-
Nature Communications Jun 2024Differentiation of male gametocytes into flagellated fertile male gametes relies on the assembly of axoneme, a major component of male development for mosquito...
Differentiation of male gametocytes into flagellated fertile male gametes relies on the assembly of axoneme, a major component of male development for mosquito transmission of the malaria parasite. RNA-binding protein (RBP)-mediated post-transcriptional regulation of mRNA plays important roles in eukaryotic sexual development, including the development of female Plasmodium. However, the role of RBP in defining the Plasmodium male transcriptome and its function in male gametogenesis remains incompletely understood. Here, we performed genome-wide screening for gender-specific RBPs and identified an undescribed male-specific RBP gene Rbpm1 in the Plasmodium. RBPm1 is localized in the nucleus of male gametocytes. RBPm1-deficient parasites fail to assemble the axoneme for male gametogenesis and thus mosquito transmission. RBPm1 interacts with the spliceosome E complex and regulates the splicing initiation of certain introns in a group of 26 axonemal genes. RBPm1 deficiency results in intron retention and protein loss of these axonemal genes. Intron deletion restores axonemal protein expression and partially rectifies axonemal defects in RBPm1-null gametocytes. Further splicing assays in both reporter and endogenous genes exhibit stringent recognition of the axonemal introns by RBPm1. The splicing activator RBPm1 and its target introns constitute an axonemal intron splicing program in the post-transcriptional regulation essential for Plasmodium male development.
Topics: Introns; RNA Splicing; RNA-Binding Proteins; Animals; Protozoan Proteins; Male; Axoneme; Female; Gametogenesis; Spliceosomes; Plasmodium berghei; Malaria; Plasmodium
PubMed: 38824128
DOI: 10.1038/s41467-024-49002-9 -
Developmental Dynamics : An Official... Jun 2024Approximately 7% of the males exhibit reduced fertility; however, the regulatory genes and pathways involved remain largely unknown. TBC1 domain family member 21...
BACKGROUND
Approximately 7% of the males exhibit reduced fertility; however, the regulatory genes and pathways involved remain largely unknown. TBC1 domain family member 21 (TBC1D21) contains a conserved RabGAP catalytic domain that induces GDP/GTP exchange to inactivate Rabs by interacting with microtubules. We previously reported that Tbc1d21-null mice exhibit severe sperm tail defects with a disrupted axoneme, and that TBC1D21 interacts with RAB10. However, the pathological mechanisms underlying the Tbc1d21 loss-induced sperm tail defects remain unknown.
RESULTS
Murine sperm from wild-type and Tbc1d21-null mice were comparatively analyzed using proteomic assays. Over 1600 proteins were identified, of which 15 were significantly up-regulated in Tbc1d21-null sperm. Notably, several tektin (TEKT) family proteins, belonging to a type of intermediate filament critical for stabilizing the microtubular structure of cilia and flagella, were significantly up-regulated in Tbc1d21 sperm. We also found that TBC1D21 interacts with TEKT1. In addition, TEKT1 co-localized with RAB10 during sperm tail formation. Finally, we found Tbc1d21-null sperm exhibited abnormal accumulation of TEKT1 in the midpiece region, accompanied by disrupted axonemal structures.
CONCLUSIONS
These results reveal that TBC1D21 modulates TEKTs protein localization in the axonemal transport system during sperm tail formation.
PubMed: 38822685
DOI: 10.1002/dvdy.716 -
Disease Models & Mechanisms May 2024Vertebrate photoreceptors are highly specialized retinal neurons that have cilium-derived membrane organelles called outer segments (OS), which function as platforms for...
Vertebrate photoreceptors are highly specialized retinal neurons that have cilium-derived membrane organelles called outer segments (OS), which function as platforms for phototransduction. Male germ cell-associated kinase (MAK) is a cilium-associated serine/threonine kinase, and its genetic mutation causes photoreceptor degeneration in mice and retinitis pigmentosa in humans. However, the role of MAK in photoreceptors is not fully understood. Here, we report that zebrafish mak mutants show rapid photoreceptor degeneration during embryonic development. In mak mutants, both cone and rod photoreceptors completely lack OSs and undergo apoptosis. Interestingly, zebrafish mak mutants fail to generate axonemes during photoreceptor ciliogenesis, whereas basal bodies are specified. These data suggest that MAK contributes to axoneme development in zebrafish, in contrast to mouse Mak mutants, which have elongated photoreceptor axonemes. Furthermore, the kinase activity of MAK is critical in ciliary axoneme development and photoreceptor survival. Thus, MAK is required for ciliogenesis and OS formation in zebrafish photoreceptors to ensure intracellular protein transport and photoreceptor survival.
