Actin Filaments as Tension Sensors
2012; Elsevier BV; Volume: 22; Issue: 3 Linguagem: Inglês
10.1016/j.cub.2011.12.010
ISSN1879-0445
AutoresVitold E. Galkin, Albina Orlova, Edward H. Egelman,
Tópico(s)Polymer Surface Interaction Studies
ResumoThe field of mechanobiology has witnessed an explosive growth over the past several years as interest has greatly increased in understanding how mechanical forces are transduced by cells and how cells migrate, adhere and generate traction. Actin, a highly abundant and anomalously conserved protein, plays a large role in forming the dynamic cytoskeleton that is so essential for cell form, motility and mechanosensitivity. While the actin filament (F-actin) has been viewed as dynamic in terms of polymerization and depolymerization, new results suggest that F-actin itself may function as a highly dynamic tension sensor. This property may help explain the unusual conservation of actin's sequence, as well as shed further light on actin's essential role in structures from sarcomeres to stress fibers. The field of mechanobiology has witnessed an explosive growth over the past several years as interest has greatly increased in understanding how mechanical forces are transduced by cells and how cells migrate, adhere and generate traction. Actin, a highly abundant and anomalously conserved protein, plays a large role in forming the dynamic cytoskeleton that is so essential for cell form, motility and mechanosensitivity. While the actin filament (F-actin) has been viewed as dynamic in terms of polymerization and depolymerization, new results suggest that F-actin itself may function as a highly dynamic tension sensor. This property may help explain the unusual conservation of actin's sequence, as well as shed further light on actin's essential role in structures from sarcomeres to stress fibers. Actin is a central player in many aspects of cell biology and has been intensively studied for more than 60 years, but surprisingly we continue to realize how little we still understand about this protein. While actin was first studied in muscle, most research on actin today is focused on the crucial roles that actin plays in the cytoskeleton and in non-muscle motility. The burgeoning field of mechanobiology [1Mammoto T. Ingber D.E. Mechanical control of tissue and organ development.Development. 2010; 137: 1407-1420Crossref PubMed Scopus (630) Google Scholar] addresses questions of how mechanical forces are sensed and generated by cytoskeletal elements, and it has become clear that the transduction of such mechanical signals [2Ehrlicher A.J. Nakamura F. Hartwig J.H. Weitz D.A. Stossel T.P. Mechanical strain in actin networks regulates FilGAP and integrin binding to filamin A.Nature. 2011; 478: 260-263Crossref PubMed Scopus (264) Google Scholar] is as important as the sensing of molecules. The cell has elaborate mechanisms for generating different actin networks in different parts of the cell, each with distinct binding proteins and functions, and our understanding of the mechanisms responsible for such specialization is still unfolding [3Michelot A. Drubin D.G. Building distinct actin filament networks in a common cytoplasm.Curr. Biol. 2011; 21: R560-R569Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar]. New areas of study, such as the role of actin in the nucleoskeleton, have recently emerged, while less than 10 years ago the existence of actin within the nucleus was fiercely debated. Advances in cryo-electron microscopy (cryo-EM; Figure 1) have provided unprecedented insights into actin filament structure and dynamics [4Fujii T. Iwane A.H. Yanagida T. Namba K. Direct visualization of secondary structures of F-actin by electron cryomicroscopy.Nature. 2010; 467: 724-728Crossref PubMed Scopus (284) Google Scholar, 5Galkin V.E. Orlova A. Schroder G.F. Egelman E.H. Structural polymorphism in F-actin.Nat. Struct. Mol. Biol. 2010; 17: 1318-1323Crossref PubMed Scopus (147) Google Scholar]. One of the most striking features about actin, in addition to its abundance, has been its exquisite degree of sequence conservation. From chickens to humans, an evolutionary distance of more than 300 million years, every one of the 375 residues in the skeletal muscle isoform has been conserved. If one looks at an evolutionary distance of more than 1 billion years, around 90% of the residues are identical between yeast actin and the cytoplasmic isoform of human actin. While suggestions have been made about why almost all actin residues might be under selective pressure, we have no definitive answer at this point. One possibility for actin's anomalous sequence conservation is that the interaction of actin with more