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 Ti me, 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. form transient bundles that fall apart rapidly (not shown). (E) Accumulation of TMR-fascin on the actin bundle over time. Although structurally very different, we conclude that fascin and a-actinin bundle F-actin cooperatively and with similar drive membrane protrusions such as filopodia, microvilli, and ster- efficiency. eocilia. These networks are associated with one or more compact bundlers such as fimbrin/plastin, espin/forked, and fascin [8]. The Fascin and a-Actinin Segregate to Different F-Actin second type of network contains widely spaced filaments of Networks on Biomimetic Beads mixed polarity. These networks make up contractile elements Because fascin and a-actinin make bundles with unique prop- such as stress fibers, muscle sarcomeres, and cytokinetic rings. erties and associate with different F-actin networks in cells, we

2698 Current Biology 26, 2697–2706, October 24, 2016 Fascin + Figure 2. Fascin and a-Actinin Compete and ACFascin D α-Actinin ESegregate to Different F-Actin Networks Re- constituted on Motile Beads 3 α-Actinin (A) Low-speed (10,000 3 g) sedimentation of α 2.5 F-actin assembled from 3.0 mM Mg-ATP-actin 2 monomers with 600 nM fascin and a range of F a-actinin concentrations. Coomassie-stained SDS- A 1.5 polyacrylamide gel (top) and graph (bottom) of the Actin Actin 1 amount of actin (A), fascin (F), or a-actinin (a)in 1 α-Actinin pellets. Error bars indicate SEM; n R 2. Fascin 0.5 Fascin in comet tail, a.u. (B–G) Addition of GST-pWA-coated beads to 0.8 0 m F +α-Actinin polymerization reactions containing 4 M Mg-ATP- 1 actin (1% Oregon green-actin), 5 mM profilin, 0.6 100 nM Arp2/3 complex, 20 nM capping protein, 0.8 0.4 and 150 nM Cy5-fascin (blue), with and without Fascin Fascin 0.6 50 nM TMR-a-actinin (red). 0.2 (B) Cartoon of comet tail and protrusive networks 0.4 formed on a motile bead.

Protein in pellet, normalized 0 (C and D) Fluorescence confocal micrographs of 0 0.25 0.5 0.75 1.0 0.2 [ α- Actinin dimers], μM beads in the presence of (C) Cy5-fascin or (D) Cy5- Fascin in protrusions, a.u. 0 fascin and TMR-a-actinin. Shown are representa- B +α-Actinin G 1 tive single planes taken from the center of a confocal α-Actinin z stack with brightness enhanced in the insets for pWA-coated 0.8 visualization of protrusions. Scale bar, 2 mm. bead (E and F) Comparison of the ratio of Cy5-fascin to Comet tail 0.6 Oregon green-actin fluorescence in (E) comet tails α-Actinin / 0.4 or (F) protrusions in the absence and presence of Protrusions a Comet Tail

Protrusions -actinin. Error bars indicate SE; n = 2 experiments

Fascin 0.2

Fluorescence ratio with >20 beads each. α-Actinin Fascin a 0 (G) Ratio of Cy5-fascin to TMR- -actinin fluores- cence in protrusions and comet tails.

tested whether they compete for F-actin in simplified systems Fascin and a-Actinin Separate into Mutually Exclusive using purified components. When mixed in sedimentation Domains on F-Actin Bundles assays, increasing concentrations of a-actinin reduce the To better understand the mechanisms by which a-actinin and amount of fascin in F-actin bundles, revealing a competitive fascin segregate, we employed multi-color TIRFM to directly relationship (Figure 2A). We then asked whether fascin and visualize bundle formation of elongating actin filaments (15% a-actinin intrinsically segregate to different F-actin networks Oregon green-actin). To simplify analysis, we looked exclusively assembled in vitro, free of cellular signaling networks (Figures at two-filament bundle formation. A two-filament bundle con- 2B–2G) [17]. We generated a mixture of short-branched and tains an unbundled leading barbed end (marked by arrowheads protruding filopodia-like F-actin networks on biomimetic in all figures) and a trailing barbed end (marked by arrows) of beads (Figure 2B). Actin (1% Oregon green-actin) was assem- the bundle (see Figure 3A). With TMR-fascin alone, the trailing bled from beads coated with the Arp2/3 complex activator barbed end is rapidly bundled, as there is no resolvable lag be- GST-pWA in reactions containing capping protein, profilin, tween TMR-fascin signal and the elongating trailing barbed and fascin in either the absence or presence of a-actinin. In end (Figures 3A and 3B; Movie S1). TMR-fascin associates these conditions, beads form long comet tails (Figures 2B with the entire length of the two-filament bundle (Figures 3A– and 2C) and become motile (not shown). The periphery con- 3C). Conversely, when unlabeled a-actinin was included in the tains protrusive F-actin networks that are detectable with reaction, TMR-fascin separates into discrete domains of the enhanced image brightness (Figure 2C, insets). In the absence two-filament bundle (Figures 3D–3F; Movie S1). In the presence of a-actinin, cyanine5 (Cy5)-fascin associates with both net- of a-actinin, fascin-decorated two-filament bundles are punctu- works (Figure 2C). When tetramethylrhodamine (TMR)-a-acti- ated by long stretches of bundle devoid of fascin (Figures 3D and nin is included in the reaction, comet tails are shorter and 3E, asterisks). Line scans revealed that TMR-fascin signal within TMR-a-actinin localizes more intensely to comet tails than these undecorated regions is not above levels associated with protrusions (Figure 2D). Importantly, in the presence of a-acti- either single filaments or background binding (Figure 3F). Elon- nin, fascin is depleted in comet tails and enriched in protru- gation rates of the trailing filament barbed ends are reduced sions (Figures 2D–2G). Therefore, a-actinin and fascin appear 10% in fascin domains but not in a-actinin domains (Figure S1), to have intrinsic properties that are sufficient to drive their perhaps because of a structural difference in fascin bundles [18, segregation in the absence of the complex cellular environ- 19] or because the dense packing of filaments in fascin bundles ment. Because we are observing large-scale network effects blocks access of actin monomers. Intrinsic segregation is a at low resolution and filament architecture is complex, the conserved property because TMR-fascin also forms domains mechanism(s) driving segregation is difficult to elucidate using in the presence of Caenorhabditis elegans a-actinin (Figures biomimetic bead assays. S2A and S2B; Movie S2).

