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):  
Signal Transduction ◽  
Male Infertility ◽  
Male Fertility ◽  
Gene Encoding ◽  
Ciliary Beating ◽  
Radial Spoke ◽  
Evolutionarily Conserved ◽  
Radial Spokes ◽  
Cilia And Flagella ◽  
Mammalian Spermatozoa

Cilia and flagella are ancient structures that achieve controlled motor functions through the coordinated interaction based on microtubules, and some attached projections. Radial spokes (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 radial spoke proteins (RSP1-23), with the roles of some radial spoke proteins remained unknown. Recently, RSP15 has been reported to be located to the stalk of RS2, but its homolog in mammals has not been explored. Herein, we show that Lrrc23 is an evolutionarily conserved testis-enriched gene encoding an RSP15 homolog in mice. We found that LRRC23 localizes to the RS complex within murine sperm flagella and interacts with RSPH3A/B. The knockout of Lrrc23 resulted in male infertility due to RS disorganization and impaired motility in murine spermatozoa, whereas the ciliary beating was unaffected significantly. 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.

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.

scholarly journals Ccdc113/Ccdc96 complex, a novel regulator of ciliary beating that connects radial spoke 3 to dynein g and the nexin link

PLoS Genetics ◽  
2021 ◽  
Vol 17 (3) ◽  
pp. e1009388
Author(s):  
Rafał Bazan ◽  
Adam Schröfel ◽  
Ewa Joachimiak ◽  
Martyna Poprzeczko ◽  
Gaia Pigino ◽  
...  
Keyword(s):  
Protein Composition ◽  
Ciliary Beating ◽  
Radial Spoke ◽  
Beat Pattern ◽  
Evolutionarily Conserved ◽  
Radial Spokes ◽  
Central Apparatus ◽  
And Function ◽  
Beating Frequency

Ciliary beating requires the coordinated activity of numerous axonemal complexes. The protein composition and role of radial spokes (RS), nexin links (N-DRC) and dyneins (ODAs and IDAs) is well established. However, how information is transmitted from the central apparatus to the RS and across other ciliary structures remains unclear. Here, we identify a complex comprising the evolutionarily conserved proteins Ccdc96 and Ccdc113, positioned parallel to N-DRC and forming a connection between RS3, dynein g, and N-DRC. Although Ccdc96 and Ccdc113 can be transported to cilia independently, their stable docking and function requires the presence of both proteins. Deletion of either CCDC113 or CCDC96 alters cilia beating frequency, amplitude and waveform. We propose that the Ccdc113/Ccdc96 complex transmits signals from RS3 and N-DRC to dynein g and thus regulates its activity and the ciliary beat pattern.

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 Biallelic variants in MAATS1 encoding CFAP91, a calmodulin-associated and spoke-associated complex protein, cause severe astheno-teratozoospermia and male infertility

2020 ◽  
Vol 57 (10) ◽  
pp. 708-716 ◽  
Author(s):  
Guillaume Martinez ◽  
Julie Beurois ◽  
Denis Dacheux ◽  
Caroline Cazin ◽  
Marie Bidart ◽  
...  
Keyword(s):  
Male Infertility ◽  
Sperm Cells ◽  
Gene Encoding ◽  
Infertile Men ◽  
Transmission Electron ◽  
Complex Protein ◽  
Radial Spokes ◽  
Gene Defects ◽  
Primary Male ◽  
And Function