than 100–200 actin-binding proteins might constrain many residues. But this argument ignores the fact that many actin-binding proteins have significantly diverged over the same evolutionary distances (for example, from yeast to humans). Further, the residues in actin that are not absolutely conserved [6Egelman E.H. Actin allostery again?.Nat. Struct. Biol. 2001; 8: 735-736Crossref PubMed Scopus (33) Google Scholar] are mainly on the surface of the filament where they would directly interact with actin-binding proteins. A quite different argument comes from the observation that highly expressed proteins evolve slowly [7Drummond D.A. Bloom J.D. Adami C. Wilke C.O. Arnold F.H. Why highly expressed proteins evolve slowly.Proc. Natl. Acad. Sci. USA. 2005; 102: 14338-14343Crossref PubMed Scopus (589) Google Scholar], presumably as a means to prevent protein misfolding. This argument may explain some of the anomalous sequence conservation, since actin is one of the most highly expressed proteins in many cells, but is unlikely to explain why every amino acid appears to be under rather intense selective pressure. We would like to advance a different hypothesis in this Minireview, one supported by a series of recent papers [4Fujii T. Iwane A.H. Yanagida T. Namba K. Direct visualization of secondary structures of F-actin by electron cryomicroscopy.Nature. 2010; 467: 724-728Crossref PubMed Scopus (284) Google Scholar, 5Galkin V.E. Orlova A. Schroder G.F. Egelman E.H. Structural polymorphism in F-actin.Nat. Struct. Mol. Biol. 2010; 17: 1318-1323Crossref PubMed Scopus (147) Google Scholar, 8Galkin V.E. Orlova A. Kudryashov D. Solodukhin A. Reisler E. Schroeder G.N. Egelman E.H. Remodeling of actin filaments by ADF/cofilin proteins.Proc. Nat. Acad. Sci. USA. 2011; 108: 20568-20572Crossref PubMed Scopus (161) Google Scholar, 9Hayakawa K. Tatsumi H. Sokabe M. Actin filaments function as a tension sensor by tension-dependent binding of cofilin to the filament.J. Cell Biol. 2011; 195: 721-727Crossref PubMed Scopus (219) Google Scholar, 10Suarez C. Roland J. Boujemaa-Paterski R. Kang H. McCullough B.R. Reymann A.C. Guerin C. Martiel J.L. De La Cruz E.M. Blanchoin L. Cofilin tunes the nucleotide state of actin filaments and severs at bare and decorated segment boundaries.Curr. Biol. 2011; 21: 862-868Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 11Uyeda T.Q. Iwadate Y. Umeki N. Nagasaki A. Yumura S. Stretching actin filaments within cells enhances their affinity for the myosin II motor domain.PLoS ONE. 2011; 6: e26200Crossref PubMed Scopus (111) Google Scholar], suggesting that cooperative and allosteric properties of the actin filament are essential for its cellular function, and that the internal networks within the actin subunit needed to maintain such allosteric linkages [12Suel G.M. Lockless S.W. Wall M.A. Ranganathan R. Evolutionarily conserved networks of residues mediate allosteric communication in proteins.Nat. Struct. Biol. 2003; 10: 59-69Crossref PubMed Scopus (658) Google Scholar] have placed every residue under selective pressure. Allosteric interactions may explain why buried residues in actin, which cannot interact with actin-binding proteins in muscle such as myosin, tropomyosin, troponin and α-actinin, are responsible for hereditary myopathies [13Feng J.J. Marston S. Genotype-phenotype correlations in ACTA1 mutations that cause congenital myopathies.Neuromuscul. Disord. 2009; 19: 6-16Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar]. For example, it has been shown that replacing residue 372 in yeast actin with the residue found at this position in vertebrate muscle actin (the H372R mutation) led to severe growth defects [14McKane M. Wen K.K. Boldogh I.R. Ramcharan S. Pon L.A. Rubenstein P.A. A mammalian actin substitution in yeast actin (H372R) causes a suppressible mitochondria/vacuole phenotype.J. Biol. Chem. 2005; 280: 36494-36501Crossref PubMed Scopus (22) Google Scholar]. However, substitution of four amino-terminal muscle actin residues into the amino terminus of yeast actin restored the viability of cells with the H372R mutation [14McKane M. Wen K.K. Boldogh I.R. Ramcharan S. Pon L.A. Rubenstein P.A. A mammalian actin substitution in yeast actin (H372R) causes a suppressible mitochondria/vacuole phenotype.J. Biol. Chem. 2005; 280: 36494-36501Crossref PubMed Scopus (22) Google Scholar]. Since these two regions are widely separated in both G- and F-actin (Figure 1B), the best explanation for this effect involves an allosteric linkage between these regions [14McKane M. Wen K.K. Boldogh I.R. Ramcharan S. Pon L.A. Rubenstein P.A. A mammalian actin substitution in yeast actin (H372R) causes a suppressible mitochondria/vacuole phenotype.J. Biol. 