Vascular disease-causing mutation R258C in ACTA2 disrupts dynamics and interaction with

Hailong Lua, Patricia M. Fagnanta, Carol S. Bookwaltera, Peteranne Joela, and Kathleen M. Trybusa,b,1

aDepartment of Molecular Physiology and Biophysics, University of Vermont, Burlington, VT 05405; and bCardiovascular Research Institute of Vermont, University of Vermont, Burlington, VT 05405

Edited by Edward D. Korn, National Heart, Lung, and Blood Institute, Bethesda, MD, and approved June 15, 2015 (received for review April 17, 2015)

Point mutations in vascular α-actin (SM α-actin), Little is known about the underlying biochemical mechanisms encoded by the ACTA2, are the most prevalent cause of famil- by which mutations in SM α-actin trigger pathways that ulti- ial thoracic aortic aneurysms and dissections (TAAD). Here, we pro- mately result in aortic cell loss or in the cell proliferation typical vide the first molecular characterization, to our knowledge, of the of occlusive diseases in small muscular arteries. Here, we present effect of the R258C mutation in SM α-actin, expressed with the an in vitro characterization of the defects caused by the R258C α baculovirus system. Smooth muscles are unique in that force gener- mutation in SM -actin. To our knowledge, we are the first to α ation requires both interaction of stable actin filaments with myosin express the human SM -actin isoform successfully using the and polymerization of actin in the subcortical region. Both aspects baculovirus/insect cell system, which is a critical aspect of this of R258C function therefore need investigation. Total internal re- study because the effect of point mutations can vary depending on the isoform in which they are present. We investigated the flection fluorescence (TIRF) microscopy was used to quantify the effect of the R258C mutation first because of its prevalence in growth of single actin filaments as a function of time. R258C fila- patients (6), its relatively poor prognosis (median life expectancy ments are less stable than WT and more susceptible to severing by of ∼35 y of age), and high penetrance (5), and because it causes cofilin. Smooth muscle offers little protection from TAAD as well as moyamoya-like disease, an occlusive disease of cofilin cleavage, unlike its effect on WT actin. Unexpectedly, the cerebral vasculature. binds tighter to the R258C monomer, which will increase the pool of The actin monomer consists of two major domains, with ATP globular actin (G-actin). In an in vitro motility assay, smooth muscle bound in the cleft between them. In the typical view of monomeric myosin moves R258C filaments more slowly than WT, and the slow- globular actin (G-actin), R258 is located in a helix on the backside ing is exacerbated by smooth muscle tropomyosin. Under loaded of subdomain 4 (Fig. 1A). [Note that the R258 mutation corre- conditions, small ensembles of myosin are unable to produce force sponds to amino acid R256 in the actin , due to post- on R258C actin-tropomyosin filaments, suggesting that tropomyosin translational processing that removes both the N-terminal Met occupies an inhibitory position on actin. Many of the observed de- and Cys residues (7)]. In filamentous actin (F-actin), the outer fects cannot be explained by a direct interaction with the mutated domain consists of subdomains 1 and 2, as well as the inner do- residue, and thus the mutation allosterically affects multiple regions main of subdomains 3 and 4. The two helical strands that compose of the monomer. Our results align with the hypothesis that defec- the filament are stabilized by interactions between protomers both tive contractile function contributes to the pathogenesis of TAAD. within a strand and between strands. R258C lies at the interface between the two strands (Fig. 1 B and C). Upon polymerization, actin | myosin II | smooth muscle | thoracic aortic aneurysms | the actin protomer undergoes a conformational change: The two vascular disease major domains, which are twisted in monomeric G-actin, become flatter with respect to one another in F-actin. Another feature of F-actin is that it can adopt multiple states in which the protomers horacic aortic aneurysms and dissections (TAAD) are the adopt varied twists and tilts with respect to one another, as well as T18th most common cause of death in individuals in the having different loop conformations (8–11). United States (1). The high degree of mortality is partly due to the fact that aneurysms tend to be asymptomatic until a life- Significance threatening acute aortic dissection occurs. Familial TAAD is an autosomal dominant disorder with variable penetrance, which is α characterized by enlargement or dissection of the thoracic aorta Point mutations in vascular smooth muscle -actin are the most (reviewed in 2). The most prevalent genetic cause of familial prevalent cause of familial thoracic aortic aneurysms leading to TAAD, responsible for ∼15% of all cases, are mutations in acute dissections, yet the molecular mechanism by which these vascular smooth muscle α-actin (SM α-actin), encoded by mutations affect actin function is unknown. An underlying the gene ACTA2. More than 40 mutations in ACTA2 have been cause of the disease is thought to be contractile dysfunction, identified to date (3–5). Intriguingly, ACTA2 mutations also which initiates adaptive pathways to repair the defects in the differentially predispose individuals to occlusive vascular dis- smooth muscle cells. Here, we investigate the effects of the eases, such as premature and (6). R258C mutation, a prevalent mutation in humans with a poor ACTA2 mutations thus can lead to either dilation of large elastic prognosis. The mutant actin shows multiple defects, including arteries like the aorta or occlusion of smaller muscular arteries. impaired interaction with myosin, formation of less stable fil- SM α-actin is the most abundant protein in vascular smooth aments, and enhanced levels of monomer. These defects are muscle cells, constituting ∼40% of the total protein and ∼70% of likely to decrease cellular force production and initiate aberrant the total actin, with the rest composed of β-andγ-cytoplasmic mechanosensing pathways that culminate in the disease. actin. Actin is critical for contraction and force production by smooth muscle cells, as well as for their proliferation and migra- Author contributions: H.L. and K.M.T. designed research; H.L., P.M.F., C.S.B., and P.J. performed tion. Dissected aortas show several characteristic features, namely, research; H.L., P.M.F., and K.M.T. analyzed data; and H.L. and K.M.T. wrote the paper. loss and disarray of the smooth muscle cells in the medial layer, The authors declare no conflict of interest. loss of elastic fibers, and proteoglycan accumulation in the medial This article is a PNAS Direct Submission. space (reviewed in 2). The compromised integrity of the aortic wall See Commentary on page 9500. allows progression to dissection. In contrast, the vascular pathology 1To whom correspondence should be addressed. Email: [email protected]. ACTA2 in the occluded arteries of patients with mutations is This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. characterized by enhanced numbers of smooth muscle cells. 1073/pnas.1507587112/-/DCSupplemental.

