Tropomyosin variants describe distinct functional subcellular domains in differentiated vascular...

Tropomyosin variants describe distinct functional subcellular domains indifferentiated vascular smooth muscle cells

Cynthia Gallant,1* Sarah Appel,1* Philip Graceffa,2 Paul Leavis,2 Jim Jung-Ching Lin,3

Peter W. Gunning,4 Galina Schevzov,4 Christine Chaponnier,5 Jon DeGnore,6 William Lehman,7

and Kathleen G. Morgan1

1Department of Health Sciences, Boston University, Boston, Massachusetts; 2Boston Biomedical Research Institute,Watertown, Massachusetts; 3Department of Biology, University of Iowa, Iowa City, Iowa; 4Department of Pharmacology,School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia; 5Department of Pathologyand Immunology, Faculty of Medicine, University of Geneva, Centre Médical Universitaire (CMU), Geneva, Switzerland;6Tufts University School of Medicine, Boston, Massachusetts; and 7Department of Physiology and Biophysics, BostonUniversity School of Medicine, Boston, Massachusetts

Submitted 5 November 2010; accepted in final form 31 January 2011

Gallant C, Appel S, Graceffa P, Leavis P, Lin JJ, Gunning PW,Schevzov G, Chaponnier C, DeGnore J, Lehman W, Morgan KG.Tropomyosin variants describe distinct functional subcellular domainsin differentiated vascular smooth muscle cells. Am J Physiol CellPhysiol 300: C1356–C1365, 2011. First published February 2, 2011;doi:10.1152/ajpcell.00450.2010.—Tropomyosin (Tm) is known to bean important gatekeeper of actin function. Tm isoforms are encodedby four genes, and each gene produces several variants by alternativesplicing, which have been proposed to play roles in motility, prolif-eration, and apoptosis. Smooth muscle studies have focused ongizzard smooth muscle, where a heterodimer of Tm from the �-gene(Tmsm-�) and from the �-gene (Tmsm-�) is associated with contrac-tile filaments. In this study we examined Tm in differentiated mam-malian vascular smooth muscle (dVSM). Liquid chromatography-tandem mass spectrometry (LC MS/MS) analysis and Western blotscreening with variant-specific antibodies revealed that at least fivedifferent Tm proteins are expressed in this tissue: Tm6 (Tmsm-�) andTm2 from the �-gene, Tm1 (Tmsm-�) from the �-gene, Tm5NM1from the �-gene, and Tm4 from the �-gene. Tm6 is by far mostabundant in dVSM followed by Tm1, Tm2, Tm5NM1, and Tm4.Coimmunoprecipitation and coimmunofluorescence studies demon-strate that Tm1 and Tm6 coassociate with different actin isoforms anddisplay different intracellular localizations. Using an antibody specificfor cytoplasmic �-actin, we report here the presence of a �-actincortical cytoskeleton in dVSM cells. Tm1 colocalizes with corticalcytoplasmic �-actin and coprecipitates with �-actin. Tm6, on the otherhand, is located on contractile bundles. These data indicate that Tm1and Tm6 do not form a classical heterodimer in dVSM but ratherdescribe different functional cellular compartments.

actin isoforms; liquid chromatography-tandem mass spectrometryanalysis; cytoplasmic �-actin

TROPOMYOSIN (Tm) is an abundant F-actin binding proteinknown to regulate actomyosin interactions in adult striated(overviews in Refs. 5, 17, 24, and 62) and smooth muscle(overview in Ref. 33). The four Tm genes (overview in Refs.11 and 29), TPM1 (�), TPM2 (�) TPM3 (�), and TPM4 (�),lead to the production of multiple variants by alternativesplicing (55). Smooth muscle Tm, based on work primarilyusing gizzard tissue, has been focused essentially exclusively

on the heterodimers of the smooth muscle-specific Tmsm-�variant (from the �-gene) and the Tmsm-� variant (from the�-gene) (6, 59, 60), associated with the smooth muscle actin ofthe contractile filaments of gizzard. In contrast, �40 mamma-lian variants have been described and the majority of them areexpressed in nonmuscle cells (12, 25, 55, 56, 58). It is knownthat the differentiation status of smooth muscle cells affects theexpression pattern of Tm proteins. The TPM1 gene encodes fora smooth muscle-specific exon, exon 2a, which is only ex-pressed in the differentiated state. Upon a switch to a syntheticphenotype, exon 2a is no longer utilized during splicing andinstead exon 2b is used, creating the fibroblast-type Tm protein(9, 58).

In striated muscle, the function of Tm has been intensivelystudied and it is known that Tm proteins act in concert withtroponin to regulate a Ca2�-dependent activation of actinfilaments (overview in Ref. 5). In smooth muscle, however,there is no troponin, and contraction is mainly controlled byphosphorylation of the myosin light chain (LC20) (49) and thephosphorylation status of h-caldesmon, a smooth muscle-spe-cific actin-binding protein (overview in Ref. 23). Here Tmmight act as a gatekeeper for actin function by controlling theaccessibility of other actin-binding proteins to actin filaments,and thereby regulating both actin polymerization as well as theinteraction of actin and myosin.

Our lab and others have previously shown that actin netpolymerization is important in regulating contractility of dif-ferentiated vascular smooth muscle cells (dVSMCs; 20–22).We have previously found that �-adrenergic agonist-inducedcontractility is primarily associated with a change in net po-lymerization of �-actin-containing filaments, not with the �-ac-tin filaments contained in the contractile filament bundles. Onthe basis of indirect evidence as well as work in nonmusclecells, we predicted that �-actin might be localized in the cellcortex. Unfortunately, we were not able to specifically imagefor �-actin due to the lack of a specific antibody. A specificanti-cytoplasmic �-actin antibody has now been developed (4),and we show here that cytoplasmic Beta, not �-actin is local-ized in a cortical F-actin network in dVSMCs.

