Allele-specific Effects of Thoracic Aortic Aneurysm and Dissection α-Smooth Muscle Actin Mutations on Actin Function
2011; Elsevier BV; Volume: 286; Issue: 13 Linguagem: Inglês
10.1074/jbc.m110.203174
ISSN1083-351X
AutoresSarah E. Bergeron, Elesa W. Wedemeyer, Rose Lee, Kuo‐Kuang Wen, Melissa McKane, Alyson R. Pierick, Anthony P. Berger, Peter A. Rubenstein, Heather L. Bartlett,
Tópico(s)Connective tissue disorders research
ResumoTwenty-two missense mutations in ACTA2, which encodes α-smooth muscle actin, have been identified to cause thoracic aortic aneurysm and dissection. Limited access to diseased tissue, the presence of multiple unresolvable actin isoforms in the cell, and lack of an animal model have prevented analysis of the biochemical mechanisms underlying this pathology. We have utilized actin from the yeast Saccharomyces cerevisiae, 86% identical to human α-smooth muscle actin, as a model. Two of the known human mutations, N115T and R116Q, were engineered into yeast actin, and their effect on actin function in vivo and in vitro was investigated. Both mutants exhibited reduced ability to grow under a variety of stress conditions, which hampered N115T cells more than R116Q cells. Both strains exhibited abnormal mitochondrial morphology indicative of a faulty actin cytoskeleton. In vitro, the mutant actins exhibited altered thermostability and nucleotide exchange rates, indicating effects of the mutations on monomer conformation, with R116Q the most severely affected. N115T demonstrated a biphasic elongation phase during polymerization, whereas R116Q demonstrated a markedly extended nucleation phase. Allele-specific effects were also seen on critical concentration, rate of depolymerization, and filament treadmilling. R116Q filaments were hypersensitive to severing by the actin-binding protein cofilin. In contrast, N115T filaments were hyposensitive to cofilin despite nearly normal binding affinities of actin for cofilin. The mutant-specific effects on actin behavior suggest that individual mechanisms may contribute to thoracic aortic aneurysm and dissection. Twenty-two missense mutations in ACTA2, which encodes α-smooth muscle actin, have been identified to cause thoracic aortic aneurysm and dissection. Limited access to diseased tissue, the presence of multiple unresolvable actin isoforms in the cell, and lack of an animal model have prevented analysis of the biochemical mechanisms underlying this pathology. We have utilized actin from the yeast Saccharomyces cerevisiae, 86% identical to human α-smooth muscle actin, as a model. Two of the known human mutations, N115T and R116Q, were engineered into yeast actin, and their effect on actin function in vivo and in vitro was investigated. Both mutants exhibited reduced ability to grow under a variety of stress conditions, which hampered N115T cells more than R116Q cells. Both strains exhibited abnormal mitochondrial morphology indicative of a faulty actin cytoskeleton. In vitro, the mutant actins exhibited altered thermostability and nucleotide exchange rates, indicating effects of the mutations on monomer conformation, with R116Q the most severely affected. N115T demonstrated a biphasic elongation phase during polymerization, whereas R116Q demonstrated a markedly extended nucleation phase. Allele-specific effects were also seen on critical concentration, rate of depolymerization, and filament treadmilling. R116Q filaments were hypersensitive to severing by the actin-binding protein cofilin. In contrast, N115T filaments were hyposensitive to cofilin despite nearly normal binding affinities of actin for cofilin. The mutant-specific effects on actin behavior suggest that individual mechanisms may contribute to thoracic aortic aneurysm and dissection. IntroductionAneurysm and dissection of the thoracic aorta is a major cause of mortality, accounting for 0.5–1% of deaths annually in the United States, and the incidence is increasing, affecting 9–16 in 100,000 individuals/year (1Olsson C. Thelin S. Ståhle E. Ekbom A. Granath F. Circulation. 