E4168–E4177 | PNAS | Published online July 7, 2015 www.pnas.org/cgi/doi/10.1073/pnas.1507587112 Downloaded by guest on September 28, 2021 A B expressed actin on a nickel-chelate column. The thymosin-β4–HIS PNAS PLUS 2 4 tag is then cleaved from the C terminus of actin by proteolytic di- gestion with chymotrypsin, whose primary cleavage site is after the last native residue of actin, a Phe. Following ion exchange chroma- tography to remove the thymosin-β4 and any actin from which the tag was not cleaved, pure actin with no nonnative residues at either the N or C terminus was obtained. This strategy worked equally well for WT and the R258C mutant actin. SDS gels of the purified WT and R258C show the purity of the preparations (Fig. 2A). The WT actin is shown before and

after removal of the thymosin-β4–His tag (Fig. 2A, lanes 2 and 3). SEE COMMENTARY 1 3 R258C actin is shown after final purification (Fig. 2A, lane 4). The C two actins were run on a 1D isoelectric focusing gel (Fig. 2B). As expected, the R258C migrated faster than WT because the mu- tation replaced a positively charged residue with an uncharged residue. Their relative mobilities were consistent with the calcu- lated isoelectric points of 5.24 for WT and 5.16 for R258C. Av- erage yields of purified actin were ∼0.5 mg of actin for WT and R258 ∼0.4 mg for R258C actin per 109 Sf9 cells (200-mL culture). E197 The WT and mutant SM α-actins were compared with regard to their intrinsic ability to polymerize, their interaction with key K115 actin binding (e.g., smooth muscle tropomyosin, profi- lin, cofilin), and their ability to be moved by smooth muscle myosin under both unloaded and loaded conditions. The muta- Fig. 1. Location of R258 in monomeric and F-actin. (A) Ribbon representation tion significantly affected most of these properties of actin. of one protomer from a model of F-actin obtained from high-resolution electron cryomicroscopy [Protein Data Bank (PDB) ID code 3J8A] (11). The R258C Forms a Less Stable Actin Filament. Polymerization of in- four subdomains of actin are indicated. R258, whose side chain is indicated dividual SM α-actin filaments was visualized as a function of time

by a red stick, is located in subdomain 4. Note that R258C is the gene BIOCHEMISTRY numbering for the mutated residue, and that the residue is amino acid R256 using total internal reflection fluorescence (TIRF) microscopy in the processed protein. (B) Ribbon representation of three protomers from [10 mM imidazole (pH 7.5), 50 mM KCl, 4 mM MgCl2,1mM a model of F-actin (PDB ID code 3J8A) (11). Three residues (indicated with EGTA, 2 mM MgATP at 37 °C]. The actin filament is polarized spheres) are involved in salt bridges: R258 (red) and E197 (blue) in the same such that the two ends differ. The association and dissociation of monomer and K115 (cyan) from the cross-strand monomer. (C) Close-up subunits at the barbed end are intrinsically much faster than at view of the three residues forming salt bridges shown in B. the pointed end. Growth from the barbed end is 10- to 20-fold faster than from the pointed end, and thus we measure total growth from both ends. A series of images at various times il- Here, we compare the biochemical properties of expressed WT lustrate growth of individual filaments (Fig. 3A and Movie S1). and R258C SM α-actin, with the goal of understanding how the mutation affects some of the basic functions of actin, because this initial insult ultimately culminates in vascular disease. In smooth muscle cells, force production requires both proper interaction of AB actin with myosin in the contractile domain and polymerization of kD actin in the cytoskeletal domain, where it strengthens the mem- 225 - brane for force transmission to the ECM (reviewed in 12). The 150 WT R258C mutation adversely affects interactions with myosin and 100 R258C weakens filament stability. Our results predict that cells expressing the R258C mutation will have decreased force output and a larger 75 + pool of monomeric actin. These dysfunctions could trigger aber- 1432 50 rant mechanosensing pathways that culminate in compromised C WT R258C aortic muscle tissue (13). Increasing the monomer/polymer ratio of actin may also have an impact on cellular phenotype, which could have implications for occlusive vascular disease. 35 Results Expression of Human Vascular SM α-Actin. Recombinant human SM 25 α -actin was expressed using the baculovirus/Sf9 insect cell expres- 1432 1 2 3 4 5 6 7 8 sion system. Several challenges needed to be overcome to achieve this goal: (i) The purified actin could not retain a tag, because both Fig. 2. Gel characterization of the expressed purified actin. (A) SDS gels of the N and C termini are near the myosin binding site; (ii) con- purified expressed human SM α-actin (lane 1, molecular mass standards; lane tamination by endogenous Sf9 cell actin must be avoided; and 2, expressed WT actin following HIS-affinity purification and before removal (iii) polymerization of actin during expression in the Sf9 cells of thymosin-β4; lane 3, final purified WT actin following cleavage of thymosin-β4 needed to be prevented because it lowers the protein yield. In ad- with chymotrypsin and ion exchange chromatography; lane 4, final purified dition, expression levels of SM α-actin are considerably lower than R258C actin). (B) One-dimensional isoelectric focusing gels (lane 1, WT actin; what we obtain for β-orγ-cytoplasmic actin. Most of these prob- lanes 2 and 3, comigration of WT and R258C actin; lane 4, R258C actin). (C) Native gel showing similar interactions of WT and R258C actin with lems were surmounted by using a method that was designed for Dictyostelium DNase. The faster migrating bands are free actin, and the slower migrating expression of toxic actin mutants in (14). The C ter- band (arrowhead) is the actin–DNase complex (lane 1, WT actin; lane 2, minus of actin was fused to a 43-aa actin-monomer sequestering R258C actin; lanes 3–5, WT actin plus DNase; lanes 6–8, R258C actin plus protein, thymosin-β4, followed by a HIS tag. Binding of thymosin-β4 DNase). Actin is constant at 10 μM, and DNase is at 10, 5, and 2.5 μM in lanes to the cleft between subdomains 1 and 3 renders the expressed actin 3–5 and 6–8. In this gel system, the R258C consistently runs with a slightly monomeric in the Sf9 cell. The HIS tag allows purification of the faster mobility than WT actin.

Lu et al. PNAS | Published online July 7, 2015 | E4169 Downloaded by guest on September 28, 2021 obtained for the two homopolymers, but skewed more toward A 4 m the rate observed for WT actin (Fig. 4A and Table 2). To fit the data, it is assumed that R258C monomers add onto the filament more slowly only when the filament end adopts a “mutant-like” 0s conformation. This simple model is described in more detail in Materials and Methods and Discussion (Fig. 4B).

