The generation and evolution of Tm isoforms.
The number of Tm genes varies from one and two in the fungi Schizosaccharomyces pombe
and Saccharomyces cerevisiae
, respectively, four in mammals and six in zebra fish (Danio rerio
lists the genes identified in 6 commonly studied animal model systems together with the chromosome location and the genes found adjacent to the Tm loci. Interestingly, the Tm loci are found adjacent to a few common genes including talin and Rab8a. For the most part the focus of this report will be on mammalian Tm genes and isoforms. A schematic representation of the intronexon organization of the four mammalian genes is shown in . The overall organization of exons and splicing patterns in the mammalian genes is conserved between mammals, avian, amphibians and fish. The genes in simpler species follow similar principles but differ substantially in the details of alternative exons and their evolutionary relationship to the specific genes in vertebrates.27
The human Tm genes have been named TPM1, TPM2, TPM3 and TPM4, however, due to the lack of a unified nomenclature for the gene names or the isoforms, alternative names documented in the literature are listed in .
Ensemble identification numbers of the Tm genes from different species, chromosome location and adjacent genes
Figure 1 Schematic representation of the organization of the mammalian Tm genes. Colored boxes represent the protein coding exons numbered 1 to 9 and lines the introns. Unshaded boxes signify untranslated sequences and black boxes are exons common to all the genes (more ...)
Alternative names for mammalian Tms
show, for each gene, the amino acid sequences of each of the exons compared between mammals, birds, amphibians and fish. The consensus sequence across species is shown below each exon. The most striking feature of this comparison is the extraordinary conservation of primary structure across over 500 million years of evolution. This emphasizes that these proteins are under great selective pressure to maintain a precise primary sequence. Selection is unlikely to reflect the structural demands of maintaining the coiled coil because there are few absolute primary sequence requirements necessary to generate a coiled coil.28
Rather, the selection is more likely to reflect isoform specific functional demands1
possibly underpinned by subtle structural features as described recently in reference 29
and/or positioning in the major groove of the actin filament.30,31
Figure 2 Amino acid sequence comparison of the TPM1 gene from six animal species. Amino acid alignment of all exons contained within the TPM1 gene for selected members of chordata. Species included are human (Homo sapiens), mouse (Mus musculus) and rat (Rattus (more ...)
Figure 5 Amino acid sequence comparison of the TPM4 gene from six animal species. Amino acid alignment of all exons contained within the TPM4 gene for selected members of chordata. Species included are human (Homo sapiens), mouse (Mus musculus) and rat (Rattus (more ...)
Sequence diversity between the isoforms results from both sequence divergence between alternative exons in the same gene and between the same exons in different genes (). Comparison of the amino acid sequences of each of the 4 human genes, exon by exon, shows that there is a high degree of sequence similarity between the genes although this varies between exons (). Some exons, like 1a, 2b, 3, 4, 5, 6b, 7, 8 and 9a show a high degree of similarity between the genes whereas exons 1b, 6a, 9c and 9d show substantial differences which would be expected to contribute to isoform divergence (). The greatest source of primary sequence divergence comes from alternative promoter use and alternative splicing. There are two types of N-termini which result from the use of different promoters (). Exon 1a plus either exon 2a or 2b gives rise to the N-terminus of high molecular weight tropomyosins (approximately 284 aa). The alternative N-terminus derives from the use of exon 1b and gives rise to the shorter LMW isoforms (approximately 248 aa). In addition to the obvious length difference between the choices of N-terminus, there is also considerable sequence divergence () between these alternative N-termini. Thus the similarity between genes for the same exons is much greater than it is between alternative exons in the same gene. This trend is also seen for exons 6a vs. 6b and also for the C-terminal exons 9a, 9b, 9c and 9d. In both cases, it is almost impossible to detect any homology between the alternative exons in the same gene (). This divergence has provided an effective strategy for the generation of antibodies which recognize specific subsets of isoforms (see below).
Figure 6 Exon alignment of the human Tm genes. A comparison highlighting the differences in amino acid composition between an alignment of corresponding exons from human TPM1, TPM2, TPM3 and TPM4 genes and an alignment of the exons responsible for generating isoforms; (more ...)