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In contrast, the bacterial actin homologs have diverged considerably in sequence, so much so that many of them are as different from each other as they are from actin [16Derman A.I. Becker E.C. Truong B.D. Fujioka A. Tucey T.M. Erb M.L. Patterson P.C. Pogliano J. Phylogenetic analysis identifies many uncharacterized actin-like proteins (Alps) in bacteria: regulated polymerization, dynamic instability and treadmilling in Alp7A.Mol. Microbiol. 2009; 73: 534-552Crossref PubMed Scopus (97) Google Scholar]. While it is still an open question as to whether all or even most of these bacterial proteins form filaments, the filaments formed by all bacterial actin-like proteins studied thus far are significantly different from F-actin [17Galkin V.E. Orlova A. Rivera C. Mullins R.D. Egelman E.H. Structural polymorphism of the ParM filament and dynamic instability.Structure. 2009; 17: 1253-1264Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 18Polka J.K. Kollman J.M. Agard D.A. 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If our hypothesis is correct, the bacterial actin-like filaments will not display the cooperativity and allostery observed for F-actin [6Egelman E.H. Actin allostery again?.Nat. Struct. Biol. 2001; 8: 735-736Crossref PubMed Scopus (33) Google Scholar]. Some specific predictions can therefore be made in this review about how the bacterial actin-like filaments will behave differently from F-actin. To explore this idea of a highly tuned actin filament that has emerged from extensive evolutionary selection, we must start by abandoning the notion that F-actin is merely a passive cable, existing in a single state, to which other proteins can bind. The notion that physical stresses on the actin filament might modulate the interaction with actin-binding proteins has appeared in a number of studies. A recent paper on the interaction of formins with actin [22Mizuno H. Higashida C. Yuan Y. Ishizaki T. Narumiya S. Watanabe N. Rotational movement of the formin mDia1 along the double helical strand of an actin filament.Science. 2011; 331: 80-83Crossref PubMed Scopus (84) Google Scholar] concluded: “Our data have opened up the possibility that actin elongation and remodeling could be regulated by axial torsion in the filament.” It has previously been shown that nucleation of an actin filament by formins can cause long-range conformational changes in the actin filament [23Papp G. Bugyi B. Ujfalusi Z. Barko S. Hild G. Somogyi B. Nyitrai M. Conformational changes in actin filaments induced by formin binding to the barbed end.Biophys. J. 2006; 91: 2564-2572Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar], just as nucleation of actin filaments by gelsolin has been shown to cause such long-range changes [24Orlova A. Prochniewicz E. Egelman E.H. Structural dynamics of F-actin. II. Co-operativity in structural transitions.J. Mol. Biol. 1995; 245: 598-607Crossref PubMed Scopus (146) Google Scholar, 25Prochniewicz E. Zhang Q. Janmey P.A. Thomas D.D. Cooperativity in F-actin: binding of gelsolin at the barbed end affects structure and dynamics of the whole filament.J. Mol. Biol. 1996; 260: 756-766Crossref PubMed Scopus (93) Google Scholar]. These long-range perturbations tell us that the different conformational states accessible by actin must be comparable energetically, so that nucleation by a particular protein is able to bias the distribution of states. Nucleation of actin filaments by different proteins thus provides a means for the cell to differentiate one actin filament from another [3Michelot A. Drubin D.G. Building distinct actin filament networks in a common cytoplasm.Curr. Biol. 2011; 21: R560-R569Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar]. A less static and more active picture of the actin filament has arisen from new insights into the dynamic properties of actin filaments. Actin-based structures that were considered to be static, such as the core of the stereocilium (responsible for the mechanotransduction of sound), have now been shown to be dynamic, at least in the sense that there is a continuous flux of actin subunits through these filaments [26Schneider M.E. Belyantseva I.A. Azevedo R.B. Kachar B. Rapid renewal of auditory hair bundles.Nature. 2002; 418: 837-838Crossref PubMed Scopus (153) Google Scholar]. Consider muscle, where a passive view of actin has been dominant historically: the regulation of myosin heads binding to F-actin, and therefore the generation of force, has been viewed as being largely due to tropomyosin strands moving across a fixed actin surface. But a number of papers have shown that actin can be modified, either chemically [27Prochniewicz E. Yanagida T. Inhibition of sliding movement of F-actin by crosslinking emphasizes the role of actin structure in the mechanism of motility.J. Mol. 