Fascin- and A-Actinin-Bundled Networks Contain Intrinsic Structural Features That Drive Protein Sorting
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Article Fascin- and a-Actinin-Bundled Networks Contain Intrinsic Structural Features that Drive Protein Sorting Graphical Abstract Authors Jonathan D. Winkelman, Cristian Suarez, Glen M. Hocky, ..., Gregory A. Voth, James R. Bartles, David R. Kovar Correspondence [email protected] In Brief Winkelman et al. address how actin- binding proteins segregate to functionally distinct F-actin networks. They show that purified F-actin-bundling proteins contain intrinsic properties that are sufficient to drive their sorting to distinct reconstituted F-actin networks by establishing particular network architectures. Highlights d Purified bundling proteins intrinsically sort within reconstituted actin networks d Fascin and a-actinin segregate to F-actin bundles with different filament spacing d Fascin and a-actinin facilitate their own binding while inhibiting the other d The compact bundlers fascin, espin, and fimbrin co- segregate away from a-actinin Winkelman et al., 2016, Current Biology 26, 2697–2706 October 24, 2016 ª 2016 Elsevier Ltd. http://dx.doi.org/10.1016/j.cub.2016.07.080 Current Biology Article Fascin- and a-Actinin-Bundled Networks Contain Intrinsic Structural Features that Drive Protein Sorting Jonathan D. Winkelman,1,8 Cristian Suarez,1,8 Glen M. Hocky,2,3 Alyssa J. Harker,4 Alisha N. Morganthaler,1 Jenna R. Christensen,1 Gregory A. Voth,2,3,5,6 James R. Bartles,7 and David R. Kovar1,4,9,* 1Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637, USA 2Department of Chemistry, The University of Chicago, Chicago, IL 60637, USA 3James Franck Institute, The University of Chicago, Chicago, IL 60637, USA 4Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA 5Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL 60637, USA 6Computation Institute, The University of Chicago, Chicago, IL 60637, USA 7Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA 8Co-first author 9Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cub.2016.07.080 SUMMARY bundled domains that are specifically recognized by other actin-binding proteins. Cells assemble and maintain functionally distinct actin cytoskeleton networks with various actin fila- ment organizations and dynamics through the coor- INTRODUCTION dinated action of different sets of actin-binding pro- Cells simultaneously assemble and disassemble multiple actin teins. The biochemical and functional properties of filament (F-actin) networks from a common pool of proteins [1]. diverse actin-binding proteins, both alone and in The filament organization and dynamics of these networks are combination, have been increasingly well studied. tailored to facilitate different fundamental cellular processes Conversely, how different sets of actin-binding pro- such as motility, cytokinesis, and endocytosis. Functionally teins properly sort to distinct actin filament networks diverse F-actin networks are optimized to perform specific tasks in the first place is not nearly as well understood. through the coordinated actions of numerous actin-binding Actin-binding protein sorting is critical for the self-or- proteins (ABPs) with complementary biochemical properties ganization of diverse dynamic actin cytoskeleton net- [1]. F-actin networks obtain their particular properties by recruit- works within a common cytoplasm. Using in vitro ing unique sets of ABPs that specifically shape the networks reconstitution techniques including biomimetic as- by directly regulating F-actin geometric organization, turnover says and single-molecule multi-color total internal dynamics, and protein composition [2]. Although the behavior of individual ABPs has been well stud- reflection fluorescence microscopy, we discovered ied, we know far less about how they segregate to the proper that sorting of the prominent actin-bundling proteins F-actin network in the first place. At least three non-mutually a fascin and -actinin to distinct networks is an intrinsic exclusive mechanisms have been suggested [2]. First, signaling behavior, free of complicated cellular signaling cas- cascades, such as through Rho-family GTPases, control the cades. When mixed, fascin and a-actinin mutually formation of different types of networks by regulating ABPs. exclude each other by promoting their own recruit- For example, the Arp2/3 complex nucleation-promoting factors ment and inhibiting recruitment of the other, resulting WASP/WAVE and Diaphanous-related formins induce branched in the formation of distinct fascin- or a-actinin- or straight filament organizations in response to different signals bundled domains. Subdiffraction-resolution light mi- [3, 4]. Second, the F-actin binding affinities of ABPs may be croscopy and negative-staining electron microscopy altered locally, such as through the local chemical environment revealed that fascin domains are densely packed, (e.g., pH or