BackgroundMultiple morphological abnormalities of the flagella (MMAF) consistently lead to male infertility due to a reduced or absent sperm motility defined as asthenozoospermia. Despite numerous genes recently described to be recurrently associated with MMAF, more than half of the cases analysed remain unresolved, suggesting that many yet uncharacterised gene defects account for this phenotypeMethodsExome sequencing was performed on 167 infertile men with an MMAF phenotype. Immunostaining and transmission electron microscopy (TEM) in sperm cells from affected individuals were performed to characterise the ultrastructural sperm defects. Gene inactivation using RNA interference (RNAi) was subsequently performed in Trypanosoma.ResultsWe identified six unrelated affected patients carrying a homozygous deleterious variants in MAATS1, a gene encoding CFAP91, a calmodulin-associated and spoke-associated complex (CSC) protein. TEM and immunostaining experiments in sperm cells showed severe central pair complex (CPC) and radial spokes defects. Moreover, we confirmed that the WDR66 protein is a physical and functional partner of CFAP91 into the CSC. Study of Trypanosoma MAATS1’s orthologue (TbCFAP91) highlighted high sequence and structural analogies with the human protein and confirmed the axonemal localisation of the protein. Knockdown of TbCFAP91 using RNAi impaired flagellar movement led to CPC defects in Trypanosoma as observed in humans.ConclusionsWe showed that CFAP91 is essential for normal sperm flagellum structure and function in human and Trypanosoma and that biallelic variants in this gene lead to severe flagellum malformations resulting in astheno-teratozoospermia and primary male infertility.

scholarly journals Structural insights into the cause of human RSPH4A primary ciliary dyskinesia

2021 ◽  
pp. mbc.E20-12-0806
Author(s):  
Yanhe Zhao ◽  
Justine Pinskey ◽  
Jianfeng Lin ◽  
Weining Yin ◽  
Patrick R. Sears ◽  
...  
Keyword(s):  
Primary Ciliary Dyskinesia ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Radial Spoke ◽  
Radial Spokes ◽  
Cilia And Flagella ◽  
Tract Infections ◽  
Ciliary Dyskinesia

Cilia and flagella are evolutionarily conserved eukaryotic organelles involved in cell motility and signaling. In humans, mutations in Radial Spoke Head Protein 4 homolog A ( RSPH4A) can lead to primary ciliary dyskinesia (PCD), a life-shortening disease characterized by chronic respiratory tract infections, abnormal organ positioning, and infertility. Despite its importance for human health, the location of RSPH4A in human cilia has not been resolved, and the structural basis of RSPH4A-/- PCD remains elusive. Here, we present the native, three-dimensional structure of RSPH4A-/- human respiratory cilia using samples collected non-invasively from a PCD patient. Using cryo-electron tomography and subtomogram averaging, we compared the structures of control and RSPH4A-/- cilia, revealing primary defects in two of the three radial spokes (RSs) within the axonemal repeat and secondary (heterogeneous) defects in the central pair complex. Similar to RSPH1-/- cilia, the radial spoke heads of RS1 and RS2, but not RS3, were missing in RSPH4A-/- cilia. However, RSPH4A-/- cilia also exhibited defects within the arch domains adjacent to the RS1 and RS2 heads, which were not observed with RSPH1 loss. Our results provide insight into the underlying structural basis for RSPH4A-/- PCD and highlight the benefits of applying cryo-ET directly to patient samples for molecular structure determination. [Media: see text]

Novel homozygous CFAP69 mutations in humans and mice cause severe asthenoteratospermia with multiple morphological abnormalities of the sperm flagella

2018 ◽  
Vol 56 (2) ◽  
pp. 96-103 ◽  
Author(s):  
Xiaojin He ◽  
Weiyu Li ◽  
Huan Wu ◽  
Mingrong Lv ◽  
Wangjie Liu ◽  
...  
Keyword(s):  
Male Infertility ◽  
Sanger Sequencing ◽  
Han Chinese ◽  
Human Reproduction ◽  
Chinese Family ◽  
Loss Of Function ◽  
Gene Encoding ◽  
Morphological Abnormalities ◽  
Cilia And Flagella ◽  
Experimental Findings