Effect of Tropomyosin on Filament Stability. Tropomyosin, an α-helical coiled-coil protein that spans seven actin protomers, is a key component of the smooth muscle thin filament, and it also 60s affects the binding of other actin binding proteins. A cosedi- mentation assay was used to measure the binding of smooth muscle tropomyosin to WT and mutant filaments. The binding constant (Kapp) for tropomyosin to the R258C filaments was 6 −1 similar (3.3 × 10 M ) to the Kapp of the WT filaments (2.3 × 6 −1 120s 10 M ) (Fig. 5). When the TIRF polymerization assay was repeated in the presence of smooth muscle tropomyosin, both the assembly and B disassembly rates for WT and R258C actin were reduced, but the WT critical concentration was similar to the critical concentration seen in the absence of tropomyosin (Table 1). This result implies 30 that tropomyosin stabilizes the filament by slowing both the addition to and removal of actin monomers from the filament 20 R258C but has no effect on nucleation because tropomyosin does not bind to actin monomers or small nucleating oligomers. Despite 10 the presence of tropomyosin, the R258C filaments remain less stable than WT. 0 Rate (subunits/s) 12 3 Profilin Binding Is Strengthened by the R258C Mutation. One of the ways in which actin polymerization is regulated is via formins, Actin (M) which enhance filament formation. Formins contain two con- Fig. 3. Quantification of single actin filament growth observed by TIRF served domains: Formin homology domain 1 (FH1) binds profilin, microscopy. (A) Growth of 1.5 μM WT G-actin as a function of time. Each and formin homology domain 2 (FH2) binds actin (reviewed in 15, panel is separated by 20 s. A yellow arrowhead follows the growth of one 16). A TIRF polymerization experiment was performed in the filament with time. (B) Rate of polymerization of WT (blue circles) and R258C presence of the constitutively active FH1-FH2 fragment of mouse (red triangles) as a function of actin concentration. Data were obtained from diaphanous 1 (mDia1). The presence of formin had no effect on five experiments using three independent protein preparations each of WT the rate of polymerization of WT, R258C, R258H, or an equi- and R258C actin. Values (assembly rate, disassembly rate, and critical con- molar mixture of WT and R258C, but it did help nucleate all of centration) obtained from the fits to a line are provided in Table 1. Error bars them, based on the observed increase in the number of filaments are SE. (Fig. 6A). The mutation thus has little or no effect on the in- teraction of actin with formin. We compared R258C with R258H in some assays because both mutations lead to the same disease The filaments were not visibly different by eye, and filament phenotype in humans. breaking was rarely observed for either WT or R258C actin. Strikingly, when profilin was added, either alone or in combi- Neither showed delayed polymerization, suggesting that nucle- nation with formin, the R258C or R258H actin did not polymerize ation was normal for both of them. To extract kinetic param- under our experimental conditions (Fig. 6A). We thus investigated eters, the observed increase in filament length was plotted as a the effect of profilin alone in more detail. The rate of filament B function of actin concentration (Fig. 3 ). The slope of each growth was quantified as a function of actin concentration in the y linear plot defines the polymerization rate. The -intercept is the presence of 3 μM profilin, using the TIRF polymerization assay rate at which actin subunits disassemble from the filament ends. (Fig. 6B, dashed lines). Profilin decreased the polymerization rate x − − The -intercept is the critical concentration (i.e., the concentra- for both WT and R258C actin (from 16 to 3.5 subunits per μM 1·s 1 − − tion of monomeric actin in equilibrium with polymer). for WT and from 11 to 3.8 subunits per μM 1·s 1 for R258C) Average data from five experiments, performed with three (Table 3), suggesting that profilin-actin adds more slowly onto independent protein preparations each of WT and R258C actin, are summarized in Table 1. The assembly rate of R258C actin ∼ μ −1· −1 is 30% slower than WT (11 vs. 16 subunits per M s ), Table 1. Polymerization rates of WT and R258C actin whereas the disassembly rate is approximately fourfold faster − (2.7 vs. 0.7 subunits per s 1), suggesting that the mutant fila- Assembly Disassembly ments are less stable than WT filaments. The critical concen- rate, rate, Critical subunits subunits concentration, tration for polymerization of R258C actin is approximately − − − fivefold higher than for WT (238 nM vs. 48 nM for WT), which Actin TM per ·μM 1·s 1 per s 1 nM will increase the G-actin concentration in the cell. The defects in WT − 15.9 ± 3.4 0.7 ± 0.6 47.8 ± 44.2 polymerization are consistent with the fact that R258 is involved R258C − 11.2 ± 2.9 2.7 ± 1.0 238.2 ± 49.1 in salt-bridge interactions that stabilize cross-strand interactions + ± ± in the filament, based on the position of this residue in the WT* 6.9 0.7 0.5 0.9 76 + ± ± pseudoatomic model of the actin filament (Fig. 1 B and C). R258C* 6.4 0.8 1.5 1.4 229 A human heterozygous for the R258C mutation expresses Data were obtained from five experiments using three independent both WT and mutant actin, and thus we quantified the poly- protein preparations each of WT and R258C actin. Conditions: 10 mM im-

merization of an equimolar ratio of WT and R258C. The rate idazole (pH 7.5), 50 mM KCl, 4 mM MgCl2, and 1 mM EGTA (37 °C). TM, of elongation of a 50:50 mixture of WT and R258C as a function tropomyosin. of actin concentration was intermediate between the values *Data were obtained from one experiment. Errors are SE of the fit.

E4170 | www.pnas.org/cgi/doi/10.1073/pnas.1507587112 Lu et al. Downloaded by guest on September 28, 2021 A assay (Fig. 7 and Movie S2): Filaments were severed internally, and PNAS PLUS WT they also shortened from either end, with one end much faster than the other, until the filament disappeared. These two behaviors were 80 50:50 not mutually exclusive; namely, a given filament could exhibit one or both behaviors. Both processes depended on cofilin concentra- 60 tion and resulted in the destruction of actin filaments. We first assessed the effect of cofilin on actin filament short- 40 ening. As little as 10 nM cofilin induced shortening of R258C R258C filaments (Fig. 7A). At 100 nM cofilin, the process was so fast 20 that no R258C filaments were observed by the time the slide was Rate (subunits/s) mounted on the microscope. In contrast, WT filaments only SEE COMMENTARY started to slowly shorten at 100 nM cofilin. 1234 WT filaments were also more resistant than R258C filaments to severing by cofilin (Fig. 7B). WT filaments showed virtually no Actin (M) severing at 100 nM cofilin, whereas with 75 nM cofilin, severing events were frequently observed with R258C filaments. B Binding of cofilin is competitive with tropomyosin (reviewed in 17). Tropomyosin reduced the rate of shortening and strongly suppressed the severing effects of cofilin with WT actin filaments (Fig. 7). Even at 1.2 μM cofilin, there was minimal shortening WT and almost no severing. In contrast, the R258C filaments were R258C much more vulnerable to attack by cofilin despite the presence of tropomyosin. Smooth muscle tropomyosin binds at least as tightly to R258C filaments as to WT filaments (Fig. 5), so low binding affinity does not explain the result.

DNase and Binding Are Unaffected. To assess if the R258C mutation affects its binding to DNase and gelsolin segment-1 BIOCHEMISTRY faster slower qualitatively, actin was incubated with varying molar ratios of actin to binding protein, and actin complexes were separated association association from monomers on native gels. Fig. 2C shows the results with rate rate DNase, but essentially identical gels were obtained with gelsolin Fig. 4. Copolymerization of equal amounts of WT and R258C actin. (A) Rate segment-1. In both cases, there was no obvious difference in the of polymerization of an equimolar mixture of WT and R258C actin (black amount of complex formed with WT and the R258C actin. circles). Data for the homopolymers (WT, blue circles; R258C, red triangles) were obtained at the same time. Table 2 tabulates the values obtained from R258C Actin Filaments Are Propelled at Slower Speeds than WT. An the fits (solid lines). Error bars are SE. (B) Schematic to illustrate polymeri- unloaded in vitro motility assay was used to measure the speed zation of a 50:50 mixture of WT (white circles) and R258C (red circles) actin. atwhichsmoothmusclemyosinmovesWTvs.R258Cactin. A WT monomer can add onto an existing filament that has either a WT or We first performed the typical motility assay using filaments mutant protomer at the end; likewise, a R258C monomer can add onto an that were stabilized and visualized using rhodamine-phalloidin. existing filament that has either a WT or mutant protomer at the end. The Mutant and WT filaments were moved at the same speed (Fig. 8A). rate of assembly is slower only if the filament end adopts a mutant-like In the presence of smooth muscle tropomyosin, WT actin conformation, which skews the curve toward WT values. Details of the moved ∼40% faster than bare actin. In contrast, the speed at model are described in Materials and Methods. Table 2 compares the cal- culated and experimental values. which myosin moved the R258C filaments was not enhanced by tropomyosin. This increase in speed resulted in WT filaments moving ∼1.7-fold faster than R258C filaments in the presence filament ends compared with the free actin monomer. The dis- of tropomyosin. assembly rate for both WT and R258C was faster in the presence The in vitro motility was repeated in the absence of phalloidin of profilin compared with its absence (increased from 0.7 to 3 to test if this compound, which stabilizes the filament, moderates − − subunits per s 1 for WT and from 2.7 to 8.2 subunits per s 1 for the defects caused by the mutation. To visualize the filaments, WT R258C) (Table 3), which may be due to the unstable configuration actin was directly labeled with rhodamine and incorporated into of actin at the end of filament when profilin is still bound. Addi- the filament as 25% of the total actin. The speed of filament tion of profilin also increases the critical concentration from ∼50 movement slowed as the percentage of R258C actin in the filament to 330 nM for WT actin because it binds monomeric actin. Un- expectedly, the critical concentration for polymerization of R258C Table 2. Polymerization rates of mixtures of WT and R258C actin increased to a much greater extent in the presence of 3 μM profilin, from ∼240 to 1,680 nM (Table 3). This increase may have Assembly Disassembly Critical cellular implications because the level of free actin monomer is rate, subunits rate, subunits concentration, tightly controlled in vivo. The data were fitted to a simple model Actin per μM·s per s nM (Materials and Methods) in which both free actin monomer and WT 20.4 0.70 34.4 profilin-actin can take part in the polymerization process. The R258C 11.5 1.91 165.9 parameters obtained from the fit show that profilin binds to R258C actin with ∼20-fold higher affinity than to WT (Table 3). 50% WT/50% 18.2 1.48 81.5 R258C R258C Filaments Are More Sensitive to the Action of Cofilin. Cofilin is an Calculated 18.2 1.31 71.9 important regulator of actin dynamics. It has a filament severing from model*