RT-PCR analysis has identified over 40 Tm isoforms that are coded by the mammalian genes.5,32,33
Evidence for the presence of transcripts detected at the level of northern blot analysis or most preferable at the protein level only really exists for half of these isoforms and the majority are listed in . Tm isoforms found in association with the contractile apparatus of skeletal, cardiac and smooth muscle cells are referred to as muscle Tms whereas those associated with the cytoskeleton of all cells are referred to as the cytoskeletal isoforms (often referred to, incorrectly, as non-muscle isoforms in the literature).
TPM1 and TPM2 are also referred to as the αTm and βTm gene respectively34
(). The TPM1 gene has been found by RT-PCR to code for 20 isoforms in rat adult tissues and cultured cells.32
Included among the encoded isoforms is the αTm expressed in striated muscle, the recently discovered TPM1κ in human hearts,35
the isoforms preferentially enriched in the central nervous system, TmBr1, TmBr2 and TmBr3 and Tm2, Tm3, Tm5a and Tm5b. The TPM2 gene codes for the skeletal muscle βTm isoform and, thus far, only one cytoskeletal isoform, Tm1, has been detected. Tm1 has been found to be significantly downregulated in transformed cells and restoring Tm1 expression has been shown to reverse transformation in ras-transformed cells.36
The TPM3 gene codes for the slow-twitch skeletal muscle isoform (αs
Tm) and at least 9 LMW cytoskeletal isoforms referred to as Tm5NM1 to Tm5NM11.33,37–39
Isoforms NM3 and NM6 are identical at the protein level but have unique 3′UTRs, the same is for isoforms NM5 and NM11 (). The TPM4 gene40,41
codes for the cytoskeletal Tm4 isoform and a HMW Tm4 found in many species but not rodents.42
Post translational modifications.
To date post translational modifications of Tms are largely an unexplored area of investigation. The most reported forms of post translational modifications in the literature are NH2-terminal acetylation and phosphorylation although evidence of S-nitrosylation and disulfide-crosslinked Tm has been documented.
Acetylation has been shown to significantly enhanced Tm affinity for actin. Unacetylated mammalian skeletal Tm is unable to bind actin in the absence of troponin43,44
and unacetylated α-smooth muscle Tm has much weaker affinity for actin than the acetylated form.45
In addition, acetylation has been shown to increase the end-to-end interactions between Tm dimers9,44,46
and in Schizosaccharomyces pombe
to significantly enhance the ability of Tm to regulate myosin activity.47
At least in yeast, distinct spatial segregation of acetylated and nonacetylated Tm-containing actin filaments has been observed suggesting the possibility that different forms of Tm can regulate specific myosins.17
Phosphorylation of Tm has been found in both skeletal and cardiac muscle of many different species.48–54
The phosphorylation of Tm has been shown to enhance the ability of Tm to form head-to-tail interactions and promote activation of myosin Mg2+
Phosphorylation of cytoskeletal Tms has also been documented and postulated to be required for the remodelling of the actin cytoskeleton.57,58
Finally, endothelial cells exposed to shear flow were shown to have more than 12 proteins that had significantly increased S-nitrosylation among them Tm at Cys 170 located in the hydrophobic motif.59
The authors suggest that such modification may allow for the remodelling together with maintaining the integrity of the endothelial cells under flow conditions.
Evaluating the specificity of Tm antibodies.
Many of the Tm antibodies commercially available are sold with minimal information regarding the antigen used to raise the antibody and hence the isoform specificity. This greatly limits their use and in some cases leads to confusion in the literature. We previously described the characterization of 10 Tm antibodies.60
Here, we report an additional 9 Tm antibodies. is a comprehensive list of all the Tm antibodies that we have extensively characterized together with the isoform specificity, published references and commercial availability. The majority of the antibodies were generated using peptides corresponding to part or all of a specific exon. There are exceptions where the initial antibodies were made by using whole tissue tropomyosin, for example chicken gizzard, and the epitopes are yet to be identified. shows the name of the antibody below the exon encoding the epitope and the peptide used as immunogen for these antibodies is shown in –
. Comparison of the peptide sequence containing the epitope cross species indicates the likelihood that many of these antibodies will be reactive across many model systems (–
Summary of Tm antibodies, exon and isoform specificity
The initial evaluation on the specificity of the antibodies was performed by protein gel blot analysis on panels of recombinant Tm proteins. The corresponding cDNAs of rat (Tm1, 2, 3, 4, 5a, 5b, Br1, Br2, Br3 and Tm5NM1) and human (Tm1, 2, 3, 4, NM1, NM2, NM4, NM5, NM6, NM7) isoforms were cloned into the pPROEX HT prokaryotic or pET expression system and induction of protein was performed as described in Materials and Methods.
