Fascin- and α-Actinin-Bundled Networks Contain Intrinsic Structural Features that Drive Protein Sorting.
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.
Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637, USA.
Jonathan D
JD
Winkelman
Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637, USA.
Cristian
C
Suarez
Department of Chemistry, The University of Chicago, Chicago, IL 60637, USA; James Franck Institute, The University of Chicago, Chicago, IL 60637, USA.
Glen M
GM
Hocky
Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.
Alyssa J
AJ
Harker
Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637, USA.
Alisha N
AN
Morganthaler
Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637, USA.
Jenna R
JR
Christensen
Department of Chemistry, The University of Chicago, Chicago, IL 60637, USA; James Franck Institute, The University of Chicago, Chicago, IL 60637, USA; Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL 60637, USA; Computation Institute, The University of Chicago, Chicago, IL 60637, USA.
Gregory A
GA
Voth
Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.
James R
JR
Bartles
Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637, USA; Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA. Electronic address: drkovar@uchicago.edu.
David R
DR
Kovar
D008841:Actin Cytoskeleton / Q000378:metabolism; D000185:Actinin / Q000378:metabolism*; D000818:Animals; D017173:Caenorhabditis elegans / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism*; D021381:Protein Transport
C111453:ACTN4 protein, human; D002352:Carrier Proteins; C502658:FSCN1 protein, human; D008840:Microfilament Proteins; D000185:Actinin
Arp2/3 complex; TIRF microscopy; actin; cytoskeleton; espin; filopodia; fimbrin; plastin; tropomyosin
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