Tissue-specific Tropomyosin Isoform Composition
Oncology Research Unit (GS,BV,NSB,SE,GMO,PWG) and Discipline of Paediatrics and Child Health (GS,GMO,PWG), University of Sydney, Sydney, Australia; John Douglas Centre for Structural Pathology (MRQ) and Institute for Neuromuscular Research (NY), The Children's Hospital at Westmead, Sydney, Australia; and Children's Cancer Institute Australia for Medical Research, Randwick, Australia (NMV,MK)
Correspondence to: Peter Gunning, Oncology Research Unit, The Children's Hospital at Westmead, Locked Bag 4001, Westmead, NSW, Sydney, Australia. E-mail: peterg3{at}chw.edu.au
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Summary |
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Key Words: non-muscle tropomyosin actin immunohistochemistry cytoskeleton
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Introduction |
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Subcellular localization studies using isoform-specific antibodies further support isoform-specific functions of Tms. Immunohistochemical studies, both in vivo and in cells in culture, have clearly demonstrated that Tm isoforms mark distinct populations of actin microfilaments (reviewed in Gunning et al. 1998a,b
). Such distinct intracellular compartments defined by the association of Tm isoforms may allow for independent regulation of specific microfilaments. A comprehensive characterization study of both actin and Tm antibodies is described in this report, using Western blotting, immunohistochemistry of mouse tissues, and immunofluorescence staining of mouse fibroblasts in culture. These antibodies should have broad utility in the evaluation of tropomyosin function and regulation of the actin cytoskeleton.
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Materials and Methods |
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Gel Electrophoresis and Immunoblotting
Six-month-old ARC mice were euthanized by cervical dislocation. The major organs were quickly dissected and frozen in liquid nitrogen. The stomach was cut open and its contents removed, and rinsed several times with cold PBS before being frozen. The tissue was dissociated in a sufficient volume of 50 mM Tris-Cl, pH 8, using a Polytron blender. Protein concentration was estimated using a BCA protein detection kit (Pierce; Rockford, IL). The proteins were precipitated as previously described (Wessel and Flugge 1984) and solubilized in SDS solubilization buffer (1x buffer contains 0.125 M Tris, pH 6.8, 0.5% SDS, 5% glycerol, 5% 2-mercaptoethanol, and 0.005% bromophenol blue). SDS-PAGE was performed according to Laemmli (1970)
on 12.5% acrylamide and 0.1% bis-acrylamide. Prestained molecular-mass markers were used (BenchMark PreStained Protein Ladder; Invitrogen). Proteins were transferred to Immobilon-P PVDF (Millipore Corp.; Bedford, MA) for 2 hr at 80 V according to Towbin et al. (1979)
. A 5% low-fat skim milk in TBS (100 mM Tris-Cl, pH 7.5, 150 mM NaCl) solution was used to block nonspecific binding on the blot. Primary and secondary (anti-rabbit, anti-sheep, and anti-mouse Ig-conjugated horseradish peroxidase; Amersham Biosciences Pty, Ltd.; Sydney, Australia) antibodies were incubated for 1 to 2 hr each, and 4 x 15 min washes with TTBS (TBS with 0.05% Tween 20) were carried out following each antibody incubation. Blots were developed with the Western Lighting Chemiluminescence Reagent (PerkinElmer Life Sciences; Boston, MA) and exposed to Fuji X-ray Film (Kodak; Rochester, NY). Equal protein loading on the Western blots was verified by staining the polyvinylidene fluoride membrane, following transfer, with 0.5% Ponceau-S red/1% acetic acid solution for 1 min. Blot was destained with Milli-Q-grade water (Millipore) to the desired contrast.