A total of 13 antibodies have been generated that detect Tm isoforms from the TPM1 and 2 genes with three of these Tm antibodies having been generated as both polyclonals and monoclonals. The epitope for the commercially available TM311 antibody has been mapped to exon 1a of the TPM1 gene and thus it detects the striated muscle isoform plus Tms 2, 3, 6, Br1 and Tm1 plus the striated muscle isoform from the TPM2 gene ( and
). Isoforms containing exon 1b (Tm5a, 5b, Br2 and Br3) are detected with the α/1b antibody (). Antibodies with epitopes directed to the C-terminal exon of the αTm gene include the α/9b antibody specific for TmBr2 () and the α/9c (clone #554) which preferentially detects TmBr1 and TmBr3 (). It is notable that α/9c does not see Tm5NM4 which contains exon 9c from the TPM3 gene. The α/9d antibody raised against a peptide directed to the entire exon 9d from the TPM1 gene has a high degree of specificity for Tm1, 2, 3, 5a and 5b and, we predict, Tm6 (). Although exons 9d from the TPM1, 3 and 4 genes have a high degree of similarity, the α/9d antibody exhibits no cross reactivity with either Tm4 from the TPM4 gene nor Tm5NM1 and Tm5NM2 from the TPM3 gene (). The CG1 and CGβ6 antibodies were originally raised against chicken gizzard Tm.61
CG1 has strong reactivity to both rat and human Tm1 and also detects Tm4 (). The epitope for the CGβ6 antibody has been mapped to the C-terminal end of human Tm2 and Tm3 62
and as expected it detects both human and mouse Tm2 and Tm3 and also shows weak reactivity with Tm5a ().
Figure 7 Evaluation of the specificity of TPM1 and TPM2 genes directed antibodies with bacterially produced Tm proteins. Protein gel blot analysis of 0.5 µg recombinant Tms probed with (A) TM311, (B) α/1b, (C) α/9b, (D) α/9c, (E) (more ...)
Peptide sequences corresponding to the alternatively spliced C-terminal exons of the TPM3 gene (exons 9a, 9c and 9d) were used to generate the γ/9a, γ/9c and γ/9d antibodies (). The characterization of these antibodies raised in sheep has been previously documented in reference 60
. Here we report on the complementary antibodies generated using the identical peptide sequences but raised as mouse monoclonals. These three antibodies show the predicted isoform specificity (). The γ/9d antibody does exhibit minor cross reactivity with Tm1, 2, 3, 5a and 5b (). The LC1 antibody is shown to have a high degree of specificity for the human isoforms Tm5NM1, NM2, NM4 and 7 with no cross reactivity to the mouse Tm5NM1 (). Although the epitope for this antibody is yet to be mapped our data and that reported by Sung et al. strongly indicate that it resides in the first few N-terminal end amino acids of the human exon 1b from the TPM3 gene. Amino acid sequence alignment demonstrates that the only amino acid difference between the mouse/rat and human sequences is the fourth amino acid, isoleucine in human and serine in mouse and rat (). We expect that this residue must be in the LC1 epitope. The CG3 antibody with its epitope mapped to exon 1b amino acids 29–44 64
detects all the cytoskeletal products from this gene both human and mouse (). It has also been found to detect the striated muscle isoform from this gene via cross reaction with the TPM3 exons 1a or 2b.65
Figure 8 Evaluation of the specificity of TPM3 gene directed monoclonal antibodies with bacterially produced Tm proteins. Protein gel blot analysis of 0.5µg recombinant Tms probed with (A) γ/9a, (B) γ/9c, (C) γ/9d, (D) LC1 and (E) (more ...)
Figure 4 Amino acid sequence comparison of the TPM3 gene from six animal species. Amino acid alignment of all exons contained within the TPM3 gene for selected members of chordata. Species included are human (Homo sapiens), mouse (Mus musculus) and rat (Rattus (more ...)
