Structural and biophysical features of α- and β-actin isoforms in native and inactivated states.

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Actin isoforms organize into distinct networks that are essential for the normal function of eukaryotic cells. Despite a high level of sequence and structure conservation, subtle differences in their design principles determine the interaction with myosin motors and actin-binding proteins. Therefore, identifying how the structure of actin isoforms relates to function is important for our understanding of normal cytoskeletal physiology. Here, we report the high-resolution structures of filamentous skeletal muscle α-actin (3.37 Å), cardiac muscle α-actin (3.07 Å), ß-actin (2.99 Å), and γ-actin (3.38 Å) in the Mg2+·ADP state with their native post-translational modifications. The structures revealed isoform-specific conformations of the N-terminus that shift closer to the filament surface upon myosin binding, thereby establishing isoform-specific interfaces. Collectively, the structures of single-isotype, post-translationally modified bare skeletal muscle α-actin, cardiac muscle α-actin, ß-actin, and γ-actin reveal general principles, similarities, and differences between isoforms. They complement the repertoire of known actin structures and allow for a comprehensive understanding of in vitro and in vivo functions of actin isoforms. Editor's evaluation This study presents four high quality cryo-EM structures of ADP-actin filaments formed from skeletal α-, cardiac α-, cytoplasmic β- and cytoplasmic γ-actin. These structures are important for understanding the functional differences among these actin isoforms. This work is of significant general interest, because actin filaments, composed of different actin isoforms, have a critical role in a number of physiological processes from muscle contraction to cell migration and division. https://doi.org/10.7554/eLife.82015.sa0 Decision letter eLife's review process eLife digest The protein actin is important for many fundamental processes in biology, from contracting muscle to dividing a cell in two. As actin is involved in such a variety of roles, human cells have slightly different versions of the protein, known as isoforms. For example, alpha-actin is vital for contracting muscle, while beta- and gamma-actin drive cellular processes in non-muscle cells. In order to carry out its various functions, actin interacts with many other proteins inside the cell, such as myosin motors which power muscle contraction. These interactions rely on the precise chain of building blocks, known as amino acids, that make up the actin isoforms; even subtle alterations in this sequence can influence the behavior of the protein. However, it is not clear how differences in the amino acid sequence of the actin isoforms impact actin’s interactions with other proteins. Arora et al. addressed this by studying the structure of four human actin isoforms using a technique called cryo-electron microscopy, where the proteins are flash-frozen and bombarded with electrons. These experiments showed where differences between the amino acid chains of each isoform were located in the protein. Arora et al. then compared their structures with previous work showing the structure of actin bound to myosin. This revealed that the tail-end of the protein (known as the N-terminus) differed in shape between the four isoforms, and this variation may influence how actin binds to others proteins in the cell. These results are an important foundation for further work on actin and how it interacts with other proteins. The structures could help researchers design new tools that can be used to target specific isoforms of actin in different types of laboratory experiments. Introduction Actin isoforms are among the most ubiquitous and abundant structural proteins that facilitate the functional organization of the cytoplasm of eukaryotic cells (Blanchoin et al., 2014; Pollard, 2016). Humans express six actin genes in a tissue-specific and developmentally regulated manner (Kashina, 2020). The gene products are structurally and functionally highly conserved among vertebrates and can be grouped into four muscle actins: skeletal muscle α-actin, smooth muscle α-actin (vascular), cardiac muscle α-actin, smooth muscle γ-actin (enteric), and two nonmuscle actins (ß-actin and γ-actin; Otey et al., 1987; Vandekerckhove and Weber, 1978). Most cells maintain a defined ratio of actin isoforms with muscle and nonmuscle actins representing the main isoforms in muscle and nonmuscle cells, respectively (Kashina, 2020; Otey et al., 1987; Vandekerckhove and Weber, 1978; Kee et al., 2009; Tondeleir et al., 2009; Patrinostro et al., 2017). Actin isoforms have specific and redundant roles in cells and display different biochemistries, cellular localization, and interactions with myosin motors and actin-binding proteins (ABPs; Pollard, 2016; Kashina, 2020; Perrin and Ervasti, 2010; Vedula et al., 2021; Varland et al., 2019; Bunnell et al., 2011; Tondeleir et al., 2012; Baranwal et al., 2012; Diensthuber et al., 2011; Müller et al., 2013; Lee and Dominguez, 2010; Harris et al., 2020; Lappalainen, 2016). Driven by the dominating action of ABPs and signaling proteins, differences between actin isoforms may facilitate the formation of diverse cellular actin networks with distinct compositions, architectures, dynamics, and mechanics that enable fundamental cell functions including adhesion, migration, and contractility (Blanchoin et al., 2014; Tondeleir et al., 2009; Vedula et al., 2021). The altered expression and mutation of the genes encoding for actin isoforms have been linked to human diseases (Tondeleir et al., 2009; Chaponnier and Gabbiani, 2004; Parker et al., 2020). Actin isoforms share high sequence identity at the protein level (~93–99%) and the propensity to self-assemble into helical, polarized filaments (F-actin) from monomers (G-actin; Pollard, 2016; Perrin and Ervasti, 2010; Dominguez and Holmes, 2011; Arnesen et al., 2018). Since the publication of the first crystal structure of G-actin in complex with DNaseI ~30 years ago, extensive studies have advanced our understanding of the structure of monomeric and filamentous actin, polymerization mechanisms, post-translational modifications (PTMs), interaction with drugs, myosin motors, and ABPs at ever-increasing resolution (Dominguez and Holmes, 2011; Holmes et al., 1990; Kabsch et al., 1990; von der Ecken et al., 2016; Chou and Pollard, 2019; Zsolnay et al., 2020; Chou and Pollard, 2020; Belyy et al., 2020; Merino et al., 2018; Mentes et al., 2018; Mei et al., 2020; Ducka et al., 2010; Lee et al., 2007; Otomo et al., 2005; Oda et al., 2009; Egelman et al., 1982; Ali et al., 2022; Oda et al., 2020; Gong et al., 2022). Although isoform-specific mechanisms with myosin motors and ABPs that drive functional distinction are widely described, they are poorly understood at the structural level. To address how the structure contributes to the functional distinction of actin isoforms, we employed a combination of recombinant post-translationally modified actins and actins purified from native source to obtain pure, single-isotype preparations of individual actin isoforms to perform cryo-electron microscopy (cryo-EM) analyses. Specifically, we used our previously established Pick-ya actin method to recombinantly produce human ß-actin and γ-actin in an engineered Pichia pastoris strain that expresses the human N-acetyl transferase NAA80 and histidine methyl transferase SETD3 to ensure uniform Nt-acetylation and methylation of H72/H73, a conserved PTM profile of vertebrate actins (Hatano et al., 2020). Skeletal muscle α-actin and cardiac muscle α-actin were purified from rabbit skeletal muscle and the left ventricle of a bovine heart, respectively. At the protein level, all actin isoforms are conserved across vertebrates, allowing us to compare our structures to previous structures of filamentous actin from other vertebrate species in the correct physiological context (Figure 1—figure supplement 1; Pollard, 2016; Perrin and Ervasti, 2010; Dominguez and Holmes, 2011). Our 2.99–3.38 Å resolution structures of filamentous actin isoforms show that the N-termini of bare muscle and nonmuscle actins have different orientations that contribute to distinct binding interfaces for myosin motors and likely other ABPs. Results High-resolution structures and general principles of actin isoforms To determine the structural characteristics and differences between actin isoforms, we solved the high-resolution structures of single-isotype skeletal muscle α-actin (3.37 Å), cardiac muscle α-actin (3.07 Å), ß-actin (2.99 Å), and γ-actin (3.38 Å) in the filamentous state using cryo-EM (Figure 1A–D, Figure 1—figure supplement 1, Table 1). The structures show local resolutions ranging from 2.1 to 3.5 Å for skeletal muscle α-actin, 1.9–3.3 Å for cardiac muscle α-actin, 1.5–3.0 Å for ß-actin, and 2.1–4.4 Å for γ-actin (Figure 1—figure supplement 2). For all actin isoforms, the highest local resolutions were obtained