-
Cell Jun 2023Sperm motility is crucial to reproductive success in sexually reproducing organisms. Impaired sperm movement causes male infertility, which is increasing globally. Sperm...
Sperm motility is crucial to reproductive success in sexually reproducing organisms. Impaired sperm movement causes male infertility, which is increasing globally. Sperm are powered by a microtubule-based molecular machine-the axoneme-but it is unclear how axonemal microtubules are ornamented to support motility in diverse fertilization environments. Here, we present high-resolution structures of native axonemal doublet microtubules (DMTs) from sea urchin and bovine sperm, representing external and internal fertilizers. We identify >60 proteins decorating sperm DMTs; at least 15 are sperm associated and 16 are linked to infertility. By comparing DMTs across species and cell types, we define core microtubule inner proteins (MIPs) and analyze evolution of the tektin bundle. We identify conserved axonemal microtubule-associated proteins (MAPs) with unique tubulin-binding modes. Additionally, we identify a testis-specific serine/threonine kinase that links DMTs to outer dense fibers in mammalian sperm. Our study provides structural foundations for understanding sperm evolution, motility, and dysfunction at a molecular level.
Topics: Male; Animals; Cattle; Sperm Tail; Sperm Motility; Semen; Microtubules; Axoneme; Spermatozoa; Mammals
PubMed: 37327785
DOI: 10.1016/j.cell.2023.05.026 -
American Journal of Human Genetics Jan 2022Asthenoteratozoospermia, defined as reduced sperm motility and abnormal sperm morphology, is a disorder with considerable genetic heterogeneity. Although previous...
Asthenoteratozoospermia, defined as reduced sperm motility and abnormal sperm morphology, is a disorder with considerable genetic heterogeneity. Although previous studies have identified several asthenoteratozoospermia-associated genes, the etiology remains unknown for the majority of affected men. Here, we performed whole-exome sequencing on 497 unrelated men with asthenoteratozoospermia and identified DNHD1 bi-allelic variants from eight families (1.6%). All detected variants were predicted to be deleterious via multiple bioinformatics tools. Hematoxylin and eosin (H&E) staining revealed that individuals with bi-allelic DNHD1 variants presented striking abnormalities of the flagella; transmission electron microscopy (TEM) further showed flagellar axoneme defects, including central pair microtubule (CP) deficiency and mitochondrial sheath (MS) malformations. In sperm from fertile men, DNHD1 was localized to the entire flagella of the normal sperm; however, it was nearly absent in the flagella of men with bi-allelic DNHD1 variants. Moreover, abundance of the CP markers SPAG6 and SPEF2 was significantly reduced in spermatozoa from men harboring bi-allelic DNHD1 variants. In addition, Dnhd1 knockout male mice (Dnhd1) exhibited asthenoteratozoospermia and infertility, a finding consistent with the sperm phenotypes present in human subjects with DNHD1 variants. The female partners of four out of seven men who underwent intracytoplasmic sperm injection therapy subsequently became pregnant. In conclusion, our study showed that bi-allelic DNHD1 variants cause asthenoteratozoospermia, a finding that provides crucial insights into the biological underpinnings of this disorder and should assist with counseling of affected individuals.
Topics: Alleles; Animals; Asthenozoospermia; Axoneme; Computational Biology; DNA Mutational Analysis; Disease Models, Animal; Dyneins; Flagella; Gene Frequency; Genetic Association Studies; Genetic Predisposition to Disease; Humans; Infertility, Male; Male; Mice; Mice, Knockout; Mitochondria; Mutation; Pedigree; Phenotype; Semen Analysis; Sperm Tail; Exome Sequencing
PubMed: 34932939
DOI: 10.1016/j.ajhg.2021.11.022 -
Proceedings of the National Academy of... Aug 2022Cilium formation and regeneration requires new protein synthesis, but the underlying cytosolic translational reprogramming remains largely unknown. Using ribosome...