By antibody screening and liquid chromatography-tandemmass spectrometry (LC MS/MS), we have identified a total offive Tm variants in adult vascular smooth muscle. In additionto �-smooth muscle Tm6, Tm2 was identified from the �-gene.

* C. Gallant and S. Appel contributed equally to this work.Address for reprint requests and other correspondence: K. G. Morgan,

Health Sciences Dept., Boston Univ., 635 Commonwealth Ave., Boston, MA02215 (e-mail: [email protected]).

Am J Physiol Cell Physiol 300: C1356–C1365, 2011.First published February 2, 2011; doi:10.1152/ajpcell.00450.2010.

0363-6143/11 Copyright © 2011 the American Physiological Society http://www.ajpcell.orgC1356

Additionally, from dVSM homogenates, evidence was foundfor Tm1 from the �-gene, Tm5NM1 from the �-gene, and Tm4from the �-gene. We show here that Beta-Tm1 is less abundantthan Tm6, is primarily localized in the cortex of differentiatedsmooth muscle cells, and coimmunoprecipitates with �-actin.�-Smooth muscle Tm6 is not colocalized with Tm1, but doescolocalize with �-smooth muscle actin and �-actin in contrac-tile filament bundles.

MATERIALS AND METHODS

Tissue preparation. All procedures in this study were performedaccording to protocols approved by the Institutional Care and UseCommittee of Boston University. Ferrets (Marshall Farms, NorthRose, NY) were euthanized by an overdose of isoflurane, and the aortawas removed and placed in an oxygenated physiological salt solution(PSS; in mM: 120 NaCl, 5.9 KCl, 1.2 NaH2PO4, 25 NaHCO3, 11.5dextrose, 1 CaCl2, and 1.4 MgCl2, pH 7.4). Aorta tissue was dissectedunder a microscope in oxygenated (95% O2 and 5% CO2) PSS toremove the adventitia and the endothelium. On the basis of previousstudies showing that the normal intima and media of all mammalianarteries contain only smooth muscle cells (42), we assumed that thetissue we were working with was not contaminated with fibroblasts orother cell types. After 1 h of equilibration, tissue viability was testedby stimulation for 10 min with 51 mM KCl-PSS (51 mM of NaClreplaced with 51 mM KCl). After washing the tissue and relaxationfor 1 h, tissue was used for further experiments. Finally, tissue wasquick frozen in a dry ice-acetone slurry containing 10 mM DTT.

Identification of Tm variants in aorta via LC MS/MS analysis. Forpurification of Tm, a protocol from Bretscher (1) was used. Briefly,smooth muscle tissue from freshly isolated ferret aorta was homoge-nized in extraction buffer (0.3 mM KCl, 1 mM EGTA, 0.5 mMMgCl2, 50 mM imidazole-HCl, pH 6.9, and protease inhibitors) onice. The homogenate was then heated in a boiling water bath for 10min before chilled on ice for 10 min. The material was next cleared bycentrifugation for 30 min at 47,000 g at 4°C. The supernatant,containing heat-stable proteins like Tm and caldesmon, was analyzedon a 10% SDS-Gel and proteins were detected via Coomassie stain-ing. Five protein bands in the size range of 42 to 28 kDa (where Tmproteins were expected) were excised from the gel and subjected toin-gel reduction, alkylation, and enzymatic digestion (Roche AppliedScience, Indianapolis, IN) in a HEPA-filtered hood to reduce keratinbackground. LC MS/MS analysis was performed on the in-gel digestextracts using an Agilent (Santa Clara, CA) 1100 binary pump directlycoupled to a mass spectrometer. Six microliters of sample wereinjected on column using a LC Packings (Sunnyvale, CA) FAMOSautosampler. Nanobore electrospray columns were constructed from360 �m outer diameter, 75 �m inner diameter fused silica capillarywith the column tip tapered to a 10-�m opening (New Objective,Woburn, MA). The columns were packed with 200 Å 5 �m C18 beads(Michrom BioResources, Auburn, CA), a reverse-phase packing ma-terial, to a length of 10 cm. The flow through the column was splitprecolumn to achieve a flow rate of 320 nl/min. The mobile phaseused for gradient elution consisted of 1) 0.3% acetic acid-99.7% waterand 2) 0.3% acetic acid-99.7% acetonitrile. Tandem mass spectra (LCMS/MS) were acquired on a Thermo LTQ ion trap mass spectrometer(Thermo, San Jose, CA). Needle voltage was set to 3 kV, isolationwidth was 3 Da, relative collision energy was 30%, and dynamicexclusion was used to exclude recurring ions. Ion signals above apredetermined threshold automatically triggered the instrument toswitch from MS to MS/MS mode for generating fragmentation spec-tra. The MS/MS spectra were searched against the National Center forBiotechnology Information nonredundant protein sequence databaseusing the SEQUEST computer algorithm (61) to produce a list ofproteins identified in each sample. LC MS/MS analysis was per-formed at both Tufts University and Boston University Core facilities.

Isolation of single differentiated vascular smooth muscle cells fromaorta. The method used for isolating dVSMCs was described previ-ously (21). Briefly, ferret aorta tissue was cut into small pieces andincubated in Ca2� and Mg2�-free HBSS containing 100 U/ml type IIcollagenase (Worthington), 1 U/ml elastase (Roche), and 0.2% BSA(Sigma). Tissue pieces were then filtered on a nylon mesh membrane(pore size 500 �m) and washed with Ca2� and Mg2�-free HBSS and0.2% BSA. The wash buffer containing the isolated differentiatedvascular smooth muscle cells was poured onto glass coverslips on iceunder a 100% oxygen atmosphere, stored in PSS, and used forimmunofluorescence staining.

