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

Jonathan D. Winkelman, Cristian Suarez, Glen M. Hocky, Alyssa J. Harker, Alisha N. Morganthaler, Jenna R. Christensen, Gregory A. Voth, James R. Bartles, David R. Kovar

Research output: Contribution to journalArticle

Abstract

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 total internal reflection fluorescence 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, whereas 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.

Original languageEnglish (US)
Pages (from-to)2697-2706
Number of pages10
JournalCurrent Biology
Volume26
Issue number20
DOIs
StatePublished - Oct 24 2016

Keywords

  • Arp2/3 complex
  • TIRF microscopy
  • actin
  • cytoskeleton
  • espin
  • filopodia
  • fimbrin
  • plastin
  • tropomyosin

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

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    Winkelman, J. D., Suarez, C., Hocky, G. M., Harker, A. J., Morganthaler, A. N., Christensen, J. R., Voth, G. A., Bartles, J. R., & Kovar, D. R. (2016). Fascin- and α-Actinin-Bundled Networks Contain Intrinsic Structural Features that Drive Protein Sorting. Current Biology, 26(20), 2697-2706. https://doi.org/10.1016/j.cub.2016.07.080