in the central core region. Lower local resolutions were obtained in the most surface-exposed and flexible regions such as the N-terminus and the D-loop as expected. All actin structures were solved in the Mg2+·ADP state (Figure 1E, Figure 2) and have been obtained without the use of stabilizing drugs that may interfere with the filament structure (Diensthuber et al., 2011; Isambert et al., 1995; Zimmermann et al., 2015). Figure 1 with 8 supplements see all Download asset Open asset Cryo-electron microscopy (cryo-EM) filament structures of actin isoforms. (A) Helical reconstruction of skeletal muscle α-actin, (B) cardiac muscle α-actin, (C) β-actin, and (D) γ-actin. Views in (B–D) are according to (A). Four individual actin protomers in the filament are shown and denoted with italic numbers. The pointed (−) and barbed (+) ends are indicated. (E) Representative key regions of actin isoforms with corresponding cryo-EM densities in transparent surface representation are shown. The protein backbone and amino acid side chains are shown in licorice and stick representation, respectively. Throughout this work, amino acids are numbered according to the sequence of mature actin isoforms (Figure 1—figure supplement 1C). Figure 2 with 1 supplement see all Download asset Open asset Conserved nucleotide-binding cleft active site in actin isoforms. (A–D) Coordination of Mg2+·ADP in the nucleotide-binding cleft of skeletal muscle α-actin (A), cardiac muscle α-actin (B), β-actin (C), and γ-actin (D). Underlines indicate locations of amino acid substitutions between actin isoforms. The protein backbone and side chains are shown in licorice and stick representation, respectively. ADP is shown in cyan-colored stick representation. Electron densities for key amino acids in the nucleotide-binding cleft active site of actin isoforms are shown. Schematic representations of key interactions in the nucleotide-binding cleft active sites of the respective actin isoforms are shown in the right panel. The schematics are not drawn to scale. Table 1 Data collection, image processing, and structure characteristics summary. MapSkeletal muscle α-actinCardiac muscle α-actinβ-actinγ-actinData collectionMicroscopeFEI Titan Krios G3iFEI Titan Krios G3iFEI Titan Krios G3iFEI Titan Krios G3iVoltage (kV)300300300300DetectorGatan K3Gatan K3Gatan K3Gatan K3Automation softwareEPUEPUEPUEPUEnergy filter slit width (eV)20202020Recording modeSuper-resolutionSuper-resolutionSuper-resolutionSuper-resolutionMagnification (nominal)81,00081,00081,00081,000Movie micrograph pixel size (Å)0.8910.8910.8910.891Total Dose rate (e−/Å2)65605065Defocus range (µm)–0.5 to –2.5–0.5 to –2.5–0.5 to –2.5–0.5 to –2.5Spherical aberration (mm)0.010.010.010.01Movies2046144413522952Total extracted particles261,195657,300279,1201,249,379Total # of refined particles185,406657,041263,9111,009,372ReconstructionEMDB codeEMD-27548EMD-27549EMD-27572EMD-27565Box size350256256256SymmetryhelicalhelicalC1C1Map sharpening B-factor (Å2)–90–149–81–201Resolution (global) (Å)3.373.072.993.38Structure building and validationPDB ID8 DMX8DMY8DNH8DNFModel buildingCootCootCootCootRefinement programPhenixPhenixPhenixPhenixRefinement targetReal-spaceReal-spaceReal-spaceReal-spaceRMSD from ideal valuesBond length (Å)0.020.020.040.03Bond Angles (0)0.4930.4940.7610.711Ramachandran favored (%)97.6196.6896.2196.20Ramachandran allowed (%)2.393.323.793.46Ramachandran outliers (%)0000.34MolProbity Score1.421.421.721.72Structures CharacteristicsSpeciesRabbitBovineHumanHumanAmino acid resolved4–3752–3751–3741–374PTMs resolvedH73H73D1/H72E1/H72 Our cryo-EM maps allowed us to build unambiguous models of actin isoforms in which secondary structure information including the side chains, the nucleotide and associated cation (Mg2+·ADP), and PTMs were apparent from the densities (Figure 1E, Figure 1—figure supplement 2). This allowed us to resolve the N-terminus of actin isoforms that is often disordered or missing in prior structures (Kudryashov and Reisler, 2013). For ß-actin and γ-actin, we could resolve the entire N-terminus starting from amino acids D1 and E1, respectively. For skeletal muscle α-actin and cardiac muscle α-actin, we could resolve the N-terminus starting from amino acids E4 and D2, respectively. The lack of resolvable density for the very first amino acid of cardiac muscle α-actin and the first three amino acids of skeletal muscle α-actin may be attributed to a nonuniform PTM pattern of native actin isoforms prepared from muscle compared to our recombinant nonmuscle actin isoforms with uniform PTM pattern in that the entire N-terminus region could be resolved (Figure 1E, Figure 1—videos 1–5). Consistent with the high sequence conservation across actin isoforms (Figure 1—figure supplement 1B–C), our reconstructions show the characteristic double-stranded actin helix with a helical rise of ~27.6 Å to 28 Å and a helical twist of ~–166.5° to –168° (Figure 1A–D, Figure 1—figure supplement 1A) that has been observed in numerous previous structural studies, including previous high-resolution cryo-EM studies (Holmes et al., 1990; Chou and Pollard, 2019; Mei et al., 2020; Oda et al., 2009; Egelman et al., 1982; Ali et al., 2022; Fujii et al., 2010; Galkin et al., 2015). The actin filament itself is composed of G-actin (42 kDa) protomers that are oriented in the same direction (Holmes et al., 1990). Each protomer folds into four subdomains that are referred to as SD1–SD4 (Figure 1, Figure 1—figure supplement 1A; Kabsch et al., 1990). SD1 and SD2 form the outer domain, and SD3 and SD4 form the inner domain (Figure 1). This domain arrangement results in the formation of two clefts – the nucleotide-binding cleft and the barbed end groove (Figure 1, Figure 1—figure supplement 1A; Pollard, 2016; Dominguez and Holmes, 2011; Merino et al., 2020). The nucleotide-binding cleft between SD2 and SD4 harbors the active site that is occupied by Mg2+·ADP in our structures (Figure 1E, Figure 2, Figure 1—figure supplement 1A). The barbed end groove between SD1 and SD3 represents a major binding interface for myosins and ABPs (von der Ecken et al., 2016). It further mediates longitudinal interfaces within the actin filament. SD2 and SD4 of an actin protomer are at the pointed end, and SD1 and SD3 are at the barbed end (Figure 1—figure supplement 1A; Pollard, 2016; Dominguez and Holmes, 2011; Merino et al., 2020). The longitudinal interface between two adjacent actin protomers involves the extended D-loop located in SD2 of one actin protomer that interacts with amino acids located in SD1 and SD3 of another protomer. Both the N- and C-terminus of the actin protomer are in SD1 (Pollard, 2016; Dominguez and Holmes, 2011; Merino et al., 2020). Similarities and differences between actin isoforms The superposition of all actin isoform structures shows a root-mean-square deviation (RMSD) between 0.83Å to 1.04Å for Cα atoms, indicating an overall similar topology. No significant differences in the pitch of the actin helix were observed between our structures of actin isoforms, emphasizing their overall conserved filamentous structure in the absence of myosin motors or ABPs. Actin isoforms differ by conservative and nonconservative substitutions (Figure 1—figure supplement 1C) that contribute to their distinct biochemical and in vivo functions (Blanchoin et al., 2014; Tondeleir et al., 2009; Vedula et al., 2021). Overall, the amino acid sequence is more conserved among muscle actins than between muscle and nonmuscle actins (Figure 1—figure supplement 1C). A structural comparison of our cryo-EM reconstructions shows amino acid substitutions across isoforms with the positions of substituted amino acids highlighted (Figure 3). Actin isoforms show the largest divergence at the acidic N-terminus within SD1 (Figure 1E, Figure 1—figure supplement 1C, Figure 3A). Of note, amino acids 1–3 in our structure of skeletal muscle α-actin and the first amino acid in our structure of cardiac muscle α-actin are not resolved and therefore not shown in Figure 3. Other substitutions are within SD1 (Figure 3B, Figure 3—figure supplement 1), SD3 (Figure 3C, Figure 3—figure supplement 1), and SD4 (Figure 3D, Figure 3—figure supplement 1). There are no substitutions in SD2, the smallest and most flexible subdomain (Kudryashov and Reisler, 2013). Figure 3 with 1 supplement see all Download asset Open asset Similarities and differences between actin isoforms. (A) Sequence variations at the N-terminus located in SD1 of actin isoforms. (B) Sequence variations in SD1 of actin isoforms. (C) Sequence variations in SD3 of actin isoforms. (D) Sequence variations in SD4 of actin isoforms. SD2 is conserved between actin isoforms. The identical and nonidentical amino acids at sites of substitutions within the actin protomer across isoforms are shown for skeletal muscle α-actin (orange), cardiac muscle α-actin (yellow), β-actin (purple), and γ-actin (teal) as spheres. Note that the first three amino acids of skeletal muscle α-actin and the first amino acid of cardiac muscle α-actin are unresolved in our structures. The