2006; 114: 2611-2618Crossref PubMed Scopus (591) Google Scholar, 2Hoyert D.L. Arias E. Smith B.L. Murphy S.L. Kochanek K.D. Natl. Vital Stat. Rep. 2001; 49: 1-113Google Scholar). Aortic aneurysms tend to be asymptomatic until dissection, contributing to the high degree of morbidity and mortality. Aortic aneurysms can occur in the absence of systemic findings of a connective tissue disorder, complicating diagnosis (3Hiratzka L.F. Bakris G.L. Beckman J.A. Bersin R.M. Carr V.F. Casey Jr., D.E. Eagle K.A. Hermann L.K. Isselbacher E.M. Kazerooni E.A. Kouchoukos N.T. Lytle B.W. Milewicz D.M. Reich D.L. Sen S. Shinn J.A. Svensson L.G. Williams D.M. Circulation. 2010; 121: e266-e369Crossref PubMed Scopus (1857) Google Scholar). Twenty percent of affected patients inherit the disorder, and the vast majority have an autosomal dominant pattern of inheritance. However, variable penetrance further hobbles patient identification in patients with familial thoracic aortic aneurysm and dissection (TAAD) 2The abbreviations used are: TAADthoracic aortic aneurysm and dissectionα-SM actinα-smooth muscle actinϵ-ATP1,N6-ethenoadenosine 5′-triphosphate. (4Albornoz G. Coady M.A. Roberts M. Davies R.R. Tranquilli M. Rizzo J.A. Elefteriades J.A. Ann. Thorac. Surg. 2006; 82: 1400-1405Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar).Multiple genes and loci have been associated with familial TAAD (5Pearson G.D. Devereux R. Loeys B. Maslen C. Milewicz D. Pyeritz R. Ramirez F. Rifkin D. Sakai L. Svensson L. Wessels A. Van Eyk J. Dietz H.C. Circulation. 2008; 118: 785-791Crossref PubMed Scopus (92) Google Scholar, 6Milewicz D.M. Guo D.C. Tran-Fadulu V. Lafont A.L. Papke C.L. Inamoto S. Kwartler C.S. Pannu H. Annu. Rev. Genomics Hum. Genet. 2008; 9: 283-302Crossref PubMed Scopus (311) Google Scholar). Mutations in ACTA2, which encodes α-smooth muscle actin (α-SM actin), are the most common genetic cause of familial TAAD, responsible for 15% of cases (7Guo D.C. Pannu H. Tran-Fadulu V. Papke C.L. Yu R.K. Avidan N. Bourgeois S. Estrera A.L. Safi H.J. Sparks E. Amor D. Ades L. McConnell V. Willoughby C.E. Abuelo D. Willing M. Lewis R.A. Kim D.H. Scherer S. Tung P.P. Ahn C. Buja L.M. Raman C.S. Shete S.S. Milewicz D.M. Nat. Genet. 2007; 39: 1488-1493Crossref PubMed Scopus (617) Google Scholar). Interestingly, mutations in ACTA2 are associated with an array of cardiovascular diseases from congenital to premature acquired heart disease. Clinical problems include patent ductus arteriosus, occlusive strokes, and coronary artery disease in addition to thoracic aortic aneurysms (8Guo D.C. Papke C.L. Tran-Fadulu V. Regalado E.S. Avidan N. Johnson R.J. Kim D.H. Pannu H. Willing M.C. Sparks E. Pyeritz R.E. Singh M.N. Dalman R.L. Grotta J.C. Marian A.J. Boerwinkle E.A. Frazier L.Q. LeMaire S.A. Coselli J.S. Estrera A.L. Safi H.J. Veeraraghavan S. Muzny D.M. Wheeler D.A. Willerson J.T. Yu R.K. Shete S.S. Scherer S.E. Raman C.S. Buja L.M. Milewicz D.M. Am. J. Hum. Genet. 