Structural diversity of axonemes across mammalian motile cilia

Nature, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 1, 2025

Abstract Reproduction, development and homeostasis depend on motile cilia, whose rhythmic beating is powered by a microtubule-based molecular machine called the axoneme. Although an atomic model of axoneme available for alga Chlamydomonas reinhardtii 1 , structures mammalian axonemes are incomplete 1–5 . Furthermore, we do not fully understand how vary across motile-ciliated cell types in body. Here use cryoelectron microscopy, tomography proteomics to resolve 96-nm modular repeat axonemal doublet microtubules (DMTs) from both sperm flagella epithelial cilia oviduct, brain ventricles respiratory tract. We find that DMTs most specialized, with having only minor differences tissues. build DMT, defining positions interactions 181 proteins including 34 newly identified proteins. elucidate composition radial spoke 3 uncover binding sites kinases associated regeneration ATP regulation ciliary motility. discover sperm-specific, axoneme-tethered T-complex protein ring complex (TRiC) chaperone may contribute construction or maintenance long sperm. dyneins their prestroke states, illuminating conformational changes occur during movement. Our results illustrate elements chemical mechanical embedded within axoneme, providing valuable resources understanding aetiology ciliopathy infertility, exemplifying discovery power modern structural biology.

Language: Английский

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De novo protein identification in mammalian sperm using in situ cryoelectron tomography and AlphaFold2 docking

Cell, Journal Year: 2023, Volume and Issue: 186(23), P. 5041 - 5053.e19

Published: Oct. 20, 2023

To understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization, and determine structures in vitro. Here, we combined cryoelectron tomography (cryo-ET) AlphaFold2 modeling address these questions how mammalian sperm are built situ. Our cryo-ET subtomogram averaging provided 6.0-Å reconstructions axonemal microtubule structures. The well-resolved tertiary allowed us unbiasedly match sperm-specific densities with 21,615 AlphaFold2-predicted protein models mouse proteome. We identified Tektin 5, CCDC105, SPACA9 as novel microtubule-associated proteins. These proteins form an extensive interaction network crosslinking lumen doublet microtubules, suggesting roles modulating mechanical properties filaments. Indeed, Tekt5 −/− possess more deformed flagella 180° bends. Together, our studies presented a visual proteomics workflow shed light on vivo functions 5.

Language: Английский

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DomainFit: Identification of protein domains in cryo-EM maps at intermediate resolution using AlphaFold2-predicted models

Structure, Journal Year: 2024, Volume and Issue: 32(8), P. 1248 - 1259.e5

Published: May 15, 2024

Language: Английский

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Development of functional spermatozoa in mammalian spermiogenesis

Development, Journal Year: 2024, Volume and Issue: 151(14)

Published: July 15, 2024

ABSTRACT Infertility is a global health problem affecting one in six couples, with 50% of cases attributed to male infertility. Spermatozoa are gametes, specialized cells that can be divided into two parts: the head and flagellum. The contains vesicle called acrosome undergoes exocytosis flagellum motility apparatus propels spermatozoa forward components, axonemes accessory structures. For fertilize oocytes, must formed correctly. In this Review, we describe comprehensively how functional develop mammals during spermiogenesis, including formation acrosomes, structures by focusing on analyses mouse models.

Language: Английский

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Systematic screens for fertility genes essential for malaria parasite transmission reveal conserved aspects of sex in a divergent eukaryote

Cell Systems, Journal Year: 2024, Volume and Issue: 15(11), P. 1075 - 1091.e6

Published: Nov. 1, 2024

Sexual reproduction in malaria parasites is essential for their transmission to mosquitoes and offers a divergent eukaryote model understand the evolution of sex. Through panel genetic screens Plasmodium berghei, we identify 348 sex transmission-related genes define roles unstudied as putative targets transmission-blocking interventions. The functional data provide deeper understanding female metabolic reprogramming, meiosis, axoneme. We complex SUN domain protein (SUN1) allantoicase (ALLC1) that male fertility by linking microtubule organizing center nuclear envelope enabling mitotic spindle formation during gametogenesis. Both proteins have orthologs mouse testis, raise possibility an ancient role atypical coupling nucleus Altogether, our unbiased picture molecular processes underpin parasite transmission. A record this paper's transparent peer review process included supplemental information.