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Alteration in crossbridge kinetics caused by mutations in actin.Nature. 1990; 348: 440-442Crossref PubMed Scopus (47) Google Scholar] or by proteolysis [32Schwyter D.H. Kron S.J. Toyoshima Y.Y. Spudich J.A. Reisler E. Subtilisin cleavage of actin inhibits In vitro siding movement of actin filaments over myosin.J. Cell Biol. 1990; 111: 465-470Crossref PubMed Scopus (71) Google Scholar], in a way that inhibits myosin force generation without inhibiting either the binding of myosin to actin or the actin-induced activation of myosin's ATPase activity. The simplest explanation for these observations is that actin must undergo structural transitions during actomyosin force generation, and these modifications of actin inhibit such structural transitions. Supporting the notion of structural transitions in F-actin, we have shown that naked actin filaments in vitro exist in a multiplicity of discrete structural states [5Galkin V.E. Orlova A. Schroder G.F. Egelman E.H. Structural polymorphism in F-actin.Nat. Struct. Mol. Biol. 2010; 17: 1318-1323Crossref PubMed Scopus (147) Google Scholar]. A different picture of F-actin was presented in another recent paper [4Fujii T. Iwane A.H. Yanagida T. Namba K. Direct visualization of secondary structures of F-actin by electron cryomicroscopy.Nature. 2010; 467: 724-728Crossref PubMed Scopus (284) Google Scholar], where it was argued that F-actin is quite homogeneous structurally, and that “F-actin is not so flexible” in contrast to the large literature showing that the helical twist of F-actin can be quite variable [33Hanson J. Axial period of actin filaments: electron microscope studies.Nature. 1967; 213: 353-356Crossref Scopus (60) Google Scholar, 34Egelman E.H. DeRosier D.J. Image analysis shows that variations in actin crossover spacings are random, not compensatory.Biophys. J. 1992; 63: 1299-1305Abstract Full Text PDF PubMed Scopus (37) Google Scholar, 35Schmid M.F. Sherman M.B. Matsudaira P. Chiu W. Structure of the acrosomal bundle.Nature. 2004; 431: 104-107Crossref PubMed Scopus (72) Google Scholar, 36McGough A. Pope B. Chiu W. Weeds A. Cofilin changes the twist of F-actin: Implications for actin filament dynamics and cellular function.J. Cell Biol. 1997; 138: 771-781Crossref PubMed Scopus (580) Google Scholar]. For example, the protein cofilin changes the average twist of F-actin (Figure 1) by ∼5° per subunit [36McGough A. Pope B. Chiu W. Weeds A. Cofilin changes the twist of F-actin: Implications for actin filament dynamics and cellular function.J. Cell Biol. 1997; 138: 771-781Crossref PubMed Scopus (580) Google Scholar], while in bundles with the actin crosslinking protein scruin the twist of actin subunits ranged from 142.5° to 176.5° [35Schmid M.F. Sherman M.B. Matsudaira P. Chiu W. Structure of the acrosomal bundle.Nature. 2004; 431: 104-107Crossref PubMed Scopus (72) Google Scholar], deviating widely from the average twist within these filaments of ∼167°. In the actin angle-layered aggregate [37Egelman E.H. Francis N. DeRosier D.J. Helical disorder and the filament structure of F-actin are elucidated by the angle-layered aggregate.J. Mol. Biol. 1983; 166: 605-629Crossref PubMed Scopus (29) Google Scholar], which is formed in solution prior to specimen preparation for EM, the angular disorder is locked into the structure, so specimen preparation can be discounted as a source of the variability in twist of actin. These aggregates yielded a root mean square (rms) deviation of ∼6° per subunit [37Egelman E.H. Francis N. DeRosier D.J. Helical disorder and the filament structure of F-actin are elucidated by the angle-layered aggregate.J. Mol. Biol. 1983; 166: 605-629Crossref PubMed Scopus (29) Google Scholar]. How can these very different observations, of variable twist and polymorphic filaments, versus relatively fixed twist and a single structure, be reconciled? We think that the answer lies in specimen preparation for cryo-EM, and that understanding the differences between the results obtained is likely to have great biological significance. In preparing a sample for cryo-EM, filaments in buffer are applied to a holey carbon film on an EM grid, and then blotted so that a thin film is formed prior to plunging into a cryostat for vitrification. In the process of creating this film, the filaments can be subjected to very large forces due to both fluid flow and transverse compression. Fujii et al. [4Fujii T. Iwane A.H. Yanagida T. Namba K. Direct visualization of secondary structures of F-actin by electron cryomicroscopy.Nature. 