The antibodies known to specifically detect the cytoskeletal Tm4 isoform from the TPM4 gene are δ/1b, WD4/9d and LC24. The δ/1b antibody was raised against the last 19 amino acids of exon 1b from the TPM4 gene. This antibody is shown to detect both rat and human Tm4 and shows no cross reactivity to other exon 1b containing isoforms from the other Tm genes (Tm5a, 5b, Br2, Br3, NM1, NM2, NM4, NM7) (). It does however, show weak reactivity with human Tm1. The WD4/9d generated in rabbit using a partial amino acid sequence from exon 9d of the TPM4 gene has been previously characterized.60
It detects Tm4 but does cross react with Tm1 from the βTm gene (on a longer exposure, data not shown), and also Tm5NM1 and Tm5NM2 from the γTm gene (). The LC24 was generated against bacterially produced human Tm4 with only the C-terminal end used.62
This antibody detects both rat and human Tm4 but also cross reacts with Tm1 from the TPM2 gene ().
Evaluation of the specificity of TPM4 gene directed antibodies with bacterially produced Tm proteins. Protein gel blot analysis of 0.5 µg recombinant Tms probed with (A) δ/1b, (B) WD4/9d and (C) LC24 antibodies.
The use of Tm antibodies to identify the Tm isoform expression profiles in cell lines and mouse tissues.
The selective repertoire of Tm isoform expression, including the number and type of isoforms expressed in different cell types is strictly regulated by as yet not fully understood mechanisms. Tm isoform expression profiles in different cell types have previously been reviewed in reference 6
. Here we report the use of the Tm antibodies described above to profile isoform expression in three different cell types from three different mammalian species, human, mouse and rat and further evaluate the specificity of these tools. On a SDS PAGE gel the HMW Tm isoforms have an apparent molecular weight of 34 to 40 kDa and the LMW isoforms of 28 to 33 kDa. Migration of Tms in SDS gels is not predictable based on MW and in our experience, optimal resolution of Tm isoforms is best achieved on SDS-PAGE gels with a low concentration of bisacrylamide (12.5% acrylamide and 0.1% bisacrylamide).66
The antibodies known to detect isoforms from the TPM1 and TPM2 genes including TM311, α/9d, 1 and CGβ6 all perform well on total protein lysates isolated from cell lines (). The initial observation made on the protein gel blots is the differences in the profiles of isoforms and the quantitative amounts expressed by the three cell lines. Both the human and mouse cell lines express Tm1 as detected with the TM311, α/9d and CG1 antibody (, C and D
). The rat B35 cells show no evidence of Tm1 or Tm3 expression whereas Tm2 and Tm3 are detected in both the human and mouse cells and only Tm2 in the rat B35 cells ( and E
). The α/1b antibody detects very low levels of Tm5a and Tm5b but there is also reactivity with unknown bands (). Antibodies directed to Tm isoforms enriched in brain tissue (TmBr1, Br2 and Br3) and smooth muscle (Tm6) were as predicted absent from these cell lines (data not shown).
Figure 10 Expression of Tm isoforms coded by the TPM1 and TPM2 genes in three cell lines. Equal loading (20 µg) of total cellular protein isolated from human primary fibroblasts, mouse primary fibroblasts and rat neuroblastoma B35 cells, was electrophoresed (more ...)
All the TPM3 antibodies tested on the cell lines were found to detect the expected cytoskeletal LMW isoforms coded by the gene (). The abundance of isoforms detected with the γ/9a antibody was found to be low as a very long exposure time was used to generate the blot shown in . Some additional unknown bands were also detected. The γ/9d antibody detects the predicted 30 kDa corresponding to Tm5NM1/NM2 in all the cell lines (). As expected the LC1 antibody, like CG3, detects a band in all the human cell lines as its epitope is in exon 1b, but CG3 also sees the same band in mouse and rat unlike LC1 which is human specific (). The exon 9c containing isoforms identified with the γ/9c antibody were found to be undetected by protein gel blotting (data not shown). Exon 9c containing isoforms are primarily detected in central nervous system cells.67,68
Figure 11 Expression of Tm isoforms coded by the TPM3 gene in three cell lines. Equal loading (20 µg) of total cellular protein isolated from human primary fibroblasts, mouse primary fibroblasts and rat neuroblastoma B35 cells, was electrophoresed on 12.5% (more ...)
Analysis of expression of the cytoskeletal Tm4 isoform with the δ/1b, WD4/9d and LC24 antibodies is shown in . The δ/1b is very specific to Tm4 (). In contrast, the WD4/9d detects in addition to Tm4 a HMW isoform predicted to be Tm1 () which is relatively abundant in the human and mouse cells, as seen with the TM311 and α/9d antibodies ( and C). Surprisingly, the LC24 antibody detects Tm4 in human and rat cells but not in the mouse fibroblasts. Further confirmation of the failure of this antibody to detect the mouse cytoskeletal Tm4 isoform is demonstrated in other mouse cells later in this report.
