PT  - JOURNAL ARTICLE
AU  - Lin Mei
AU  - Matthew J. Reynolds
AU  - Damien Garbett
AU  - Rui Gong
AU  - Tobias Meyer
AU  - Gregory M. Alushin
TI  - Structural mechanism for bi-directional actin crosslinking by T-plastin
AID  - 10.1101/2021.12.07.471696
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.12.07.471696
4099  - http://biorxiv.org/content/early/2021/12/09/2021.12.07.471696.1.short
4100  - http://biorxiv.org/content/early/2021/12/09/2021.12.07.471696.1.full
AB  - To fulfill the cytoskeleton’s diverse functions in cell mechanics and motility, actin networks with specialized architectures are built by crosslinking proteins, which bridge filaments to control micron-scale network geometry through nanoscale binding interactions via poorly defined structural mechanisms. Here, we introduce a machine-learning enabled cryo-EM pipeline for visualizing active crosslinkers, which we use to analyze human T-plastin, a member of the evolutionarily ancient plastin/fimbrin family of tandem calponin-homology domain (CHD) proteins. We define a sequential bundling mechanism which enables T-plastin to bridge filaments in both parallel and anti-parallel orientations. Our structural, biochemical, and cell biological data highlight inter-CHD linkers as key structural elements underlying flexible but stable crosslinking which are likely to be disrupted by mutations causing hereditary bone diseases. Beyond revealing how plastins are evolutionary optimized to crosslink dense actin networks with mixed polarity, our cryo-EM workflow will broadly enable analysis of the structural mechanisms underlying cytoskeletal network construction.One sentence summary Cryo-EM, biochemical, and cellular studies reveal how the crosslinking protein T-plastin bridges actin filaments in two opposing orientations.Competing Interest StatementThe authors have declared no competing interest.