2010; 467: 724-728Crossref PubMed Scopus (284) Google Scholar] explicitly stated that the high resolution they achieved was due in part to the use of very thin films which improved the signal to noise ratio, and that blotting conditions were chosen to make “F-actin as straight as possible.” We normally think of the straightness of filaments in solution, in the absence of external forces, as arising from only two physical parameters: the temperature of the solution (T) and the flexural rigidity of the polymer (a). Thus, the persistence length λ for a filament is simply given by λ = a/kT. If blotting conditions are changing the observed flexibility or straightness of these filaments, it is a prima facie argument that forces are being introduced. The use of fluid flow to intentionally stretch and straighten polymers is not novel and has been used in many experiments involving DNA. But such straightening may also arise, in the case of F-actin, from the compressive forces perpendicular to the filament axis when the filament experiences the surface tension resulting from a very thin film. Tomographic reconstructions of axonemes in thin ice showed extreme flattening, with the suggestion that the flattening arose from this large surface tension [38McEwen B.F. Marko M. Hsieh C.E. Mannella C. Use of frozen-hydrated axonemes to assess imaging parameters and resolution limits in cryoelectron tomography.J. Struct. Biol. 2002; 138: 47-57Crossref PubMed Scopus (42) Google Scholar]. Most importantly, observations have already been made by Greene et al. [39Greene G.W. Anderson T.H. Zeng H. Zappone B. Israelachvili J.N. Force amplification response of actin filaments under confined compression.Proc. Natl. Acad. Sci. USA. 2009; 106: 445-449Crossref PubMed Scopus (26) Google Scholar] on F-actin filaments confined between two mica surfaces (Figure 2), which appear to be a good analog for the thin films being used for cryo-EM. Surprisingly, these filaments become anomalously stiff under compression, which is consistent with the structural homogeneity and straightening of the actin filaments seen in Fujii et al. [4Fujii T. Iwane A.H. Yanagida T. Namba K. Direct visualization of secondary structures of F-actin by electron cryomicroscopy.Nature. 2010; 467: 724-728Crossref PubMed Scopus (284) Google Scholar]. Greene et al. [39Greene G.W. Anderson T.H. Zeng H. Zappone B. Israelachvili J.N. Force amplification response of actin filaments under confined compression.Proc. Natl. Acad. Sci. USA. 2009; 106: 445-449Crossref PubMed Scopus (26) Google Scholar] proposed a two-state model for actin, and suggested that compression leads to the stress-stiffening of filaments by forcing subunits into a state that is stiffer than the one normally populated. This is consistent with all of the EM results. We may extend the assumption of Greene et al. [39Greene G.W. Anderson T.H. Zeng H. Zappone B. Israelachvili J.N. Force amplification response of actin filaments under confined compression.Proc. Natl. Acad. Sci. USA. 2009; 106: 445-449Crossref PubMed Scopus (26) Google Scholar] to suggest that actin filaments may exist in at least three states of macroscopic flexibility. When filaments are under axial tension or transverse compression there is a stiff state, while in the absence of forces there is an ensemble average over a number of states [5Galkin V.E. Orlova A. Schroder G.F. Egelman E.H. Structural polymorphism in F-actin.Nat. Struct. Mol. Biol. 2010; 17: 1318-1323Crossref PubMed Scopus (147) Google Scholar] that yields the persistence length for ‘normal’ F-actin. However, it has been shown that, under conditions where subdomain 2 of actin becomes disordered, the filaments become anomalously flexible [40Orlova A. Egelman E.H. A conformational change in the actin subunit can change the flexibility of the actin filament.J. Mol. Biol. 1993; 232: 334-341Crossref PubMed Scopus (173) Google Scholar], which we can treat as a third macroscopic state. As we have shown [8Galkin V.E. Orlova A. Kudryashov D. Solodukhin A. Reisler E. Schroeder G.N. Egelman E.H. Remodeling of actin filaments by ADF/cofilin proteins.Proc. Nat. Acad. Sci. USA. 2011; 108: 20568-20572Crossref PubMed Scopus (161) Google Scholar], cofilin can both substantially displace subdomain 2 of actin as well as cause it to be disordered. One would thus expect that cofilin binding to F-actin might make it more flexible, and the cofilin-induced increase in F-actin flexibility has already been reported [41McCullough B.R. Blanchoin L. Martiel J.L. De La Cruz E.M. Cofilin increases the