Generation of antibodies against different Tm variants. Tm variant-specific polyclonal antibodies have been previously described (47):�9d (sheep polyclonal) recognizes the peptide sequence encoded byexon 9d from the TPM1 gene and detects Tm1, 2, 3, 5a, 5b; �2a(sheep polyclonal) recognizes the peptide sequence encoded by exon2a from the TPM1 gene and detects Tm6; �9d (sheep polyclonal)recognizes the peptide sequence encoded by exon 9d from the TPM3gene (both human and mouse) corresponding to Tm5NM1 andTm5NM2; �9d (rabbit polyclonal) recognizes the peptide sequenceencoded by exon 9d from the TPM4 gene and detects Tm4 (13).

Monoclonal antibodies, CG1 and CG-�6, were generated againstchicken gizzard Tms and characterized previously (15, 16, 28, 30). Inaddition to chicken Tms, the CG1 specifically recognizes human Tmisoform, hTm1, whereas the CG-�6 recognizes the epitope in theCOOH-terminal region of hTM2 and hTM3 (7, 29, 30). Monoclonalantibodies LC24 and LC1, generated against bacterially producedhTM5/4 (NH2-terminal of hTM5 fused to the C-terminal of hTM4)fusion protein, specifically recognize the hTm4 and hTM5, respec-tively (29, 32, 53, 57).

Generation of Tm standards. The entire coding regions of hTm1,hTm2, hTm3, hTm4, or hTm5 cDNAs (38) were subcloned into theexpression vector pET8c/s as described previously (39). The resultingplasmids were separately transformed to BL21(DE3)LysS cells for theproduction of respective Tm variants. The bacterially produced Tmwas purified as described previously with FPLC monoQ and hydroxy-lapatite column chromatographs as the final steps. The purified Tmvariants were used for quantification of Tms in dVSMCs. Chickengizzard Tm, used as standard for quantifying Tm6 in dVSMCs, wasprepared as described previously (19).

Immunofluorescence analysis. For immunofluorescence analysis ofTm1, Tm6, �-actin, and �-actin (costaining with Tm6), differentiatedvascular smooth muscle cells were fixed with 4% paraformaldehydefor 10 min. To remove residual paraformaldehyde, two washing stepsfor 5 min with 0.1 mM glycine and 1% BSA in HBSS followed. Forpermeabilizing the cells we used 0.1% Triton X-100 in HBSS for 10min. After blocking with 10% goat serum (for Tm1, �-actin), respec-tively, donkey serum (for Tm6), 0.05% Triton X-100, and 1% BSAfor 30 min, cells were incubated, depending on the experiment, withthe sheep polyclonal anti-Tm6 antibody (Sh X Tropomyosin, SmoothMuscle; Millipore) at 1:200 dilution, the mouse monoclonal anti-Tm1antibody [CG1; (7, 15, 16, 28)] at 1:500 dilution, the mouse mono-clonal anti-�-actin antibody (Sigma) at 1:10,000 dilution, or themouse monoclonal anti-�-actin antibody (Sigma) at 1:400 dilutionovernight. For immunofluorescence staining of �-actin (single stain-ing) and cytoplasmic �-actin, differentiated vascular smooth musclecells were fixed with 1% paraformaldehyde for 20 min followed byice-cold methanol for 3 min. Cells were washed with HBSS contain-ing 0.075% BSA and then permeabilized with 0.1% TX-100 for 10min. Cells were then incubated with the mouse monoclonal anti-�-actin antibody (4) at 1:100 dilution or the mouse monoclonal anti-cytoplasmic �-actin antibody (4) at 1:500 dilution over night. Assecondary antibodies, Alexa488-conjugated goat anti-mouse IgG(Molecular Probes), Alexa568-conjugated goat anti mouse IgG (Mo-lecular Probes), Alexa488-conjugated donkey anti-sheep IgG (Molec-ular Probes), or Alexa568-conjugated donkey anti-sheep or antimouse IgG (Molecular Probes) was used at 1:400 dilution for 30 min.

C1357TROPOMYOSIN VARIANTS IN VASCULAR SMOOTH MUSCLE

AJP-Cell Physiol • VOL 300 • JUNE 2011 • www.ajpcell.org

Total F-actin was stained with Alexa568-labeled phalloidin (Molec-ular Probes) at 1:5,000 dilution, and nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; Sigma). Cells were examined witha Nikon TE 2000-E2 Perfect Focus EpiFluorescence Microscope anda �60, 1.4 numerical aperture oil immersion objective. For deconvo-lution, three-dimensional images were acquired and recorded with aCoolSNAP HQ2, Photometrics camera. Out-of-focus fluorescent blurwas removed by deconvolution using the NIS-Elements AR 3.0software (Richardson-Lucy algorithm, constrained iterative-maxi-mum likelihood estimation algorithm). Images were transferred withAdobe Photoshop CS4 software.

SDS-PAGE and Western blot analysis. Quick-frozen tissue washomogenized in homogenization buffer (20 mM MOPS, pH 6.8, 10%glycerol, 4% SDS, 1% Triton X-100, 3 mM DTT, 67 �M ZnCl2, 29.6mM �-glycerophosphate, protease inhibitors), and the homogenatewas analyzed via SDS-PAGE according to standard procedures andelectrophoretically transferred onto polyvinylidene difluoride mem-branes (Millipore). Residual protein-binding sites on the membranewere blocked with blocking buffer (Odyssey, LI-COR Biosciences,Lincoln, NB). The membranes were incubated either with the sheeppolyclonal anti-Tm6 antibody (Sh X Tropomyosin) at 1:300 dilution,the mouse monoclonal anti-Tm1 antibody [CG1; (7, 15, 16, 28)] at1:2,000 dilution, the mouse monoclonal anti-Tm2 and Tm3 antibody[CG-�6; (28, 30)] at 1:500 dilution, the mouse monoclonal anti-Tm4antibody [LC24; (57)] at 1:500 dilution, the mouse monoclonalanti-Tm5NM1 antibody [LC1; (53, 57)] at 1:500 dilution, the sheeppolyclonal �9d used at 1:400, the sheep polyclonal �2a used at 1:100,the sheep polyclonal �9d used at 1:100, the rabbit polyclonal �9d usedat 1:250 , the rabbit monoclonal anti-�-actin antibody (Epitomics) at1:3,000 dilution, the mouse monoclonal anti-�-actin antibody (Sigma)at 1:100 dilution or the mouse monoclonal anti-�-actin antibody(Santa Cruz) at 1:500 dilution. As secondary antibodies, IRDye680-conjugated donkey anti-sheep IgG (Odyssey, LI-COR Biosciences) orIRDye800CM-conjugated goat anti mouse IgG (Odyssey, LI-CORBiosciences) were used at 1:1,000 dilution. Bound antibodies weredetected with an Odyssey 1400 Infrared Imager (Odyssey, LI-CORBiosciences).