BackgroundMale infertility is a major issue of human reproduction health. Asthenoteratospermia can impair sperm motility and cause male infertility. Asthenoteratospermia with multiple morphological abnormalities of the flagella (MMAF) presents abnormal spermatozoa with absent, bent, coiled, short and/or irregular-calibre flagella. Previous studies on MMAF reported that genetic defects in cilia-related genes (eg, AKAP4, DNAH1, CFAP43, CFAP44 and CFAP69) are the major cause of MMAF. However, the known MMAF-associated genes are only responsible for approximately 30% to 50% of human cases. We further investigated the cases with MMAF in search of additional genes mutated in this condition.Methods and resultsWe conducted whole exome sequencing in a male individual with MMAF from a consanguineous Han Chinese family. Sanger sequencing was also conducted in additional individuals with MMAF. Intriguingly, a homozygous frameshift mutation (p.Leu357Hisfs*11) was identified in the gene encoding CFAP69 (cilia and flagella-associated protein 69), which is highly expressed in testis. The subsequent Sanger sequencing of the CFAP69 coding regions among 34 additional individuals with MMAF revealed a case with homozygous nonsense mutation (p.Trp216*) of CFAP69. Both of these CFAP69 loss-of-function mutations were not present in the human population genome data archived in the 1000 Genomes Project and ExAC databases, nor in 875 individuals of two Han Chinese control populations. Furthermore, we generated the knockout model in mouse orthologue Cfap69 using the CRISPR-Cas9 technology. Remarkably, male Cfap69-knockout mice manifested with MMAF phenotypes.ConclusionOur experimental findings elucidate that homozygous loss-of-function mutations in CFAP69 can lead to asthenoteratospermia with MMAF in humans and mice.

scholarly journals CFAP61 is required for sperm flagellum formation and male fertility in human and mouse

2021 ◽  
Author(s):  
Siyu Liu ◽  
Jintao Zhang ◽  
Zine Eddine Kherraf ◽  
Shuya Sun ◽  
Xin Zhang ◽  
...  
Keyword(s):  
Male Fertility ◽  
Primary Ciliary Dyskinesia ◽  
Exon Skipping ◽  
Intraflagellar Transport ◽  
Transport Proteins ◽  
Radial Spoke ◽  
Sperm Flagellum ◽  
Cilia And Flagella ◽  
Ciliary Dyskinesia ◽  
Human And Mouse

Defects in the structure or motility of cilia and flagella may lead to severe diseases such as primary ciliary dyskinesia (PCD), a multisystemic disorder with heterogeneous manifestations affecting primarily respiratory and reproductive functions. We report that CFAP61 is a conserved component of the Calmodulin and radial Spoke associated Complex (CSC) of cilia. We find that a CFAP61 splice variant, c.143+5G>A, causes exon skipping in human, inducing a multiple morphological abnormalities of the flagella (MMAF) phenotype. We generated Cfap61 knockout mice that recapitulate the infertility phenotype of the human CFAP61 mutation, but without other symptoms usually observed in PCD. We find that CFAP61 interacts with the CSC, radial spoke stalk and RS head. During early stages of Cfap61-/- spermatid development, the assembly of RS components is impaired. With the progress of spermiogenesis, the axoneme in Cfap61-/- cells becomes unstable and scatters, and the distribution of intraflagellar transport proteins is disrupted.

scholarly journals Bend propagation drives central pair rotation in Chlamydomonas reinhardtii flagella

2004 ◽  
Vol 166 (5) ◽  
pp. 709-715 ◽  
Author(s):  
David R. Mitchell ◽  
Masako Nakatsugawa
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Wild Type ◽  
Central Pair ◽  
Radial Spoke ◽  
Passive Response ◽  
Mutant Strains ◽  
Radial Spokes ◽  
Presence And Absence ◽  
Cilia And Flagella ◽  
Pair Interactions

Regulation of motile 9+2 cilia and flagella depends on interactions between radial spokes and a central pair apparatus. Although the central pair rotates during bend propagation in flagella of many organisms and rotation correlates with a twisted central pair structure, propulsive forces for central pair rotation and twist are unknown. Here we compared central pair conformation in straight, quiescent flagella to that in actively beating flagella using wild-type Chlamydomonas reinhardtii and mutants that lack radial spoke heads. Twists occur in quiescent flagella in both the presence and absence of spoke heads, indicating that spoke–central pair interactions are not needed to generate torque for twisting. Central pair orientation in propagating bends was also similar in wild type and spoke head mutant strains, thus orientation is a passive response to bend formation. These results indicate that bend propagation drives central pair rotation and suggest that dynein regulation by central pair–radial spoke interactions involves passive central pair reorientation to changes in bend plane.