Cilium formation and regeneration requires new protein synthesis, but the underlying cytosolic translational reprogramming remains largely unknown. Using ribosome footprinting, we performed global translatome profiling during cilia regeneration in and uncovered that flagellar genes undergo an early transcriptional activation but late translational repression. This pattern guided our identification of sphingolipid metabolism enzymes, including serine palmitoyltransferase (SPT), as essential regulators for ciliogenesis. Cryo-electron tomography showed that ceramide loss abnormally increased the membrane-axoneme distance and generated bulged cilia. We found that ceramides interact with intraflagellar transport (IFT) particle proteins that IFT motors transport along axoneme microtubules (MTs), suggesting that ceramide-IFT particle-IFT motor-MT interactions connect the ciliary membrane with the axoneme to form rod-shaped cilia. SPT-deficient vertebrate cells were defective in ciliogenesis, and SPT mutations from patients with hereditary sensory neuropathy disrupted cilia, which could be restored by sphingolipid supplementation. These results reveal a conserved role of sphingolipid in cilium formation and link compromised sphingolipid production with ciliopathies.
Topics: Axoneme; Ceramides; Chlamydomonas; Cilia; Flagella; Protein Transport; Regeneration; Sphingolipids
PubMed: 35895683
DOI: 10.1073/pnas.2201096119 -
Nature Jun 2023Motile cilia and flagella beat rhythmically on the surface of cells to power the flow of fluid and to enable spermatozoa and unicellular eukaryotes to swim. In humans,...
Motile cilia and flagella beat rhythmically on the surface of cells to power the flow of fluid and to enable spermatozoa and unicellular eukaryotes to swim. In humans, defective ciliary motility can lead to male infertility and a congenital disorder called primary ciliary dyskinesia (PCD), in which impaired clearance of mucus by the cilia causes chronic respiratory infections. Ciliary movement is generated by the axoneme, a molecular machine consisting of microtubules, ATP-powered dynein motors and regulatory complexes. The size and complexity of the axoneme has so far prevented the development of an atomic model, hindering efforts to understand how it functions. Here we capitalize on recent developments in artificial intelligence-enabled structure prediction and cryo-electron microscopy (cryo-EM) to determine the structure of the 96-nm modular repeats of axonemes from the flagella of the alga Chlamydomonas reinhardtii and human respiratory cilia. Our atomic models provide insights into the conservation and specialization of axonemes, the interconnectivity between dyneins and their regulators, and the mechanisms that maintain axonemal periodicity. Correlated conformational changes in mechanoregulatory complexes with their associated axonemal dynein motors provide a mechanism for the long-hypothesized mechanotransduction pathway to regulate ciliary motility. Structures of respiratory-cilia doublet microtubules from four individuals with PCD reveal how the loss of individual docking factors can selectively eradicate periodically repeating structures.
Topics: Humans; Male; Artificial Intelligence; Axonemal Dyneins; Axoneme; Cilia; Cryoelectron Microscopy; Flagella; Mechanotransduction, Cellular; Microtubules; Chlamydomonas reinhardtii; Ciliary Motility Disorders; Movement; Protein Conformation
PubMed: 37258679
DOI: 10.1038/s41586-023-06140-2 -
Cold Spring Harbor Perspectives in... Jan 2017The axoneme is the main extracellular part of cilia and flagella in eukaryotes. It consists of a microtubule cytoskeleton, which normally comprises nine doublets. In... (Review)
Review
The axoneme is the main extracellular part of cilia and flagella in eukaryotes. It consists of a microtubule cytoskeleton, which normally comprises nine doublets. In motile cilia, dynein ATPase motor proteins generate sliding motions between adjacent microtubules, which are integrated into a well-orchestrated beating or rotational motion. In primary cilia, there are a number of sensory proteins functioning on membranes surrounding the axoneme. In both cases, as the study of proteomics has elucidated, hundreds of proteins exist in this compartmentalized biomolecular system. In this article, we review the recent progress of structural studies of the axoneme and its components using electron microscopy and X-ray crystallography, mainly focusing on motile cilia. Structural biology presents snapshots (but not live imaging) of dynamic structural change and gives insights into the force generation mechanism of dynein, ciliary bending mechanism, ciliogenesis, and evolution of the axoneme.