Current Biology 26, 2697–2706, October 24, 2016 2699 100 nM TMR-Fascin 100 nM TMR-Fascin A No α-actinin D + 400 nM α-Actinin G 400 nM α-Actinin dimers J Actin+Fascin+α-Actinin 50 nM Fascin Actin

Fascin α-Actinin

500 nM Fascin K 0 sec Actin Time

Fascin 8 H Data Simple model 6 Coop. model 220 4 α-Actinin B E 2 Length, μm

Fascin domain 0 Time 0 0.4 0.8 1.2 1.6 I [Fascin]/[α-Actinin] Length 1.2 L Fascin C Length F Length α-Actinin Background Background 8

Fascin, no α-Actinin 4 Fascin domain + α-Actinin Actinin domain Fascin

Intensity, a.u. 0 Fluor. Intensity, a.u. 0481216 0481216 a..u Fascin Fluor./μm, 0801600510 Length, μm Length, μm [TMR-Fascin], nM Length, μm

Figure 3. Fascin and a-Actinin Segregate into Mutually Exclusive Domains on Two-Filament Bundles (A–I) Two-color TIRFM of 1.5 mM actin (15% Oregon green-actin) with TMR-fascin (red). (A–F) 100 nM TMR-fascin (red) in the absence (A–C) or presence (D–F) of 400 nM a-actinin dimers. Arrowheads and arrows mark leading and trailing barbed ends of parallel two-filament bundles, respectively. See also Movies S1 and S2. (A and D) Merged time-lapse micrographs. Scale bar, 2 mm. (B and E) Merged kymographs of filament length (x axis scale bar, 2 mm) over time (y axis scale bar, 50 s). (C and F) Line scans of TMR-fascin intensity across the two-filament bundles at 300 s. Blue line scans were generated from fascin intensity on single filaments. (G–I) 400 nM a-actinin dimers with a range of TMR-fascin (red) concentrations. (G) Merged micrographs in the presence of 50 nM (top) or 500 nM (bottom) TMR-fascin. Scale bar, 2 mm. Arrows indicate TMR-fascin domains on two-filament bundles. (H) Dependence of TMR-fascin domain length on TMR-fascin concentration. Error bars indicate SEM; n = 2 experiments each containing >10 domains. The dashed blue line indicates domain sizes predicted from a simple kinetic model of competition for binding sites, whereas the solid blue line includes a kinetic barrier to exchange of 4.8 kBT (see the Supplemental Experimental Procedures). (I) Dependence of TMR-fascin fluorescence intensity on TMR-fascin concentration within either fascin- (red circles) or a-actinin domains (blue circles) of composite bundles or pure fascin bundles (cyan circles). (J–L) Three-color TIRFM of 1.5 mM actin (15% Oregon green-actin), 200 nM TMR-fascin (red), and 400 nM Cy5-a-actinin dimers (cyan). See also Movie S3. (J) Micrographs of actin (top) with fascin and a-actinin (bottom). Scale bar, 2 mm. Arrows indicate the leading edge of a two-filament bundle. (K) Kymographs of filament length (x axis scale bar, 2 mm) over time (y axis scale bar, 50 s) for Oregon green-actin (top), TMR-fascin (middle), and Cy5-a-actinin (bottom). The arrowhead and arrow indicate leading and trailing barbed ends, respectively. (L) Line scan of TMR-fascin and Cy5-a-actinin intensity across the two-filament bundle from (J). See also Figures S1–S5.

Although fascin and a-actinin may directly compete for bind- line), whereas including cooperativity by adding an energetic ing sites, domain formation cannot be fully explained by this barrier of 4.8 kBT (product of the Boltzman constant [kB] and tem- type of simple mass action competition. Control reactions with perature [T]) for switching from one type of crosslinker to the both TMR- and Cy5-fascin illustrate that binding site competition other gives a reasonable fit to the experimental data (Figure 3H, does not produce resolvable domains (Figures S2C and S2D). solid blue line; Figure S3) (see the Experimental Procedures). We next mixed a-actinin with a range of TMR-fascin concentra- Interestingly, although the fascin domains become longer, the tions to determine the dependence of fascin domain length on density of fascin within the domains stays constant as TMR-fas- TMR-fascin concentration. Fascin domain length is exponen- cin concentration increases (Figure 3I). Because increasing the tially distributed (Figures S2E and S2F), and higher TMR-fascin ratio of fascin to a-actinin increases domain length while having concentrations form longer domains, as expected (Figures 3G no effect on the density of fascin binding within domains, we and 3H; Figures S2E and S2F). Fascin domains are significantly conclude that competition occurs at the leading edge of the trail- longer than those predicted from a Monte Carlo simulation ing barbed end as the two-filament bundle assembles rather based on simple mass action kinetics (Figure 3H, dashed blue than within a domain once it is formed.

2700 Current Biology 26, 2697–2706, October 24, 2016 A B C α-Actinin DE Fascin 30 OG-Actin 6 nm 37 nm Alexa-phalloidin 20 10 nm 20 25 )

) 34 nm 2 2 15 15 20 Fascin domain 15 10 α-Actinin domain 10 Fascin

10 α-Actinin Intensity, (X10 Intensity, 5 (X10 Intensity, 5 5 Interfilament distance (nm) 0 0 0 200 600 1000 200 600 1000 Distance, nm Distance, nm F GH 35 35

Fascin 30 30 25 α-Actinin only 25 (mixed with fascin) Fascin only 20 20 (mixed with α-Actinin) 15

