Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA.
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.
Cell. 2023 Nov 9;186(23):5041-5053.e19. doi: 10.1016/j.cell.2023.09.017. Epub 2023 Oct 20.
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.
为了了解细胞通路的分子机制，当代的工作流程通常需要多种技术来鉴定蛋白质、追踪其定位，并在体外确定其结构。在这里，我们结合细胞冷冻电子断层扫描（cryo-ET）和 AlphaFold2 建模来解决这些问题，并了解哺乳动物精子是如何在体内构建的。我们的细胞冷冻电子断层扫描和亚断层平均化提供了轴丝微管结构的 6.0-Å 重建。这些高度解析的三级结构使我们能够无偏地将精子特异性密度与小鼠蛋白质组的 21615 个 AlphaFold2 预测蛋白模型相匹配。我们鉴定出 Tektin 5、CCDC105 和 SPACA9 为新型微管相关蛋白。这些蛋白形成了一个广泛的相互作用网络，交联轴丝双微管的内腔，表明它们在调节纤维的机械性能方面发挥作用。事实上，Tekt5-/-精子的鞭毛具有更多的 180°弯曲，变形更为严重。总之，我们的研究提出了一种细胞可视化蛋白质组学工作流程，并揭示了 Tektin 5 的体内功能。
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