protein backbone is shown in licorice representation, and the substituted amino acids are shown in spheres representation. Amino acid substitutions at subdomain interfaces, such as the nucleotide-binding cleft active site of actin isoforms, are likely to influence protein function. To evaluate their possible impact on nucleotide coordination and the structural organization of the active site, we performed a comparative structural analysis. Our cryo-EM reconstructions show that the nucleotide-binding cleft active site is conserved between actin isoforms (Figure 1E and Figure 2). The densities for Mg2+ and ADP were assigned without ambiguity and revealed interactions with amino acids located in SD2 (Q58/Q59 and Y68/Y69) and SD4 (E206/E207, R209/R210, K212/K213, and E213/E214) but also with amino acids located in SD1 (M15/L16, K17/K18, Q136/Q137, and Y336/Y337) and SD3 (D156/D157, M304/M305, Y305/Y306, and K335/K336; Figure 2). While most of the interactions are polar and electrostatic, amino acid Y305/Y306 forms π-π interactions with the adenine ring of ADP. The superimposition of the nucleotide cleft active sites of our four structures of actin isoforms shows small differences in the positions of the nucleotide (RMSD ~0.44–0.47 Å) and the bound Mg2+ (RMSD ~0.5–1.3 Å), especially in the position of the β-phosphate group relative to the α-phosphate group (Figure 2, Figure 2—figure supplement 1). The position of the Mg2+ moves relative to the position of the β-phosphate group (Figure 2—figure supplement 1). The superimposition of the nucleotide-binding cleft active sites further shows small local rearrangements of side chains of conserved amino acids (Figure 2—figure supplement 1), including R182/R183 and K335/K336. Amino acids M15/L16 and M304/M305 form hydrophobic interactions in the nucleotide-binding cleft active site (Figure 2). Amino acid substitution M15/L16 between nonmuscle and muscle actin does not alter the overall topology of the nucleotide-binding site. Instead, the longer side chain of M15 in nonmuscle actins, located in a loop that protrudes into the nucleotide-binding cleft, acts as an extended lid that flanks the active site and shields the phosphate groups of ADP (Figure 2, Figure 2—figure supplement 1). Near the nucleotide-binding cleft active site, amino acids C10 and V17 in muscle actins are substituted with V9/I9 and C16 in β/γ-actin (Figure 3, Figure 1—figure supplement 1C). These reciprocal amino acid substitutions maintain the overall oxidation-reduction environment within filamentous actin isoforms which is important for its dynamic properties and the interaction with some regulatory proteins (Farah et al., 2011; Lassing et al., 2007; Wilson et al., 2016; Terman and Kashina, 2013). The analysis of interprotomer interfaces in our four structures of actin isoforms (Figure 4, Figure 5, Figure 4—figure supplement 1) showed that longitudinal interactions are mainly mediated by hydrophilic amino acids that are likely to enable interactions with water molecules that were recently shown to mediate interprotomer contacts within the filament core (Figure 4, Figure 4—figure supplement 1; Reynolds et al., 2022). Amino acid substitutions at the longitudinal interprotomer interface (also called long pitch helix interface) include L175/M176, T200/V201, Q224/N225, C271/A272, F278/Y279, and V286/I287. These substitutions may influence the stability of the promoters based on their ability to interact with the solvent and other protomer residues (Reynolds et al., 2022). The transverse interprotomer interface (also called short pitch helix interface) is formed through hydrophilic and hydrophobic interactions (Figure 5, Figure 4—figure supplement 1). In contrast to interactions at the longitudinal interface, most of the transverse interprotomer interactions are direct and not mediated by solvent. A single amino acid substitution (V286/I287) is at the transverse interprotomer This amino acid substitution is located the of the longitudinal and transverse interprotomer interfaces (Figure 3—figure supplement 1). Amino acid is in hydrophobic with and in muscle actins and a surface of the interaction between and in nonmuscle actins is hydrophobic and a surface of in the transverse interprotomer interface (Figure 4—figure supplement and with the D-loop located in SD2 are conserved in our structures of actin isoforms, the critical and conserved role of the D-loop to mediate interprotomer At the of the longitudinal and transverse interprotomer interfaces, and with as central Figure with 1 supplement see all Download asset Open asset structural analysis of the longitudinal interprotomer (A–D) residues at the interprotomer interface of skeletal muscle α-actin (A), cardiac muscle α-actin (B), β-actin (C), and γ-actin (D). protomers in actin isoforms are oriented according to Figure 4—figure supplement Underlines indicate locations of amino acid substitutions between actin isoforms. The protein backbone and side chains are shown in licorice and stick representation, respectively. Figure Download asset Open asset structural analysis of the transverse interprotomer (A–D) residues at the interprotomer interface of skeletal muscle α-actin (A), cardiac muscle α-actin (B), β-actin (C), and γ-actin (D). protomers in actin isoforms are oriented according to Figure 4—figure supplement Underlines indicate locations of amino acid substitutions between actin isoforms. The protein backbone and amino acid side chains are shown in licorice and stick representation, respectively. PTMs are not essential for actin and but also for their interaction with myosin motors and et al., 2019; et al., et al., 2018). Here, we on two widely and highly conserved PTMs of mature vertebrate actins that are important for actin structure and the post-translational of the N-terminus by NAA80 and methylation of by domain protein 3 Kabsch et al., 1990; Terman and Kashina, 2013; et al., 2018; et al., 2019; et al., Both PTMs are in actin prepared from vertebrate and our preparations of recombinant human β- and γ-actin in an engineered Pichia pastoris strain (Hatano et al., 2020). The quality of our density maps allowed us to resolve key PTMs (Figure 1E, Figure 5, Figure 1—figure supplement 3). Specifically, the of resolvable density allowed us to the entire N-terminus including the D1 and the in the cryo-EM reconstructions of β- and γ-actin (Figure The site on the N-terminus is on the filament surface and an to the N-terminus (Figure 1—figure supplement 1C). in previous cryo-EM structures of muscle are no resolvable densities for the very including in our density maps of skeletal muscle α-actin and cardiac muscle α-actin (Figure to nonuniform PTM of muscle In to Nt-acetylation in β- and γ-actin, we could resolve the in all cryo-EM reconstructions of actin isoforms (Figure 1—figure supplement 3). The of key PTMs in our cryo-EM reconstructions that our recombinant human actins post-translationally mature nonmuscle actins (Hatano et al., 2020). actin filament structure motors actin in a manner to and and The myosin can be into with and and and actin and 2009; and In the a binding interface is established between actin and the myosin domain (von der Ecken et al., 2016; and 2021; and Holmes, To determine myosin binding to actin may actin filament we compared our structures of filamentous bare actin isoforms with previous high-resolution cryo-EM structures of compared the structure of nonmuscle bound to γ-actin, the structure of bound to skeletal muscle α-actin and the structure of bound to skeletal muscle α-actin with our structures of bare actin isoforms (Figure also compared the structure of cardiac bound to the cardiac filament muscle α-actin with and Figure supplement 1) with our structure of bare cardiac muscle The structures were they distinct binding and of myosin motors with different et al., 2018; and and 2011). Figure with 2 supplements see all Download asset Open asset The (A) of bare actin isoform structures in the Mg2+·ADP state superimposition of actin isoforms structures and of bare actin structures are shown. (B) of the interface at the D-loop region. For involved in the binding of myosins are highlighted in the respective The between the structures in the two is by a of SD2 in compared to bare actin structures. binding to actin filaments subtle in the filament (Figure of the actin structures showed that the Cα of bare and actin protomers with an of These are similar for myosins from different and of the nucleotide state of the respective domain (Figure The superposition of structures of bare actin isoforms and known structures of actins shows that SD2 a different (Figure and Figure supplement 1, Figure 1). The of the located in SD2, with a Cα of Å (Figure and of bare γ-actin with complex shows the subtle of the D-loop with a Cα of Å in the that subtle in the barbed end groove that not the between and The of the D-loop (RMSD Å) is also observed in the structure of cardiac myosin bound to the filament compared to our structure of bare cardiac muscle α-actin (Figure supplement 1),