α-Actinin 15 L = 142 +/- 52 nm 10 10 Distance between filaments (nm) 5

Transverse repeat (nm) Transverse 5 0

Fascin 0 α-Actinin α-Actinin Fascin α-Actinin Fascin

Figure 4. Actin Filaments Are Spaced Differently in Bundles Formed by Fascin and a-Actinin (A–E) Three-color TIRFM of bundles formed with 100 nM TMR-fascin and 400 nM a-actinin between a preassembled filament stabilized with Alexa 647-phalloidin (magenta) and 1.5 mM spontaneously assembled Mg-ATP-actin (33% Oregon green-actin). (A and C) Schematics of predicted outcomes (top) [8, 14] and merged micrographs of two-filament bundles (bottom) for (A) fascin and (C) a-actinin. Scale bars, 2 mm. (B and D) Plots of average fluorescence intensities across a normal line (white lines in dashed boxes [A and C]) for two-filament bundles formed by (B) fascin and (D) a-actinin. Distances between the centroids of the bundled filaments were revealed by curve fits to a Gaussian. (E) Average distance between filaments within a-actinin or fascin domains. Error bars indicate SEM; n R 6 bundles. (F–H) Electron microscopy of F-actin bundles negatively stained with uranyl acetate, which were formed from 1.5 mM actin. (F) Micrographs of bundles with 1 mM fascin (top), 800 nM a-actinin (middle), or both (1 mM a-actinin, 0.25 mM fascin; bottom). Magenta and blue arrowheads indicate fascin and a-actinin molecules, respectively. L, length of the transition zone. Scale bar, 30 nm. (G) Distance of transverse repeat in fascin and a-actinin bundles. Error bars indicate SEM; n R 10 bundles. (H) Distance between filaments in fascin and a-actinin bundles. Error bars indicate SEM; n R 8 bundles.

To better understand the intrinsic sorting of a-actinin and fas- proteins and that fascin is highly selective for fascin- over a-ac- cin, we used three-color TIRFM to simultaneously visualize both tinin-bundled domains. bundlers on two-filament actin bundles (Figures 3J–3L; Movie S3). Indeed, fascin and a-actinin segregate to mutually exclusive Fascin and a-Actinin Form Bundles with Different Actin sections of two-filament F-actin bundles. Whereas fascin only Filament Spacing binds parallel bundles, a-actinin also binds antiparallel bundles How do fascin and a-actinin mutually exclude each other on and weakly to single filaments (Figure S4). We examined sin- F-actin bundles? Ultrastructure studies revealed that fascin gle-molecule dynamics of fascin and a-actinin on bundles to and a-actinin form bundles with filaments spaced significantly determine the basis for enhanced association with particular do- differently (8 and 35 nm, respectively) [11, 14]. We therefore mains (Figure S5). Both fascin and a-actinin are highly dynamic, visualized two-filament bundles to determine filament spacing with similar residence times of 5 s on two-filament bundles in our assays containing mixtures of a-actinin and fascin (Fig- (Figures S5D and S5E). Although we observed a-actinin mole- ure 4). First, we employed subdiffraction-resolution light micro- cules binding to single filaments and fascin-bundled domains, scopy on two-filament bundles generated by assembling new we failed to calculate a reliable koff for these binding modes actin monomers (33% Oregon green-actin) onto preassembled due to the presence of high background in those experiments. filaments labeled with Alexa 647-phalloidin (magenta) (Figures On the other hand, we never observed association of fascin 4A and 4C). Filament distances were measured by fitting a with single filaments or a-actinin-bundled domains, even at Gaussian function to each filament peak from line scans perpen- very fast imaging rates (10 frames/s) (Figures S5A–S5C). When dicular to the bundle (Figures 4A–4D), similar to a previously mixed with a-actinin, single molecules of Cy5-fascin were only described method [20]. The average filament distances are 5 observed binding to TMR-fascin domains (Figures S5A–S5C). and 25 nm for fascin and a-actinin domains, respectively (Fig- We conclude that fascin and a-actinin are dynamic bundling ure 4E), which are probably underestimated because bundles