Figure 12 Expression of Tm isoforms coded by the TPM4 gene in three cell lines. Equal loading (20 µg) of total cellular protein isolated from human primary fibroblasts, mouse primary fibroblasts and rat neuroblastoma B35 cells, was electrophoresed on 12.5% (more ...)
Total protein lysates were generated from both mouse and rat tissues and probed with the Tm antibodies described above. Even though individual tissues are comprised of many different cell types the expression of the major Tm isoforms expressed can be evaluated by protein gel blot analysis. The sheep α/1b antibody detects a prominent band of about 30 kDa in brain, liver, lung, kidney and spleen (). Since the isoforms detected by this antibody all have similar or the same apparent molecular weights, it is difficult to distinguish whether they correspond to Tm5a, 5b or Br3. Comparison of the Tm expression profile generated with the use of the α/9d antibody shows that Tm5a/5b are most abundant in kidney, liver, lung and spleen whereas these isoforms are undetected in the adult brain at the level of sensitivity of protein gel blotting (). Hence, we can deduce that the α/1b antibody is detecting TmBr3 in the brain. TmBr3 is one of the major isoforms expressed in mature neurons.69–71
Figure 13 Expression profile of Tm isoforms coded by the TPM1 and 2 genes in a panel of mouse tissues. Equal loading (20 µg) of total cellular protein isolated from different mouse and rat tissues was electrophoresed on 12.5% SDS gels and individual protein (more ...)
The α/2a monoclonal antibody is raised against a peptide that corresponds to the unique exon 2a found only in the TPM1 gene. Exon 2a containing isoforms are the Tmsmα also known as Tm6 in addition to hTmsmα-1 and hTmskα1-1 identified in human tissues.42
The Tmsmα (Tm6) and hTmsmα-1 are enriched in smooth muscle such as placenta, uterus42 and stomach (). The expression of the exon 2a containing isoform(s) is highly enriched in mouse stomach (). Note also the slower mobility of the exon 2a containing isoform in skeletal muscle; possibly Tmskα1-1 ( and
The monoclonal antibody α/9d was raised against a peptide corresponding to the entire exon 9d of the TPM1 gene with predicted recognition of isoforms Tm1, 2, 3, 5a, 5b and 6. On a panel of mouse tissues, the expression of the HMW isoforms Tm1/6 are most highly enriched in the kidney, lung, spleen and stomach (). Significant levels of Tm2 are detected in kidney, spleen and stomach and on a longer blot exposure (data not shown) Tm2 is also detected in lung. Expression of Tm3 was limited to the stomach sample. The LMW isoforms Tm5a/5b were enriched in the kidney, lung and spleen and to a lesser extent in the brain, liver and stomach, seen only on a longer exposure (data not shown).
The CG1 antibody, shown in to detect Tm1 but also cross-reacts with Tm4, detects a prominent HMW band in skeletal muscle most likely to correspond to βTm (). In the kidney, lung and spleen the prominent HMW band most likely corresponds to Tm1 as Tm1 detected with the α/9d antibody is highly expressed in these tissues ( compared to C). The brain and heart also show a faint HMW band and both tissues also express Tm1 but at relatively lower levels ( compared to C). A LMW band is also detected in the kidney, lung and spleen (). This may correspond to Tm4, as later shown, these tissues express relative high levels of Tm4 detected with the two antibodies ( and C).
Figure 15 Expression profile of Tm isoforms coded by the TPM4 gene in a part of mouse and rat tissues. Equal loading (20 µg) of total cellular protein isolated from different mouse (A–D) and rat (E) tissues was electrophoresed on 12.5% SDS gels (more ...)