The protein actin is important for many fundamental processes in biology, from contracting muscle to dividing a cell in two. As actin is involved in such a variety of roles, human cells have slightly different versions of the protein, known as isoforms. For example, alpha-actin is vital for contracting muscle, while beta- and gamma-actin drive cellular processes in non-muscle cells. In order to carry out its various functions, actin interacts with many other proteins inside the cell, such as myosin motors which power muscle contraction. These interactions rely on the precise chain of building blocks, known as amino acids, that make up the actin isoforms; even subtle alterations in this sequence can influence the behavior of the protein. However, it is not clear how differences in the amino acid sequence of the actin isoforms impact actin’s interactions with other proteins. Arora et al. addressed this by studying the structure of four human actin isoforms using a technique called cryo-electron microscopy, where the proteins are flash-frozen and bombarded with electrons. These experiments showed where differences between the amino acid chains of each isoform were located in the protein. Arora et al. then compared their structures with previous work showing the structure of actin bound to myosin. This revealed that the tail-end of the protein (known as the N-terminus) differed in shape between the four isoforms, and this variation may influence how actin binds to others proteins in the cell. These results are an important foundation for further work on actin and how it interacts with other proteins. The structures could help researchers design new tools that can be used to target specific isoforms of actin in different types of laboratory experiments.