Current Biology 26, 2697–2706, October 24, 2016 2701 may not always lie flat on the surface. We also visualized bundles ure 5F). We conclude that fimbrin, espin 2B, and fascin recog- by electron microscopy of negatively stained actin filaments (Fig- nize a similar bundle type that is different from those formed ures 4F–4H). On their own, bundles are formed with the expected by a-actinin, which facilitates their intrinsic segregation into spacing of 8 nm for fascin and 32 nm for a-actinin [11, 14]. distinct bundled domains. Importantly, fimbrin, espin, and a-ac- The distance between bound fascin or a-actinin molecules is tinin all bind to single filaments with a much lower affinity than about 35 nm, corresponding to a turn of F-actin (Figures 4F bundled filaments. and 4G). When fascin and a-actinin were mixed together, we Fascin, a-actinin, espin, and fimbrin all have a significantly observed transitions from filaments spaced 8 nm apart by fascin higher affinity for bundled filaments than single filaments (for to 32 nm apart by a-actinin (Figure 4F, lower). As expected, we example, see Figure 1D). A sorting model based on filament saw the same characteristic interfilament distances when both spacing distance predicts that ABPs that bind only one filament bundlers were present in the same reaction (Figure 4H). would not be sensitive to spacing. We tested this possibility by looking at labeled skeletal muscle a/b-tropomyosin. Whereas Fimbrin and Espin Bind to Fascin but Not a-Actinin tropomyosin competes with and inhibits both fascin and a-acti- Bundles nin binding at high concentrations (Figure 5I), at lower concentra- To test the idea that filament spacing is important for segrega- tions tropomyosin binds to both fascin and a-actinin domains as tion of fascin and a-actinin, we investigated other F-actin- predicted (Figures 5G and 5H). bundling proteins that often co-segregate with fascin in cells. Fimbrin is a globular protein like fascin with an actin-bundling DISCUSSION core 6–8 nm in diameter (Figure 5A) [21], and is also often pre- sent with fascin in densely packed protrusive actin structures Fascin and a-Actinin Contain Intrinsic Properties that such as microvilli, filopodia, and hair-cell stereocilia [22, 23]. Drive Their Segregation However, fimbrin is actually in the same bundling-protein family Our work directly shows that a-actinin and fascin contain as a-actinin because they share a conserved tandem calponin intrinsic properties that drive their segregation in the absence homology (CH) actin-binding domain (ABD) [24], and evidence of complex cellular signaling pathways. It has been appreciated suggests they stabilize a similar actin filament structural confor- that actin filaments within different networks are not identical mation [25–27]. An important difference between fimbrin and because combinations of different ABPs promote particular fila- a-actinin is that a-actinin’s ABDs are separated by a long ment and network properties. Here we show that bundlers do not (25-nm) spectrin-repeat rod (Figure 5A) [28]. We first looked generically crosslink filaments together but rather generate spe- at fimbrin and fascin in the absence of a-actinin using three-co- cific bundled features that result in both cooperative and lor TIRF microscopy. Fascin and fimbrin do not mutually competitive binding effects that influence the association of exclude each other but instead co-localize on parallel two-fila- other ABPs. ment bundles (Figure S6A). Fascin binding to parallel bundles decreases as a function of TMR-fimbrin concentration, but we Mechanism of Segregation never observed segregation of fascin and fimbrin into domains Bundlers generally bind to bundled F-actin with a much higher (Figures S6A and S6B). We hypothesized that fimbrin and fascin affinity than single filaments, presumably because within the co-localize on two-filament bundles due to their similar size (5- particular bundles both actin-binding sites are easily engaged to 8-nm diameter), and that fimbrin, like fascin, does not bind to adjacent filaments. We propose that filament spacing within well to bundles facilitated by a-actinin. Therefore, we used bundles may facilitate the engagement of both binding sites for three-color TIRFM to follow the assembly of actin (15% Oregon specific bundlers, and therefore recruit or inhibit the binding of green-actin) with TMR-fascin, unlabeled a-actinin, and Cy5-fim- bundler classes. a-actinin-4 and fascin 1 both bind in exclusive brin (Figures 5B and 5C; Movie S4). 6-fold more fimbrin associ- runs that are 503 longer than predicted by simulations based ates with fascin bundles than with parallel a-actinin bundles on simple mass action competition for binding sites (Figure 3H). (Figures 5B and 5C). A low amount of fimbrin does associate Our results could be explained by a mechanism whereby a-acti- with a-actinin bundles, but this is most likely due to association nin and fascin specifically promote their own binding by stabiliz- with single filaments given that fimbrin binds similarly to a-acti- ing different filament structural conformations [2]. However, nin parallel bundles and single filaments (Figure 5C). To deter- fimbrin may actually stabilize an F-actin conformation similar to mine whether this segregation feature extends to other proteins a-actinin [23–25], yet they segregate from each other on two-fila- that generate compact bundles, we also looked at espin using ment bundles, suggesting that individual filament conformation the same three-color TIRFM assay. Espin associates with pro- is not the primary mechanism of sorting. Therefore, we hypothe- trusive F-actin structures, and in hair-cell stereocilia it co-local- size that filament spacing, rather than filament conformation, is izes with both fimbrin and fascin to generate tight parallel bun- the dominant feature, for several reasons (Figure 6). First, both dles [29](Figure 5A). In the absence of a-actinin, espin isoform fimbrin and espin make very condensed bundles with similar fila- 2B co-localizes (and competes at high concentrations) with fas- ment spacing to fascin bundles (10 nm), and both are highly se- cin in parallel bundles (data not shown). When a-actinin is lective for fascin bundles over a-actinin bundles. Second, fimbrin included in the reaction, Cy5-espin 2B selectively associates and espin display affinities for a-actinin two-filament bundles with fascin domains over a-actinin domains (Figures 5D–5F; that are similar to their affinities for single, non-bundled filaments Movie S5). Additionally, espin 2B enhances fascin’s ability to (Figures 5C and 5E), which suggests that these bundlers asso- compete with a-actinin. As increasingly more espin 2B is ciate with only one of the filaments within the two-filament a-ac- included in the reaction, the fascin domain length expands (Fig- tinin bundle. Third, filaments would need to be highly bent to

2702 Current Biology 26, 2697–2706, October 24, 2016 Fascin + ABα-Actinin + Fimbrin C4 Fimbrin Actin ime

Espin T Actin 3 m F-actin Fascin Fascin μ 2 Fascin

α-Actinin Fimbrin molecules/ 1 in Fimbrin 0 Length Fimbr Single Parallel Parallel D Fascin + α-Actinin + Espin EFfilaments α-Actinin Fascin Actin 1

Time 6 5 0.8 Fascin 4 0.6 3 0.4 2

Espin 1 total bundle length 0.2 Fascin domain length/ 0 Espin molecules/um of F-actin 0 Single Parallel 0 0.5 1 1.5 2 2.5 Length filaments α-Actinin Fascin [Snap-647-Espin], μM G Fascin + α-Actinin + Tropomyosin H I Actin Time α-Actinin 1 1 Fascin Tropomyosin Fascin 0.8 0.8 0.6 0.6

0.4 0.4 Tropomyosin 0.2 0.2

Tropomyosin fluorescence, a.u. 0 0 Single Parallel Parallel Fraction of bundler bound 02468 10 12 [Tropomyosin], uM filaments α-Actinin Fascin