Language: Английский

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CRISPR/Cas9-mediated knockout of Tektin 4-like gene (TEKT4L) causes male sterility of Cydia pomonella

Insect Biochemistry and Molecular Biology, Journal Year: 2025, Volume and Issue: 177, P. 104257 - 104257

Published: Jan. 5, 2025

Language: Английский

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Undocking of an extensive ciliary network induces proteostasis and cell fate switching resulting in severe primary ciliary dyskinesia

Science Translational Medicine, Journal Year: 2025, Volume and Issue: 17(783)

Published: Jan. 29, 2025

Primary ciliary dyskinesia is a rare monogenic syndrome that associated with chronic respiratory disease, infertility, and laterality defects. Although more than 50 genes causative of primary have been identified, variants in the encoding coiled-coil domain-containing 39 (CCDC39) CCDC40 particular cause severe disease not explained by loss motility alone. Here, we sought to understand consequences these on cellular functions beyond impaired motility. We used human cells pathogenic CCDC39 CCDC40, Chlamydomonas reinhardtii genetics, cryo-electron microscopy, proteomics define perturbations assembly cilia stability, as well multiple motility-independent pathways. Analysis from patient identified absence axonemal CCDC39/CCDC40 heterodimer resulted network 90 structural proteins, including 14 were defined address recognition which provide docking for missing structures. The microtubule architecture, activated cell quality control pathways, switched multiciliated fate mucus-producing defective periciliary barrier. In variant cells, phenotypes reversed through expression normal transgene. These findings indicate scaffold support an extensive whose results both motility-dependent may explain severity disease. Gene therapy might be potential treatment option explored future studies.

Language: Английский

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Cryo-ET suggests tubulin chaperones form a subset of microtubule lumenal particles with a role in maintaining neuronal microtubules

Proceedings of the National Academy of Sciences, Journal Year: 2025, Volume and Issue: 122(5)

Published: Jan. 31, 2025

The functional architecture of the long-lived neuronal microtubule (MT) cytoskeleton is maintained by various MT-associated proteins (MAPs), most which are known to bind MT outer surface. However, electron microscopy (EM) has long ago revealed presence particles inside lumens MTs, yet unknown identity and function. Here, we use cryogenic tomography (cryo-ET) analyze three-dimensional (3D) organization structures lumenal in primary hippocampal neurons, human induced pluripotent stem cell-derived differentiated P19 cells. We obtain situ density maps several from respective cells detect common structural features underscoring their potential overarching functions. Mass spectrometry-based proteomics combined with modeling suggest that a subset could be tubulin-binding cofactors (TBCs) bound tubulin monomers. A different smaller particles, remains unidentified, exhibits densities bridge across protofilaments. show increased particle concentration within MTs concomitant differentiation correlates higher curvatures. Enrichment around lattice defects at freshly polymerized open-ends suggests protective role. Together identified resemblance TBCs, these results hint role local proteostasis for maintenance MTs.

Language: Английский

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Microtubule inner proteins of Plasmodium are essential for transmission of malaria parasites

Proceedings of the National Academy of Sciences, Journal Year: 2025, Volume and Issue: 122(6)

Published: Feb. 5, 2025

Microtubule inner proteins (MIPs) are microtubule-associated that bind to tubulin from the luminal side. MIPs can be found in axonemes stabilize flagellar beat or within cytoplasmic microtubules. Plasmodium spp. causative agents of malaria feature different parasite forms across a complex life cycle with both unique and divergent microtubule-based arrays. Here, we investigate four rodent for their role transmission mosquito. We show by single double gene deletions SPM1 TrxL1, associated subpellicular microtubules, dispensable vertebrate host mosquito back. In contrast, FAP20 FAP52, gametes, essential mosquitoes but only if genes deleted. absence B-tubule axoneme partly detaches A-tubule, resulting deficiency axonemal beating hence gamete formation egress. Our data suggest high level redundancy ensures microtubule stability transmissive stages , which is important transmission.

Language: Английский

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In-cell structural insight into the stability of sperm microtubule doublet

Cell Discovery, Journal Year: 2023, Volume and Issue: 9(1)

Published: Nov. 21, 2023

The propulsion for mammalian sperm swimming is generated by flagella beating. Microtubule doublets (DMTs) along with microtubule inner proteins (MIPs) are essential structural blocks of flagella. However, the intricate molecular architecture intact DMT remains elusive. Here, in situ cryo-electron tomography, we solved in-cell structure mouse at 4.5-7.5 Å resolutions, and built its model 36 kinds MIPs 48 nm periodicity. We identified multiple copies Tektin5 that reinforce Tektin bundle, different periodicities anchor bundle to tubulin wall. This contributes a superior stability A-tubule than B-tubule DMT, which was revealed comparison DMTs from deformed axonemes. Our work provides an overall picture periodicity understand mechanism motility as well related ciliopathies.

Language: Английский

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