2009; 84: 617-627Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). α-SM actin is one of six highly homologous isoactins expressed in mammals. Mutations in the actin family have recently been associated with disease, the vast majority of which behave in a dominant fashion. Over the past decade, mutations in α-cardiac (ACTC), α-skeletal (ACTA1), and γ-cytoplasmic actin (ACTG1) have been associated with cardiomyopathies, skeletal myopathies, and deafness, respectively. As such, the role for the α-SM actin in human disease is not surprising.In vascular smooth muscle cells, α-SM actin has many roles, including maintenance of cell wall integrity, force transduction, and regulation of vascular smooth muscle cell proliferation (9Ayscough K.R. Stryker J. Pokala N. Sanders M. Crews P. Drubin D.G. J. Cell Biol. 1997; 137: 399-416Crossref PubMed Scopus (635) Google Scholar). α-SM actin is the most abundant protein in vascular smooth muscle cells, making up 40% of total cellular protein and over 70% of the total actin (10Fatigati V. Murphy R.A. J. Biol. Chem. 1984; 259: 14383-14388Abstract Full Text PDF PubMed Google Scholar). The other two actin isoforms in the vascular smooth muscle cells are β- and γ-cytoplasmic actin. Histopathology from individuals with α-SM actin mutations noted loss or disorganization of the vascular smooth muscle cells in the tunica media of the aorta (8Guo D.C. Papke C.L. Tran-Fadulu V. Regalado E.S. Avidan N. Johnson R.J. Kim D.H. Pannu H. Willing M.C. Sparks E. Pyeritz R.E. Singh M.N. Dalman R.L. Grotta J.C. Marian A.J. Boerwinkle E.A. Frazier L.Q. LeMaire S.A. Coselli J.S. Estrera A.L. Safi H.J. Veeraraghavan S. Muzny D.M. Wheeler D.A. Willerson J.T. Yu R.K. Shete S.S. Scherer S.E. Raman C.S. Buja L.M. Milewicz D.M. Am. J. Hum. Genet. 2009; 84: 617-627Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). Analysis of aortic vascular smooth muscle cells showed diminished α-SM actin, with substantially fewer polymerized actin fibers that did not extend completely across the cell body. α-SM actin staining did not co-localize with polymerized actin fibers and was found clumped along the cell wall or nuclear wall.To gain insight into how ACTA2 mutations cause disease, the functional changes in the context of the structure of the actin molecule must be established. The actin monomer can be divided into two large domains. An adenine nucleotide binds between the two halves, bridging the cleft to impart stability to the protein, and plays a major role in controlling protein dynamics. Each of the domains can each be further divided into two subdomains, as shown in Fig. 1. Actin polymerizes to form a polar filament with what is termed a barbed and a pointed end. The barbed end is the preferred site for monomer addition during filament elongation and, as such, is where much of actin assembly regulation occurs. The asymmetry can be imparted to the actin monomer as well. Subdomains 1 and 3 constitute the monomer barbed end, whereas subdomains 2 and 4 make up the pointed end.At least 22 autosomal dominant missense mutations in α-SM actin have now been identified in familial TAAD, and they are distributed across all four subdomains (7Guo D.C. Pannu H. Tran-Fadulu V. Papke C.L. Yu R.K. Avidan N. Bourgeois S. Estrera A.L. Safi H.J. Sparks E. Amor D. Ades L. McConnell V. Willoughby C.E. Abuelo D. Willing M. Lewis R.A. Kim D.H. Scherer S. Tung P.P. Ahn C. Buja L.M. Raman C.S. Shete S.S. Milewicz D.M. Nat. Genet. 2007; 39: 1488-1493Crossref PubMed Scopus (617) Google Scholar, 11Morisaki H. Akutsu K. Ogino H. Kondo N. Yamanaka I. Tsutsumi Y. Yoshimuta T. Okajima T. Matsuda H. Minatoya K. Sasaki H. Tanaka H. Ishibashi-Ueda H. Morisaki T. Hum. Mutat. 2009; 30: 1406-1411Crossref PubMed Scopus (77) Google Scholar, 12Yoo E.H. Choi S.H. Jang S.Y. Suh Y.L. Lee I. Song J.K. Choe Y.H. Kim J.W. Ki C.S. Kim D.K. Ann. Clin. Lab. Sci. 2010; 40: 278-284PubMed Google Scholar, 13Disabella E. Grasso M. Gambarin F.I. Narula N. Dore R. Favalli V. Serio A. Antoniazzi E. Mosconi M. Pasotti M. Odero A. Arbustini E. Heart. 