Immunofluorescence Staining
Primary mouse embryonic fibroblasts were prepared for immunofluorescence staining by first culturing them in eight-well glass chamber slides (5000 cells/well). Cells were fixed in 4% paraformaldehyde in PBS for 15 min (except for
/9a, see method below), rinsed three times with PBS, and then permeabilized with chilled methanol for 15 min. For the
/9a antibody, cells were fixed for 45 min in a mixture of 4% paraformaldehyde and 0.1% glutaraldehyde (Sigma Aldrich) in PBS followed by permeabilization with chilled methanol as described above. The primary fibroblasts were then incubated in blocking solution/wash solution (2% fetal bovine serum in PBS) for 30 min and then incubated with the primary antibody diluted in PBS for 1 hr at room temperature. The cells were rinsed three times with wash solution and then incubated with the secondary antibody for 1 hr. Cells were finally rinsed three times in PBS before mounting a coverslip onto the chamber slides with 1,2-diazabicyclo (2.2.2) octane (Sigma Aldrich). Slides were examined with a Leica TCS SP2 laser scanning confocal microscope (Leica Microsystems Pty, Ltd; Wetzlar, Germany) using the x63 objective. Primary fibroblasts were optically sectioned in the x-y plane, and serial sections of 0.5 µm-thickness were taken with 10-scan averaging. Images were processed using the Image Pro Plus 4.1 (Media Cybernetics; Singapore, Republic of Singapore).
Immunohistochemistry
Immunohistochemistry was performed essentially as described by Weinberger et al. (1996). Major mouse organs were dissected from 6-month-old ARC mice, fixed in 10% neutral-buffered formalin, and embedded in paraffin, and 8-µm parasagittal sections were cut onto poly-L-lysinecoated glass slides (Lomb Scientific; Sydney, Australia). The sections were blocked with normal goat serum for 30 min or preimmune serum as a negative control. Primary antibody was incubated at the same dilution as that used for immunofluorescence staining for 2 hr, followed by secondary antibody for 1 hr at 1/1000 (donkey anti-sheep; goat anti-rabbit, or goat anti-mouse conjugated to alkaline phosphatase; Jackson ImmunoResearch). Immunoreactivity was visualized by nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Invitrogen, La Jolla, CA) for 4060 min. Slides were counterstained using nuclear fast red for 1 min prior to dehydration. A number of serial sections were analyzed, and the different antibodies were incubated and developed concurrently. General tissue morphology was visualized by hematoxylin and eosin stain. Phase-contrast images were captured using a Spot II cooled CCD digital camera (Diagnostic Instruments Inc.; Sterling Heights, MI) mounted on an Olympus Bx50 microscope (Olympus Australia Pty, Ltd, Melbourne, Australia).
Tropomyosin Expression Constructs
The pPROEX HT prokaryotic expression system (Gibco BRL, Invitrogen; Melbourne, Australia) was used for the production of recombinant Tm isoform proteins. The Tm cDNAs (Tm2, 3, 5a, 5b, and NM1 in pGEX plasmids originally supplied by D. Helfman and Tm1, Tm4, TmBr1, Br2, Br3, and Tm5NM2) were PCR amplified and cloned into the pPROEX HT vector using restriction enzymes engineered into the PCR primers. All Tm constructs were verified by sequencing. Induction of recombinant pPROEX HT clones was done according to the protocols outlined by Gibco BRL, Invitrogen; Melbourne, Australia. In brief, DH5 strain of Escherichia coli carrying the Tm constructs was cultured in the presence of 100 µg/ml of ampicillin to a density of A590 of 0.51.0. To induce expression of the recombinant Tm proteins, isopropyl-ß-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.6 mM. A 1-ml aliquot of cells was removed after 2 hr induction.
Preparation of Recombinant Tm Isoforms
IPTG-induced bacterial cells were pelleted, resuspended in lysis buffer (50 mM Tris, pH 8, 1 mM EDTA, 100 mM NaCl), and sonicated. The suspension was centrifuged to remove cell debris. The supernatant was heated at 95C for 10 min and cooled on ice. The denatured proteins were removed by centrifugation, and the remaining supernatant was enriched for recombinant Tm proteins. Protein concentration was estimated using a BCA protein detection kit (Pierce).