In vertebrate skeletal muscle, the proliferating myoblasts synthesize nonmuscle isoforms of actin, and the cells begin to express muscle-specific actin isoforms during their myogenic differentiation. To study the distributions of the actin isoforms in myogenic cells and fully differentiated skeletal muscle, we prepared a peptide antibody specific for the skeletal alpha isoform of actin and used this antibody along with an antibody specifically reactive with nonmuscle gamma actin to stain cultured myotubes and adult skeletal myofibrils by double-indirect immunofluorescence. At this level of resolution, no differences in isoform localization were seen: Both muscle and nonmuscle actins were detected in the myotubes and in the striations of mature myofibrils. Myotubes were also double-stained using immunogold electron microscopy, and the isoform distributions were determined quantitatively by counting the two sizes of gold particles that corresponded to labeling with each antibody. A quantitative analysis of immunoreactivity revealed that, although both forms were present in all actin-containing structures, nonmuscle actin was relatively more prevalent along the edges (cortical microfilaments) of the myotubes, whereas the muscle isoform predominated in the interior regions (containing forming myofibrils). Thus, we have found evidence of a heterogeneous distribution of muscle and nonmuscle actin isoforms in differentiating myogenic cells, and we have demonstrated that a nonmuscle actin isoform is a component of the muscle contractile apparatus.