Figure 5. Fimbrin and Espin Segregate to Filaments Bundled by Fascin Three-color TIRFM of two-filament bundles formed by fascin and a-actinin with (A–C) fimbrin, (D–F) espin, or (G and H) muscle tropomyosin. Red and white lines indicate fascin and a-actinin domains, respectively. Arrowheads and arrows indicate leading and trailing barbed ends, respectively. (A) Schematic of bundles formed by fimbrin, espin, fascin, and a-actinin [8, 14]. (B and C) 1.5 mM actin (15% Oregon green-actin), 400 nM a-actinin dimers, 75 nM Cy5-fascin (red), and 15 nM TMR-fimbrin (cyan). See also Movie S4. (B) Micrographs (left) and corresponding kymographs (right) of a parallel two-filament bundle, with Oregon green-actin (top), TMR-fascin (middle), and Cy5- fimbrin (bottom). Scale bars, 2 mm. (C) Cy5-fimbrin molecules on single filaments (n = 6), two-filament parallel a-actinin bundles (n = 23), and two-filament fascin bundles (n = 16). Error bars indicate SEM. (D–F) 1.5 mM actin (15% OG labeled), 100 nM Cy5-fascin (red), 400 nM a-actinin dimers, and a range of concentrations of SNAP-647-espin-2B (cyan). See also Movie S5. (D) Micrographs (left) and corresponding kymographs (right) of a parallel two-filament bundle, with Oregon green-actin (top), TMR-fascin (middle), and SNAP- 647-espin-2B (bottom). Scale bars, 2 mm. (E) Number of SNAP-647-espin-2B molecules per mm of single filament (n = 15), parallel a-actinin bundles (n = 15), or fascin bundles (n = 17). Error bars indicate SEM. (F) Ratio of the total domain length of TMR-fascin two-filament parallel bundles over the total length of two-filament parallel bundles as a function of SNAP-647- espin-2B concentration. Error bars indicate SEM; n = 2 movies. (G and H) 1.5 mM actin (10% Alexa 488 labeled), 100 nM Cy5-fascin (red), 150 nM a-actinin, and 100 nM skeletal muscle a/b-TMR-tropomyosin (cyan). (G) Micrographs (left) and corresponding kymographs (right) of a parallel two-filament bundle, with Alexa 488-actin (top), Cy5-fascin (middle), and TMR- tropomyosin (bottom). Scale bars, 2 mm. (H) Normalized tropomyosin fluorescence on control actin filament and a-actinin and fascin parallel bundles. Error bars indicate SEM. (I) High-speed co-sedimentation assays with 5 mM Mg-ATP-actin, 600 nM fascin (red) or a-actinin (blue), and an increasing concentration of skeletal muscle a/b-tropomyosin. Gels (inset) show fascin or a-actinin depletion from the pellet with increasing tropomyosin. The graph shows the fraction of total fascin or a-actinin in the pellet as a function of tropomyosin concentration. Normalized curves were fit to a single exponential decay function. See also Figure S6. accommodate heterogeneous binding of long and short bun- This rapid binding can be observed in the cooperative binding of dlers (Figures 6C and 6D). The interfilament spacing of an a-ac- these bundlers (Figures 1B–1E), which is most likely due to the tinin bundle (Figure 6A) is about the same distance (35 nm) that newly assembled binding site being well-oriented for fascin to separates binding sites along a filament (Figure 6B). Fascin bun- make contacts with both filaments. Conversely, due to their dles the trailing filament rapidly (Figures 1E and 3A–3F) and, as small size, fascin (and other small bundlers) can only associate new binding sites appear on growing filaments (35 nm from with one of the two filaments in an a-actinin bundle (Figure 6D). the last binding site), they are rapidly bound by fascin (Figure 6C). Small bundlers such as fimbrin and espin can exchange readily

Current Biology 26, 2697–2706, October 24, 2016 2703 D F C B A

E

Figure 6. Model for Segregation of Fascin and a-Actinin on F-Actin Bundles (A) Fascin (red) and a-actinin (blue) form bundles with interfilament spacing of 8 and 35 nm, respectively [11, 14]. (B) The transverse repeat (distance between proteins along the length within a bundle) of both fascin and a-actinin is 35 nm, about the same distance as a complete turn of the actin filament. (C) Fascin molecules add cooperatively to a polymerizing fascin bundle, whereas a-actinin’s affinity for fascin bundles is much lower. (D) a-actinin molecules add cooperatively to a polymerizing a-actinin bundle, whereas fascin molecules do not. (E) Short filament spacing bundlers such as fimbrin and espin (cyan) associate well within fascin bundles but not within a-actinin bundles. (F) Fascin and a-actinin drive the formation of square- and hexagonally packed multi-filament bundles. within fascin bundles, where they can make connections to both We propose that filament spacing significantly contributes to filaments (Figure 6E). the segregation of ABPs to different F-actin networks, which is a previously unappreciated mechanism. We imagine that other Scaling of Segregation to Multi-filament Bundles signals are necessary to initiate either fascin or a-actinin net- Much of our work is based on two-filament bundles. Although we works. Phosphorylation of both fascin and a-actinin regulates do not know how these principles scale with bundles containing their bundling properties [9, 35], and a-actinin bundling is greater than two filaments, we found that these proteins segregate sensitive to Ca2+ [9]. Furthermore, actin assembly factors may efficiently in our reconstituted biomimetic system where bundles generate filament conformations and/or filament spacing, which presumably contain many filaments (Figures 2B–2G). However, promote binding of particular bundlers. For example, densely it is difficult in the biomimetic system to know the mechanism of branched networks of short filaments generated by the Arp2/3 segregation. Looking at two-filament bundles shows that these complex in lamellipodia may not be favorable to fascin binding, bundlers segregate intrinsically, and we therefore reasonably whereas formin and/or Ena/VASP activity may allow filaments speculate that similar mechanisms are involved in the biomimetic to grow long enough to become bundled by fascin. For example, assay. However, filaments in the different parts of the network may we recently discovered that Ena/VASP and fascin have a syner- display different levels of alignment. Filaments in the comet tail gistic relationship [36]. Conversely, a-actinin can bind to Arp2/3 may be more highly branched than those in protrusions, and a-ac- complex-generated networks in lamellipodia [32], which could tinin may crosslink unaligned filaments more efficiently than fascin position it to become incorporated into transverse arcs [37]. [30]. Bundlers may also differently recognize branched filament However, our work shows that once fascin- or a-actinin-bundled architectures. For example, fimbrin does not efficiently bundle networks are initiated, they promote their own binding along with short-branched 2D networks [31], but we imagine that filament specific sets of ABPs. architectures are far more complicated in 3D networks. There are several ways we imagine segregation could scale to EXPERIMENTAL PROCEDURES multi-filament bundles. First, a particular actin conformation could be recognized by ABPs that are incorporating new fila- Protein Purification and Labeling ments into the bundle. Second, multi-filament bundles pack Fission yeast fimbrin SpFim1 was purified as described [38]. Human a-actinin- into very distinct lattice structures. Fascin bundles at steady 4, C. elegans a-actinin, and SNAP-espin-2B were expressed in Escherichia state have 20–30 filaments in vitro and assemble into highly or- coli BL21-CodonPlus(DE3)-RP (Stratagene) cells and purified with affinity dered hexagonally packed bundles [32], whereas a-actinin gen- chromatography. Actin was purified from rabbit muscle acetone powder erates a square lattice with filaments spaced 30 nm apart [33] (Pel-Freez Biologicals) and labeled on Cys374 with Oregon green (OG) (Life Technologies) as described [39, 40]. Mouse capping protein, human fascin (Figure 6F). We imagine that either type of bundled structure is 1, and human profilin HPRO1 were expressed in bacteria and purified as a stable, whereas a mixed structure is not. -actinin crosslinks described [41–43]. Arp2/3 complex was purified from calf thymus by may impose unfavorable and energetically costly defects in hex- WASp(VCA) affinity chromatography [44]. WASP fragment construct GST-hu- agonally packed structures, and vice versa. Classic experiments man WASp pWA was purified by glutathione-Sepharose affinity chromatog- showed that multiple compact bundlers coordinate to efficiently raphy [45]. Human fascin and both human and C. elegans a-actinin were assemble large protrusive F-actin structures such as bristles and labeled with either Cy5-monomaleimide (GE Healthcare) or TMR-6-maleimide (Life Technologies). Skeletal muscle tropomyosin was purified from rabbit microvilli [8, 34]. For example, in the assembly of Drosophila bris- muscle acetone powder (Pel-Freez Biologicals) and labeled as described tles, the espin homolog forked is thought to generate F-actin [46]. Proteins containing SNAP fusions were labeled with SNAP-surface 549 bundles early on that are poised in an orientation to be maximally or SNAP-surface 647 (New England BioLabs). Proteins were incubated with crosslinked by fascin [34]. the dye overnight at 4C according to the manufacturer’s protocols.

