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 The CSC connects three major axonemal complexes involved in dynein regulation

2012 ◽  
Vol 23 (16) ◽  
pp. 3143-3155 ◽  
Author(s):  
Thomas Heuser ◽  
Erin E. Dymek ◽  
Jianfeng Lin ◽  
Elizabeth F. Smith ◽  
Daniela Nicastro
Keyword(s):  
Electron Tomography ◽  
Structural Heterogeneity ◽  
Flagellar Motility ◽  
Wild Type ◽  
Localization Model ◽  
Cryo Electron Tomography ◽  
Radial Spokes ◽  
Regulatory Complex ◽  
Health And Development ◽  
Cilia And Flagella

Motile cilia and flagella are highly conserved organelles that play important roles in human health and development. We recently discovered a calmodulin- and spoke-associ­ated complex (CSC) that is required for wild-type motility and for the stable assembly of a subset of radial spokes. Using cryo–electron tomography, we present the first structure-based localization model of the CSC. Chlamydomonas flagella have two full-length radial spokes, RS1 and RS2, and a shorter RS3 homologue, the RS3 stand-in (RS3S). Using newly developed techniques for analyzing samples with structural heterogeneity, we demonstrate that the CSC connects three major axonemal complexes involved in dynein regulation: RS2, the nexin–dynein regulatory complex (N-DRC), and RS3S. These results provide insights into how signals from the radial spokes may be transmitted to the N-DRC and ultimately to the dynein motors. Our results also indicate that although structurally very similar, RS1 and RS2 likely serve different functions in regulating flagellar motility.

scholarly journals LRRC23 is a conserved component of the radial spoke that is necessary for sperm motility and male fertility in mice

2021 ◽  
Author(s):  
Xin Zhang ◽  
Jiang Sun ◽  
Yonggang Lu ◽  
Jintao Zhang ◽  
Keisuke Shimada ◽  
...  
Keyword(s):  
Male Fertility ◽  
Gene Encoding ◽  
Ciliary Beating ◽  
Evolutionarily Conserved ◽  
Radial Spokes ◽  
Regulatory Complex ◽  
Dynein Arms ◽  
Cilia And Flagella ◽  
Pass Through ◽  
Mammalian Spermatozoa

Cilia and flagella are ancient structures that achieve controlled motor functions through the coordinated interaction of structures including dynein arms, radial spokes (RSs), microtubules, and the dynein regulatory complex (DRC). RSs facilitate the beating motion of these organelles by mediating signal transduction between dyneins and a central pair (CP) of singlet microtubules. RS complex isolation from Chlamydomonas axonemes enabled the detection of 23 different proteins (RSP1-23), with the roles of RSP13, RSP15, RSP18, RSP19, and RSP21 remained poorly understood. Herein, we show that Lrrc23 is an evolutionarily conserved testis-enriched gene encoding an RSP15 homolog in mice. Through immunoelectron microscopy, we demonstrate that LRRC23 localizes to the RS complex within murine sperm flagella. We further found that LRRC23 was able to interact with RSHP9 and RSPH3A/B. The knockout of Lrrc23 resulted in RS disorganization and impaired motility in murine spermatozoa, whereas the ciliary beating was unaffected by the loss of this protein. Spermatozoa lacking LRRC23 were unable to efficiently pass through the uterotubal junction and exhibited defective zona penetration. Together these data indicate that LRRC23 is a key regulator underpinning the integrity of RS complex within the flagella of mammalian spermatozoa, whereas it is dispensable in cilia.

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 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.

Introductory Remarks

1980 ◽  
Vol 38 ◽  
pp. 598-599
Author(s):  
H. Mohri
Keyword(s):  
Muscle Contraction ◽  
Experimental Evidence ◽  
Sea Urchin ◽  
Constant Length ◽  
Thin Filaments ◽  
Bridge Formation ◽  
Thick Filaments ◽  
Sliding Mechanism ◽  
Radial Spokes ◽  
Cilia And Flagella

In 1959, Afzelius observed the presence of two rows of arms projecting from each outer doublet microtubule of the so-called 9 + 2 pattern of cilia and flagella, and suggested a possibility that the outer doublet microtubules slide with respect to each other with the aid of these arms during ciliary and flagellar movement. The identification of the arms as an ATPase, dynein, by Gibbons (1963)strengthened this hypothesis, since the ATPase-bearing heads of myosin molecules projecting from the thick filaments pull the thin filaments by cross-bridge formation during muscle contraction. The first experimental evidence for the sliding mechanism in cilia and flagella was obtained by examining the tip patterns of molluscan gill cilia by Satir (1965) who observed constant length of the microtubules during ciliary bending. Further evidence for the sliding-tubule mechanism was given by Summers and Gibbons (1971), using trypsin-treated axonemal fragments of sea urchin spermatozoa. Upon the addition of ATP, the outer doublets telescoped out from these fragments and the total length reached up to seven or more times that of the original fragment. Thus, the arms on a certain doublet microtubule can walk along the adjacent doublet when the doublet microtubules are disconnected by digestion of the interdoublet links which connect them with each other, or the radial spokes which connect them with the central pair-central sheath complex as illustrated in Fig. 1. On the basis of these pioneer works, the sliding-tubule mechanism has been established as one of the basic mechanisms for ciliary and flagellar movement.