2011; 97: 321-326Crossref PubMed Scopus (54) Google Scholar). The focus of this paper is on two of these mutations, N117T and R118Q, which are adjacent to one another in a secondary structural element, near the barbed end of the protein. The location is predicted to be involved in intermonomer interactions, as shown in Fig. 1. Of interest, the two mutations have different clinical phenotypes. The N117T mutation has been found in both α-skeletal muscle and α-SM actins and is associated primarily with aneurysms of the thoracic aorta (7Guo D.C. Pannu H. Tran-Fadulu V. Papke C.L. Yu R.K. Avidan N. Bourgeois S. Estrera A.L. Safi H.J. Sparks E. Amor D. Ades L. McConnell V. Willoughby C.E. Abuelo D. Willing M. Lewis R.A. Kim D.H. Scherer S. Tung P.P. Ahn C. Buja L.M. Raman C.S. Shete S.S. Milewicz D.M. Nat. Genet. 2007; 39: 1488-1493Crossref PubMed Scopus (617) Google Scholar, 8Guo D.C. Papke C.L. Tran-Fadulu V. Regalado E.S. Avidan N. Johnson R.J. Kim D.H. Pannu H. Willing M.C. Sparks E. Pyeritz R.E. Singh M.N. Dalman R.L. Grotta J.C. Marian A.J. Boerwinkle E.A. Frazier L.Q. LeMaire S.A. Coselli J.S. Estrera A.L. Safi H.J. Veeraraghavan S. Muzny D.M. Wheeler D.A. Willerson J.T. Yu R.K. Shete S.S. Scherer S.E. Raman C.S. Buja L.M. Milewicz D.M. Am. J. Hum. Genet. 2009; 84: 617-627Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 14Sparrow J.C. Nowak K.J. Durling H.J. Beggs A.H. Wallgren-Pettersson C. Romero N. Nonaka I. Laing N.G. Neuromuscul. Disord. 2003; 13: 519-531Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). R118Q is the second most common mutation identified in α-SM actin, and remarkably, 75% of patients with this mutation have premature coronary artery disease in addition to aortic aneurysms. The varied clinical phenotypes suggest that although all 22 of these mutations cause TAAD, the molecular mechanisms by which different mutations lead to aortic disease are allele-specific.Understanding of the effects of α-SM actin mutations at the molecular level in the context of the aortic wall is hampered by the difficulty in obtaining sufficient samples from patients for biochemical studies. Even if one were to introduce the mutations into model animals to establish cultured vascular smooth muscle cell preparations, the amount of material available for biochemical analysis would be limited. Additionally, the presence of three essentially unresolvable isoforms of actin in the vascular smooth muscle cells would hinder direct assessment of the effects of the mutations on α-SM actin function.An attractive model system for studying the effects of actin mutations is the budding yeast, Saccharomyces cerevisiae. Yeast actin is 86% identical and 94% similar to α-SM actin and is encoded by a single essential gene, ACT1 (15Ng R. Abelson J. Proc. Natl. Acad. Sci. U.S.A. 1980; 77: 3912-3916Crossref PubMed Scopus (348) Google Scholar). More importantly, the two residues at which the TAAD mutations occur in α-SM actin are identical in yeast actin (N117T and R118Q in α-SM actin correspond to N115T and R116Q in yeast actin, and for the remainder of this work, the yeast numbering system will be used). In addition, actin regulation is well conserved between species. Many of the actin-binding proteins in mammalian cells are also found in yeast (16Drubin D.G. Cell Motil. Cytoskeleton. 1990; 15: 7-11Crossref PubMed Scopus (34) Google Scholar, 17Bretscher A. Drees B. Harsay E. Schott D. Wang T. J. Cell Biol. 1994; 126: 821-825Crossref PubMed Scopus (59) Google Scholar), and yeast actin will interact with many of the mammalian isoforms of actin-binding proteins due to the high degree of homology between actins (18Chen W. Wen K.K. Sens A.E. Rubenstein P.A. Biophys. J. 2006; 90: 1308-1318Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 19Wen K.K. Rubenstein P.A. J. Biol. Chem. 2005; 280: 24168-24174Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Mutations can be readily introduced into the actin gene by