scholarly journals The CSC is required for complete radial spoke assembly and wild-type ciliary motility

2011 ◽  
Vol 22 (14) ◽  
pp. 2520-2531 ◽  
Author(s):  
Erin E. Dymek ◽  
Thomas Heuser ◽  
Daniela Nicastro ◽  
Elizabeth F. Smith
Keyword(s):  
Calcium Binding ◽  
Critical Role ◽  
Artificial Microrna ◽  
Structural Studies ◽  
Wild Type ◽  
Radial Spoke ◽  
Radial Spokes ◽  
Dynein Arms ◽  
Cilia And Flagella

The ubiquitous calcium binding protein, calmodulin (CaM), plays a major role in regulating the motility of all eukaryotic cilia and flagella. We previously identified a CaM and Spoke associated Complex (CSC) and provided evidence that this complex mediates regulatory signals between the radial spokes and dynein arms. We have now used an artificial microRNA (amiRNA) approach to reduce expression of two CSC subunits in Chlamydomonas. For all amiRNA mutants, the entire CSC is lacking or severely reduced in flagella. Structural studies of mutant axonemes revealed that assembly of radial spoke 2 is defective. Furthermore, analysis of both flagellar beating and microtubule sliding in vitro demonstrates that the CSC plays a critical role in modulating dynein activity. Our results not only indicate that the CSC is required for spoke assembly and wild-type motility, but also provide evidence for heterogeneity among the radial spokes.

scholarly journals Flagellar Motility Contributes to Cytokinesis in Trypanosoma brucei and Is Modulated by an Evolutionarily Conserved Dynein Regulatory System

2006 ◽  
Vol 5 (4) ◽  
pp. 696-711 ◽  
Author(s):  
Katherine S. Ralston ◽  
Alana G. Lerner ◽  
Dennis R. Diener ◽  
Kent L. Hill
Keyword(s):  
Cell Division ◽  
Trypanosoma Brucei ◽  
Genetic Interaction ◽  
Regulatory System ◽  
Flagellar Motility ◽  
Central Pair ◽  
Radial Spoke ◽  
Flagellar Beat ◽  
Evolutionarily Conserved ◽  
Radial Spokes

ABSTRACT The flagellum of Trypanosoma brucei is a multifunctional organelle with critical roles in motility and other aspects of the trypanosome life cycle. Trypanin is a flagellar protein required for directional cell motility, but its molecular function is unknown. Recently, a trypanin homologue in Chlamydomonas reinhardtii was reported to be part of a dynein regulatory complex (DRC) that transmits regulatory signals from central pair microtubules and radial spokes to axonemal dynein. DRC genes were identified as extragenic suppressors of central pair and/or radial spoke mutations. We used RNA interference to ablate expression of radial spoke (RSP3) and central pair (PF16) components individually or in combination with trypanin. Both rsp3 and pf16 single knockdown mutants are immotile, with severely defective flagellar beat. In the case of rsp3, this loss of motility is correlated with the loss of radial spokes, while in the case of pf16 the loss of motility correlates with an aberrant orientation of the central pair microtubules within the axoneme. Genetic interaction between trypanin and PF16 is demonstrated by the finding that loss of trypanin suppresses the pf16 beat defect, indicating that the DRC represents an evolutionarily conserved strategy for dynein regulation. Surprisingly, we discovered that four independent mutants with an impaired flagellar beat all fail in the final stage of cytokinesis, indicating that flagellar motility is necessary for normal cell division in T. brucei. These findings present the first evidence that flagellar beating is important for cell division and open the opportunity to exploit enzymatic activities that drive flagellar beat as drug targets for the treatment of African sleeping sickness.

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