Developmental changes in actin and myosin heavy chain isoform expression in smooth muscle

Actin is a highly conserved protein although many isoforms exist. In vertebrates and insects the different actin isoforms can be grouped by their amino acid sequence and tissue-specific gene expression into muscle and nonmuscle actins, suggesting that the different actins may have a functional significance. We ask here whether atomic models for G- and F-actins may help to explain this functional diversity. Using a molecular graphics program we have mapped the few amino acids that differ between isoactins. A small number of residues specific for muscle actins are buried in internal positions and some present a remarkable organization. Within the molecule, the replacements observed between muscle and nonmuscle actins are often accompanied by compensatory changes. The others are dispersed on the protein surface, except for a cluster located at the N-terminus which protrudes outward. Only a few of these residues specific for muscle actins are present in known ligand binding sites except the N-terminus, which has a sequence specific for each isoactin and is directly implicated in the binding to myosin. When we simulated the replacements of side chains of residues specific for muscle actins to those specific for nonmuscle actins, the N-terminus appears to be less compact and more flexible in nonmuscle actins. This would represent the first conformational grounds for proposing that muscle and nonmuscle actins may be functionally distinguishable. The rest of the molecule is very similar or identical in all the actins, except for a possible higher internal flexibility in muscle actins. We propose that muscle actin genes have evolved from genes of nonmuscle actins by substitutions leading to some conformational changes in the protruding N-terminus and the internal dynamics of the main body of the protein.