Immunoprecipitation. Quick frozen tissue was homogenized inimmunoprecipitation (IP) buffer (50 mM Tris·HCl pH 7.4, 1 mMEGTA, 50 mM NaCl, 3 mM MgCl2, 1% Nonident P-40, 1% sodiumdeoxycholate, 3 mM ATP, 3 mM DTT, 67 �M ZnCl2, 29.6 mM�-glycerophosphate, protease inhibitors) and then precleared withprotein G-coupled dynabeads (Invitrogen). To reduce background, theantibodies used in this assay were cross-linked to protein G-coupleddynabeads (Invitrogen) using the cross-linking reagent Bis(sulfosuc-cinimidyl)suberate (BS3) following the manufacturer’s instructions.The precleared cell lysates were incubated for 16 h at room temper-ature with either the sheep polyclonal anti-Tm6 antibody (Sh XTropomyosin) or the mouse monoclonal anti-Tm1 antibody [CG1; (7,15, 16, 28)], cross-linked to protein G-coupled dynabeads. As nega-tive control, the mouse monoclonal anti-green fluorescent proteinantibody (Clontech) cross-linked to protein G-coupled dynabeads wasused. Precipitated proteins were washed three times with IP buffer andeluted from the beads by boiling for 5 min in sample buffer. Sampleswere separated on SDS-PAGE and further analyzed by Westernblotting.

Statistical analysis. All values in the text are means SE. Differ-ences between means were evaluated using a two-tailed Student’st-test. Significant differences were taken at the P 0.05 level.

RESULTS

Protein expression levels of Tm variants in dVSMCs. Tounderstand if and how Tm proteins control actin functions indVSMCs, we first investigated which Tm isoforms are ex-pressed in this cell type. Therefore, smooth muscle tissue fromfreshly isolated aorta was homogenized and analyzed via

Western blotting. A previously described set of polyclonalantibodies (47), raised against different regions of Tm proteins,was used as an initial screen for Tm isoforms present in aorticvascular smooth muscle tissue. As shown in Fig. 1A, an �9dantibody [raised against the peptide sequence encoded by exon9d of the TPM1 (�) gene], detects Tm6, Tm1, Tm2, andTm5a/5b. The antibody �2a raised against the peptide se-quence encoded by exon 2a of the TPM1 (�) gene, detects Tm6in aorta. The antibody �9d raised against the peptide sequenceencoded by exon 9d of the TPM3 (�) gene, binds to threedifferent Tm isoforms: Tm6, Tm1, and Tm5NM1/2. Andfinally, the �9d antibody raised against the peptide sequenceencoded by exon 9d of TPM4 (�) gene, detects Tm4 in aorta.The antibody also recognizes a higher molecular weight ofunknown identity.

We also probed the aortic vascular smooth muscle homog-enates with a set of previously described monoclonal antibod-ies (7, 28, 30, 32, 53). For comparison, we loaded recombinantTm proteins. As can be seen in Fig. 1Ba, the antibody CG-�6(raised against chicken gizzard Tm) reacts with recombinantTm2 and Tm3 from the �-gene (7, 30) and detects a proteinband in an aorta lysate (see arrow) running at about the samesize as the Tm2 standard. The CG1 antibody (raised againstchicken gizzard Tm), shown in Fig. 1Bb, is specific for recom-binant Tm1 from the �-gene (7) and stains a specific band ofthe same size in aorta homogenates (see arrow). Figure 1Bcshows staining with the LC24 antibody [raised against ahTm5/4 fusion protein and specifically recognized hTm4 fromnonmuscle human cells; (32, 57)]. This antibody detects bothrecombinant Tm1 from the �-gene and the short form Tm4from the �-gene. In aorta lysates, the antibody stains two bandsof the same size as recombinant Tm1 and Tm4 (see arrows)although the intensity of staining of Tm4 is far less intense inaorta, suggesting a lesser abundance. In Fig. 1Bd, staining withthe LC1 antibody [raised against a hTm5/4 fusion protein andspecifically recognized hTm4 from nonmuscle human cells;(32, 57)] is pictured, showing that it detects a single band inaorta of �28 kDa (lane 1, see arrow) which likely representsthe short form Tm5NM1 from the �-gene since it runs at thesame height as recombinant Tm5NM1 (lane 2).