Two-dimensional Gel Electrophoresis
Human acute lymphoblastic leukemia CCRF-CEM cell pellets were resuspended in lysis buffer (7 M urea, 2 M thiourea, 2% CHAPS, 1% sulfobetaine 310, 1% amidosulfobetaine-14, 2 mM tributyl phosphine, 65 mM dithiothreitol, 1% carrier ampholyte 310, 1% carrier ampholyte 46, 0.01% bromophenol blue) as previously described (Verrills et al. 2003). Narrow-range immobilized pH gradient strips, pH 4.55.5 (Pharmacia; Uppsala, Sweden) were rehydrated in 500 µl lysis buffer, cup-loaded with 100 µg of total cellular protein, and isoelectrically focused before separation in the second dimension as previously described in detail (Verrills et al. 2003
). Proteins were either transferred onto membranes and probed with the corresponding antibodies, ß-actin,
-actin, and C4 (total actin antibody), or stained with SYPRO Ruby.
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Results |
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Ten Tm antibodies have been generated and are listed in Table 1. Figure 1 shows the name of the antibody below the exon where the epitope is found. To confirm the specificity of the antibodies, recombinant Tm proteins were used. As described in Materials and Methods, the cDNA of each corresponding Tm isoform was cloned into the pPROEX HT prokaryotic expression system. Expression of the recombinant Tm proteins was induced, and the total bacterial lysate was heated at 95C for 10 min to enrich for the recombinant Tm proteins.
The mouse monoclonal TM311 antibody detects the amino terminal exon 1a of the Tmfast, ßTm, and
Tm genes; hence, it detects recombinant Tm1, 2, 3, and Br-1 (Figure 2A) but none of the products with an exon 1b N terminus. The sheep
/9d antibody was raised against a peptide corresponding to the entire exon 9d of the
Tmfast gene (Figure 1). Consequently, the antibody detects Tm1, 2, 3, 5a, and 5b and exhibits no cross-reactivity with exon 9d containing Tm isoform from the
(Tm4) gene but very low cross-reactivity with the isoforms from the
(Tm5NM1, Tm5NM2) Tm gene (Figure 2B). The rabbit WS
/9c antibody raised against a peptide corresponding to exon 9c of the
Tmfast gene only detects TmBr-1 and TmBr-3 (Figure 2C). The rabbit WD4/9d antibody raised against 18 amino acids of exon 9d of the
Tm gene preferentially detects Tm4 but exhibits cross-reactivity with Tm1 from the ßTm gene, Tm2 and 3 from the
Tmfast, and very weak reactivity with Tm5NM1 and Tm5NM2 (Figure 3D). The epitope for the mouse monoclonal CG3 antibody has been mapped to exon 1b amino acids 2944 of the
Tm gene (Novy et al. 1993
), and it detects all products from this gene including Tm5NM1 and NM2 (Figure 2E). The sheep
/9d antibody raised against a peptide corresponding to the entire exon 9d of the
Tm gene was found to detect both Tm5NM1 and NM2 (Figure 2G). The two bands seen correspond to the his-tagged bacterial products. The
/9d antibody was also found to show slight cross-reactivity with the other exon 9d containing isoforms from both the
Tmfast and ßTm gene, including Tm1, 2, 3, 5a, and 5b. Due to the high degree of homology among exon 9d of the
and
Tm genes, the
/9d and
/9d antibodies recognize the same set of Tm isoforms but with different avidity. Finally, the sheep
/9c antibody raised against exon 9c of the
Tm gene was found to show slight cross-reactivity with the other exon 9c containing isoforms from the
Tmfast, TmBr-1, and TmBr-3, 34, and 30 kDa, respectively (Figure 2F). However, it does detect the appropriate product in brain extracts of 30 kDa (Vrhovski et al. 2003
).
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The WS/9c antibody detected the brain-specific isoforms TmBr-1 and Br-3 only in the brain, with apparent molecular mass of 34 and 30 kDa, respectively (Figure 3C).