2704 Current Biology 26, 2697–2706, October 24, 2016 Actin Filament Sedimentation Assay AUTHOR CONTRIBUTIONS Actin filaments were preassembled for 45 min at 25C from 15 mM Mg-ATP- actin monomers. Assembled actin was then incubated with an accessory pro- J.D.W., C.S., and D.R.K. designed research; J.D.W., C.S., G.M.H., A.J.H., tein(s) for 20 min at 25C and spun at 10,000 3 g (low-speed) for 20 min at A.N.M., J.R.C., and J.R.B. performed and analyzed research; J.D.W., C.S., 25C. In a-actinin-fascin competition assays, a-actinin was introduced first G.M.H., G.A.V., J.R.B., and D.R.K. interpreted data; and J.D.W. and D.R.K. and incubated for 20 min and then fascin was added and incubated for an wrote the manuscript with input from C.S., G.M.H., G.A.V., and J.R.B. additional 20 min. Equal volumes of supernatant and pellet were separated by 12.5% SDS-PAGE, stained with Coomassie blue for 20 min, and destained ACKNOWLEDGMENTS for 16 hr. Gels were analyzed by densitometry on an Odyssey infrared imager (LI-COR Biosciences). This work was supported by NIH R01 GM079265 and ACS RSG-11-126-01- CSM (to D.R.K.), NIH MCB training grant T32 GM0071832 (to A.J.H. and TIRFM Assay J.R.C.), NSF graduate student fellowship DGE-1144082 (to A.J.H. and TIRF microscopy and flow chamber construction were performed as J.R.C.), NIH Ruth L. Kirschstein NRSA F32 GM113415-01 (to G.M.H.), and described previously [36]. NIH R01 DC004314 (to J.R.B.). Additional support was provided to D.R.K. and G.A.V. by the Chicago MRSEC, which is funded by the NSF through grant Bead Motility Assay DMR-1420709. We thank Ben Glick and GSL Biotech for the use of SnapGene Polystyrene microspheres (Polysciences) were coated with GST-pWA [47]. for plasmid construction, Jared Winkelman for helpful discussions, and Joe Motile beads were imaged after 15 min of polymerization on an inverted micro- Austin and Yimei Chen of The University of Chicago Advanced Electron Micro- scope (Ti-E; Nikon) with a confocal scan head (CSU-X; Yokogawa Electric), scopy Facility. 491, 561, and 642 laser lines (Spectral Applied Research), and an HQ2 CCD camera (Roper Scientific). Z stacks were acquired, reconstructed, and Received: April 18, 2016 analyzed using ImageJ (NIH). Fluorescence ratios were determined using the Revised: July 4, 2016 central, single plane of motile beads. Background-subtracted fluorescence Accepted: July 28, 2016 values were measured for each fluorescent protein in the comet tail region Published: September 22, 2016 and in the protrusion region. The fluorescence ratios (Figures 2E and 2F) were determined by dividing fascin fluorescence by actin fluorescence. The ra- REFERENCES tio for fascin/a-actinin fluorescence was determined similarly. 1. Blanchoin, L., Boujemaa-Paterski, R., Sykes, C., and Plastino, J. (2014). Interfilament Distance Measurement by TIRFM Actin dynamics, architecture, and mechanics in cell motility. Physiol. To generate two-filament bundles where each filament was a different color, we Rev. 94, 235–263. first let 5 mM Mg-ATP-actin assemble for 45 min at room temperature. These fil- 2. Michelot, A., and Drubin, D.G. (2011). Building distinct actin filament net- aments were then incubated with an equimolar amount of Alexa 647-phalloidin, works in a common cytoplasm. Curr. Biol. 21, R560–R569. diluted in TIRF buffer, and flowed into the chamber. We then initiated assembly of 1.5 mM actin, 33% OG labeled, plus TMR-fascin and a-actinin, and gently flowed 3. Hall, A. (1998). Rho GTPases and the actin cytoskeleton. Science 279, this mixture into the chamber. A fraction of the bundles that formed contained 509–514. one Alexa 647 filament and one OG-actin filament. If the two-filament bundle 4. Ridley, A.J. (2006). Rho GTPases and actin dynamics in membrane protru- had TMR signal, we scored it as a fascin bundle; if it had no TMR signal, we sions and vesicle trafficking. Trends Cell Biol. 16, 522–529. scored it as an a-actinin bundle. The distance between actin filaments within 5. Michelot, A., Costanzo, M., Sarkeshik, A., Boone, C., Yates, J.R., III, and both fascin and a-actinin bundles is expected to be less than the pixel size Drubin, D.G. (2010). Reconstitution and protein composition analysis of (<100 nm) of our images. To calculate this subpixel value, we analyzed the endocytic actin patches. Curr. Biol. 20, 1890–1899. data similar to a previously described method [20]. 6. Brawley, C.M., and Rock, R.S. (2009). Unconventional myosin traffic in cells reveals a selective actin cytoskeleton. Proc. Natl. Acad. Sci. USA Negative-Staining Electron Microscopy 106, 9685–9690. Actin (1.5 mM) was polymerized with either 250–500 nM a-actinin, 1–2 mM fascin, 7. Nagy, S., and Rock, R.S. (2010). Structured post-IQ domain governs or both bundling proteins for 30 min. This solution was then applied to formvar- selectivity of myosin X for fascin-actin bundles. J. Biol. Chem. 285, and carbon-coated 400 mesh copper grids for 1 min, washed, and negatively 26608–26617. stained with 1% (w/v) uranyl acetate for 1 min, blotted, and dried. Visualization of the bundles using transmission electron microscopy was performed on an FEI 8. Bartles, J.R. (2000). Parallel actin bundles and their multiple actin-bundling Tecnai G2 Spirit microscope at 120 kV. Images were captured on a Gatan 2k 3 proteins. Curr. Opin. Cell Biol. 12, 72–78. 2k CCD camera. Bundle parameters were measured using ImageJ. 9. Foley, K.S., and Young, P.W. (2014). The non-muscle functions of actinins: an update. Biochem. J. 459, 1–13. Cooperative Kinetic Model 10. Chen, L., Yang, S., Jakoncic, J., Zhang, J.J., and Huang, X.-Y. (2010). We chose to model the extension of the bundle as a simple kinetic process on Migrastatin analogues target fascin to block tumour metastasis. Nature a lattice of binding sites where only the next open site can be modified. For the 464, 1062–1066. data in Figure 3H, we created a lattice of 400 virtual binding sites, correspond- 11. Jansen, S., Collins, A., Yang, C., Rebowski, G., Svitkina, T., and ing to two actin filaments coming together of length 400 3 37 nm 15 mm. In Dominguez, R. (2011). Mechanism of actin filament bundling by fascin. the simulations, there are two types of crosslinkers that can occupy a site, A A J. Biol. Chem. 286, 30087–30096. and B. We associate with each a rate of addition to a bundle of like type, kon B 12. Sedeh, R.S., Fedorov, A.A., Fedorov, E.V., Ono, S., Matsumura, F., Almo, and kon. In the case where a crosslinker of type A occupies a site, the rate of B ðe=kB TÞ S.C., and Bathe, M. (2010). Structure, evolutionary conservation, and addition for crosslinker B is changed to kone , where kB is Boltzmann’s constant and T is temperature. Similarly, occupancy of site B means the addi- conformational dynamics of Homo sapiens fascin-1, an F-actin crosslink-