activity that increases the number of filament ends, and it de- Conditions: 10 mM imidazole (pH 7.5), 50 mM KCl, 4 mM MgCl2,and polymerizes filaments from their minus, pointed ends (reviewed in 1 mM EGTA (37 °C). 17). We observed both types of behavior in a TIRF polymerization *Details of the model are described in Discussion.

Lu et al. PNAS | Published online July 7, 2015 | E4171 Downloaded by guest on September 28, 2021 1.0 increases the monomeric pool of actin. How these in vitro de- WT fects affect smooth muscle cell function must be viewed in light R258C of the many roles that SM α-actin plays in vascular smooth muscle cells. These roles include production of contractile force, main- tenance of the integrity of the submembrane , cellular 0.5 mechanosensing, and regulation of cell differentiation and pro- liferation. Genetic mutations that predispose patients to TAAD include mutations in the contractile proteins and their regulators, Fraction bound ECM structural components of the aortic wall, and proteins in- volved in mechanosensing. It has been proposed that smooth 0.125 0.25 0.5 1 2 muscle cell contractile dysfunction is one of the primary insults Tropomyosin (µM) that activates the stress and strain pathways that initiate malad- aptive remodeling of the smooth muscle cell, ultimately culmi- Fig. 5. Affinity of WT and R258C actin for smooth muscle tropomyosin. A nating in an aortic aneurysm (reviewed in 2, 13). sedimentation assay was used to measure the binding affinity of actin for Actin is found in two distinct domains in smooth muscle cells. tropomyosin at 200 mM NaCl. Maximal binding was normalized to 1. Data were The “contractile” domain of smooth muscle cells consists of in- × 6 −1 fit to the Hill equation. The Kapp forWTis2.3 10 M ,andtheKapp for R258C terdigitating myosin and stable actin filaments that cyclically interact 6 −1 is 3.3 × 10 M . The Hill coefficient is 1.9 for WT, and the Hill coefficient is 2.3 to generate tension, controlled by phosphorylation of the regulatory for R258C. Data obtained from three independent pelleting assays are shown. light chain of smooth muscle myosin. These filaments are anchored

increased from 50–75% (Fig. 8B). The slowing of speed by the mutant actin was accentuated in the presence of tropomyosin. A In some patients, Arg-258 is mutated to His instead of Cys. We WT expressed and purified R258H to test if this mutation had sim- WT-R258C ilar effects to R258C. In filaments containing 75% mutant actin, R258C speeds were comparable whether the Arg residue was mutated to 20 R258H Cys or His (minus tropomyosin: 0.47 ± 0.13 for R258C vs. 0.50 ± 0.10 for R258H; plus tropomyosin: 0.31 ± 0.08 for R258C vs. 0.32 ± 0.09 for R258H). 10 To determine if the slowing of speed with the mutant actin

filament was due to a change in the rate of release of ADP from Rate (subunits/s) myosin, a stopped-flow experiment was performed. Acto-smooth heavy meromyosin (HMM)-ADP was mixed with 2 mM MgATP. Under these conditions, the rate of acto-HMM dissociation is rate-limited by ADP dissociation. The rate of ADP release from − 160 the mutant and WT filament was the same (134 ± 1s 1 for WT − actin and 127 ± 1s 1 for R258C actin). 120

Actomyosin Force Generation Is Severely Impaired When Tropomyosin 80 Is Bound to the R258C Filament. To test if the R258C mutation af- 40 fects force generation by myosin, we carried out a one-bead laser trap experiment to investigate the force/velocity relationship of a Filament number small ensemble of smooth muscle heavy meromyosin with WT and Actin Actin Actin Actin R258C actin filaments (Fig. 9A). When studying actin mutants, an formin formin --- advantage of the one-bead setup is that the actin filaments do not --- profilin require phalloidin for stabilization because they are attached to the profilin coverslip. In the more traditional three-bead assay, the actin is sus- B pended between two beads and stabilized with rhodamine-phalloidin. We first compared the force/velocity relationship obtained with 80 bare WT and R258C actin filaments. A force-feedback system is 60 used to clamp the force at five levels between 1 and 5 pN. The velocity at any force level was obtained by fitting the raw data to a 40 line (Fig. 9B). The force/velocity relationship was then fitted to the Hill equation. As expected, velocity slowed as force increased 20 (Fig. 9C). There was virtually no difference between the curves

obtained with bare WT vs. bare R258C filaments. In the presence Rate (subunits/s) 0 of smooth muscle tropomyosin, the WT filament showed the same 1234567 force/velocity relationship as in its absence. In contrast, velocity was greatly reduced at all force levels for R258C actin-tropomyosin filaments. Fig. 6. Profilin binds more strongly to R258C than to WT actin. (A) Rate of Discussion polymerization and total filament number (in a 54 × 54-μm area after 2 min) α observed by TIRF microscopy. The polymerization was done with pure actin, Multiple facets of SM -actin function are quite severely affected actin-formin, actin-formin-profilin, or actin-profilin (WT actin, blue bars; by the R258C mutation. Defects induced by the R258C mutation i equimolar mix of WT and R258C, gray bars; R258C actin, red bars; R258H, include ( ) slower and less productive interactions with smooth green bars). Error bars are SE. Conditions are 1 μM actin, 50 nM formin muscle myosin, particularly in the presence of tropomyosin, that [mouse diaphanous 1 (mDia1) FH1-FH2], and 3 μM profilin. (B) Rate of po- lead to reduced force output; (ii) formation of a less stable fil- lymerization as a function of actin concentration in the absence or presence ament with greater susceptibility to cleavage by cofilin even in of 3 μM profilin (WT, blue circles; R258C, red triangles). Solid lines (no pro- the presence of tropomyosin, which enhances actin filament filin) are linear fits, whereas the dashed lines (plus 3 μM profilin) are the fits dynamics; and (iii) higher affinity bindingtoprofilin,which to a model described in Materials and Methods. Error bars are SE.

