<i>In situ</i> cryo-electron tomography reveals the asymmetric architecture of mammalian sperm axonemes

Chen, Zhe; Greenan, Garrett; Shiozaki, Momoko; Liu, Yanxin; Skinner, Will M.; Zhao, Xiaowei; Zhao, Shumei; Yan, Rui; Guo, Caiying; Yu, Zhiheng; Lishko, Polina V.; Agard, David A.; Vale, Ronald D.

doi:10.1101/2022.05.15.492011

Cited by 3 publications

(6 citation statements)

References 37 publications

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“…These four-helix bundles are stacked next to one along the helical pitch of the microtubule, consistent with the previously reported density striations at lower resolutions (Fig. 1e) 9 . Together, these data reveal that the mammalian sperm doublets have the most extensive MIP network of any microtubule structure observed so far.…”

Section: In Situ Structures Of Sperm Doublets At Subnanometer Resolut...supporting

confidence: 91%

“…1b and Extended Data Fig. 3a, b) 9 . Individual α-helices for the tubulins and MIPs are clearly resolved in both maps.…”

Section: Introductionmentioning

confidence: 92%

“…Three-dimensional classification and refinement of subtomograms corresponding to the 96 nm-repeating units were performed as reported previously (Extended Data Fig. 1) 9 .…”

Section: In Situ Structures Of Sperm Doublets At Subnanometer Resolut...mentioning

confidence: 99%

“…Tilt series were recorded using a 300 kV cryo transmission electron microscope (cryoTEM) and a dose-symmetric scheme with a tilting increment of 4°. The 4° tilt increment, instead of the commonly used 1-3° 7-9 , was used to improve the signal-to-noise ratio of each tilt image while keeping the same angular range and total dose. Three-dimensional classification and refinement of subtomograms corresponding to the 96 nm-repeating units were performed as reported previously (Extended Data Fig.…”

Section: Introductionmentioning

confidence: 99%

“…Three-dimensional classification and refinement of subtomograms corresponding to the 96 nm-repeating units were performed as reported previously (Extended Data Fig. 1) 9 . We then performed focused refinement on the 48 nm-repeating structures of doublets focusing on the microtubules, aiming to reach the highest possible resolution (see the workflow shown in Extended Data Fig.…”

Section: Introductionmentioning

confidence: 99%

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De novo protein identification in mammalian sperm using high-resolution in situ cryo-electron tomography

Chen

Shiozaki

Haas

et al. 2022

Preprint

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Understanding molecular mechanisms of cellular pathways requires knowledge of the identities of participating proteins, their cellular localization and their 3D structures. Contemporary workflows typically require multiple techniques to identify target proteins, track their localization using fluorescence microscopy, followed by in vitro structure determination. To identify mammal-specific sperm proteins and understand their functions, we developed a visual proteomics workflow to directly address these challenges. Our in situ cryo-electron tomography and subtomogram averaging provided 6.0-angstrom resolution reconstructions of axonemal microtubules and their associated proteins. The well-resolved secondary and tertiary structures allowed us to computationally match, in an unbiased manner, novel densities in our 3D reconstruction maps with 21,615 AlphaFold2-predicted protein models of the mouse proteome. We identified Tektin 5, CCDC105 and SPACA9 as novel microtubule inner proteins that form an extensive network crosslinking the lumen of microtubule and existing proteins. Additional biochemical and mass spectrometry analyses helped validate potential candidates. The novel axonemal sperm structures identified by this approach form an extensive interaction network within the lumen of microtubules, suggesting they have a role in the mechanical and elastic properties of the microtubule filaments required for the vigorous beating motions of flagella.