ion concentration). Third, actin filaments within different networks may selectively bind different ABPs through whereas a-actinin domains consist of widely spaced recognition of different F-actin features (e.g., branched versus parallel actin filaments. Importantly, other actin-bind- straight architecture) or specific F-actin conformations [2, 5–7]. ing proteins such as fimbrin and espin show high Metazoan cells contain two general types of bundled F-actin specificity between these two bundle types within networks that display different mechanical properties as a conse- the same reaction. Here we directly observe that fas- quence of their association with unique sets of ABPs. The first type cin and a-actinin intrinsically segregate to discrete of network contains tightly packed, parallel-aligned filaments that Current Biology 26, 2697–2706, October 24, 2016 ª 2016 Elsevier Ltd. 2697 a A Fascin α-Actinin (dimer) They are often associated with the bundling protein -actinin, an 6 nm 35 nm elongated but rigid homodimer [9]. Because these bundling proteins facilitate the formation of F-actin networks with fundamentally different mechanical prop- erties and vastly different architectures, we posit that their segre- 1 250285 406 518 632 767 gation could occur without the need for any complex signaling 1 138 259 383 493 ABD SR1 SR2 SR3 SR4 1234 mechanisms. We demonstrate through in vitro reconstitution and multi-color total internal reflection fluorescence microscopy B D (TIRFM) imaging that self-segregation is an intrinsic property of Fascin fascin and a-actinin. Fascin and a-actinin compete for F-actin not through simple mass action competition but rather each F 0 sec A drives the formation of F-actin bundles that enhance their own recruitment while excluding the other. Therefore, there is a segregation mechanism intrinsically built into these classes of 5 0.15 bundlers driving them to different types of F-actin networks. Additionally, we show that other ABPs discriminate between 0.1 these types of bundles, most likely through recognition of a 10 structural or architectural feature of the F-actin bundle. We pro- 0.05 vide evidence that suggests this feature is the spacing between the actin filaments. KD = 272 nM Bound Fascin/F-actin Hill coefficient = 2.3 15 0 1.5 μM OG-actin + 100 nM TMR-Fascin RESULTS 0 0.4 0.8 1.2 [Free Fascin], μM a C 20 Fascin and -Actinin Bundle F-Actin Cooperatively and α-Actinin with Similar Efficiency α Fascin1 and a-actinin-4 are widely expressed F-actin-bundling proteins with very different structural organization. Fascin is a 25 A globular protein (diameter 5–6 nm) that uses four tandem 0.08 b-trefoil domains to bind F-actin (Figure 1A) [10–13], whereas a-actinin is a dimer consisting of two calponin homology 0.06 E 30 F-actin-binding domains separated by a rod formed of spec- trin repeats producing an elongated molecule on the order of 1 a 0.04 0.8 35 nm (Figure 1A) [14–16]. Given that fascin and -actinin Actinin/F-actin are found at very different types of bundled F-actin networks α- 0.6 0.02 0.4 in cells, we considered whether their segregation is an intrinsic KD = 290 nM 0.2 property or requires other cellular factors. Therefore, we Bound Hill coefficient = 2.5 0 0 analyzed their behavior when mixed in F-actin-bundling as- Fascin Fluor. Int.,a.u. Fluor. Fascin 0 0.4 0.8 1.2 04080 says in vitro. We first investigated the bundlers alone. Fascin [Free α-Actinin dimers], μM Time, sec and a-actinin bundle F-actin with moderate cooperativity (Hill coefficient 2.3 and 2.5, respectively) and with similar bundling Figure 1. Fascin and a-Actinin Each Bundles F-Actin Cooperatively affinity (KD 272 and 290 nM, respectively) in low-speed sedi- (A) Structural fold (top) and domain organizations (bottom) showing the four mentation assays (Figures 1B and 1C). Cooperative F-actin b-trefoil domains of fascin (PDB: 3P53 [11]) (left) and an a-actinin dimer (PDB: bundling was directly visualized with two-color TIRFM assays. 1SJJ [15]) (right). ABD, actin-binding domain; SR, spectrin repeat. (B and C) Low-speed (10,000 3 g) sedimentation assays. F-actin was Fascin-mediated bundling initiates slowly from discrete spots assembled from 3.0 mM Mg-ATP-actin monomers with increasing concen- (Figure 1D) but then progresses rapidly, with fascin intensity trations of (B) fascin (F) or (C) a-actinin (A). Coomassie-stained SDS-poly- saturating within seconds (Figure 1E). This suggests two acrylamide gels (top) and graphs (bottom) of the amount of actin in pellets. limiting steps in bundling, whereby filaments must come into Error bars indicate SEM; n = 2. close proximity by diffusion and bundler concentration must m (D and E) Two-color TIRFM of 1.5 M actin (15% Oregon green-actin) and be above a critical threshold. This bundling threshold is 100 nM TMR-fascin. a (D) Merged time-lapse micrographs. Scale bar, 2 mm. Arrows indicate two 25–50 nM for fascin and -actinin as lower concentrations initial TMR-fascin loading events.