E4172 | www.pnas.org/cgi/doi/10.1073/pnas.1507587112 Lu et al. Downloaded by guest on September 28, 2021 Table 3. Polymerization data in the presence of 3 μM profilin mutations in both the myosin heavy chain (MYH11)andmyosin PNAS PLUS MLCK Fit parameters WT R258C light chain kinase ( ), which phosphorylates the regulatory light chain of myosin and is required for actomyosin interactions, Assembly rate, subunits per μM−1·s−1 3.5 3.8 also cause autosomal dominant inheritance of TAAD (24, 25). − Disassembly rate, subunits per s 1 3.1 8.2 encoding proteins involved in either smooth muscle cell β Kd, μM 3.0 0.16 contraction or the TGF- signaling pathway are the predominant Critical concentration, nM 330 1,680 causes of familial TAAD.

Conditions: 10 mM imidazole (pH 7.5), 50 mM KCl, 4 mM MgCl2, and R258C Destabilizes the Filament. Although R258C was not specif- 1 mM EGTA (37 °C). ically looked at, immunofluorescence of cultured smooth muscle ACTA2 cells from individuals with mutations showed substan- SEE COMMENTARY tially fewer actin filaments than controls, suggesting that these into dense bodies, a structure characteristic of smooth muscle cells. “ ” mutations may interfere with actin assembly (3). Our in vitro A second cytoplasmic domain located in the subcortical region results support this idea. R258C actin has a higher critical con- has a more dynamic pool of actin that is not associated with myosin. centration for assembly, caused by a slower rate of assembly and At the membranous dense plaques, this pool of actin attaches to a faster rate of disassembly. Based on the location of R258 in the the cytoplasmic tails of transmembrane integrins via focal adhesion model of the actin filament structure (Fig. 1), a polymerization proteins, which transduce mechanical force across the plasma mem- defect was predictable. Recent advances in electron cryomicro- brane by linking the ECM with the cytoskeleton. scopy have led to the highest resolution structures (3.7–4.7 Å) of A unique feature of tension development in smooth muscle the actin filament to date (9, 11). In these structures, R258 cells is that the independently regulated contractile domain and (R256 in protein) forms a salt bridge with E197 (E195 cytoskeletal domains need to collaborate to produce force. This in protein) in the same protomer, which, in turn, forms a salt view is supported by numerous studies showing that in response to stimulation, the amount of F-actin in the cell increases, and if bridge with K115 (K113 in protein) on the cross-strand actin this new actin polymerization is blocked, tension is not de- veloped (reviewed in 12, 18–20). The mechanism by which actin polymerization facilitates force production may include an en- A hancement of the linkage of the actin filaments to integrins, which would strengthen force transduction between the con- BIOCHEMISTRY tractile domain and the ECM. Approximately 10% of the actin 20 undergoes polymerization, so that a resting cell has ∼70–80% - F-actin, which increases to ∼80–90% after stimulation. Whether + both smooth and cytoskeletal actin isoforms undergo polymeri- 15 - zation upon stimulation remains unresolved (21, 22). Moreover, + when the cell contains a mutant SM α-actin, such as R258C, TM 10 which appears to be more dynamic than WT α-actin, it may more readily become part of both the contractile and cytoskeletal pools of actin. Our results suggest that the R258C mutation has 5 the potential to disrupt the function of actin in both domains. 1200

R258C Mutation Decreases Contractile Function by Perturbing Interactions (subunits/s) rate Shortening WT 800 with Both Myosin and Tropomyosin. Under unloaded conditions, the R258C 400 presence of R258C in the actin filament slowed the speed of myosin-powered movement in an in vitro motility assay in a dose- dependent fashion. Phalloidin stabilization of the filament sup- Cofilin (nM) presses this defect. Two general mechanisms can lead to slower B movement. One is a change in myosin kinetics, and the other is via a change in the properties of the actin filament per se. The kinetic step in myosin that controls actin motility speed is ADP release. Because we observed no change in the rate of ADP -3 20 release, we favor a mechanism whereby the structure of the mutant filament does not allow the full displacement per work- 16 ing of myosin to be realized, resulting in less displacement 12 per MgATP hydrolyzed, and thus a slower speed. Tropomyosin occupies a more inhibitory position on R258C 8 actin filaments. In the absence of myosin and , the latter of which is not present in smooth muscle cells, all tropomyosins 4 occupy either the blocked or closed state on actin (23). In the blocked state, tropomyosin sterically prevents myosin from frequency Severing 120 binding to actin. In the closed state, myosin can bind weakly, and WT 0 800 additional binding of myosin cooperatively shifts tropomyosin to R258C R258CR 400 the open state, which fully exposes the myosin binding site. In the 258C presence of tropomyosin, the difference in speed between WT and mutant filaments was accentuated. More strikingly, once Cofilin (nM) load was applied, myosin was essentially unable to interact with Fig. 7. R258C filaments are more susceptible to cofilin-induced shortening R258C actin-tropomyosin filaments. In the loaded assay, only a and cleavage than WT actin, both in the presence and absence of smooth small number of myosin heads interact with the actin-tropomyosin muscle tropomyosin (TM). (A) Rate of actin filament shortening induced by filament at any time, and this number appears to be insufficient to cofilin (WT, blue bars; R258C, red bars). Lighter shaded bars are in the displace tropomyosin from an inhibitory position. presence of tropomyosin. Error bars are SE. (B) Frequency of cleavage by That defects in force output from the contractile domain can cofilin for WT and R258C actin filaments. Frequency is reported as the lead to TAAD is further supported by the fact that loss-of-function number of events per 1 min per 1 μm of actin filament. Error bars are SE.

Lu et al. PNAS | Published online July 7, 2015 | E4173 Downloaded by guest on September 28, 2021 disassembly rate). As shown in Table 2, the calculated results from A minus TM B 0.8 WT this model agree with the experimentally observed values. R258C 0.10 0.6 Actin Filament Dynamics Regulated by Binding Proteins. In nonmuscle