Topics: Animals; Axoneme; Chlamydomonas; Cilia; Crystallography, X-Ray; Dyneins; Microscopy, Electron; Proteins
PubMed: 27601632
DOI: 10.1101/cshperspect.a028076 -
Cell Nov 2023To understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization,...
To understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization, and determine their structures in vitro. Here, we combined cellular cryoelectron tomography (cryo-ET) and AlphaFold2 modeling to address these questions and understand how mammalian sperm are built in situ. Our cellular cryo-ET and subtomogram averaging provided 6.0-Å reconstructions of axonemal microtubule structures. The well-resolved tertiary structures allowed us to unbiasedly match sperm-specific densities with 21,615 AlphaFold2-predicted protein models of the mouse proteome. We identified Tektin 5, CCDC105, and SPACA9 as novel microtubule-associated proteins. These proteins form an extensive interaction network crosslinking the lumen of axonemal doublet microtubules, suggesting their roles in modulating the mechanical properties of the filaments. Indeed, Tekt5 -/- sperm possess more deformed flagella with 180° bends. Together, our studies presented a cellular visual proteomics workflow and shed light on the in vivo functions of Tektin 5.
Topics: Animals; Male; Mice; Axoneme; Cryoelectron Microscopy; Flagella; Microtubules; Semen; Spermatozoa; Proteome
PubMed: 37865089
DOI: 10.1016/j.cell.2023.09.017 -
Cell Oct 2019The axoneme of motile cilia is the largest macromolecular machine of eukaryotic cells. In humans, impaired axoneme function causes a range of ciliopathies. Axoneme...
The axoneme of motile cilia is the largest macromolecular machine of eukaryotic cells. In humans, impaired axoneme function causes a range of ciliopathies. Axoneme assembly, structure, and motility require a radially arranged set of doublet microtubules, each decorated in repeating patterns with non-tubulin components. We use single-particle cryo-electron microscopy to visualize and build an atomic model of the repeating structure of a native axonemal doublet microtubule, which reveals the identities, positions, repeat lengths, and interactions of 38 associated proteins, including 33 microtubule inner proteins (MIPs). The structure demonstrates how these proteins establish the unique architecture of doublet microtubules, maintain coherent periodicities along the axoneme, and stabilize the microtubules against the repeated mechanical stress induced by ciliary motility. Our work elucidates the architectural principles that underpin the assembly of this large, repetitive eukaryotic structure and provides a molecular basis for understanding the etiology of human ciliopathies.
Topics: Axoneme; Cell Movement; Cilia; Ciliopathies; Cryoelectron Microscopy; Humans; Microtubule Proteins; Microtubules; Stress, Mechanical
PubMed: 31668805
DOI: 10.1016/j.cell.2019.09.030 -
Journal of Cell Science Dec 2022The assembly and maintenance of most cilia and eukaryotic flagella depends on intraflagellar transport (IFT), the bidirectional movement of multi-megadalton IFT trains... (Review)
Review
The assembly and maintenance of most cilia and eukaryotic flagella depends on intraflagellar transport (IFT), the bidirectional movement of multi-megadalton IFT trains along the axonemal microtubules. These IFT trains function as carriers, moving ciliary proteins between the cell body and the organelle. Whereas tubulin, the principal protein of cilia, binds directly to IFT particle proteins, the transport of other ciliary proteins and complexes requires adapters that link them to the trains. Large axonemal substructures, such as radial spokes, outer dynein arms and inner dynein arms, assemble in the cell body before attaching to IFT trains, using the adapters ARMC2, ODA16 and IDA3, respectively. Ciliary import of several membrane proteins involves the putative adapter tubby-like protein 3 (TULP3), whereas membrane protein export involves the BBSome, an octameric complex that co-migrates with IFT particles. Thus, cells employ a variety of adapters, each of which is substoichiometric to the core IFT machinery, to expand the cargo range of the IFT trains. This Review summarizes the individual and shared features of the known cargo adapters and discusses their possible role in regulating the transport capacity of the IFT pathway.