Cultured fibroblasts develop several biochemical and morphological properties of smooth muscle cells, particularly the expression of alpha-smooth muscle actin, the actin isoform typical of vascular smooth muscle cells. They resemble modified fibroblasts or myofibroblasts observed in granulation tissue during wound repair and in fibrotic situations. We have analysed by immunolabeling the fate of exogenous epitope-tagged actin isoforms by transfection of the corresponding cDNAs into fibroblasts cultured from rat subcutaneous tissue. Tagged muscle actins were efficiently integrated into stress fibers and did not produce obvious changes in cell shape of transfected cells. Transfected nonmuscle actins in contrast changed the morphology and were not or poorly incorporated into stress fibers. These cultured subcutaneous fibroblasts behave similarly to smooth muscle cells when transfected with the same actin encoding cDNAs, indicating another common characteristic of these two cell types in sorting and targeting actin isoforms. Subcutaneous fibroblasts transfected with muscle and nonmuscle actin isoforms provide a good in vitro model to analyze the intracellular sorting of isoactins and to improve our knowledge of myofibroblast characterization and differentiation during tissue repair as well as to understand the relationships between modifications of actin cytoskeleton, adhesion and extracellular matrix proteins.

In many vertebrate nonmuscle cells, the microfilament subunit protein, actin, exists as two isoforms, called beta and gamma, whose sequences differ only in their amino-terminal regions. We have prepared a peptide antibody specifically reactive with the amino-terminal sequence of gamma actin. This antibody reacted with nonmuscle actin as determined by Western blots of SDS gels, and reacted with the gamma, but not the beta, nonmuscle actin isoform as shown by Western blots of isoelectric focusing gels. In immunofluorescence experiments, the gamma peptide antibody stained microfilament bundles, ruffled edges, and the contractile ring of a variety of cultured cells, including mouse L cells, which have previously been reported to contain only the beta actin isoform (Sakiyama, S., S. Fujimura, and H. Sakiyama, 1981, J. Biol. Chem., 256:31-33). Double immunofluorescence experiments using the gamma peptide antibody and an antibody reactive with all actin isoforms revealed no differences in isoform localization. Thus, at the level of resolution of light microscopy, we have detected the gamma actin isoform in all microfilament-containing structures in cultured cells, and have observed no subcellular sorting of the nonmuscle actin isoforms.

We characterized the form and distribution of muscle and nonmuscle actin within retinal pericytes. Antibodies with demonstrable specificities for the actin isoforms were used in localization and immunoprecipitation experiments to identify those cellular domains that were enriched or deficient in one or several actin isoforms. Living pericyte behavior was monitored with phase-contract video microscopy before fixation to identify those cellular areas that might preferentially be stained with either of the fluorescent antiactins or phallotoxins. Antibody and phallotoxin staining of pericytes revealed that nonmuscle actin is present within membrane ruffles, pseudopods, and stress fibers. In contrast, muscle actin could be convincingly localized in stress fibers, but not within specific motile areas of pericyte cytoplasm. To confirm and quantitatively extend the results obtained by fluorescence microscopy, nonionic and ionic detergents were used to selectively extract the motile or immobilized (stress fiber- containing) regions of biosynthetically labeled pericyte cytoplasm. Immunoprecipitated actins that were present within these discrete cellular domains were subjected to isoelectric focusing in urea- polyacrylamide gels before fluorographic analysis. Scanning laser densitometry of the focused actins could not reveal any detectable alpha-actin within those beta- and gamma-actin-enriched motile regions extracted with nonionic detergents. Moreover, when pericyte stress fibers are completely dissolved by ionic detergent lysis, three actin isoforms can be quantified to be present in a ratio of 1:2.75:3 (alpha:beta:gamma). These biochemical findings on biosynthetically labeled and immunoprecipitated pericyte actins confirm the fluorescent localization studies. While the regulatory events governing this actin sorting are unknown, it seems possible that such events may play important roles in controlling cell shape, adhesion, or the promotion of localized cell spreading.

Regulation of Actin Isoforms by Nitric Oxide

Genes representing six different actin isoforms were isolated from a chicken genomic library. Cloned actin cDNAs as well as tissue-specific mRNAs enriched in different actin species were used as hybridization probes to group individual actin genomic clones by their relative thermal stability. Restriction maps showed that these actin genes were derived from separate and nonoverlapping regions of genomic DNA. Of the six isolated genes, five included sequences from both the 5' and 3' ends of the actin-coding area. Amino acid sequence analysis from both the NH2- and COOH-terminal regions provided for the unequivocal identification of these genes. The striated isoforms were represented by the isolated alpha-skeletal, alpha-cardiac, and alpha-smooth muscle actin genes. The nonmuscle isoforms included the beta-cytoplasmic actin gene and an actin gene fragment which lacked the 5' coding and flanking sequence; presumably, this region of DNA was removed from this gene during construction of the genomic library. Unexpectedly, a third nonmuscle chicken actin gene was found which resembled the amphibian type 5 actin isoform (J. Vandekerckhove, W. W. Franke, and K. Weber, J. Mol. Biol., 152:413-426). This nonmuscle actin type has not been previously detected in warm-blooded vertebrates. We showed that interspersed, repeated DNA sequences closely flanked the alpha-skeletal, alpha-cardiac, beta-, and type 5-like actin genes. The repeated DNA sequences which surround the alpha-skeletal actin-coding regions were not related to repetitious DNA located on the other actin genes. Analysis of genomic DNA blots showed that the chicken actin multigene family was represented by 8 to 10 separate coding loci. The six isolated actin genes corresponded to 7 of 11 genomic EcoRI fragments. Only the alpha-smooth muscle actin gene was shown to be split by an EcoRI site. Thus, in the chicken genome each actin isoform appeared to be encoded by a single gene.