m/sec) cells, the actin cytoskeleton is highly dynamic, and controlled as- µ 0.4 0.05 sembly and disassembly of actin play roles both in cell migration 0.2 Speed ( QD A plus TM minus TM plus TM Probability 0.10 Laser trap 0.05 Polystyrene bead Smooth muscle HMM 0.4 0.8 Speed (µm/s) Actin Fig. 8. Smooth muscle myosin moves R258C filaments more slowly in an in vitro motility assay. (A) Actin filaments were stabilized and visualized with rhodamine- phalloidin. Speeds were determined by a semiautomated tracking program and fitted to a Gaussian distribution. (Upper, minus tropomyosin) Myosin moved WT − actinataspeedof0.53± 0.18 μm·s 1 (n = 2,742) and R258C filaments at a speed − of 0.50 ± 0.16 μm·s 1 (n = 3,415). The difference in speeds was statistically sig- B nificant (Student’s t test, P < 0.01) because of the large number of filaments analyzed, but it has no physiological relevance. (Lower, plus tropomyosin) In the 340 presence of smooth muscle tropomyosin, myosin moved WT actin at a speed of − 0.74 ± 0.19 μm·s 1 (n = 1,294) and R258C filaments at the slower speed of 0.43 ± − 300 0.15 μm·s 1 (n = 3,743) (Student’s t test, P < 0.01). Data from five independent actin preparations were pooled. (B) Speed of movement of WT and R258C actin 260 filaments that were not stabilized with phalloidin. Filaments were visualized by incorporation of 25% WT actin that was directly labeled with rhodamine (solid Bead position (nm) blue bar, 100% WT; striped red bar, 50% WT and 50% R258C; solid red bar, 25% WT and 75% R258C). (Left) Speed of movement of WT filaments (0.61 ± 0.13 − − μm·s 1; n = 196) decreased to 0.53 ± 0.12 μm·s 1 (n = 160) with 50% R258C and − 2 pN 1 pN to 0.47 ± 0.13 μm·s 1 (n = 180) with 75% R258C. The difference between all pairs 5 pN 4 pN 3 pN was statistically significant (Student’s t test, P < 0.01). (Right) Same comparison in Force 5.4 5.5 5.6 the presence of smooth muscle tropomyosin (TM). The speed of movement of Time (s) WT filaments (0.60 ± 0.10 μm·s−1; n = 170) decreased to 0.43 ± 0.09 μm·s−1 (n = − 80) with 50% R258C and to 0.31 ± 0.081 μm·s 1 (n = 142) with 75% R258C.The C difference between all pairs was statistically significant (Student’s t test, P < 0.01). Data from five independent actin preparations were pooled. Filament speed was WT tracked manually. Temperature, 30 °C. 300 R258C WT + tropomyosin (Fig. 1 B and C). This stabilizing salt bridge triad will be dis- R258C + tropomyosin rupted upon mutation of R258 to either Cys or His, which 200 changes both side-chain size and charge. Mutation of R258 to either Cys or His causes similar patient phenotypes. Filament destabilization as a result of an R256H mutation (equivalent to 100 Velocity (nm/s) Velocity residue Arg-258 in ACTA2) was first observed in the backbone of Saccharomyces cerevisiae actin by Malloy et al. (26). Ionic cross- strand stabilization that involves this residue thus appears to be a 12345 property common to SM α-actin and budding yeast actin. Copolymerization of equal amounts of WT and mutant actin Force (pN) tempers the polymerization defect. The observation that polymer- ization as a function of actin concentration was skewed toward the Fig. 9. Force dependence of smooth muscle myosin HMM interacting with WT or R258C filaments in the presence or absence of smooth muscle tropo- WT values could be explained by a simple model if we make the Materials and Methods myosin. (A) Schematic of experimental setup. Smooth muscle myosin HMM, following two assumptions (Fig. 4 and ). First, with a biotin tag at the C terminus, was attached to 1-μm polystyrene beads only when an R258C monomer adds onto a filament that adopts a coated with neutravidin. A myosin-coated bead was then captured by the laser mutant-like conformation (e.g., a filament with a mutant protomer trap and allowed to interact with actin immobilized on the surface. A force- atthebarbedend)willitassembleataslowratesimilartotherate feedback mechanism was used to keep force constant once an interaction was observed for the R258C homopolymer. In all other scenarios, the detected. QD, quadrant detector. (B) Sample trace showing the bead motion monomer adds onto the filament with a faster rate that approxi- under different constant loads, varying from 1 to 5 pN. The solid yellow lines are linear fits to the raw data, shown in black. (C) Force-velocity curves for mates the WT assembly rate. Second, the disassembly rate depends HMM interacting with WT (blue) or R258C (red) filaments in the presence solely on the actin type (i.e., WT dissociates with the WT disas- (dashed lines, open symbols) or absence (solid lines, filled symbols) of tropo- sembly rate, R258C dissociates from filaments with the R258C myosin. The lines are fit to the Hill equation. Error bars are SE.

E4174 | www.pnas.org/cgi/doi/10.1073/pnas.1507587112 Lu et al. Downloaded by guest on September 28, 2021 WT WT-R258C than to WT. This tight binding decreases formin-mediated actin PNAS PLUS polymerization. The net result is that in cells containing R258C actin, we predict that the pool of monomeric actin is considerably larger than in WT cells. Another prediction is that more WT monomers will be incorporated into the actin filaments than the Force mutant in cells expressing both, potentially attenuating the detri- Speed mental effect of the mutant actin. The tighter binding to profilin is unexpected because the profilin binding site to actin is far from the location of the R258C mutation. This tight binding indicates the R258C mutation changes not only the actin filament structure

but also the conformation of monomeric actin. SEE COMMENTARY WT actin A previous study investigated the effect of mutating this same R258C actin smooth muscle myosin Arg residue to His in the backbone of budding yeast actin, which is only 86% identical to SM α-actin (26). The major defect those cofilin smooth muscle tropomyosin investigators observed in vitro was that yeast formin (Bni1) did not enhance polymerization but, instead, strongly capped the filament profilin integrin end. This result was consistent with their cellular results in which budding yeast cells expressing the mutant actin were delayed in Fig. 10. Summary of likely differences in cells containing only WT actin vs. their ability to rebuild their actin cytoskeleton following a chal- cells expressing both WT and R258C actin. The left-hand portion of the lenge by the actin-depolymerizing drug latrunculin A. Although schematic cell illustrates that myosin interacts productively with WT actin- tropomyosin to produce force and motion. Most actin is filamentous, with we also observed intrinsic polymerization defects with this muta- tion, and altered interaction of R258C with profilin and cofilin, only a small pool of monomeric actin. The filaments that are decorated with ’ tropomyosin are resistant to severing and shortening by cofilin. In contrast, our results did not show a defect in the mutant actin s interaction cells expressing both WT and R258C actin would be expected to show slower with formin for either R258H or R258C. We conclude that the filament sliding and have a lower force output. Moreover, it is expected that effect of vascular disease-causing mutations is best studied in the the pool of monomeric actin will increase relative to WT cells, because R258 SM α-actin backbone rather than a heterologous system. has a higher critical concentration, a higher affinity for profilin, and is more A potential downstream effect of increasing the amount of susceptible to severing and shortening by cofilin. monomeric actin in the cell is altering smooth muscle cell phe-