The /2a antibody was raised against a peptide corresponding to the unique exon 2a of the
Tmfast gene, not found in any other Tm isoform. It detects
-smooth Tm isoform (apparent molecular mass, 40 kDa) expressed at high levels in the stomach but also seen in the spleen and, to a lesser extent, in the lung (Figure 3D). In Figure 4, a sample of stomach was probed with the
/9d,
/2a, and TM311 antibodies. The
/2a antibody unambiguously recognizes Tm6, also known as
-smooth Tm, (40 kDa) in the stomach but not Tm1 seen with both the
/9d and TM311 antibodies. The Tm6 band was found to be heat stable (data not shown). This does not absolutely prove that there is only
-smooth Tm in the Tm6 band, but it does unambiguously show that the
-smooth Tm migrates at the position of Tm6. A complete characterization of the
/2a antibody is published separately (Vrhovski et al. 2005
) because this is the first time that such an antibody has become available. The conclusion reached from the Vrhovski et al. data is that both Western blots and immunohistochemistry on human and mouse lung with the
-smooth Tm antibody (
/2a) showed an identical profile and tissue colocalization with
-smooth actin.
The antibody specific to Tm4, WD4/9d, detects Tm4 (30 kDa) in all the mouse tissues examined except for the sample of quadriceps muscle, where Tm4 was undetectable at this sensitivity (Figure 5A). A higher-molecular-mass band expressed in lung, spleen, stomach, and embryonic fibroblasts was also detected. This band is proposed to be either Tm1, inasmuch as a similar pattern is seen with the /9d antibody that also detects Tm1, (Figure 3A) or an unknown isoform. To confirm the identity of this 38-kDa band, protein extracts from a Tm1 stably transfected cell line (Yager et al. 2003
) was probed with the WD4/9d and
/9d antibodies. The WD4/9d antibody failed to detect the 38-kDa Tm1 band (Figure 5B) seen with the
/9d antibody in the Tm1-expressing cells (Figure 5C).
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The /9d antibody detects a significant level of a 30-kDa protein in most tissues, with even quadriceps muscle showing a very faint band, seen in a longer exposure (not shown) (Figure 6D). In addition, a higher-molecular-mass band is also detected, 38 kDa, in lung, spleen, and stomach (Figure 6D). Interestingly, the high-molecular-mass bands detected with the
/9a,
/9c, and
/9d antibodies are not seen by the CG3 antibody. This observation means that these isoforms lack exon 1b; however, they may contain exons 1a or 2b of the
Tm gene. Due to the slight cross-reactivity of the
/9d antibody with Tm1 (Figure 2G) and the relatively high levels of expression of Tm1 in these tissues as seen with the
/9d antibody (Figure 3B), it is also possible that this 38-kDa band is Tm1.
Expression Profile of Actin Isoforms in Mouse Tissues
The non-muscle actin isoforms ß- and -actin differ by four amino acids within the first 10 coding amino acids. Both the ß- and
-actin antibodies have been generated with peptides that span these 10 amino acids. The specificity of the
-actin antibody was confirmed by Western blotting of two-dimensional SDS polyacrylamide gels (Figure 7) and mass spectrometry as previously reported (Verrills et al. 2003
). Both the ß-actin and the
-actin antibodies are specific for the corresponding actin isoforms.
In the different mouse tissues, -actin was found to be the most widely expressed non-muscle isoform as compared with ß-actin (Figures 8A and 8B). The presence of ß-actin was undetected in the muscle samples like heart, quadriceps muscle, and stomach under these conditions (Figure 8A). In contrast, detectable levels of
-actin were seen in all tissues examined. The stomach sample showed numerous lower-molecular-mass bands (Figure 8B). These bands were also detected with the
-smooth-muscle actin antibody and may correspond to degraded actin (Figure 8C).