PubMed: 38813692
DOI: 10.1242/dmm.050618 -
Journal of Cell Science Oct 2024The definitive demonstration of protein localization on primary cilia has been a challenge for cilia biologists. Primary cilia are solitary thread-like projections that...
The definitive demonstration of protein localization on primary cilia has been a challenge for cilia biologists. Primary cilia are solitary thread-like projections that have a specialized protein composition, but as the ciliary structure overlays the cell membrane and other cell parts, the identity of ciliary proteins are difficult to ascertain by conventional imaging approaches like immunofluorescence microscopy. Surface scanning electron microscopy combined with immunolabeling (immuno-SEM) bypasses some of these indeterminacies by unambiguously showing protein expression in the context of the three-dimensional ultrastructure of the cilium. Here, we apply immuno-SEM to specifically identify proteins on the primary cilia of mouse and human pancreatic islets, including post-translationally modified tubulin, intraflagellar transport (IFT)88, the small GTPase Arl13b, as well as subunits of axonemal dynein. Key parameters in sample preparation, immunolabeling and imaging acquisition are discussed to facilitate similar studies by others in the cilia research community.
Topics: Cilia; Animals; Humans; Mice; Islets of Langerhans; Microscopy, Electron, Scanning
PubMed: 38804679
DOI: 10.1242/jcs.262038 -
Journal of Physiological Investigation Mar 2024Uterine adenomyosis is an estrogen-dependent chronic inflammatory condition and may cause painful symptoms, abnormal uterine bleeding, and/or subfertility/infertility.... (Review)
Review
Uterine adenomyosis is an estrogen-dependent chronic inflammatory condition and may cause painful symptoms, abnormal uterine bleeding, and/or subfertility/infertility. It is characterized by the presence of endometrial glands and stroma within the myometrium causing enlargement of the uterus as a result of reactive hyperplastic and/or hypertrophic change of the surrounding myometrium. Similar to endometriosis, adenomyosis has a negative impact on female fertility. Abnormal uterotubal sperm transport, tissue inflammation, and the toxic effect of chemical mediators have been proposed as contributing factors. Inflammation-induced damage of the mucosal cilia in the fallopian tube has been reported. Besides other proposed mechanisms, our most recent study with transmission electron microscopy analysis indicated that microvilli damage and an axonemal alteration in the apical endometria occur in response to endometrial inflammation. This may be involved in the negative fertility outcome in women with adenomyosis. We present a critical analysis of the literature data concerning the mechanistic basis of infertility in women with adenomyosis and its impact on fertility outcome.
Topics: Humans; Female; Adenomyosis; Infertility, Female; Endometrium; Cilia
PubMed: 38780290
DOI: 10.4103/ejpi.EJPI-D-24-00032 -
Cytoskeleton (Hoboken, N.J.) May 2024In this review, we provide a condensed overview of what is currently known about the mechanical functioning of the flagellar/ciliary axoneme. We also present a list of... (Review)
Review
In this review, we provide a condensed overview of what is currently known about the mechanical functioning of the flagellar/ciliary axoneme. We also present a list of 10 specific areas where our current knowledge is incomplete and explain the benefits of further experimental investigation. Many of the physical parameters of the axoneme and its component parts have not been determined. This limits our ability to understand how the axoneme structure contributes to its functioning in several regards. It restricts our ability to understand how the mechanics of the structure contribute to the regulation of motor function. It also confines our ability to understand the three-dimensional workings of the axoneme and how various beating modes are accomplished. Lastly, it prevents accurate computational modeling of the axoneme in three-dimensions.
PubMed: 38780123
DOI: 10.1002/cm.21879 -
Journal of Assisted Reproduction and... May 2024In this study, we investigated the role of a newly identified homozygous variant (c.1245 + 6T > C) in the CFAP61 gene in the development of multiple...
In this study, we investigated the role of a newly identified homozygous variant (c.1245 + 6T > C) in the CFAP61 gene in the development of multiple morphologically abnormal flagella (MMAF) in an infertile patient. Using exome sequencing, we identified this variant, which led to exon 12 skipping and the production of a truncated CFAP61 protein. Transmission electron microscopy analysis of the patient's spermatozoa revealed various flagellar abnormalities, including defective nuclear chromatin condensation, axoneme disorganization, and mitochondria embedded in residual cytoplasmic droplets. Despite a fertilization rate of 83.3% through ICSI, there was no successful pregnancy due to poor embryo quality.Our findings suggest a link between the identified CFAP61 variant and MMAF, indicating potential disruption in radial spokes' assembly or function crucial for normal ciliary motility. Furthermore, nearly half of the observed sperm heads displayed chromatin condensation defects, possibly contributing to the low blastulation rate. This case underscores the significance of genetic counseling and testing, particularly for couples dealing with infertility and MMAF. Early identification of such genetic variants can guide appropriate interventions and improve reproductive outcomes.