LC MS/MS analysis of Tm variants in dVSMCs. To corrob-orate the findings for Tm variants present in smooth musclecells by using specific antibodies on a Western blot, wepartially purified Tms by a modified version of the heattreatment method of Bretscher (1) (see MATERIALS AND METHODS

for details). Aorta tissue was homogenized and boiled toremove heat-unstable proteins. Heat-stable proteins (primarilycaldesmon and Tms) were separated on a 10% SDS-gel andvisualized via Coomassie staining, and five bands in the sizerange of 42 to 28 kDa were analyzed by LC MS/MS (see Fig. 2A).The peptides resulting from trypsin digestion were aligned to ahuman protein database because of the close similarity of theferret to the human (2, 48, 54). The uppermost band at 37 kDa(band 2) was matched with a 326 aa long form of Tm6, alsoknown as isoform 2 CRA_f of the TPM1 (�) gene (83%coverage). This was surprising, since this long Tm6 form hasnot been previously described in protein form and has onlybeen previously reported as a transcript (AK131384). Thismatch was based on peptides corresponding to both exon 2aand exon 2b from the �-gene; however, we did not find anypeptide corresponding to the transition region of exon 2a/2b

C1358 TROPOMYOSIN VARIANTS IN VASCULAR SMOOTH MUSCLE


which would confirm that those two exons exist in the sameprotein. Moreover, previous studies have excluded the possi-bility that exons 2a and 2b from the �-gene can be splicedtogether, in theory, because the intron is thought to be too shortto accommodate the splicing machinery (51). A sequencealignment of exons 2b of the �- and �-gene, compared with thepeptide identified, moreover ruled out that the band contains amix of Tm6 (�-gene) and Tm1 (�-gene) (Fig. 2B, underlinedpeptide; MS/MS fragmentation for this particular peptide isshown in Fig. 2D). Therefore, it is more likely that the band cutfrom this region of the gel contains a mix of two different Tmvariants from the �-gene, containing exon 2a or exon 2b.Combined with our Western blot staining using antibodies �9dand CG-�6 (Fig. 1, A and Ba), it seems likely that this band

contains Tm6 plus Tm2. Furthermore, the fact that we detectedpeptides only from exon 6b but not 6a from the �-gene makesit more likely that we have Tm2 and not Tm3 in the examinedtissue.

The next band (band 3), of slightly lower molecular mass,contained Tm1 [isoform 2 of the TPM2 (�) gene] (55%coverage), with peptides corresponding to exons 1a, 2b, 3, 4, 5,6a, 7, 8, and 9d from the �-gene.

Band 5 was identified as a mix of two short Tm isoforms;Tm4 from the � (38% coverage) and a variant from the �-genecontaining exons 1b and 6a (38% coverage). Since no peptidesmatching exon 9 from the �-gene were detected by LC MS/MS, the specific Tm �-variant is not determined by thismethod. Western blot staining with either the �9d (Fig. 1A) or

Fig. 1. Western blot analysis of tropomyosin (Tm)variants in differentiated vascular smooth muscle(dVSM). A: aorta tissue homogenates were separated onSDS-PAGE and further examined by Western blottingand staining with different polyclonal anti-Tm antibod-ies as indicated. Ba–Bd: 10 �g aortic homogenates wereexamined via Western blotting and probing with a set ofanti-Tm monoclonal antibodies as indicated. For com-parison, recombinant human Tm proteins were alsoloaded. The blank space between the lanes of Ba indi-cates that these are nonadjacent lanes of the same gel.



the LC1 antibody (Fig. 1Bd) however, indicates that the shortTm variant from the �-gene is Tm5NM1/2. Since Tm5NM1and Tm5NM2 differ only in the use of exon 6a versus 6b, weconclude, on the basis of the LC MS/MS data for exon 6a fromthe TPM3 (�) gene, that Tm5NM1 is present in aortic smoothmuscle.

Band 4 was identified as the smooth muscle isoform ofcalponin (h1 calponin, basic calponin), which was not surpris-ing since this protein has been described previously to be heatstable (52).

The faint band at �42 kDa (band 1), which we expected tocontain Tm6 because of the fact that the 2a antibody recognizesa specific band of that size in ferret aorta lysates (see Fig. 2C,lane 1 “Input”), was mainly composed of actin. Under the Tmpurification conditions used here, it appears that the proteinrecognized by the Tm6 antibody is less heat-soluble than theother isoforms and was precipitated in the pellet (Fig. 2C, lane2) during the purification process. To test this hypothesis, wealso examined the 42-kDa band of a total aorta lysate via LCMS/MS to see whether Tm6 is present in this band. The topmatch for this band was again actin, but peptides matching thetransition of exons 1a/2a and 2a/3 from the TPM1 (�) genewere also detected (data not shown), indicating the presence ofTm6. This indicates that 1) most of the Tm6 is insoluble withthe Bretscher (1) protocol used here, and 2) Tm6 seems to shiftto 37 kDa during the purification process since we were able to

identify Tm6 in band 2 (upper band of 37 kDa doublet) of thepurified Tm sample and the anti-Tm6 antibody does detect this37-kDa band in the purified Tm fraction (Fig. 2C, lane 3,“sup”). Others have also reported that this 37-kDa Tm6 proteinoften runs at 40–42 kDa, indicating a gel shift which appearsto be seen in a variable manner under different conditions (47).The fact that we found peptides matching the transition of exon2a/3 in this 42-kDa band furthermore rules out the possibilitythat the putative long form of Tm6 (containing both exon 2aand 2b) is present in aortic smooth muscle.

The Coomassie-stained gels that were used for LC MS/MSanalysis suggested initially that the proteins from the 37-kDadoublet (band 2: Tm6 and Tm2; band 3: Tm1) exist in aboutequal amounts. However, since the vast majority of the Tm6 isnot soluble by this protocol, in the native tissue, Tm6 may bein excess of Tm1. Thus we approached the question of therelative abundance of Tm6 by calibration with standard pro-teins below.