A ðe=kB TÞ ing protein. J. Mol. Biol. 400, 589–604. tion rate of A will be changed to kone . 13. Yang, S., Huang, F.-K., Huang, J., Chen, S., Jakoncic, J., Leo-Macias, A., SUPPLEMENTAL INFORMATION Diaz-Avalos, R., Chen, L., Zhang, J.J., and Huang, X.-Y. (2013). Molecular mechanism of fascin function in filopodial formation. J. Biol. Chem. 288, Supplemental Information includes Supplemental Experimental Procedures, 274–284. six figures, and five movies and can be found with this article online at 14. Hampton, C.M., Taylor, D.W., and Taylor, K.A. (2007). Novel structures for http://dx.doi.org/10.1016/j.cub.2016.07.080. a-actinin:F-actin interactions and their implications for actin-membrane

Current Biology 26, 2697–2706, October 24, 2016 2705 attachment and tension sensing in the cytoskeleton. J. Mol. Biol. 368, 32. Svitkina, T.M., Bulanova, E.A., Chaga, O.Y., Vignjevic, D.M., Kojima, S., 92–104. Vasiliev, J.M., and Borisy, G.G. (2003). Mechanism of filopodia initiation 15. Liu, J., Taylor, D.W., and Taylor, K.A. (2004). A 3-D reconstruction of by reorganization of a dendritic network. J. Cell Biol. 160, 409–421. smooth muscle a-actinin by CryoEm reveals two different conformations 33. Pelletier, O., Pokidysheva, E., Hirst, L.S., Bouxsein, N., Li, Y., and Safinya, at the actin-binding region. J. Mol. Biol. 338, 115–125. C.R. (2003). Structure of actin cross-linked with a-actinin: a network of 16. Ribeiro, E. de A., Jr., Pinotsis, N., Ghisleni, A., Salmazo, A., Konarev, P.V., bundles. Phys. Rev. Lett. 91, 148102. Kostan, J., Sjo¨ blom, B., Schreiner, C., Polyansky, A.A., Gkougkoulia, E.A., 34. Tilney, L.G., Connelly, P.S., Vranich, K.A., Shaw, M.K., and Guild, G.M. a et al. (2014). The structure and regulation of human muscle -actinin. Cell (1998). Why are two different cross-linkers necessary for actin bundle for- 159, 1447–1460. mation in vivo and what does each cross-link contribute? J. Cell Biol. 143, 17. Achard, V., Martiel, J.-L., Michelot, A., Gue´ rin, C., Reymann, A.-C., 121–133. Blanchoin, L., and Boujemaa-Paterski, R. (2010). A ‘‘primer’’-based mech- 35. Adams, J.C. (2004). Roles of fascin in cell adhesion and motility. Curr. anism underlies branched actin filament network formation and motility. Opin. Cell Biol. 16, 590–596. Curr. Biol. 20, 423–428. 18. Claessens, M.M.E., Semmrich, C., Ramos, L., and Bausch, A.R. (2008). 36. Winkelman, J.D., Bilancia, C.G., Peifer, M., and Kovar, D.R. (2014). Ena/ Helical twist controls the thickness of F-actin bundles. Proc. Natl. Acad. VASP Enabled is a highly processive actin polymerase tailored to self- Sci. USA 105, 8819–8822. assemble parallel-bundled F-actin networks with fascin. Proc. Natl. Acad. Sci. USA 111, 4121–4126. 19. Shin, H., Purdy Drew, K.R., Bartles, J.R., Wong, G.C.L., and Grason, G.M. (2009). Cooperativity and frustration in protein-mediated parallel actin 37. Hotulainen, P., and Lappalainen, P. (2006). Stress fibers are generated by bundles. Phys. Rev. Lett. 103, 238102. two distinct actin assembly mechanisms in motile cells. J. Cell Biol. 173, 383–394. 20. James, J.R., and Vale, R.D. (2012). Biophysical mechanism of T-cell re- ceptor triggering in a reconstituted system. Nature 487, 64–69. 38. Skau, C.T., and Kovar, D.R. (2010). Fimbrin and tropomyosin competition 21. Klein, M.G., Shi, W., Ramagopal, U., Tseng, Y., Wirtz, D., Kovar, D.R., regulates endocytosis and cytokinesis kinetics in fission yeast. Curr. Biol. Staiger, C.J., and Almo, S.C. (2004). Structure of the actin crosslinking 20, 1415–1422. core of fimbrin. Structure 12, 999–1013. 39. Spudich, J.A., and Watt, S. (1971). The regulation of rabbit skeletal muscle 22. Bretscher, A., and Weber, K. (1980). Fimbrin, a new microfilament-associ- contraction. I. Biochemical studies of the interaction of the tropomyosin- ated protein present in microvilli and other cell surface structures. J. Cell troponin complex with actin and the proteolytic fragments of myosin. Biol. 86, 335–340. J. Biol. Chem. 246, 4866–4871. 