scholarly journals Tubulin glycylation controls axonemal dynein activity, flagellar beat, and male fertility

Science ◽  
2021 ◽  
Vol 371 (6525) ◽  
pp. eabd4914
Author(s):  
Sudarshan Gadadhar ◽  
Gonzalo Alvarez Viar ◽  
Jan Niklas Hansen ◽  
An Gong ◽  
Aleksandr Kostarev ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Male Fertility ◽  
Electron Tomography ◽  
Structural Basis ◽  
Cellular Functions ◽  
Flagellar Beat ◽  
Cryo Electron Tomography ◽  
Axonemal Dynein ◽  
Dynein Arms ◽  
Cilia And Flagella

Posttranslational modifications of the microtubule cytoskeleton have emerged as key regulators of cellular functions, and their perturbations have been linked to a growing number of human pathologies. Tubulin glycylation modifies microtubules specifically in cilia and flagella, but its functional and mechanistic roles remain unclear. In this study, we generated a mouse model entirely lacking tubulin glycylation. Male mice were subfertile owing to aberrant beat patterns of their sperm flagella, which impeded the straight swimming of sperm cells. Using cryo–electron tomography, we showed that lack of glycylation caused abnormal conformations of the dynein arms within sperm axonemes, providing the structural basis for the observed dysfunction. Our findings reveal the importance of microtubule glycylation for controlled flagellar beating, directional sperm swimming, and male fertility.

scholarly journals Dimeric heat shock protein 40 binds radial spokes for generating coupled power strokes and recovery strokes of 9 + 2 flagella

2008 ◽  
Vol 180 (2) ◽  
pp. 403-415 ◽  
Author(s):  
Chun Yang ◽  
Heather A. Owen ◽  
Pinfen Yang
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Adenosine Triphosphatase ◽  
Stable Conformation ◽  
Initiation And Propagation ◽  
Evolutionarily Conserved ◽  
Radial Spokes ◽  
Cilia And Flagella ◽  
Hsp 40

T-shape radial spokes regulate flagellar beating. However, the precise function and molecular mechanism of these spokes remain unclear. Interestingly, Chlamydomonas reinhardtii flagella lacking a dimeric heat shock protein (HSP) 40 at the spokehead–spokestalk juncture appear normal in length and composition but twitch actively while cells jiggle without procession, resembling a central pair (CP) mutant. HSP40− cells begin swimming upon electroporation with recombinant HSP40. Surprisingly, the rescue doesn't require the signature DnaJ domain. Furthermore, the His-Pro-Asp tripeptide that is essential for stimulating HSP70 adenosine triphosphatase diverges in candidate orthologues, including human DnaJB13. Video microscopy reveals hesitance in bend initiation and propagation as well as irregular stalling and stroke switching despite fairly normal waveform. The in vivo evidence suggests that the evolutionarily conserved HSP40 specifically transforms multiple spoke proteins into stable conformation capable of mechanically coupling the CP with dynein motors. This enables 9 + 2 cilia and flagella to bend and switch to generate alternate power strokes and recovery strokes.

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 Pcdp1 is a central apparatus protein that binds Ca2+-calmodulin and regulates ciliary motility

2010 ◽  
Vol 189 (3) ◽  
pp. 601-612 ◽  
Author(s):  
Christen G. DiPetrillo ◽  
Elizabeth F. Smith
Keyword(s):  
Primary Ciliary Dyskinesia ◽  
Calcium Concentration ◽  
Beat Frequency ◽  
Calcium Sensor ◽  
Central Pair ◽  
Ciliary Motility ◽  
Significant Impairment ◽  
Central Apparatus ◽  
Cilia And Flagella ◽  
Ciliary Dyskinesia

For all motile eukaryotic cilia and flagella, beating is regulated by changes in intraciliary calcium concentration. Although the mechanism for calcium regulation is not understood, numerous studies have shown that calmodulin (CaM) is a key axonemal calcium sensor. Using anti-CaM antibodies and Chlamydomonas reinhardtii axonemal extracts, we precipitated a complex that includes four polypeptides and that specifically interacts with CaM in high [Ca2+]. One of the complex members, FAP221, is an orthologue of mammalian Pcdp1 (primary ciliary dyskinesia protein 1). Both FAP221 and mammalian Pcdp1 specifically bind CaM in high [Ca2+]. Reduced expression of Pcdp1 complex members in C. reinhardtii results in failure of the C1d central pair projection to assemble and significant impairment of motility including uncoordinated bends, severely reduced beat frequency, and altered waveforms. These combined results reveal that the central pair Pcdp1 (FAP221) complex is essential for control of ciliary motility.

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