Quantification of Tm variant protein levels. Tms from the �-and �-gene are thought to exist in a 1:1 ratio in smooth muscle,on the basis of results from chicken gizzard (36, 40, 44). Toexamine this hypothesis, we quantified the amount of Tm1 andTm6 in aorta tissue by using purified Tms as a standard. Weloaded different amounts of human recombinant Tm1 on a gel,together with total lysate of aorta tissue. After densitometricmeasurements of the bands detected by the anti-Tm1 (CG1)

Fig. 2. LC MS/MS analysis of Tm variantsin dVSM. A: Coomassie-stained SDS gel ofthe purified Tm fraction, showing the bands1-5 that were excised from the gel and usedfor LC MS/MS analysis. The smaller boxshows the top two bands of the gel at a lowerexposure to make the two bands visible. LCMS/MS results for the 5 bands are indicatedat right and molecular mass is indicated atthe far left. B: protein sequence alignment ofhuman exon 2b derived from the TPM1 (toppeptide) or the TPM2 (bottom peptide) gene,via ClustalW. A peptide identified via LCMS/MS analysis of the uppermost 37-kDaband/band 2 (identified as a mix of Tm6 andTm2; see also D) is underlined. C: Westernblot staining of total aorta homogenate pre-pared with the extraction buffer from theBretscher protocol (1) for Tm purification(“Input”, lane 1), insoluble fraction afterhigh spin of the “Input” (“pellet”, lane 2)and soluble fraction after high spin of the“Input” (“sup”, lane 3) with antibodies (Ab)against Tm6 (�2a, top) and Tm1 (CG1, bot-tom). D: the MS/MS fragmentation of thedoubly charged (m/z 574.3) form of the LK-GTEDELDK peptide (1,146.6-Da monoiso-topic mass). The gray letters in the proteinsequence are the residues within the 81–122residue region of interest (corresponding toexon 2b). The LKGTEDELDK peptide isunderlined in the 81–122 residue region. Theb and y fragment ions observed (not allshown in MS/MS spectrum for clarity) arefrom both single and doubly charged frag-ment ions. The evidence for the unambigu-ous determination of this peptide sequence(LKGTEDELDK) is the observance of sin-gly charged fragment ions b2, b3, b4, b5, b6,b7, b9, b10 and doubly charged fragment ionsy3, y4, y5, y6, y7, y8, y9.



antibody, we calculated the total amount of Tm1 in aorticsmooth muscle cells as �1.4% of total protein in aortic smoothmuscle cells (see Fig. 3A).

For the Tm6 calibration, we used purified chicken gizzardTm as a standard, since to the best of our knowledge, norecombinant human Tmsm-�/Tm6 is available. Following den-sitometric measurement and creating a calibration curve withthe Tm standard, we calculated Tm6 in aortic smooth musclecells as 11.6% of total protein (see Fig. 3B). This is likely anoverestimate, probably because the anti-Tm6 antibody used inthis assay was raised against a mammalian (rat) sequence andlikely has a lower affinity to the chicken Tmsm-� than to theferret Tm6 (see sequence alignment of the peptide used forgenerating the �2a antibody and exons 2a from either chickenor human [close to ferret]; Fig. 3C), therefore likely artifactu-ally increasing the calculated Tm6 amount in the aorta tissue.Therefore, we cannot give a precise molar ratio of Tm1 andTm6: however, it is clear that Tm6 is present at higher levelsin this tissue than is Tm1.

As can be estimated from the Western blots shown in Fig.1B, Tm isoforms Tm2, Tm5NM1, and Tm4 were far lessabundant than Tm1 or Tm6.

�-Smooth muscle Tm6 and �-smooth muscle Tm1 binddifferent actin isoforms. The considerable difference in theamount of �-smooth muscle Tm6 and �-smooth muscle Tm1present in these samples at the protein level makes it unlikelythat these variants form 1:1 heterodimers as has been describedfor chicken gizzard smooth muscle (19, 27, 43). Thus weimmunoprecipitated Tm6 or Tm1 from aorta lysates. Probingthe samples for actin isoforms revealed that Tm1 preferentially

binds to �-actin while Tm6 binds preferentially to �-actin,previously shown to have a similar distribution as dense bodiesof contractile filament bundles (41). A typical Western blot isshown in Fig. 4A for an anti-Tm1 coimmunoprecipitation andin Fig. 4B for an anti-Tm6 coimmunoprecipitation. Densitom-etry of Western blots, derived from independent experiments,confirms that the amount of �-actin in Tm1 coimmunoprecipi-tations was significantly higher than in Tm6 coimmunoprecipi-tations (Fig. 4C), whereas the �-actin amount coprecipitatedwith Tm6 was significantly higher than in Tm1 coimmunopre-cipitations (Fig. 4D).

�-Smooth muscle actin was also probed in these coimmu-noprecipitation experiments, but we were unable to show adifferential association with the Tm variants. The pulldown of�-actin with both Tm variants may indicate a connection of the�-actin filaments to both the nonmuscle �- and �-actin net-works although other interpretations are possible (Fig. 4E).

In summary, the immunoprecipitation experiments indicatethat the two different Tm variants Tm1 and Tm6 tend toassociate with different actin isoform populations in dVSMCs.

Isoform-specific localization of actin isoforms, Tm1, andTm6 in dVSMCs. Next, imaging studies on freshly dissociatedcells from aorta were performed to demonstrate that the inter-actions shown in the coimmunoprecipitations are physiologi-cally relevant and result in colocalization. We first used actinisoform-specific antibodies to locate the actin subpopulations.Previous studies in our lab have shown that �-actin is the mostdynamic population of actin in dVSMCs in response to an�-agonist stimulus (21). However, because of the lack of a

Fig. 3. Protein calibration of Tm1 and Tm6in dVSM. A: 25 �g of ferret aorta homoge-nate were separated on a 10% SDS-gel to-gether with recombinant human Tm1 (0.05�g, 0.2 �g, 0.4 �g, 0.8 �g) and examined byWestern blotting (WB), using the anti-Tm1(CG1) antibody. The gel image was digitallyoverexposed after background subtractionfor visual display. Densitometric measure-ment of the Tm1 standards revealed that thetotal amount of Tm1 in the loaded aortasample was 0.35 �g. This corresponds to1.4% Tm1 of total protein in ferret aorta.B: 12.25 �g of ferret aorta were loaded on a10% SDS-gel in parallel with a Tm6 stan-dard (0.375 �g, 0.75 �g, 1.5 �g), purifiedfrom chicken gizzard. Densitometric analy-sis of the Western blot, probed with theanti-Tm6 (�2a) antibody, indicated that theloaded aorta sample contained 1.4 �g Tm6.This corresponds to 11.6% Tm6 of totalprotein. C: alignment of the peptide used togenerate the anti-Tm6 antibody (“2a”) andexons 2a from chicken or human Tm6.



specific antibody, the subcellular location of this dynamicsubpopulation of �-actin was not known.

