@article {68467, title = {De novo protein identification in mammalian sperm using high-resolution in situ cryo-electron tomography}, journal = {Cell}, volume = {186}, year = {2023}, month = {2023 Nov 09}, pages = {5041-5053.e19}, abstract = {

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 {\r A} 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.

}, doi = {10.1016/j.cell.2023.09.017}, url = {https://doi.org/10.1016/j.cell.2023.09.017}, author = {Zhen Chen and Momoko Shiozaki and Kelsey M. Haas and Shumei Zhao and Caiying Guo and Benjamin J. Polacco and Zhiheng Yu and Nevan J. Krogan and Robyn M. Kaake and Ronald D. Vale and David A. Agard} } @article {68082, title = {In situ cryo-electron tomography reveals the asymmetric architecture of mammalian sperm axonemes}, journal = {bioRxiv}, year = {2022}, month = {2022 May 16}, abstract = {

The flagella of mammalian sperm display non-planar, asymmetric beating, in contrast to the planar, symmetric beating of flagella from sea urchin sperm and unicellular organisms. The molecular basis of this difference is unclear. Here, we perform\ in situ\ cryo-electron tomography of mouse and human sperm axonemes, providing the highest resolution structural information to date. Our subtomogram averages reveal mammalian sperm- specific protein complexes within the outer microtubule doublets, the radial spokes and nexin-dynein regulatory complexes. The locations and structures of these complexes suggest potential roles in enhancing the mechanical strength of mammalian sperm axonemes and regulating dynein-based axonemal bending. Intriguingly, we find that each of the nine outer microtubule doublets is decorated with a distinct combination of sperm- specific complexes. We propose that this asymmetric distribution of proteins differentially regulates the sliding of each microtubule doublet and may underlie the asymmetric beating of mammalian sperm.

}, doi = {10.1101/2022.05.15.492011}, url = {https://doi.org/10.1101/2022.05.15.492011}, author = {Zhen Chen and Garrett A. Greenan and Momoko Shiozaki and Yanxin Liu and Will M. Skinner and Xiaowei Zhao and Shumei Zhao and Rui Yan and Caiying Guo and Zhiheng Yu and Polina V. Lishko and David A. Agard and Ronald D. Vale} }