PubMed: 38775994
DOI: 10.1007/s10815-024-03139-0 -
EMBO Reports Jun 2024Alpha, beta, and gamma tubulins are essential building blocks for all eukaryotic cells. The functions of the non-canonical tubulins, delta, epsilon, and zeta, however,...
Alpha, beta, and gamma tubulins are essential building blocks for all eukaryotic cells. The functions of the non-canonical tubulins, delta, epsilon, and zeta, however, remain poorly understood and their requirement in mammalian development untested. Herein we have used a spermatogenesis model to define epsilon tubulin (TUBE1) function in mice. We show that TUBE1 is essential for the function of multiple complex microtubule arrays, including the meiotic spindle, axoneme and manchette and in its absence, there is a dramatic loss of germ cells and male sterility. Moreover, we provide evidence for the interplay between TUBE1 and katanin-mediated microtubule severing, and for the sub-specialization of individual katanin paralogs in the regulation of specific microtubule arrays.
Topics: Animals; Male; Microtubules; Tubulin; Mice; Katanin; Spermatogenesis; Adenosine Triphosphatases; Germ Cells; Spindle Apparatus; Spermatozoa; Infertility, Male; Mice, Knockout; Axoneme
PubMed: 38773322
DOI: 10.1038/s44319-024-00159-w -
The Journal of Biological Chemistry Jun 2024Motile cilia on the cell surface produce fluid flows in the body and abnormalities in motile cilia cause primary ciliary dyskinesia. Dynein axonemal assembly factor 6...
Motile cilia on the cell surface produce fluid flows in the body and abnormalities in motile cilia cause primary ciliary dyskinesia. Dynein axonemal assembly factor 6 (DNAAF6), a causative gene of primary ciliary dyskinesia, was isolated as an interacting protein with La ribonucleoprotein 6 (LARP6) that regulates ciliogenesis in multiciliated cells (MCCs). In MCCs of Xenopus embryos, LARP6 and DNAAF6 were colocalized in biomolecular condensates termed dynein axonemal particles and synergized to control ciliogenesis. Moreover, tubulin alpha 1c-like mRNA encoding α-tubulin protein, that is a major component of ciliary axoneme, was identified as a target mRNA regulated by binding LARP6. While DNAAF6 was necessary for high α-tubulin protein expression near the apical side of Xenopus MCCs during ciliogenesis, its mutant, which abolishes binding with LARP6, was unable to restore the expression of α-tubulin protein near the apical side of MCCs in Xenopus DNAAF6 morphant. These results indicated that the binding of LARP6 and DNAAF6 in dynein axonemal particles regulates highly expressed α-tubulin protein near the apical side of Xenopus MCCs during ciliogenesis.
Topics: Cilia; Animals; Ribonucleoproteins; Tubulin; Xenopus laevis; Xenopus Proteins; Humans; SS-B Antigen; Autoantigens; Protein Binding; Axoneme; RNA, Messenger
PubMed: 38762183
DOI: 10.1016/j.jbc.2024.107373 -
Science China. Life Sciences May 2024The complexities of energy transfer mechanisms in the flagella of mammalian sperm flagella have been intensively investigated and demonstrate significant diversity...
The complexities of energy transfer mechanisms in the flagella of mammalian sperm flagella have been intensively investigated and demonstrate significant diversity across species. Enzymatic shuttles, particularly adenylate kinase (AK) and creatine kinase (CK), are pivotal in the efficient transfer of intracellular ATP, showing distinct tissue- and species-specificity. Here, the expression profiles of AK and CK were investigated in mice and found to fall into four subgroups, of which Subgroup III AKs were observed to be unique to the male reproductive system and conserved across chordates. Both AK8 and AK9 were found to be indispensable to male reproduction after analysis of an infertile male cohort. Knockout mouse models showed that AK8 and AK9 were central to promoting sperm motility. Immunoprecipitation combined with mass spectrometry revealed that AK8 and AK9 interact with the radial spoke (RS) of the axoneme. Examination of various human and mouse sperm samples with substructural damage, including the presence of multiple RS subunits, showed that the head of radial spoke 3 acts as an adapter for AK9 in the flagellar axoneme. Using an ATP probe together with metabolomic analysis, it was found that AK8 and AK9 cooperatively regulated ATP transfer in the axoneme, and were concentrated at sites associated with energy consumption in the flagellum. These findings indicate a novel function for RS beyond its structural role, namely, the regulation of ATP transfer. In conclusion, the results expand the functional spectrum of AK proteins and suggest a fresh model regarding ATP transfer within mammalian flagella.
PubMed: 38761355
DOI: 10.1007/s11427-023-2539-1