Structural mechanism for bidirectional actin cross-linking by T-plastin

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. 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.

Bending forces and nucleotide state jointly regulate F-actin structure

“…After initiating this project, we continued developing deep-learning-based filament particle pickers. The architectures described here have been superseded by a U-net architecture, which we found produces better segmentation with a smaller training set in a shorter time 63 .…”

Actin Bundles Dynamics and Architecture

“…Although α-actinin is the major actin-bundling protein identified in stress fibers, other bundling proteins (fascin, espin, fimbrin/plastin, and filamin) can also be present ( Table 1 , [ 47 , 48 ]). A likely role of fascin and espin in such bundles is the stabilization of a subset of filaments with uniform polarities; fimbrin/plastin, despite their compact size, can directly stabilize antiparallel actin assemblies [ 49 , 50 ]. Based on their origin, subcellular location, and protein composition, stress fibers can be grouped into four classes: ventral, dorsal, transverse arcs, and perinuclear actin caps ( Figure 2 ).…”

“…Bundling mechanism. Despite a tight arrangement of its ABDs, fimbrin/plastin can crosslink actin filaments in both parallel and antiparallel arrays [ 49 , 50 ], which correlates with its dual localization in microvilli and stereocilia (parallel bundles), and in contractile rings and the cell cortex (mixed bundles). In 2D arrays, plastin crosslinks actin filaments into dense parallel bundles with a ~120 Å [12 nm) inter-filament distance, and it is spaced after every ~13.5 actin monomers ( Figure 3 , [ 169 ]).…”

Deciphering the molecular mechanisms of actin cytoskeleton regulation in cell migration using cryo-EM

“…While the structure of several ABPs could be inferred by studying them in isolation via X-ray crystallography (see examples [4,9]), high-resolution structures of F-actin bound to most of its interactors were, for a long time, unachievable due to helical assemblies remaining refractory to crystallization [19]. However, in recent years, breakthroughs in cryo-EM methodology have led to structures up to 2 Å resolution for F-actin alone [21][22][23][24][25], or at lower resolution when bound to full-length or truncated variants of their interactors [23,24,[26][27][28][29][30][31][32], or with filament-stabilizing toxins or peptides used for filament labeling [33][34][35][36]. Specifically, structural knowledge on interactions of toxins or peptides with F-actin has the potential for structure-guided developments of new labeling compounds to facilitate visualizing actin networks in migration and beyond.…”

“…Continuing developments in cryo-EM have permitted new insights into modes of actin interaction for other side-binding proteins such as Fimbrin [ 27 , 87 ] (also referred to as Plastin or PLS3), which bundle F-actin into parallel or antiparallel assemblies and Tropomyosins [ 23 , 88 ] (coined as the master regulators of the actin cytoskeleton [ 89 ]). The above selection of cryo-EM structures contains some examples to illustrate the potential of cryo-EM integrated with additional experimental modalities to provide a better understanding of the molecular mechanisms that control the actin cytoskeleton in migratory protrusions.…”