Topics: Dyneins; Flagella; Protein Transport; Axoneme; Cilia; Biological Transport; Membrane Proteins
PubMed: 36533425
DOI: 10.1242/jcs.260408 -
Seminars in Cell & Developmental Biology Jan 2023Ciliogenesis is a complex multistep process used to describe assembly of cilia and flagella. These organelles play essential roles in motility and signaling on the... (Review)
Review
Ciliogenesis is a complex multistep process used to describe assembly of cilia and flagella. These organelles play essential roles in motility and signaling on the surface of cells. Cilia are built at the distal ends of centrioles through the formation of an axoneme that is surrounded by the ciliary membrane. As is the case in the biogenesis of other cellular organelles, regulators of membrane trafficking play essential roles in ciliogenesis, albeit with a unique feature that membranes are organized around microtubule-based structures. Membrane association with the distal end of the centriole is a critical initiating step for ciliogenesis. Studies of this process in different cell types suggests that a singular mechanism may not be utilized to initiate cilium assembly. In this review, we focus on recent insights into cilium biogenesis and the roles membrane trafficking regulators play in described ciliogenesis mechanisms with relevance to human disease.
Topics: Humans; Centrioles; Axoneme; Cilia; Microtubules; Flagella
PubMed: 35351373
DOI: 10.1016/j.semcdb.2022.03.021 -
International Journal of Molecular... Sep 2021Primary cilia are non-motile, cell cycle-associated organelles that can be found on most vertebrate cell types. Comprised of microtubule bundles organised into an... (Review)
Review
Primary cilia are non-motile, cell cycle-associated organelles that can be found on most vertebrate cell types. Comprised of microtubule bundles organised into an axoneme and anchored by a mature centriole or basal body, primary cilia are dynamic signalling platforms that are intimately involved in cellular responses to their extracellular milieu. Defects in ciliogenesis or dysfunction in cilia signalling underlie a host of developmental disorders collectively referred to as ciliopathies, reinforcing important roles for cilia in human health. Whilst primary cilia have long been recognised to be present in striated muscle, their role in muscle is not well understood. However, recent studies indicate important contributions, particularly in skeletal muscle, that have to date remained underappreciated. Here, we explore recent revelations that the sensory and signalling functions of cilia on muscle progenitors regulate cell cycle progression, trigger differentiation and maintain a commitment to myogenesis. Cilia disassembly is initiated during myoblast fusion. However, the remnants of primary cilia persist in multi-nucleated myotubes, and we discuss their potential role in late-stage differentiation and myofiber formation. Reciprocal interactions between cilia and the extracellular matrix (ECM) microenvironment described for other tissues may also inform on parallel interactions in skeletal muscle. We also discuss emerging evidence that cilia on fibroblasts/fibro-adipogenic progenitors and myofibroblasts may influence cell fate in both a cell autonomous and non-autonomous manner with critical consequences for skeletal muscle ageing and repair in response to injury and disease. This review addresses the enigmatic but emerging role of primary cilia in satellite cells in myoblasts and myofibers during myogenesis, as well as the wider tissue microenvironment required for skeletal muscle formation and homeostasis.
Topics: Animals; Axoneme; Cell Cycle; Cell Differentiation; Centrosome; Cilia; Cytoskeleton; Extracellular Matrix; Humans; Muscle Development; Muscle Fibers, Skeletal; Muscle, Skeletal; Myoblasts; Organelles; Signal Transduction
PubMed: 34502512
DOI: 10.3390/ijms22179605