Fascin- and α-Actinin-Bundled Networks Contain Intrinsic Structural Features that Drive Protein Sorting

Abstract

SUMMARY Cells assemble and maintain functionally distinct actin cytoskeleton networks with various actin filament organizations and dynamics through the coordinated action of different sets of actin binding proteins. The biochemical and functional properties of diverse actin binding proteins, both alone and in combination, have been increasingly well studied. Conversely, how different sets of actin binding proteins properly sort to distinct actin filament networks in the first place is not nearly as well understood. Actin binding protein sorting is critical for the self-organization of diverse dynamic actin cytoskeleton networks within a common cytoplasm. Using in vitro reconstitution techniques including biomimetic assays and single molecule multi-color TIRF microscopy, we discovered that sorting of the prominent actin bundling proteins fascin and α-actinin to distinct networks is an intrinsic behavior, free of complicated cellular signaling cascades. When mixed, fascin and α-actinin mutually exclude each other by promoting their own recruitment and inhibiting recruitment of the other, resulting in the formation of distinct fascin- or α-actinin-bundled domains. Subdiffraction-resolution light microscopy and negative staining electron microscopy revealed that fascin domains are densely packed, while α-actinin domains consist of widely spaced parallel actin filaments. Importantly, other actin binding proteins such as fimbrin and espin show high specificity between these two bundle types within the same reaction. Here we directly observe that fascin and α-actinin intrinsically segregate to discrete bundled domains that are specifically recognized by other actin binding proteins.

Proteomics Analysis Identifies IRSp53 and Fascin as Critical for PRV Egress and Direct Cell–Cell Transmission

“…Because fascin is involved in the motility and invasion of cancer cells, it has been widely studied as a drug target for cancer treatment . During filopodia formation, in cooperation with IRSp53, which changes the curvature of the membrane, fascin assembles the actin filaments into highly ordered hexagonally packed bundles at the filopodia initiation site . Because fascin was found in our intracellular virus preparation (six of six replicates, see Table S1, Supporting Information), we investigated whether fascin is also involved in the production of extracellular PRV virions.…”

Evolutionarily diverse LIM domain-containing proteins bind stressed actin filaments through a conserved mechanism

“…Gel-filtered actin was labeled with Alexa 488 carboxylic acid succinimidyl ester on lysine residues 57,58 . Human α-actinin IV was expressed in bacteria and purified as described 59 . Skeletal muscle myosin II was purified from chicken breast and labeled with Alexa 647 as described.…”

Computational modeling of single‐cell mechanics and cytoskeletal mechanobiology

“…Fimbrin and fascin are compact cross‐linking proteins that create parallel‐aligned actin networks that drive membrane protrusions such as filopodia . On the other hand α‐actinin and filamin‐A form networks with more widely spaced and orthogonally aligned actin filaments . Studies have shown that these cross‐linking proteins can cooperatively enhance the mechanical behavior of the cytoskeletal network.…”

“…The enhanced stiffness or force generation capacity due to the use of a combination of cross‐linkers may suggest that all cross‐linkers act cooperatively but different combinations of cytoskeletal proteins also produce different network organizations. Recent studies on reconstituted networks show that cross‐linkers may repel other types of cross‐linkers and promote cooperative binding of actin to more cross‐linkers of the same kind . Winkelman et al showed that fascin and α‐actinin repelled each other and formed different actin network topologies—this was observed in the absence of any chemical signaling or local changes to the environment (such as pH).…”

“…Recent studies on reconstituted networks show that cross‐linkers may repel other types of cross‐linkers and promote cooperative binding of actin to more cross‐linkers of the same kind . Winkelman et al showed that fascin and α‐actinin repelled each other and formed different actin network topologies—this was observed in the absence of any chemical signaling or local changes to the environment (such as pH). On the other hand, proteins that are similar to fascin, such as fimbrin, bind more cooperatively and in close proximity with fascin.…”