In the present study we used a recently characterized specificcytoplasmic �-actin antibody (4) for the first time in dVSMCsto determine the intracellular localization of this actin isoform.Deconvolution immunofluorescence microscopy revealed that,as shown in Fig. 5Aa, cytoplasmic �-actin is mainly locatedcortical in dVSMCs. In contrast, �- and �-actin are predomi-nately incorporated in stress fibers and short filaments, respec-tively, throughout the core of the cell (Fig. 5, Ab and Ac).

Since �-actin is primarily associated with Tm1 and �-actinis primarily associated with Tm6 in coimmunoprecipitationexperiments, we compared the subcellular location of thesetwo Tm variants to that of the actin isoforms described above.We also wanted to examine if these actin binding proteinsdisplay a different intracellular localization. To visualize actin,the cells were either costained with �-actin or �-actin (for Tm6labeling) or phalloidin (for Tm1 labeling, because of theincompatibility of the cytoplasmic �-actin antibody with theconditions necessary for Tm1 staining). As can be seen in Fig.5C, Tm6 partially colocalized with short bundles of �-actin(see arrows). Immunofluorescence staining of Tm6 alsoshowed that the protein was found mainly at F-actin stressfibers, as shown by the colabeling with �-actin (Fig. 5B). Incontrast, Tm1 colocalizes with cortical actin, and not withphalloidin-stained central, contractile actin filaments as can be

seen in the merged image in Fig. 5D. This staining pattern issimilar to the cytoplasmic �-actin staining shown in Fig. 5Aa.The differences in localization of Tm6 and Tm1 were con-firmed by colabeling for both proteins in the same cells (Fig.5E). The colabeled cells demonstrated Tm6-containing centralfilaments and cortical Tm1 staining. An apparent insertion ofthe Tm6-containing filaments into the Tm1-containing corticaldomain was also visible in some cases. Thus, these results areconsistent with our conclusions from the above immunopre-cipitation experiments.

DISCUSSION

In this study we have, for the first time, demonstrated that atleast five different Tm variants are expressed at the proteinlevel in one vascular cell type: Tm6 and Tm2 from the �-gene,Tm1 from the �-gene, Tm5NM1 from the �-gene, and Tm4from the �-gene. Comparison with protein standards demon-strate that, in aorta, Tm6 is the most abundant form, followedby Tm1. Tm2, Tm4, and Tm5NM1 are far less abundant.

We have also demonstrated the unexpected finding that Tm6and Tm1 can be found in different subcellular locations asdetected by immunofluorescence staining of freshly isolateddVSMCs. Tm1 colocalizes with the cortical actin network,whereas Tm6 localizes at �-actin-containing contractile fila-ment bundles in the cells. Thus, even though we are not ruling

Fig. 4. Coimmunoprecipitation analysis of actin isoforms with Tm1 and Tm6. A: aorta tissue homogenate was subjected to coimmunoprecipitation (IP) with theanti-Tm1 (CG1) antibody (lane 3). As a negative control, coimmunoprecipitation with an anti-green fluorescent protein (GFP) antibody was performed (lane 2).Two percent of total homogenate was loaded as Input control (lane 1). Precipitated proteins were separated via SDS-PAGE and transferred onto a membrane.Detection of Tm1, �-actin, �-actin, and �-actin was performed using specific antibodies. The lines between the input and IP lanes indicate where empty lanesof the same gel were deleted. B: coimmunoprecipitation with ferret aorta homogenate using the anti-Tm6 (�2a) antibody (lane 3). The anti-GFP antibody wasused as a negative control (lane 2). Two percent of the homogenate was loaded as Input (lane 1). Tm6, �-actin, �-actin, and �-actin were detected on themembrane by specific antibodies (as indicated). The lines between the input and IP lanes indicate where empty lanes of the same gel were deleted.C: densitometric analysis of copurified �-actin together with either Tm1 (right column, n � 7) or Tm6 (left column, n � 7). Shown on the y-axis is the relative�-actin amount, normalized to Input. Furthermore, the �-background signal from the GFP-control was subtracted from the �-actin signal in the immunopre-cipitation against either Tm1 or Tm6. To be able to compare different sets of immunoprecipitations, the relative copurified �-actin amount was corrected for theamount of either Tm1 or Tm6 that was pulled down in each experiment. Ctr, GFP control. Note that the difference of coprecipitated �-actin between Tm1 andTm6 is highly statistically significant (**P � 0.002). D: graph showing copurified �-actin with either Tm1 (right column, n � 7) or Tm6 (left column, n � 6).The difference is highly statistically significant (**P � 0.004). E: coprecipitated �-actin with either Tm1 (right column, n � 7) or Tm6 (left column, n � 7).



out the possibility of in vitro heterodimerization, it appearsthat, in this dVSM cell type, the majority of Tm6 and Tm1 donot form heterodimers. This is in contrast with the het-erodimers of smooth muscle �- and �-isoforms that have beenreported for gizzard smooth muscle (19, 43). It is known that

the properties of visceral smooth muscle cells differ from thoseof vascular smooth muscle cells, as has been shown for actinisoforms (6, 14); thus, it is not surprising that a heterodimer ofTmsm-�/Tmsm-� is present in gizzard but not in aorta.