site-directed mutagenesis to express mutant actin as the sole actin isoform. The impact of mutations on actin function in the cell can be assessed cytologically, and the actin can be purified for studies of the biochemical effects of the mutations on the molecule. The in vivo and in vitro data can be analyzed for alterations in molecular behavior to provide insight into the mechanisms underlying aortic disease. We have previously applied this approach to the analysis of mutations in γ-nonmuscle actin that cause autosomal dominant early onset deafness (20Bryan K.E. Rubenstein P.A. J. Biol. Chem. 2009; 284: 18260-18269Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 21Bryan K.E. Wen K.K. Zhu M. Rendtorff N.D. Feldkamp M. Tranebjaerg L. Friderici K.H. Rubenstein P.A. J. Biol. Chem. 2006; 281: 20129-20139Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 22Morín M. Bryan K.E. Mayo-Merino F. Goodyear R. Mencía A. Modamio-Høybjør S. del Castillo I. Cabalka J.M. Richardson G. Moreno F. Rubenstein P.A. Moreno-Pelayo M.A. Hum. Mol. Genet. 2009; 18: 3075-3089Crossref PubMed Scopus (56) Google Scholar).In this study, either N115T or R116Q mutant actin was expressed as the sole actin in the cell. The effects of the TAAD mutations on cell behavior and actin-related cellular functions were characterized. Actin was purified, and the impact of the mutations on actin monomer and polymer function in vitro was assessed.EXPERIMENTAL PROCEDURESMaterialsDNase I (grade D) was purchased from Worthington. DE52 DEAE-cellulose was obtained from Whatman. Micro Bio-Spin P-30 Tris columns and Affi-Gel 10-activated resin were purchased from Bio-Rad. ATP was purchased from Sigma. ϵ-ATP, rhodamine-phalloidin, FM4-64, and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Molecular Probes. The QuikChange® site-directed mutagenesis kit was from Stratagene, and oligodeoxynucleotides were purchased from Integrated DNA Technologies. Yeast cakes for wild type actin preparations were purchased from a local bakery. All other chemicals were of reagent grade quality.Construction of Mutant Yeast StrainsMutations were introduced into the centromeric plasmid pRS314 (21Bryan K.E. Wen K.K. Zhu M. Rendtorff N.D. Feldkamp M. Tranebjaerg L. Friderici K.H. Rubenstein P.A. J. Biol. Chem. 2006; 281: 20129-20139Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) containing the yeast actin coding sequence driven by the ACT1 promoter using the QuikChange® mutagenesis kit according to the manufacturer's instructions. Plasmids containing the desired mutations were introduced into a recipient yeast strain containing a deleted chromosomal ACT1 gene and a plasmid expressing wild type actin (pCENWT) as described previously (23Cook R.K. Sheff D.R. Rubenstein P.A. J. Biol. Chem. 1991; 266: 16825-16833Abstract Full Text PDF PubMed Google Scholar). Plasmid shuffling yielded viable haploid strains for each of the mutations. The plasmids containing the mutant constructs were reisolated from these strains and sequenced to confirm the presence of the desired mutation.Growth Behavior in Liquid CultureCells were grown in complete liquid YPD medium (1% yeast extract, 2% peptone, and 2% dextrose) at 30 °C on a shaking platform. Growth was determined by diluting an overnight culture of each strain into fresh medium at an initial A600 of 0.1 and following growth at 30 °C with agitation. The absorbances of the cultures were recorded as a function of time. The absorbances were back-calculated following the appropriate dilutions to lower the cell density to the linear range of the spectrometer.Growth under Stress ConditionsTemperature sensitivity of mutant actin was examined by plating four serial 10-fold dilutions of the cultures on YPD plates followed by incubation at 24, 