notype. The subcellular localization of the actin binding transcrip- BIOCHEMISTRY tional cofactor myocardin-related transcription factor (MRTF-A) and plasma membrane invagination during endocytosis and depends on the relative amount of monomeric vs. F-actin. When phagocytosis. Assembly is regulated by proteins, including formin G-actin concentration in the cytoplasm increases, it binds to and Arp2/3, that control nucleation and polymerization (reviewed MRTF-A and retains it in the cytoplasm. Conversely, upon Rho- in 15), and it is balanced by disassembly of older filaments by A–dependent actin polymerization, MRTF-A accumulates in the proteins in the ADF/cofilin family, which preferentially bind ADP- nucleus. Once in the nucleus, it interacts with serum response actin monomers (reviewed in 17). Cofilin accelerates remodeling factor, which binds to conserved CC[A/T]6GG cis-elements within of the actin network by severing actin filaments, and thus in- the smooth muscle cell-specific promoters to activate transcription creasing the concentration of ends available for elongation and of smooth muscle-specific genes (reviewed in 33, 34). Thus, a subunit exchange. Cofilin also depolymerizes filaments from the potential outcome is that cells containing R258C actin may lead to pointed ends, thereby increasing filament dynamics (27). the smooth muscle cell developing a less contractile and more Cofilin is, however, also present in striated muscle cells that are proliferative phenotype, because the increased monomeric pool considered to have considerably more stable actin filaments. In may sequester MRTF-A in the cytoplasm. striated muscle cells, cofilin is thought to be involved in precise length control of the thin filament (28, 29). In vascular smooth Allosteric Effects of the R258C Mutation. A number of the observed muscle cells, increased cofilin activation upon dephosphorylation defects in R258C were unexpected based solely on its location in with slingshot phosphatase 1 has been implicated in vascular actin. These defects include altered binding of smooth muscle smooth muscle cell migration and neointima formation following tropomyosin and tighter binding of the mutant actin to profilin. The vascular injury (30). Here, we show that R258C actin filaments are mutant residue is not directly part of the myosin, tropomyosin, or considerably more susceptible to cleavage by cofilin and show profilin binding site. These latter unexpected changes suggest new increased levels of shortening, compared with WT filaments. In pathways of allosteric communication within the actin monomer essence, the increased sensitivity of R258C to cofilin should that have been disturbed by the mutation. An allosteric connection have the same effect as having more activated, unphosphorylated between the profilin binding site and R258 is consistent with results cofilin. Our results suggest that mutation of R258 is likely to result from a hydrogen/deuterium exchange and MS study showing that in enhanced actin dynamics and cell migration. Arg-258 and residues at the profilin binding site were on a con- Although the molecular mechanism of severing is not known, it nected block of residues that display similar hydrogen/deuterium is affected by the structure and mechanical properties of the actin exchange kinetics (35). Reciprocally, a protein binding at the pro- filament (31), which differ when R258 is mutated. Moreover, al- filin site at the barbed end can propagate a conformation change though binding of smooth muscle tropomyosin protects the WT that locally alters the conformation of residue 258 at an interstrand filament from cofilin cleavage (32), the same is not true for the filament stabilization site, which may provide insight into how R258C filaments. This observation supports the idea that the po- profilin at low concentrations can facilitate actin polymerization. sition of tropomyosin on WT vs. R258C filaments differs, consis- A prevailing argument for the extreme conservation of se- tent with the inhibitory effect of tropomyosin on force and motion quence in actin is that virtually every residue has been placed generation by myosin when it interacts with the mutant actin. under selective pressure because of the internal networks within the actin filament that are needed to maintain such allosteric R258C Increases the Monomeric Pool of Actin by Binding Profilin linkages (reviewed in 36). Our results provide additional evi- Tightly. Profilin, one of the most abundant actin binding proteins dence to support this point of view. inside the cell, binds to monomeric actin with high affinity and is the predominant substrate for actin assembly in vivo. Formins Perspective. Fig. 10 shows a schematic of potential differences be- accelerate polymerization by increasing the local concentration of tween a WT smooth muscle cell and a cell expressing both WT and actin-profilin at the barbed end (reviewed in 15, 16). Here, we R258C actin. The overall goal of characterizing the functional effect show that the affinity of R258C actin for profilin is ∼20-fold higher of actin point mutations at the molecular level is to determine if the

Lu et al. PNAS | Published online July 7, 2015 | E4175 Downloaded by guest on September 28, 2021 observed defects can be correlated with vascular disease outcomes Lifeact-GFP was used to visualize the actin filaments. Lifeact binds weakly to ∼ μ μ and with patient survival rates. This study is the first step toward F-actin (Kd of 2 M), and as much as 55 M Lifeact was shown not to affect that goal. For example, both R258C and R39H lead to TAAD and the rates of polymerization and depolymerization (37). This probe allows us to moyamoya-like cerebrovascular disease, whereas other mutations use unlabeled WT and mutant actin. Previous methods for visualizing actin lead only to TAAD (e.g., W88R, G160D) and still others lead to polymerization have relied on actin that has been modified either at Cys374 or TAAD and coronary artery disease (e.g., R149C, R118Q). “Loss-of- at Lys residues (38, 39). In our study, direct modification of actin is complicated function” mutations that lead to smooth muscle cell weakness, a both by the low yields of expressed actin and by our observation that R258C actin does not label in a manner identical to the WT actin. Actin in G-buffer was failure of tension sensing, and degeneration are thought to lead to × thoracic aneurysms and aortic dissections. With R258C, we have spun for 30 min at 392,148 g. The supernatant concentration was determined by a Bio-Rad Protein Assay. The typical actin concentration is 1–2mg/mL.To evidence for loss of contractile function. In contrast, “gain- ” prepare the flow cell, 20 μLof5μg/mL N-ethyl maleimide-myosin was flowed in of-function mutations that lead to cell proliferation and increased and incubated for 2 min. The flow cell was rinsed with 20 μL of 5 mg/mL motility, or hyperplastic arterial smooth muscle cell growth, might casein, and then with 20 μL of rinse buffer. Twenty microliters of G-actin (1–5 μM, lead to occlusive vascular diseases. With R258C, pathways that lead also containing 1.64 μM Lifeact) in G-buffer was mixed with 20 μLof2× to cell proliferation could be mediated via the observed altered polymerization buffer to start the polymerization. The mixture was then interactions of R258C filaments with cofilin and profilin, leading to flowed into the flow cell (two times, 20 μL each time). Excess solution was increased pools of monomeric actin, and by the intrinsic lability of removed, and the flow cell was sealed with nail polish. The flow cell was the mutant filament. The R258C mutation affects essentially all immediately put on a Nikon ECLIPSE Ti microscope equipped with through- aspects of actin function that we measured in vitro. Cellular studies objective type TIRF and a temperature control unit (37 °C). The samples were with fibroblasts and smooth muscle cells derived from patients with excited with the TIRF field of a 488-nm laser line. The fluorescence image was the R258C mutation, or mouse models, will establish how these observedwitha100× objective and recorded on an Andor EMCCD camera defects are manifested in a cellular context. (Andor Technology USA) at a rate of one frame per second for 1–3minwith automatic focus correction. The final resolution is 0.1066 μmperpixel. Materials and Methods For experiments with profilin, 1 μLof120μM profilin (3 μM final) was added Details of cloning, expression, and purification of proteins used in this study; the to the final 40-μL mixture. For experiments with tropomyosin, various amounts tropomyosin binding assay; actin labeling and filament formation for motility; of tropomyosin were added to the mixture so that the final tropomyosin the in vitro motility assay; measurement of ADP dissociation rates; and force concentration was one-fourth the concentration of the actin monomer. For data acquisition and analysis are provided in SI Materials and Methods. experiments with cofilin, actin was first polymerized at 37 °C for a few minutes in the flow cell while being monitored on the microscope. Once the filaments μ Actin Cloning, Expression, and Purification. A chimeric construct composed of reached the desired length and density, 20 L of G-buffer containing various human SM α-actin (ACTA2 gene; accession number NP_001604) and human amounts of cofilin and 1.64 μM Lifeact was mixed with 20 μLof2× polymer- thymosin-β4 (accession number AAI41977.1) was cloned into pAcUW2B for ization buffer and then flowed into the flow cell. The flow cell was then sealed expression in the baculovirus/insect cell system. The initial Cys residue of with nail polish before observation on the microscope. actin was not included in the construct because it is not present in the mature, processed protein. The thymosin-β4 gene was separated from the Fluorescence Data Processing. Movies following actin polymerization or de- actin gene by a 14-aa linker (ASSGGSGSGGSGGA) to allow the thymosin-β4 polymerization as a function of time were imported into ImageJ (National enough flexibility to occupy the barbed end binding site on the actin Institutes of Health). The growth or shrinkage was followed manually. The monomer, thus preventing the actin from polymerizing with itself or native polymerization rate was obtained by dividing the total growth by time. Rate

Sf9 cell actin. A HIS6 tag was added at the C terminus for purification on a was converted from micrometers per second to subunits per second by nickel affinity column. The final native residue of actin is Phe, which provides multiplying by the converting factor of 370 actin subunits per micrometer. For a site for chymotrypsin cleavage to remove the linker, thymosin-β4, and the each condition, 15–50 growing filaments were measured.

