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标题：Structural mechanism for bidirectional actin cross-linking by T-plastin
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作者：Lin Mei ; Matthew J. Reynolds ; Damien Garbett 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2022
卷号：119
期号：37
DOI：10.1073/pnas.2205370119
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance To fulfill the cytoskeleton’s diverse functions in cell mechanics and motility, actin networks with specialized architectures are built by cross-linking proteins. How these cross-linkers specify cytoskeletal network geometry is poorly understood at the level of protein structure. Here, we introduce a machine-learning–enabled pipeline for visualizing cross-linkers bridging cytoskeletal filaments with cryogenic electron microscopy (cryo-EM). We apply our method to T-plastin, a member of the evolutionarily conserved plastin/fimbrin family, revealing a sequence of conformational changes that enables T-plastin to bridge pairs of actin filaments in both parallel and antiparallel orientations. This provides a structural framework for understanding how plastins can generate actin networks featuring mixed filament polarity. To orchestrate cell mechanics, trafficking, and motility, cytoskeletal filaments must assemble into higher-order networks whose local subcellular architecture and composition specify their functions. Cross-linking proteins bridge filaments at the nanoscale to control a network’s μm-scale geometry, thereby conferring its mechanical properties and functional dynamics. While these interfilament linkages are key determinants of cytoskeletal function, their structural mechanisms remain poorly understood. Plastins/fimbrins are an evolutionarily ancient family of tandem calponin-homology domain (CHD) proteins required to construct multiple classes of actin networks, which feature diverse geometries specialized to power cytokinesis, microvilli and stereocilia biogenesis, and persistent cell migration. Here, we focus on the structural basis of actin network assembly by human T-plastin, a ubiquitously expressed isoform necessary for the maintenance of stable cellular protrusions generated by actin polymerization forces. By implementing a machine-learning–enabled cryo-electron microscopy pipeline for visualizing cross-linkers bridging multiple filaments, we uncover a sequential bundling mechanism enabling T-plastin to bridge pairs of actin filaments in both parallel and antiparallel orientations. T-plastin populates distinct structural landscapes in these two bridging orientations that are selectively compatible with actin networks featuring divergent architectures and functions. Our structural, biochemical, and cell biological data highlight inter-CHD linkers as key structural elements underlying flexible but stable cross-linking that are likely to be disrupted by T-plastin mutations that cause hereditary bone diseases.
关键词：enactin cytoskeletoncryo-EMplastinfimbrin