruaki Yanagisawa ◽  
Toshiki Yagi ◽  
Masahide Kikkawa
Keyword(s):  
Radial Spokes ◽  
Axonemal Dynein ◽  
Central Apparatus

scholarly journals ptx1, a nonphototactic mutant of Chlamydomonas, lacks control of flagellar dominance.

1993 ◽  
Vol 120 (3) ◽  
pp. 733-741 ◽  
Author(s):  
C J Horst ◽  
G B Witman
Keyword(s):  
Regulatory System ◽  
Structural Defects ◽  
Cell Models ◽  
Flagellar Motility ◽  
Wild Type ◽  
Structural Localization ◽  
Calcium Dependent ◽  
Mutant Strains ◽  
Radial Spokes ◽  
Dynein Arms

A new mutant strain of Chlamydomonas, ptx1, has been identified which is defective in phototaxis. This strain swims with a rate and straightness of path comparable with that of wild-type cells, and retains the photoshock response. Thus, the mutation does not cause any gross defects in swimming ability or photoreception, and appears to be specific for phototaxis. Calcium is required for phototaxis in wild-type cells, and causes a concentration-dependent shift in flagellar dominance in reactivated, demembranated cell models. ptx1-reactivated models are defective in this calcium-dependent shift in flagellar dominance. This indicates that the mutation affects one or more components of the calcium-dependent axonemal regulatory system, and that this system mediates phototaxis. The reduction or absence of two 75-kD axonemal proteins correlates with the nonphototactic phenotype. Axonemal fractionation studies, and analysis of axonemes from mutant strains with known structural defects, failed to reveal the structural localization of the 75-kD proteins within the axoneme. The proteins are not components of the outer dynein arms, two of the three types of inner dynein arms, the radial spokes, or the central pair complex. Because changes in flagellar motility ultimately require the regulation of dynein activity, cell models from mutant strains defective in specific dynein arms were reactivated at various calcium concentrations. Mutants lacking the outer arms, or the I1 or I2 inner dynein arms, retain the wild-type calcium-dependent shift in flagellar dominance. Therefore, none of these arms are the sole mediators of phototaxis.

scholarly journals Regulation of Flagellar Dynein by Calcium and a Role for an Axonemal Calmodulin and Calmodulin-dependent Kinase

2002 ◽  
Vol 13 (9) ◽  
pp. 3303-3313 ◽  
Author(s):  
Elizabeth F. Smith
Keyword(s):  
Calcium Signaling ◽  
Signaling Pathway ◽  
Molecular Mechanism ◽  
Calcium Signal ◽  
Sliding Velocity ◽  
Flagellar Motility ◽  
Wild Type ◽  
Wild Type Level ◽  
Radial Spokes ◽  
Central Apparatus

Ciliary and flagellar motility is regulated by changes in intraflagellar calcium. However, the molecular mechanism by which calcium controls motility is unknown. We tested the hypothesis that calcium regulates motility by controlling dynein-driven microtubule sliding and that the central pair and radial spokes are involved in this regulation. We isolated axonemes from Chlamydomonasmutants and measured microtubule sliding velocity in buffers containing 1 mM ATP and various concentrations of calcium. In buffers with pCa > 8, microtubule sliding velocity in axonemes lacking the central apparatus (pf18 and pf15) was reduced compared with that of wild-type axonemes. In contrast, at pCa4, dynein activity in pf18 and pf15axonemes was restored to wild-type level. The calcium-induced increase in dynein activity in pf18 axonemes was inhibited by antagonists of calmodulin and calmodulin-dependent kinase II. Axonemes lacking the C1 central tubule (pf16) or lacking radial spoke components (pf14 and pf17) do not exhibit calcium-induced increase in dynein activity in pCa4 buffer. We conclude that calcium regulation of flagellar motility involves regulation of dynein-driven microtubule sliding, that calmodulin and calmodulin-dependent kinase II may mediate the calcium signal, and that the central apparatus and radial spokes are key components of the calcium signaling pathway.

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