30, or 37 °C. Colony size was assessed as a function of time. To assess mitochondrial function, cells were grown on media containing glycerol as the sole carbon source. Cultures were plated on YPG medium (YPD medium with the dextrose replaced with 2% glycerol) and incubated at 30 °C. To test for hyperosmolar sensitivity, cells were plated on YPD plus 0.9 m NaCl agar plates and incubated at 30 °C.CytologyCell structures were imaged with an Olympus IX81 microscope and a Hamamatsu (model C10600-10B-H) camera. Camera control and image enhancement were performed using MetaMorph version 4.5 software (Universal Image Corp., Downingtown, PA). Presentation of cell images was done using CorelDRAW 11. All cellular statistical analysis was based on cell counts of >100 for each sample. To measure cell size, mounted samples were visualized by differential interference contrast microscopy. The long axis of the cell was measured using ImageJ (Wayne Rasband, National Institutes of Health, Bethesda, MD).Mitochondria were visualized in living cells by expressing a fusion protein of green fluorescent protein (GFP) conjugated to the mitochondrial signal sequence of citrate synthase kindly provided by Dr. Liza A. Pon (24Fehrenbacher K.L. Yang H.C. Gay A.C. Huckaba T.M. Pon L.A. Curr. Biol. 2004; 14: 1996-2004Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Cells expressing the plasmid were grown to an A600 of 0.3–0.5 in Ura− synthetic medium to force retention of the URA3-marked plasmid in the otherwise Ura3− cells. An aliquot of cells was resuspended in VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA), and the cells were then observed by fluorescence microscopy as described above. Z-sections through the cell were obtained at 0.15-μm intervals. Out-of-focus light was removed by deconvolution using MetaMorph software, and each series of deconvolved images was further rendered with ImageJ.The actin cytoskeleton was visualized by fluorescence microscopy after staining fixed cells with rhodamine-phalloidin as described previously (25McKane M. Wen K.K. Boldogh I.R. Ramcharan S. Pon L.A. Rubenstein P.A. J. Biol. Chem. 2005; 280: 36494-36501Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Cytoskeletal analysis focused on budding cells when the daughter cell is less than one-half the size of the mother cell. Vacuoles were imaged following exposure of the cells to the dye FM4-64 as described previously (26Vida T.A. Emr S.D. J. Cell Biol. 1995; 128: 779-792Crossref PubMed Scopus (1127) Google Scholar). Nuclear and mitochondrial DNA was stained with DAPI as described previously (25McKane M. Wen K.K. Boldogh I.R. Ramcharan S. Pon L.A. Rubenstein P.A. J. Biol. Chem. 2005; 280: 36494-36501Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar).Latrunculin A SensitivitySterile filtered discs (0.5 cm in diameter) were presoaked in 2 μl of DMSO (control) plus 0.1, 0.5, or 1 mm of latrunculin A (9Ayscough K.R. Stryker J. Pokala N. Sanders M. Crews P. Drubin D.G. J. Cell Biol. 1997; 137: 399-416Crossref PubMed Scopus (635) Google Scholar). Soaked discs were placed on YPD plates containing 100 μl of evenly spread wild type or mutant cells (A600 = 0.1). The plates were incubated at 30 °C for 48 h, and the zone of growth inhibition around the drug eluting latrunculin A disc was assessed.