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Sorting of Tm Isoforms in Mouse Spleen
The 10 non-muscle Tm antibodies were tested on a range of paraffin wax sections of different mouse tissues. Previously published data have shown that these Tm antibodies have been successfully used to identify distinct cellular compartments, by immunohistochemistry, on a range of different tissues, including mouse kidney (Ashworth et al. 2004), mouse skeletal muscle (Kee et al. 2004
), mouse brain (Vrhovski et al. 2003
), human colon (Percival et al. 2000
), and rat duodenum (Dalby-Payne et al. 2003
). In this study, we report observations made in the spleen with four of the Tm antibodies and two non-muscle actin antibodies. The mouse spleen consists of three distinctive components: the trabecule, white pulp, and red pulp. The trabecule includes mainly fibrous tissues, the white pulp consists of lymphocytes, and the red pulp contains mainly red blood cells and macrophages. If different Tm isoforms are sorted to different cell types, the localization of these isoforms in the spleen should be different. The WD/49d and
/9d were the most widely distributed and gave the strongest staining in all three components of the spleen (Figures 9B and 9D). In contrast, the
/9d and
/9a antibodies were mainly enriched in only one component, the trabecule (Figures 9A and 9C). The ß-actin antibody was found to stain the red and white pulp but not the trabecule, whereas the
-actin antibody was found in all three components (Figures 9E and 9F). In conclusion, the distribution of Tm isoform expression between the different cell populations in the spleen is not equivalent; in particular, products of the
and ß Tm genes containing exon 9d are restricted, as are exon 9a containing products from the
Tm gene.
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These results demonstrate that different Tm isoforms can potentially sort to three different compartments within mouse primary fibroblasts, stress fibers, cell periphery/ruffling membranes, and the perinuclear region.
In summary, our bank of tropomyosin antibodies allows both the quantitation and visualization of different actin filament populations in mammalian cells, tissues, and organs.
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Discussion |
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In this study, a cohort of 10 Tm isoform-specific antibodies were fully characterized and tested on Western blotting, immunohistochemistry, and immunofluorescence staining of cells in culture. This is the first comprehensive study that clearly demonstrates that different normal mouse tissues express a very restricted repertoire of Tm isoforms (Table 2). The expression of different Tm isoforms may therefore cater for the specific functions of actin filaments within different cells. Immunohistochemical distribution of these Tm isoforms was studied in different mouse tissues, and different isoforms were found to sort to distinct regions. At the subcellular level, these Tm isoforms were found to be sorted to specific compartments within embryonic primary fibroblast cells in culture (Figure 10), including stress fibers, ruffling membranes, and the actin cytoskeleton present in the perinuclear region. Consequently, this degree of resolving power allows one to detect fundamentally distinct populations of actin filaments as opposed to visualizing and quantitating the entire actin cytoskeleton.
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It is therefore envisaged that these Tm antibodies could potentially be used as diagnostic markers of tissue dysfunction. Indeed, modifications in the expression and/or subcellular localization of specific non-muscle Tm isoforms are slowly emerging as markers of human disease. For example, alterations in the expression of specific Tm isoforms have been shown in transformed rat fibroblasts cell lines and hence have implicated Tm as suppressors of transformation (Pittenger et al. 1994). Further studies in a range of human cancers have indicated a predominant decrease in the expression of high-molecular-mass Tm isoforms, Tm1, 2, and 3. These include human breast cancer, prostate cancer, and gastric cancer (Franzen et al. 1996
,1997
; Wang et al. 1996
; Jung et al. 2000
; Raval et al. 2003
). In addition, human transitional cell carcinoma of the urinary bladder showed not only a marked reduction in the expression of Tm1 and Tm2 but also changes in the expression of isoform(s) from the
Tm gene (Pawlak et al. 2004
). Similarly, in colon cancer, aberrant expression of Tm isoform (s) derived from the
Tm gene has also been reported (Lin et al. 2002
).
Other human defects known to have altered Tm expression include ulcerative colitis, in the pathogenesis of which an autoimmune response toward hTm5 is thought to be involved (Biancone et al. 1998,2003
). And during the induction of renal ischemia-induced cell injury, breakdown of the apical microvilli in the kidney proximal tubule cells occurs, which is in turn correlated with the dissociation of specific Tm isoforms from microfilaments present in these microvilli (Ashworth et al. 2004
). Finally, Tm expression has also been linked to essential hypertension. Altered expression of Tm isoforms derived from both the
and
Tm genes in blood cell membranes of a subgroup of patients implicate these isoforms in the pathogenesis of this disease (Dunn et al. 2003
). Consequently, this cohort of Tm antibodies could potentially be used as candidate markers for early detection and prognostic indication of a wide range of pathophysiological defects.
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Acknowledgments |
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Footnotes |
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