bending flexibility of actin filaments: implications for severing and cell mechanics.J. Mol. Biol. 2008; 381: 550-558Crossref PubMed Scopus (160) Google Scholar]. This change in F-actin flexibility makes simple physical sense, since the resistance to bending will scale as the fourth power of the radius of the mass within the filament, and subdomain 2 forms the highest radius contact in the actin filament (Figure 1A). If one compares the 80 or so crystal structures of actin that now exist, the greatest structural variance is in subdomain 2 and the DNase I-binding loop within subdomain 2. Strikingly, subdomain 2 and the DNase I-binding loop are among the regions of lowest structural variance in the EM reconstruction of Fujii et al. [4Fujii T. Iwane A.H. Yanagida T. Namba K. Direct visualization of secondary structures of F-actin by electron cryomicroscopy.Nature. 2010; 467: 724-728Crossref PubMed Scopus (284) Google Scholar], in keeping with our argument that the compressive thin films are inducing this unusual structural homogeneity. The structural homogeneity of subdomain 2 is what is giving these filaments their anomalous rigidity. It is difficult to reconcile such structural homogeneity with spectroscopic observations from filaments in solution suggesting large, and discrete, changes in the conformation of subdomain 2 [42Kozuka J. Yokota H. Arai Y. Ishii Y. Yanagida T. Dynamic polymorphism of single actin molecules in the actin filament.Nat. Chem. Biol. 2006; 2: 83-86Crossref PubMed Scopus (78) Google Scholar]. What is the molecular mechanism that couples axial tension on a filament (or transverse compression) to a stabilization of subdomain 2? At this point we cannot answer the question, and we expect that this issue will motivate many studies in the future. However, it is tempting to speculate that, since the contact between subdomain 4 of one subunit and subdomain 3 of a subunit above is a relatively invariant interface in F-actin [5Galkin V.E. Orlova A. Schroder G.F. Egelman E.H. Structural polymorphism in F-actin.Nat. Struct. Mol. Biol. 2010; 17: 1318-1323Crossref PubMed Scopus (147) Google Scholar], tension along subdomains 3 and 4 within a subunit may be communicated through the hinge region separating the two major domains of actin forcing subdomain 2 into a specific vertical orientation. The hypothesis that we are proposing immediately leads to a number of testable predictions. One is that the elastic stiffening observed for F-actin [39Greene G.W. Anderson T.H. Zeng H. Zappone B. Israelachvili J.N. Force amplification response of actin filaments under confined compression.Proc. Natl. Acad. Sci. USA. 2009; 106: 445-449Crossref PubMed Scopus (26) Google Scholar] should not be seen for the filaments formed by bacterial actin-like proteins, such as ParM and AlfA. Another is that tension on an actin filament should be observable either biochemically or spectroscopically, since this tension should change the distribution of structural states. Such a result has already been observed [43Shimozawa T. Ishiwata S. Mechanical distortion of single actin filaments induced by external force: detection by fluorescence imaging.Biophys. J. 2009; 96: 1036-1044Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar], where a change in fluorescence of a probe attached to the carboxyl terminus of actin was observed as a function of tension. The resulting labeled actin filament was therefore described as a “bio-nano strain gauge”. Our explanation for this effect arises from the fact that, in the absence of tension, the carboxy-terminal region of F-actin, like subdomain 2, can exist in a number of discretely different states [5Galkin V.E. Orlova A. Schroder G.F. Egelman E.H. Structural polymorphism in F-actin.Nat. Struct. Mol. Biol. 2010; 17: 1318-1323Crossref PubMed Scopus (147) Google Scholar]. Under conditions where the filament is compressed in a thin film [4Fujii T. Iwane A.H. Yanagida T. Namba K. Direct visualization of secondary structures of F-actin by electron cryomicroscopy.Nature. 