ΔAip1p Deletion Strain StudiesUsing an Δaip1:pCENWT strain as the host (26Vida T.A. Emr S.D. J. Cell Biol. 1995; 128: 779-792Crossref PubMed Scopus (1127) Google Scholar), pRS314 plasmids containing the promoter region, the TRP1 gene, and the coding sequence for wild type or mutant yeast actin were transformed into this haploid yeast strain (27Cook R.K. Blake W.T. Rubenstein P.A. J. Biol. Chem. 1992; 267: 9430-9436Abstract Full Text PDF PubMed Google Scholar). Transformants were selected on tryptophan-deficient medium and then subjected to plasmid shuffling to eliminate the wild type actin gene. Growth curves, growth characteristics, and cytology were determined as described above for wild type and each mutant in the ΔAip1p deletion strain.Protein PurificationWild type and mutant yeast actins were purified from lysates of frozen yeast cells using a combination of DNase I-agarose affinity chromatography, DEAE-cellulose chromatography, and polymerization/depolymerization cycling as described previously (27Cook R.K. Blake W.T. Rubenstein P.A. J. Biol. Chem. 1992; 267: 9430-9436Abstract Full Text PDF PubMed Google Scholar). The quality of actin preparations was assessed using SDS-PAGE and Coomassie Blue staining of the gels. The concentration of G-actin was determined from the absorbance at 290 nm using an extinction coefficient of 0.63 m−1 cm−1. Actin was stored as G-actin in G buffer (5 mm Tris-HCl, pH 7.5, 0.1 mm ATP, pH 7.5, 0.2 mm CaCl2, and 0.2 mm dithiothreitol). All actins were stored at 4 °C and used within 4 days of purification. Yeast cofilin was purified from Escherichia coli carrying a recombinant construct for the protein according to Lappalainen et al. (20Bryan K.E. Rubenstein P.A. J. Biol. Chem. 2009; 284: 18260-18269Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 28Lappalainen P. Fedorov E.V. Fedorov A.A. Almo S.C. Drubin D.G. EMBO J. 1997; 16: 5520-5530Crossref PubMed Scopus (209) Google Scholar), and the concentration of the purified cofilin was determined by absorption at 280 nm with an extinction coefficient of 14,650 m−1 cm−1.Actin PolymerizationPolymerization of 4.8 μm G-actin in a total volume of 120 μl was induced by the addition of MgCl2 and KCl to final concentrations of 2 and 50 mm, respectively (F-salts). Polymerization at 25 °C was monitored by following the increase in light scattering of the sample in a FluoroMax-3 fluorescence spectrometer outfitted with a computer-controlled thermostatted four-position multisample exchanger (HORIBA Jobin Yvon Inc.). The excitation and emission wavelengths were both set to 360 nm with the slit widths set at 1 nm. To determine the effects of cofilin on preformed actin filaments, the desired amount of cofilin was added to the polymerized actin sample, and the resulting change in light scattering was monitored. For experiments examining the effects of different molar fractions of mutant actin on overall actin behavior, wild type and mutant actin were combined with a final total actin concentration of 4.8 μm before induction of polymerization. All polymerization experiments were performed at least three times with at least three different actin preparations.Actin Depolymerization RatesActin was polymerized to steady-state levels followed by the addition of DNase I in a 1:1 actin/DNase molar ratio. Depolymerization was monitored as a decrease in light scattering over time. Depolymerization rates were determined by fitting the data to a single exponential expression using BioKine version 3.1.Circular Dichroism MeasurementsThe apparent melting temperatures of wild type and mutant actins were determined using circular dichroism by following the change in ellipticity of the G-actin sample at 222 nm as a function of temperature between 25 and 90 °C as described previously (21Bryan K.E. Wen K.K. Zhu M. Rendtorff N.D. Feldkamp M. Tranebjaerg L. Friderici K.H. Rubenstein P.A. J. Biol. Chem. 2006; 281: 20129-20139Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Measurements were made on an Aviv 62 DS spectropolarimeter. Data were fit to a two-state model, and the apparent Tm value was determined by fitting the data to the Gibbs-Helmholtz equation to approximate the temperature at which 50% of the actin was denatured.