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Section: In Situ Structures Of Sperm Doublets At Subnanometer Resolut...supporting

confidence: 91%

“…1b and Extended Data Fig. 3a, b) 9 . Individual α-helices for the tubulins and MIPs are clearly resolved in both maps.…”

Section: Introductionmentioning

confidence: 92%

“…Three-dimensional classification and refinement of subtomograms corresponding to the 96 nm-repeating units were performed as reported previously (Extended Data Fig. 1) 9 .…”

Section: In Situ Structures Of Sperm Doublets At Subnanometer Resolut...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

De novo protein identification in mammalian sperm using high-resolution in situ cryo-electron tomography

Chen

Shiozaki

Haas

et al. 2022

Preprint

Self Cite

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Unraveling the intricate microtubule inner protein networks that reinforce mammalian sperm flagella

Leung

Roelofs

Chiozzi

et al. 2022

Preprint

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To find and fuse with the egg, mammalian sperm must complete an arduous voyage through the female reproductive tract. The sperm cell remarkable odyssey is powered by its flagellum, a microtubule-based molecular machine ornamented with accessory structures that stabilize the sperm tail in viscous media. Recently, cryo-electron tomography (cryo-ET) revealed that mammalian sperm flagella are further reinforced at the molecular scale with sperm-specific microtubule inner proteins (sperm-MIPs), but the identities of these sperm-MIPs are unknown. Here, we use cryo-electron microscopy to resolve structures of native bovine sperm doublet microtubules, thus identifying most sperm-MIPs. In the A-tubule, several copies of testis-specific Tektin-5 contribute to an extended protein network spanning nearly the entire microtubule lumen. Different copies of Tektin-5 adopt a range of conformations and organizations based on their local interactions with other MIPs. The B-tubule is in turn stabilized by sperm-MIPs that bind longitudinally along and laterally across protofilaments. We further resolve structures of endpiece singlet microtubules, revealing MIPs shared between singlets and doublets. Our structures shed light on the molecular diversity of cilia across different cell types of the vertebrate body and provide a structural framework for understanding the molecular underpinnings of male infertility.

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Swimming by spinning: spinning-top type rotations regularize sperm swimming into persistently symmetric paths in 3D

Ren

Bloomfield-Gadêlha

2023

Preprint

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Sperm modulate their flagellar symmetry to navigate through complex physico-chemical environments and achieve reproductive function. Yet it remains elusive how sperm swim forwards despite the inherent asymmetry of several components that constitutes the flagellar engine. Despite the critical importance of symmetry, or the lack of it, on sperm navigation and its physiological state, there is no methodology to date that can robustly detect the symmetry state of the beat in free-swimming sperm in 3D. How does symmetric progressive swimming emerge even for asymmetric beating, and how can beating (a)symmetry be inferred experimentally? Here, we numerically resolve the fluid mechanics of swimming around asymmetrically beating spermatozoa. This reveals that sperm spinning critically regularizes swimming into persistently symmetric paths in 3D, allowing sperm to swim forwards despite any imperfections on the beat. The sperm orientation in three-dimensions, and not the swimming path, can inform the symmetry state of the beat, eliminating the need of tracking the flagellum in 3D. We report a surprising correspondence between the movement of sperm and spinning-top experiments, indicating that the flagellum drives ''spinning-top'' type rotations during sperm swimming, and that this parallel is not a mere analogy. These results may prove essential in future studies on the role of (a)symmetry in spinning and swimming microorganisms and micro-robots, as body orientation detection has been vastly overlooked in favour of swimming path detection. Altogether, sperm rotation may provide a foolproof mechanism for forward propulsion and navigation in nature that would otherwise not be possible for flagella with broken symmetry.

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In situ cryo-electron tomography reveals the asymmetric architecture of mammalian sperm axonemes

Cited by 3 publications

References 37 publications

De novo protein identification in mammalian sperm using high-resolution in situ cryo-electron tomography

De novo protein identification in mammalian sperm using high-resolution in situ cryo-electron tomography

Unraveling the intricate microtubule inner protein networks that reinforce mammalian sperm flagella

Swimming by spinning: spinning-top type rotations regularize sperm swimming into persistently symmetric paths in 3D

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