2010; 467: 724-728Crossref PubMed Scopus (284) Google Scholar, 39Greene G.W. Anderson T.H. Zeng H. Zappone B. Israelachvili J.N. Force amplification response of actin filaments under confined compression.Proc. Natl. Acad. Sci. USA. 2009; 106: 445-449Crossref PubMed Scopus (26) Google Scholar], we suggest that this region becomes structurally homogeneous. A third prediction is that proteins that change the twist of F-actin, such as cofilin/ADF [36McGough A. Pope B. Chiu W. Weeds A. Cofilin changes the twist of F-actin: Implications for actin filament dynamics and cellular function.J. Cell Biol. 1997; 138: 771-781Crossref PubMed Scopus (580) Google Scholar], should bind much less avidly to actin filaments under tension, since tension should greatly reduce the variability in twist within these filaments. A new paper has observed precisely this expected behavior [9Hayakawa K. Tatsumi H. Sokabe M. Actin filaments function as a tension sensor by tension-dependent binding of cofilin to the filament.J. Cell Biol. 2011; 195: 721-727Crossref PubMed Scopus (219) Google Scholar]. We believe that we can explain the molecular basis for this observation and that it also provides insights into the coupling between multiple structural states and variable twist in F-actin. Just as Fujii et al. [4Fujii T. Iwane A.H. Yanagida T. Namba K. Direct visualization of secondary structures of F-actin by electron cryomicroscopy.Nature. 2010; 467: 724-728Crossref PubMed Scopus (284) Google Scholar] have observed structurally homogeneous actin filaments in contrast to the structural heterogeneity that we observe in thicker ice, they observe a distribution of twist that can be parameterized as corresponding to a random angular disorder of ∼2.5° per subunit, whereas our filaments have an observed distribution of ∼6° per subunit (Figure 3), consistent with earlier estimates from both negatively stained single actin filaments [34Egelman E.H. DeRosier D.J. Image analysis shows that variations in actin crossover spacings are random, not compensatory.Biophys. J. 1992; 63: 1299-1305Abstract Full Text PDF PubMed Scopus (37) Google Scholar] and angle-layered aggregates [37Egelman E.H. Francis N. DeRosier D.J. Helical disorder and the filament structure of F-actin are elucidated by the angle-layered aggregate.J. Mol. Biol. 1983; 166: 605-629Crossref PubMed Scopus (29) Google Scholar]. It has been shown that cofilin actually stabilizes an existing twist of F-actin that is present in vitro in naked actin filaments, rather than imposing a twist that would never be seen in the absence of cofilin [44Galkin V.E. Orlova A. Lukoyanova N. Wriggers W. Egelman E.H. Actin depolymerizing factor stabilizes an existing state of F-actin and can change the tilt of F-actin subunits.J. Cell Biol. 2001; 153: 75-86Crossref PubMed Scopus (207) Google Scholar]. Several papers have suggested that the slow initial binding of cofilin/ADF to F-actin can be explained by the limited number of sites to which cofilin can initially attach [45Cao W. Goodarzi J.P. De La Cruz E.M. Energetics and kinetics of cooperative cofilin-actin filament interactions.J. Mol. Biol. 2006; 361: 257-267Crossref PubMed Scopus (80) Google Scholar, 46Blanchoin L. Pollard T.D. Mechanism of interaction of Acanthamoeba actophorin (ADF/Cofilin) with actin filaments.J. Biol. Chem. 1999; 274: 15538-15546Crossref PubMed Scopus (242) Google Scholar], as one would expect in a structurally heterogeneous F-actin (while all actin subunits would be in identical environments in a homogeneous filament). Further, cofilin needs to shift subdomain 2 of actin when it binds to the filament [8Galkin V.E. Orlova A. Kudryashov D. Solodukhin A. Reisler E. Schroeder G.N. Egelman E.H. Remodeling of actin filaments by ADF/cofilin proteins.Proc. Nat. Acad. Sci. USA. 2011; 108: 20568-20572Crossref PubMed Scopus (161) Google Scholar]. Since we suggest that tension on a filament or transverse compression in a thin film stabilizes subdomain 2, tension should inhibit cofilin from binding to F-actin. Using both in vivo and in vitro assays, Hayakawa et al. [9Hayakawa K. Tatsumi H. Sokabe M. Actin filaments function as a tension sensor by tension-dependent binding of cofilin to the filament.J. Cell Biol. 2011; 195: 721-727Crossref PubMed Scopus (219) Google Scholar] show that, when an actin filament is under tension, cofilin binding is reduced by a factor of two to three. They used optical tweezers to apply tension to single actin filaments in vitro, and used bundles of actin filaments in vivo which could be stretched by micromanipulation. As a consequence of the reduced binding, they show that the severing of F-actin by cofilin is decreased when a filament is under tension. This has great cell biological significance, since such tension may regulate which actin filaments, whether in stress fibers, filopodia or cleavage furrows, will be severed by cofilin. The opposite prediction can also be made, which is that proteins that bind to and stretch F-actin should show a higher affinity of binding to an actin filament under tension. Remarkably, this has also now been observed [11Uyeda T.Q. Iwadate Y. Umeki N. Nagasaki A. Yumura S. Stretching actin filaments within cells enhances their affinity for the myosin II motor domain.PLoS ONE. 2011; 6: e26200Crossref PubMed Scopus (111) Google