23. Glenney, J.R., Jr., Kaulfus, P., Matsudaira, P., and Weber, K. (1981). 40. Kuhn, J.R., and Pollard, T.D. (2005). Real-time measurements of actin fila- F-actin binding and bundling properties of fimbrin, a major cytoskeletal ment polymerization by total internal reflection fluorescence microscopy. protein of microvillus core filaments. J. Biol. Chem. 256, 9283–9288. Biophys. J. 88, 1387–1402. 24. Matsudaira, P. (1991). Modular organization of actin crosslinking proteins. 41. Palmgren, S., Ojala, P.J., Wear, M.A., Cooper, J.A., and Lappalainen, P. Trends Biochem. Sci. 16, 87–92. (2001). Interactions with PIP2, ADP-actin monomers, and capping protein 25. Hanein, D., Matsudaira, P., and DeRosier, D.J. (1997). Evidence for a regulate the activity and localization of yeast twinfilin. J. Cell Biol. 155, conformational change in actin induced by fimbrin (N375) binding. 251–260. J. Cell Biol. 139, 387–396. 42. Vignjevic, D., Yarar, D., Welch, M.D., Peloquin, J., Svitkina, T., and Borisy, 26. Hodgkinson, J.L., el-Mezgueldi, M., Craig, R., Vibert, P., Marston, S.B., G.G. (2003). Formation of filopodia-like bundles in vitro from a dendritic and Lehman, W. (1997). 3-D image reconstruction of reconstituted smooth network. J. Cell Biol. 160, 951–962. muscle thin filaments containing calponin: visualization of interactions 43. Lu, J., and Pollard, T.D. (2001). Profilin binding to poly-L-proline and actin between F-actin and calponin. J. Mol. Biol. 273, 150–159. monomers along with ability to catalyze actin nucleotide exchange is 27. McGough, A., Way, M., and DeRosier, D. (1994). Determination of the required for viability of fission yeast. Mol. Biol. Cell 12, 1161–1175. alpha-actinin-binding site on actin filaments by cryoelectron microscopy and image analysis. J. Cell Biol. 126, 433–443. 44. Egile, C., Loisel, T.P., Laurent, V., Li, R., Pantaloni, D., Sansonetti, P.J., and Carlier, M.-F. (1999). Activation of the CDC42 effector N-WASP by the 28. Ylanne,€ J., Scheffzek, K., Young, P., and Saraste, M. (2001). Crystal struc- Shigella flexneri IcsA protein promotes actin nucleation by Arp2/3 complex ture of the a-actinin rod reveals an extensive torsional twist. Structure 9, and bacterial actin-based motility. J. Cell Biol. 146, 1319–1332. 597–604. 29. Shin, J.-B., Krey, J.F., Hassan, A., Metlagel, Z., Tauscher, A.N., Pagana, 45. Machesky, L.M., Mullins, R.D., Higgs, H.N., Kaiser, D.A., Blanchoin, L., J.M., Sherman, N.E., Jeffery, E.D., Spinelli, K.J., Zhao, H., et al. (2013). May, R.C., Hall, M.E., and Pollard, T.D. (1999). Scar, a WASp-related pro- Molecular architecture of the chick vestibular hair bundle. Nat. Neurosci. tein, activates nucleation of actin filaments by the Arp2/3 complex. Proc. 16, 365–374. Natl. Acad. Sci. USA 96, 3739–3744. 30. Courson, D.S., and Rock, R.S. (2010). Actin cross-link assembly and 46. Hsiao, J.Y., Goins, L.M., Petek, N.A., and Mullins, R.D. (2015). Arp2/3 disassembly mechanics for a-actinin and fascin. J. Biol. Chem. 285, complex and cofilin modulate binding of tropomyosin to branched actin 26350–26357. networks. Curr. Biol. 25, 1573–1582. 31. Skau, C.T., Courson, D.S., Bestul, A.J., Winkelman, J.D., Rock, R.S., 47. Reymann, A.-C., Suarez, C., Gue´ rin, C., Martiel, J.-L., Staiger, C.J., Sirotkin, V., and Kovar, D.R. (2011). Actin filament bundling by fimbrin is Blanchoin, L., and Boujemaa-Paterski, R. (2011). Turnover of branched important for endocytosis, cytokinesis, and polarization in fission yeast. actin filament networks by stochastic fragmentation with ADF/cofilin. J. Biol. Chem. 286, 26964–26977. Mol. Biol. Cell 22, 2541–2550.

2706 Current Biology 26, 2697–2706, October 24, 2016