HIS6 tag completely. The native codons for both actin and thymosin-β4 were Rates of polymerization were plotted against the actin monomer con- replaced with Drosophila preferred codons. centration. The data were linearly fit to Rate = a[actin] + d, in which a is the For expression of human SM α-actin, infected Sf9 cells (4 × 109) were har- second-order assembly rate of polymerization, d is the first-order rate of vested 3 d after infection and lysed in 160 mL of 1 M Tris·HCl (pH 7.5 at 4 °C), disassembly, and the critical concentration is −d/a.

0.6 M NaCl, 0.5 mM MgCl2,0.5mMNa2ATP, 1 mM DTT, 4% (vol/vol) Triton X-100, To determine the frequency of severing for experiments with cofilin, the 1 mg/mL Tween-20, and protease inhibitors [0.5 mM 4-(2-aminoethyl)benzene- total number of severing events (actin filaments that clearly broke into two sulfonyl fluoride hydrochloride, 5 μg/mL leupeptin, 0.5 mM tosyllysine chloromethyl pieces) was counted for each 1 min of video and then divided by the total actin ketone hydrochloride]. The cells were stirred for 2 h and then clarified. The filament length in the field of view at the starting frame of this 1-min video. soluble fraction was dialyzed overnight against 10 mM Hepes (pH 7.5), Shortening of actin by cofilin was traced manually and then divided by time. 0.25 mM CaCl2, 0.3 M NaCl, 7 mM β-mercaptoethanol, 0.25 mM Na2ATP, and μ – 1 g/mL leupeptin. Following dialysis, the supernatant was applied to 5 10 mL Modeling of Polymerization Data in the Presence of Profilin. The following as- – of a nickel affinity column (HIS-Select; Sigma Aldrich). Nonspecifically bound sumptions were made to model the polymerization of G-actin in the presence of contaminating proteins were washed off with dialysis buffer containing profilin. Monomeric G-actin and profilin form a profilin–G-actin complex with 10 mM imidazole (pH 7.5). Actin was eluted with 100 mM imidazole, 10 mM affinity Kd. Both G-actin and profilin–G-actin can participate in the polymeri- Hepes (pH 7.5), 0.25 mM CaCl2, 0.3 M NaCl, 7 mM β-mercaptoethanol, 0.25 mM zation process, with rates of ra and rap, respectively. Protomers dissociate from Na2ATP, and 1 μg/mL leupeptin. The peak fractions were pooled and dialyzed F-actinwithraterd. The overall scheme is described in the following equations: overnight against G-buffer [5 mM Tris (pH 8.26 at 4 °C), 0.2 mM CaCl2,0.1mM NaN , 0.5 mM DTT, 0.2 mM Na ATP, 1 μg/mL leupeptin]. The protein was Kd 3 2 profilin · actin ⇔ actin + profilin clarified by centrifugation, and the thymosin-HIS tag was cleaved from the actin with chymotrypsin (chymotrypsin/actin in a 1:80 weight ratio). The actin was − ð Þ + !ra − ð + Þ separated from the thymosin-His tag using a Mono Q 5/50 GL column (GE f actin n actin f actin n 1 Healthcare) with a gradient of 0–0.3 M NaCl in 5 mM Tris (pH 8.26 at 4 °C), − ð Þ + · rap − ð + Þ 0.2 mM CaCl2,0.1mMNaN3, 0.5 mM DTT, 0.2 mM Na2ATP, and 1 μg/mL f actin n profilin actin !f actin n 1 leupeptin, followed by a step to 0.5 M NaCl. Peak fractions were pooled, con- rd centrated, and dialyzed against 5 mM Tris (pH 8.26 at 4 °C), 0.2 mM CaCl2,0.1mM f − actinðnÞ !f − actinðn − 1Þ NaN3,0.5mMDTT,0.2mMNa2ATP (pH 7), and 1 μg/mL leupeptin. Modeling of Polymerization of Mixtures of WT and R258C. In this scheme, as il- Actin Polymerization Visualized by TIRF Microscopy. The 2× polymerization lustrated in Fig. 4B, the rate of association of G-actin to the end of an F-actin

buffer consists of 20 mM imidazole (pH 7.5), 100 mM KCl, 8 mM MgCl2,2mM filament, and the dissociation of G-actin from F-actin, depends on the com- EGTA, 0.5% methylcellulose, 4 mM MgATP, 0.26 mg/mL glucose oxidase, position of the barbed end of the F-actin polymer. For polymerization, if the 0.10 mg/mL catalase, 6 mg/mL glucose, and 20 mM DTT. The rinse buffer is last actin in F-actin is WT, then both the WT and R258C G-actin monomers add

composed of equal volumes of G-buffer [5 mM Tris (pH 8.26 at 4 °C), 0.2 mM to the F-actin with a rate of rw (WT association rate). If the end unit of F-actin CaCl2, 0.2mM Na2ATP, 0.5 mM DTT] and 2× polymerization buffer. is R258C, then a WT G-actin will add onto it with a rate of rw, but an R258C

E4176 | www.pnas.org/cgi/doi/10.1073/pnas.1507587112 Lu et al. Downloaded by guest on September 28, 2021 PNAS PLUS monomer will add to it with a rate of rm (R258C mutant–mutant association of rdm (R258C mutant dissociation rate). The following two equations de- rate). The following four equations describe the association reaction: scribe the dissociation reaction:

rw f − actinðnÞ · WT + WT !f − actinðn + 1Þ r f − actinðnÞ · WT !dw f − actinðn − 1Þ r f − actinðnÞ · R258C + WT !w f − actinðn + 1Þ r f − actinðnÞ · R258C !dm f − actinðn − 1Þ r f − actinðnÞ · WT + R258C !w f − actinðn + 1Þ

r ACKNOWLEDGMENTS. We thank Dianna Milewicz, Kristine Kamm, and f − actinðnÞ · R258C + R258C !m f − actinðn + 1Þ Susan Lowey for critical reading of the manuscript. We thank Jason Stumpff for the use of his microscope, and Alex Hodges and Elena Krementsova for For depolymerization, if the end protomer in F-actin is WT, it dissociates with their early contributions to this project. This work was supported by National arateofr (WT dissociation rate), but if it is R258C, it dissociates with a rate Institutes of Health Grant P01 HL110869. dw SEE COMMENTARY

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