Scholar]. It was shown by X-ray diffraction that the rise per subunit of actin increased by ∼0.4% when muscle goes into full tension [47Huxley H.E. Stewart A. Sosa H. Irving T. X-ray diffraction measurements of the extensibility of actin and myosin filaments in contracting muscle.Biophys. J. 1994; 67: 2411-2421Abstract Full Text PDF PubMed Scopus (399) Google Scholar]. It was subsequently shown that the binding of myosin heads in the absence of tension can elongate the actin filament by 0.2%, explaining half of the extension observed [48Tsaturyan A.K. Koubassova N. Ferenczi M.A. Narayanan T. Roessle M. Bershitsky S.Y. Strong binding of myosin heads stretches and twists the actin helix.Biophys. J. 2005; 88: 1902-1910Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar]. We also know that the conformation of actin in the rigor complex (in the absence of ATP) with myosin is quite similar to the structure of the homogeneous naked actin filament under axial tension/transverse compression [4Fujii T. Iwane A.H. Yanagida T. Namba K. Direct visualization of secondary structures of F-actin by electron cryomicroscopy.Nature. 2010; 467: 724-728Crossref PubMed Scopus (284) Google Scholar]. So the prediction is that, if an actin filament is under tension, myosin should bind more avidly, and that is exactly what Uyeda and colleagues [11Uyeda T.Q. Iwadate Y. Umeki N. Nagasaki A. Yumura S. Stretching actin filaments within cells enhances their affinity for the myosin II motor domain.PLoS ONE. 2011; 6: e26200Crossref PubMed Scopus (111) Google Scholar] have now observed in vivo. As with the cofilin result [9Hayakawa K. Tatsumi H. Sokabe M. Actin filaments function as a tension sensor by tension-dependent binding of cofilin to the filament.J. Cell Biol. 2011; 195: 721-727Crossref PubMed Scopus (219) Google Scholar], this has enormous cell biological implications, creating another means for the cell to regulate the binding of myosin to F-actin, in addition to the regulation provided by a large repertoire of other proteins such as tropomyosin, troponin, calponin, myosin binding protein C, and so on. It also has implications for further understanding stretch-activation of muscle [49Pringle J.W. The Croonian Lecture, 1977. Stretch activation of muscle: function and mechanism.Proc. R. Soc. Lond B Biol. Sci. 1978; 201: 107-130Crossref PubMed Google Scholar]. Since actin filaments, in both muscle and non-muscle cells, are associated with a large number of actin-binding proteins, including nucleators, capping proteins and cross-linking proteins, it will be extremely interesting to understand how these other proteins modulate and regulate the response of an actin filament to tension. We have proposed a hypothesis that tension on an actin filament can induce structural transitions from a multiplicity of states [5Galkin V.E. Orlova A. Schroder G.F. Egelman E.H. Structural polymorphism in F-actin.Nat. Struct. Mol. Biol. 2010; 17: 1318-1323Crossref PubMed Scopus (147) Google Scholar] to largely a single state [4Fujii T. Iwane A.H. Yanagida T. Namba K. Direct visualization of secondary structures of F-actin by electron cryomicroscopy.Nature. 2010; 467: 724-728Crossref PubMed Scopus (284) Google Scholar], and that this single state will have a higher affinity for myosin than ‘normal’ F-actin [11Uyeda T.Q. Iwadate Y. Umeki N. Nagasaki A. Yumura S. Stretching actin filaments within cells enhances their affinity for the myosin II motor domain.PLoS ONE. 2011; 6: e26200Crossref PubMed Scopus (111) Google Scholar] and a lower affinity for cofilin [9Hayakawa K. Tatsumi H. Sokabe M. Actin filaments function as a tension sensor by tension-dependent binding of cofilin to the filament.J. Cell Biol. 2011; 195: 721-727Crossref PubMed Scopus (219) Google Scholar]. This hypothesis can explain spectroscopic observations made from actin filaments under tension [43Shimozawa T. Ishiwata S. Mechanical distortion of single actin filaments induced by external force: detection by fluorescence imaging.Biophys. J. 2009; 96: 1036-1044Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar] and begins to address how allosteric relations in actin [14McKane M. Wen K.K. Boldogh I.R. Ramcharan S. Pon L.A. Rubenstein P.A. A mammalian actin substitution in yeast actin (H372R) causes a suppressible mitochondria/vacuole phenotype.J. Biol. Chem. 2005; 280: 36494-36501Crossref PubMed Scopus (22) Google Scholar] mediate such conformational transitions. This hypothesis is testable, and we think that it will provide new understanding of why actin's sequence has been anomalously conserved over one billion years of eukaryotic evolution.
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