There are four antibodies designed to identify Tm isoforms from the TPM3 gene. The γ/9a, γ/9c, γ/9d mouse monoclonal antibodies detect isoforms containing C-terminal exons 9a, 9c and 9d respectively. The CG3 antibody detects all the exon 1b containing isoforms. In the heart and skeletal muscle the γ/9a antibody detects a prominent band of 36 kDa corresponding to the exon 9a containing muscle isoforms from the TPM1, 2 and 3 genes with liver, lung and stomach expressing relatively lower levels of HMW isoform(s) (). In the brain, lung, spleen and stomach the LMW, 30 kDa, cytoskeletal exon 9a containing isoforms, may correspond to either Tm5NM5/11 or NM6/3. Stomach shows a doublet at 30 kDa which may correspond to NM8 and/or 9. Tm5NM8 and 9 result from splicing of exon 9a to 9c and result in an extension of 5 additional amino acids derived from exon 9c.33
Attempts to generate an antibody to this sequence have thus far been unsuccessful. The γ/9c antibody detects a 30 kDa band in brain, either NM4 or 7 and cross reacts with an unknown HMW band in muscle and stomach (). The γ/9d antibody that preferentially reacts with NM1 and NM2 detects a 30 kDa band in brain, kidney, liver, lung and spleen () and on a longer exposure NM1/NM2 can also be detected in the heart, skeletal muscle and stomach (data not shown). The CG3 antibody detects all the LMW cytoskeletal exon 1b containing isoforms from the TPM3 gene and a 30 kDa band is seen in all the mouse tissues () except in the gastrocnemius muscle used in this panel, even on a longer exposure (data not shown). However, the CG3 antibody does detect a 34 kDa band in extraocular and soleus, corresponding to the αs
Tm from the TPM3 gene.65
Figure 14 Expression profile of Tm isoforms coded by the TPM3 gene in a panel of mouse tissues. Equal loading (20 µg) of total cellular protein isolated from different mouse tissues was electrophoresed on 12.5% SDS gels and individual protein gel blots (more ...)
The TPM4 gene together with the TPM2 gene are the least alternatively spliced genes, each known to code for only two isoforms. We have generated two antibodies raised against peptides within the N-terminal end, exon 1b and the C-terminal end, exon 9d. On a panel of mouse tissues, the LMW Tm4 isoform, identified with the δ/1b antibody, is abundantly expressed in kidney, lung and spleen (), on a longer blot exposure a band can been seen in muscle and stomach (data not shown). There is however the presence of a slightly higher MW band seen at significant levels in the liver. In order to confirm that this unknown band corresponds to Tm, we took advantage of the fact that Tm is a heat stable protein. Following the enrichment of Tm by heating the tissues to 95°C for 10 min (see Materials and Methods) we found that the HMW bands seen in kidney and liver were now absent (). We conclude that δ/1b is detecting non-Tm proteins in mouse tissues. WD4/9d predominantly detects the LMW Tm4 isoform in all tissues examined even in brain and muscle on a longer exposure (not shown) (). The LC24 was shown to detect both the human and rat Tm1 and Tm4 recombinant proteins () however it failed to detect the Tm4 in mouse protein lysates (). Similarly, in the case of the mouse tissues the LC24 does not detect the LMW cytoskeletal Tm4 isoform (). However, a HMW band is seen which in mouse we predict to be βTm whereas in liver, lung and spleen it may correspond to Tm1 (). Surprisingly, in the rat tissues the LC24 antibody detects primarily Tm4 and a HMW isoform in muscle ().
Some of the Tm antibodies described above have been successfully used to perform immunoprecipitations. These include LC1 and WSα/9c,16
anti-sheep α/2a and CG1 72
In summary, one of the major limitations in evaluating the Tm isoform composition in either cell lines or tissues is the similarity in the apparent molecular weights when 1-D gel electrophoresis is performed. 2-D electrophoresis may allow for the identification of additional LMW isoforms as some display distinct pI values (). In some cases quantitative PCR will be the best methodology to assess the presence and quantity of different Tm isoforms, albeit at the mRNA level.
Theoretical isoelectric focusing points
Tm isoforms mark distinct actin filament populations.
