National Institutes of Health/National Institute of Dental and Craniofacial Research (NIH/NIDCR)
5DP5OD017885
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01GM141044
United States
Citation
Journal: Proc Natl Acad Sci U S A / Year: 2022 Title: Structural mechanism for bidirectional actin cross-linking by T-plastin. Authors: Lin Mei / Matthew J Reynolds / Damien Garbett / Rui Gong / Tobias Meyer / Gregory M Alushin / Abstract: To orchestrate cell mechanics, trafficking, and motility, cytoskeletal filaments must assemble into higher-order networks whose local subcellular architecture and composition specify their functions. ...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.
Name: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 5 / Formula: MG
Molecular weight
Theoretical: 24.305 Da
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Experimental details
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Structure determination
Method
cryo EM
Processing
helical reconstruction
Aggregation state
helical array
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Sample preparation
Buffer
pH: 7.5
Vitrification
Cryogen name: ETHANE
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Average exposure time: 10.0 sec. / Average electron dose: 71.02 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
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