ϵ-ATP ExchangeThe ability of G-actin to exchange bound nucleotide was assessed by first loading the actin with ϵ-ATP and quantifying the rate of displacement from actin in the presence of a large excess of ATP as described previously (21Bryan K.E. Wen K.K. Zhu M. Rendtorff N.D. Feldkamp M. Tranebjaerg L. Friderici K.H. Rubenstein P.A. J. Biol. Chem. 2006; 281: 20129-20139Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Exchange rates were determined by fitting the data to a single exponential expression using BioKine version 3.1.Cofilin Binding AssayG-actinPyrene-labeled G-actin was made according to Feng et al. (29Feng L. Kim E. Lee W.L. Miller C.J. Kuang B. Reisler E. Rubenstein P.A. J. Biol. Chem. 1997; 272: 16829-16837Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Increasing amounts of cofilin were added to a 1.5-ml sample containing 1 μm 100% pyrene-labeled G-actin, and the cofilin-dependent increase in pyrene fluorescence was recorded on a FluoroLog3 fluorescence spectrometer outfitted with a computer-controlled thermostatted sample exchanger with continuous sample mixing (HORIBA Jobin Yvon Inc.). All experiments were performed in G-buffer containing 50 mm KCl. Note that this concentration of KCl will not induce polymerization of yeast actin because polymerization of yeast actin requires Mg2+, unlike muscle actin (30Kim E. Miller C.J. Reisler E. Biochemistry. 1996; 35: 16566-16572Crossref PubMed Scopus (46) Google Scholar). The excitation and emission wavelengths were 344 and 386 nm, respectively, with the corresponding slit widths of 1 and 2 nm. Using Microsoft Excel, experimental data were fit to the quadratic binding isotherm, ΔF=Fmax[A]+[C]+Kd−([A]+[C]+Kd)2−4[A][C]2[A](Eq. 1) where ΔF represents the observed fluorescence change of the actin-cofilin complex after the fluorescence of the G-actin alone has been subtracted; Fmax is the maximum fluorescence change at complete saturation of actin with cofilin; [A] and [C] are the concentrations of G-actin and cofilin, respectively; and Kd is the observed dissociation constant. The solver function was used to minimize the difference between the experimental data and the best fit to produce the Kd.F-actinPyrene-labeled G-actin was made according to Feng et al. (29Feng L. Kim E. Lee W.L. Miller C.J. Kuang B. Reisler E. Rubenstein P.A. J. Biol. Chem. 1997; 272: 16829-16837Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Polymerization of 4.8 μm G-actin (10% pyrene-labeled and 90% unlabeled) in a total volume of 120 μl was induced by the addition of F-salts. The change in actin-pyrene fluorescence due to actin polymerization was recorded with a FluoroLog3 fluorescence spectrometer (Jobin Yvon-Spex). The excitation wavelength was 365 nm. The change in fluorescence intensity at emission wavelength 386 nm was recorded over time for kinetics analyses. After steady state was reached, an increasing amount of cofilin was added to the sample, and the cofilin-dependent quenching of the pyrene fluorescence was recorded. The excitation and emission wavelengths were 344 and 386 nm, respectively, with the corresponding slit widths of 1 and 2 nm. Using Microsoft Excel, experimental data were fit to the quadratic binding isotherm (as above).Critical Concentration DeterminationTo measure the critical concentration (Cc) of each actin, the net change in light scattering of an actin polymerization reaction was measured as a function of increasing actin concentration. Polymerization of G-actin, at concentrations between 4.8 and 1 μm, was induced by the addition of F-salts and monitored by light scattering. The final increase in light scattering for each actin concentration was reco
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