Distinct intracellular subcellular sorting of different Tm isoforms has to date been documented in various cell model systems both in vitro and in vivo (reviewed in refs. 1
). This observed sorting of Tm isoforms implies the existence of distinct populations of actin filaments, marked by Tm isoforms, influencing the dynamics and organization properties of the filaments.1
In order to further evaluate the performance of the Tm antibodies described above, immunofluorescence staining was performed on two cell lines, normal and tumor cells, mouse primary embryonic fibroblasts and the neuroblastoma B35 cells, respectively. The choice of cells is based on the fact that these cells (1) display in the case of the mouse primary fibroblasts, a well organized actin cytoskeleton,60
(2) exhibit specific structures such as ruffling membrane and filopodial, (3) according to –
express various Tm isoforms and (4) represent two commonly used cell types. At the level of resolution of an epifluorescence microscope, a clear localization of Tm to actin stress fibers was far more evident in the normal mouse primary embryonic fibroblasts than in the neuroblastoma B35 cells (compare – to –
). Subtle differences among the Tm antibodies consist primarily on whether the staining for the Tms extends to the periphery of the cell and the intensity of staining, reflecting the levels of expression. Comparison of the staining patterns of the TM311 (Tm1, 2, 3, 6, Br1) with the α/1b (Tm5a, 5b, Br2, Br3) antibodies demonstrates the diminished staining of TM311 at the periphery of the cell whereas the α/1b extends and is enriched at the cell periphery, seen in both the mouse fibroblasts ( compared to L
, arrows) and rat B35 cells ( compared to L
, arrows). This observed staining strongly suggests that Tm5a/5b are peripherally enriched isoforms and this correlates with the apical enrichment of these isoforms in epithelial cells.74
Similarly, the α/9d antibody which detects the HMW Tms, 1, 2, 3, 6 and the LMW Tm5a, 5b is also seen to extend to the cell periphery in mouse fibroblasts (, arrow) and B35 cells ( and arrow) although not as dramatically as for the α/1b antibody. The CG1 (Tm1) antibody detects prominent stress fibers in the mouse fibroblasts ( and N
, arrow) and staining is absent in the B35 cells (). The CGβ6 (Tm2, 3) antibody is seen to detect stress fibers with a striated appearance most prominent in the B35 cells ( and
, see inset). Both the γ/9d (Tm5NM1, NM2) and CG3 (all TPM3 cytoskeletal products) antibodies detect stress fibers and no enrichment of staining is seen at the cell periphery (, F and and F
, arrows). The WD4/9d antibody identifies stress fibers best seen in the mouse fibroblasts as compared to the B35 cells ( compared to 21E
) and staining is reduced at the cell periphery, most apparent in the mouse fibroblasts (, arrow). Finally, the LC24 antibody exhibits stress fiber staining in the human immortal fibroblasts and B35 cells ( and
) while no staining was detected in the mouse fibroblast cells (data not shown). There is a significant difference in the pattern of LC24 staining seen between the human immortal cells and the B35 cells, with a more even intensity of staining seen in the immortal cells while in the B35 cells the staining is significantly diminished at the cell periphery ( compare to 21F
Figure 16 Immunofluorescence staining of mouse primary embryonic fibroblasts with antibodies directed to Tm isoforms coded by the TPM1 and TPM2 genes. (A–E) Tms in green, (F–J) γactin in red and (K–O) merge images. Nuclei were visualized (more ...)
Figure 18 Immunofluorescence staining of fibroblasts with antibodies directed to Tm isoforms coded by the TPM4 gene. (A and B) Tms in green, (C and D) γactin in red and (E and F) merge images. Nuclei were visualized with DAPI. (A, C and E) correspond to (more ...)
Figure 19 Immunofluorescence staining of B35 cells with antibodies directed to Tm isoforms coded by the TPM1 and TPM2 genes. (A–E) Tms in green, (F–J) γactin in red and (K–o) merge images. Nuclei were visualized with DAPI. Arrows (more ...)
Figure 21 Immunofluorescence staining of B35 cells with antibodies directed to Tm isoforms coded by the TPM4 gene. (A and B) Tm in green, (C and D) γactin in red and (E and F) merge images. Nuclei were visualized with DAPI. Arrows in (E and F) denote the (more ...)
Figure 17 Immunofluorescence staining of mouse primary embryonic fibroblasts with antibodies directed to Tm isoforms coded by the TPM3 gene. (A and B) Tms in green, (C and D) γactin in red and (E and F) merge images. Nuclei were visualized with DAPI. Arrows (more ...)
Figure 20 Immunofluorescence staining of B35 cells with antibodies directed to Tm isoforms coded by the TPM3 gene. (A and B) Tms in green, (C and D) γactin in red and (E and F) merge images. Nuclei were visualized with DAPI. Arrows in (E and F) denote the (more ...)
In conclusion, at the level of resolution of an epifluorescence microscope the major differences among the various Tm antibody staining patterns was their enrichment at the cell periphery. In addition, subtle differences among the different cell types, normal and immortal fibroblasts, were observed.
Subcellular localization of individually tagged Tm isoforms.