Genes representing six different actin isoforms were isolated from a chicken genomic library. Cloned actin cDNAs as well as tissue-specific mRNAs enriched in different actin species were used as hybridization probes to group individual actin genomic clones by their relative thermal stability. Restriction maps showed that these actin genes were derived from separate and nonoverlapping regions of genomic DNA. Of the six isolated genes, five included sequences from both the 5' and 3' ends of the actin-coding area. Amino acid sequence analysis from both the NH2- and COOH-terminal regions provided for the unequivocal identification of these genes. The striated isoforms were represented by the isolated alpha-skeletal, alpha-cardiac, and alpha-smooth muscle actin genes. The nonmuscle isoforms included the beta-cytoplasmic actin gene and an actin gene fragment which lacked the 5' coding and flanking sequence; presumably, this region of DNA was removed from this gene during construction of the genomic library. Unexpectedly, a third nonmuscle chicken actin gene was found which resembled the amphibian type 5 actin isoform (J. Vandekerckhove, W. W. Franke, and K. Weber, J. Mol. Biol., 152:413-426). This nonmuscle actin type has not been previously detected in warm-blooded vertebrates. We showed that interspersed, repeated DNA sequences closely flanked the alpha-skeletal, alpha-cardiac, beta-, and type 5-like actin genes. The repeated DNA sequences which surround the alpha-skeletal actin-coding regions were not related to repetitious DNA located on the other actin genes. Analysis of genomic DNA blots showed that the chicken actin multigene family was represented by 8 to 10 separate coding loci. The six isolated actin genes corresponded to 7 of 11 genomic EcoRI fragments. Only the alpha-smooth muscle actin gene was shown to be split by an EcoRI site. Thus, in the chicken genome each actin isoform appeared to be encoded by a single gene.

Inhibitory effects of pectenotoxins from marine algae on the polymerization of various actin isoforms

Previous work in our laboratory has shown that microvascular pericytes sort muscle and nonmuscle actin isoforms into discrete cytoplasmic domains (Herman, I. M., and P. A. D'Amore. 1985. J. Cell Biol. 101:43- 52; DeNofrio, D.T.C. Hoock, and I. M. Herman. J. Cell. Biol. 109:191- 202). Specifically, muscle (alpha-smooth) actin is present on the stress fibers while nonmuscle actins (beta and gamma) are located on stress fibers and in regions of moving cytoplasm (e.g., ruffles, lamellae). To determine the form and function of beta actin in microvascular pericytes and endothelial cells recovering from injury, we prepared isoform-specific antibodies and cDNA probes for immunolocalization, Western and Northern blotting, as well as in situ hybridization. Anti-beta actin IgG was prepared by adsorption and release of beta actin-specific IgG from electrophoretically purified pericyte beta actin bound to nitrocellulose paper. Anti-beta actin IgGs prepared by this affinity selection procedure showed exclusive binding to beta actin present in crude cell lysates containing all three actin isoforms. For controls, we localized beta actin as a bright rim of staining beneath the erythrocyte plasma membrane. Anti-beta actin IgG, absorbed with beta actin bound to nitrocellulose, failed to stain erythrocytes. Simultaneous localization of beta actin with the entire F- actin pool was performed on microvascular pericytes or endothelial cells and 3T3 fibroblasts recovering from injury using anti-beta actin IgG in combination with fluorescent phalloidin. Results of these experiments revealed that pericyte beta actin is localized beneath the plasma membrane in association with filopods, pseudopods, and fan lamellae. Additionally, we observed bright focal fluorescence within fan lamellae and in association with the ends of stress fibers that are preferentially associated with the ventral plasmalemma. Whereas fluorescent phalloidin staining along the stress fibers is continuous, anti-beta actin IgG localization is discontinuous. When injured endothelial and 3T3 cells were stained through wound closure, we localized beta actin only in motile cytoplasm at the wound edge. Staining disappeared as cells became quiescent upon monolayer restoration. Appearance of beta actin at the wound edge correlated with a two- to threefold increase in steady-state levels of beta actin mRNA, which rose within 15-60 min after injury and returned to noninjury levels during monolayer restoration. In situ hybridization revealed that transcripts encoding beta actin were localized at the wound edge in association with the repositioned protein. Results of these experiments indicate that beta actin and its encoded mRNA are polarized at the membrane-cytoskeletal interface within regions of moving cytoplasm.

Covering the Cover

Cofilin Binding to Muscle and Non-muscle Actin Filaments: Isoform-dependent Cooperative Interactions