Owing to the lack of specific antibodies against Tm2, wewere not able to determine the intracellular localization of thisTm isoform and cannot say whether Tm2 is located at the stressfibers, at cortical actin, or both. However, the low level of Tm2compared with Tm6 and Tm1 and the preference of Tm2 toform homodimers in other tissues (8, 31) indicate that anyheterodimerization is likely to be at no more than trace levels.Given that Tm proteins need to be of the same length to be ableto dimerize (8), the possibility exists that the two short 248 aalong isoforms Tm5NM1 and Tm4 may form heterodimers inthis cell type.

Another new finding from this study is that cytoplasmic�-actin is located in a different cellular compartment, the cellcortex. This has not been previously reported for dVSM, andthe current working model for smooth muscle actin compart-mentalization comes from gizzard studies and does not includea cortical actin component (37). However, previous studies onfibroblasts showed that cytoplasmic �-actin is mainly orga-nized as a meshwork in cortical and lamellipodial structures(4). The cortical actin localization in dVSM is also of interestin comparison to our past studies on this same tissue type. Wehad previously shown that stimulation of dVSMCs with the�-agonist phenylephrine increases the ratio of F- to G-actin,indicating that contractility of dVSM is associated withchanges in actin net polymerization. Furthermore, we showedthat �-actin is the most dynamic isoform in the cells since acell-permeant �-actin decoy peptide construct selectively in-hibits phenylephrine-stimulated smooth muscle cell contrac-tion and two-dimensional gels of differential centrifugationsamples show greater changes in net polymerization of �-actin(21). Thus, the finding that this dynamic �-actin is localized inthe cell cortex, an area of high actin dynamics in nonmusclecells, is of considerable interest.

It had previously been found that an overexpression of�-actin in mouse C2 myoblasts can drive Tm2, but notTM5NM1, out of �-actin-containing stress fibers. These resultswere interpreted to indicate a preferred association betweenactin and Tm isoforms (46). However, to the best of ourknowledge, the current study is the first to clearly showpreferred association of endogenous Tms with separate actinisoforms, both by imaging and biochemical immunoprecipita-tion. Tm has been described as a gatekeeper, regulating theaccess of actin to actin-binding proteins (11, 18). This conceptis likely to apply to smooth muscle as well (10, 26). The factthat Tm1 associates predominately with �-actin at the cellcortex, whereas Tm6 is located at �/�-actin stress fibers, pointsto a model where Tm variants, as gatekeepers of actin sub-populations, might differentially control the access of differentactin-binding proteins, thereby also defining different func-tional cellular actin compartments.

The question remains as to how Tm variants are targeted todifferent intracellular locations. Tm variants might be targetedto different cellular compartments during the translation pro-cess via a signal sequence in the mRNA, also called “ZIP-code.” This mechanism was described previously for �-actin(50). Other studies, however, indicate that Tm isoforms do notpossess a sorting signal that is responsible for their different

Fig. 5. Immunofluorescence staining of Tm1, Tm6, and actin isoforms indVSM cells (dVSMCs). A: freshly isolated dVSMCs from ferret aorta, at-tached to glass coverslips, were fixed and stained with specific antibodiesagainst �-actin (a), �-actin (b), or �-actin (c). Nuclei were visualized by4,6-diamidino-2-phenylindole (DAPI). B: colabeling of Tm6 (�2a antibody, b)and �-actin (a) in dVSMCs. The nucleus was visualized using DAPI. Amerged image is displayed in c. C: dVSMCs were colabeled for Tm6 (�2aantibody, b) and �-actin (a) in dVSMCs. The nucleus was visualized usingDAPI. A merged image is displayed in c. C: dVSMCs were colabeled for Tm6(�2a antibody, b) and �-actin (a). DAPI was used to stain nuclei. A mergedimage is shown in c. D: intracellular localization of Tm1 was determined indVSMCs using the anti-Tm1 (CG1) antibody (b). Total F-actin was visualizedby phalloidin (a), and colocalization is shown in c. The nucleus was stainedwith DAPI. E: colabeling of Tm6 with the �2a antibody (a) and Tm1 with theCG1 antibody (b). Colocalization is shown in c. Scale bar for all, 10 �m.



intracellular localization. Cytochalasin D treatment, for exam-ple, disintegrates the isoform-specific Tm localization patternin several cell types (3, 45). Therefore it is more likely that Tmproteins display a variant-specific intracellular localization viaeither their differential affinities for actin-binding proteins oractin isoforms (34). The latter is supported by previous studies,showing that overexpression of nonmuscle actin isoforms al-ters the organization of Tm variants (46). Moreover, a recentstudy suggests that the entire Tm molecule serves as the unit ofsorting (35).

In summary, vascular smooth muscle not only express the“classical” Tmsm-� (Tm6) and Tmsm-� (Tm1) Tm proteins,but also at least three other Tm variants: Tm2 and the two shortforms Tm5NM1 and Tm4. Our studies further indicate thatTm6 and Tm1 do not form heterodimers in this tissue, sinceTm6 is more far abundant than Tm1 and moreover, Tm6 andTm1 associate with different actin isoforms and are located indifferent intracellular locations. The multitude of different Tmvariants in dVSM opens the possibility for complex regulationand organization of specific intracellular actin subpopulationsand associated actin-binding proteins into separate functionalcellular subcompartments.

ACKNOWLEDGMENTS

We acknowledge M. Steffen and A. Bergerat for support with LC MS/MSanalysis.

GRANTS

This study was supported by National Heart, Lung, and Blood InstituteGrants HL-80003, HL-31704, and HL-86655 (to K. G. Morgan) and SwissNational Foundation Grant 310030-125320 (to C. Chaponnier).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

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