One of the major drawbacks of using the Tm antibodies to evaluate the subcellular localization of Tms is that the majority of antibodies do detect several isoforms. In order to investigate the intracellular localization of individual Tm isoforms, specific isoforms have been cloned into plasmids containing YFP or GFP (yellow or green fluorescence protein) or hemagglutinin (HA) tags (). Surprisingly, the addition of a 27 kDa YFP or GFP protein to the N-terminal end appears not to interfere with the head-to-tail association of the Tm dimers, as these tagged proteins are quite capable of forming polymer like structures that colocalize with actin filaments.75
In addition, YFP.Tm5NM1 has been shown to have a direct role in cell migration as its expression in Tm5NM1 null cells rescued the phenotype further demonstrating the functionality of tagged Tms.76
The concern encountered with transfection studies is the fidelity of sorting, as overexpression of these tagged isoforms may lead to the saturation of pools and hence inappropriate subcellular localization. However, recent studies in U2OS cells have shown remarkable fidelity of sorting of these tagged Tms.21
Expression of fluorescently tagged Tm isoforms to study intracellular localization
Overexpression and targeted gene siRNA knockdown in vitro based methods to study Tm isoform specific function.
In order to evaluate the biological function of the different Tm isoforms, overexpression () and siRNA knockdown () methodology has been employed. The overexpression studies have shed light on the importance of Tm isoforms in defining the organizational properties and molecular composition of actin filaments ultimately influencing the overall morphology of cells.16,21,77–80
In addition, overexpression studies complemented with targeted gene siRNA knockdown experiments have demonstrated the importance of specific Tm isoforms in regulating the structure and dynamic properties of focal adhesions.76,81
Finally, recent siRNA knockdown experiments have played an important role in establishing the nonredundant roles of Tm isoforms in stress fiber assembly.21
Summary of genetically modified cell lines
Summary of published knock-down siRNA sequences
The development of mouse models to study tropomyosin.
The primary objective for the generation of Tm gene modified mice has been to study the disease process of various human myopathies. Mutations in both the TPM2 and TPM3 genes are associated with diseases of skeletal muscle such as nemaline myopathy, distal arthrogryposis and most recently “cap” disease. TPM1 gene mutations have been linked to familial hypertrophic cardiomyopathy and dilated cardiomyopathy (reviewed in refs. 82–84
). The current mouse models that recapitulated these human myopathies are listed in . It is noteworthy to mention that although the Tm mutations linked to human diseases are known to exhibit clinical abnormalities mostly restricted to the heart and skeletal muscle, these mutations are present in exons shared by numerous other Tm isoforms (see and
All known cardiomyopathies carry mutations in Tm isoforms specific to the central nervous system including TmBr1, TmBr2 and TmBr3. In the skeletal muscle myopathies, mutations in the TPM3 gene occur in exons 3, 5 and 8 which are common to all the Tm isoforms (Tm5NM1-11) encoded by this gene. Hence, it is possible that these patients may also display uncharacterized pathologies in organs other than skeletal muscle or the heart. Unfortunately, in the case of the mouse models, it is not possible to address the impact that these Tm mutations may have in other organs since the altered Tms are expressed in a tissue-specific manner. The human skeletal muscle actin or the cardiac specific α-myosin heavy chain promoters were used to generate these transgenic mice.
Tm mouse models that recapitulate human myopathies
Various other genetically modified mouse models have been generated primarily to dissect the functional differences among the isoforms and are listed in . The knockout mouse models reveal that the Tm genes are essential for life as deletion of the TPM1, 2 and 3 genes are embryonic lethal and offer further evidence for the non-redundant nature of the mammalian Tm genes.84,86–89
To date, two transgenic mouse models exist that have overexpression of the cytoskeletal Tm isoforms, Tm3 and Tm5NM1, in major organs as the transgene is driven by the human β-actin promoter.90
The Tm3 transgenic mice demonstrate dystrophic features perhaps due to increase sensitivity to contractile-induced stress.65,91
In the case of the Tm5NM1/NM2 knockout mice, lack of these isoforms leads to T-tubule dysmorphology and altered skeletal muscle function.92
Both the Tm3 and Tm5NM1 transgenics together with the complementary Tm5NM1 knockout mice have become valuable resources for the isolation of primary embryonic cells such as neuronal and fibroblast cells. Primary cortical neurons isolated from these mice have revealed the importance of Tm isoforms in the development and maintenance of neuronal morphogenesis, previously only inferred from subcellular localization studies.16,80,93
Tm mouse models generated to study the functional differences among the isoforms