Article |
Address correspondence to Edna C. Hardeman, Muscle Development Unit, Children's Medical Research Institute, Locked Bag 23, Wentworthville, New South Wales 2145, Australia. Tel.: 61-2-9687-2800. Fax: 61-2-9687-2120. email: ehardeman{at}cmri.usyd.edu.au
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: tropomyosin; muscles; muscular dystrophies; transgenic mice; sarcomeres
Abbreviations used in this paper: EDL, extensor digitorum longus; EOM, extraocular muscles; H&E, hematoxylin and eosin; MHC, myosin heavy chain; NS, nonsarcomeric; Tm, tropomyosin.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The actin filament is a double-stranded helical polymer of actin, the majority of which contain a tropomyosin polymer running along the helical groove (Flicker et al., 1982). Whereas actin is a globular protein that exists as monomer and polymer, tropomyosin is a rodlike head-to-tail dimer that is only known to exist in association with actin (Flicker et al., 1982; Matsumura et al., 1983). Both components, actin and tropomyosin, have been shown to contribute to the physical properties of the microfilament (Kojima et al., 1994).
Actin and tropomyosin are encoded by multigene families and tropomyosin is also subject to extensive alternative splicing (Herman, 1993; Pittenger et al., 1994). Genetic manipulation has demonstrated that these isoforms are not redundant but rather encode different functional information. Cardiac -actin is essential for normal cardiac function (Kumar et al., 1997), ß-actin is required for cell spreading and motility (Schevzov et al., 1992; Kislauskis et al., 1997), and
-smooth muscle actin controls the contractility of myofibroblasts (Ronnov-Jessen and Petersen, 1996). Elevated expression of
-actin disrupts stress-like organization in myoblasts (Schevzov et al., 1992) and sarcomere organization in cardiomyoctes (von Arx et al., 1995). Similarly, the Tm5NM-1 tropomyosin is required for melanoma cell motility (Miyado et al., 1996),
-fast tropomyosin is required for normal cardiac function (Thierfelder et al., 1994; Bottinelli et al., 1998; Muthuchamy et al., 1999), and the Tm1 and Tm2 tropomyosins are required to restore normal microfilament organization to cancer cells (Prasad et al., 1993; Boyd et al., 1995; Gimona et al., 1996). A specific tropomyosin isoform is required for correct mRNA targeting in Drosophila (Erdelyi et al., 1995) and the
-TM gene is essential for embryonic development and embryonic stem cell viability (Hook et al., 2004).
The tropomyosins also show a variety of isoform-specific protein properties. The strength of binding to actin differs between tropomyosin isoforms although the original observation of tighter binding of the larger sized tropomyosins (Matsumura and Yamashiro-Matsumura, 1985) does not hold for some specific smaller tropomyosins (Pittenger et al., 1995). Tropomyosin isoforms also differentially protect actin filaments from severing by gelsolin (Ishikawa et al., 1989a, b) and regulate both myosin motor mechanochemistry (Fanning et al., 1994) and the sorting of myosin motors (Bryce et al., 2003). The azimuthal position assumed by tropomyosin on an actin filament also differs between isoforms and is additionally influenced by the actin isoform (Lehman et al., 2000). Therefore, it is clear that the properties of actin filaments are likely to differ depending on both the actin and tropomyosin isoform composition of the filament.
The extensive sorting of tropomyosin and actin isoforms to different intracellular locations provides two significant advantages to the cell (Gunning et al., 1998a,b). On the one hand, it allows the cell to independently control the supply of microfilament building blocks to different cellular sites. On the other hand, it provides a mechanism to regulate the functional properties of microfilaments at these sites (Weinberger et al., 1996; Schevzov et al., 1997; Hannan et al., 1998; Percival et al., 2000). The majority of these observations concerning tropomyosins have been made in neurons both in vivo and in vitro (Gunning et al., 1998b), in fibroblasts (Lin et al., 1988), synchronized NIH3T3 cells (Percival et al., 2000, 2004), epithelial cells (Temm-Grove et al., 1998; Dalby-Payne et al., 2003), and Golgi-derived vesicle fractions from rat liver (Heimann et al., 1999). Actin isoform sorting has also been observed in skeletal muscle (Prasad et al., 1993; Rybakova et al., 2000), smooth muscle (North et al., 1994), and neurons (Weinberger et al., 1996). Thus, the combination of isoform sorting and functional differences between isoforms provides a potentially powerful mechanism to segregate and independently regulate the myriad functions of actin filaments.
Isoform sorting of actins in skeletal muscle suggests the existence of a number of separate actin filament systems. One system provides the thin filament component of the sarcomere, which interdigitates with the myosin containing thick filaments. The thin filaments, also known as sarcomeric actin filaments, are composed of specific striated muscle -actins and tropomyosins. A second filament system has been detected with a
-actin antibody. Staining for
-actin reveals its presence associated with costameres (Craig and Pardo, 1983; Rybakova et al., 2000), mitochondria (Pardo et al., 1983), and the Z-line (Nakata et al., 2001). This suggests the possibility of a
-actin containing filament system that connects the myofibrils to the costameres.
We demonstrated previously that muscle differentiation is accompanied by down-regulation of nonsarcomeric (NS) tropomyosins and induction of muscle isoforms (Gunning et al., 1990). It was noted, however, that some specific nonmuscle tropomyosins persist in adult muscle. We have used our repertoire of tropomyosin antibodies to characterize these isoforms in different adult skeletal muscles of the mouse. Two spatially distinct populations of tropomyosin-associated microfilaments are described: one in the sarcomeric compartment and the other at the myofiber periphery. Two tropomyosin isoforms, one (Tm5NM1) found in all muscles and a second novel product from the -gene (Tm5NM-34kd) found in a specific subset of muscles, localized adjacent to the Z-line in longitudinal sections. Further characterization of Tm localization in cross sections revealed that these two tropomyosins reside in separate filament systems. Tm5NM1 is particularly concentrated at the myofiber periphery and at lower amounts within the myofiber, presumably located between myofibrils. An anti
-actin antibody colocalized with this nonmuscle Tm at the periphery and within the fiber indicating that a
-actin forms the backbone of this filament system. The novel Tm isoform (Tm5NM-34kd) is located exclusively within the myofibers and in a separate filament system which does not colocalize with
-actin. We have incorporated an inappropriate Tm into the Z-line adjacent structure by overexpressing a high molecular weight NS Tm, Tm3, that is not normally present in muscle. This results in phenotypes in these mice that are characteristic of muscular dystrophy and ragged-red fibers (subsarcolemmal accumulation of mitochondria).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
Examination of Western blots probed with the 9d,
9d, and CG3 antibodies (Fig. 7 A) shows that expression of Tm3 in skeletal muscle has little effect on the expression of many other NS Tms (e.g., Tm1, Tm2, and Tm6). The only exception appears to be increased expression of the novel Tm5NM-34kd CG3 isoform in the flexor digitorum profundus muscle (Fig. 7 A).
The ectopic Tm3 protein localized to the same Z-line adjacent region as the endogenous -TM gene isoforms (Tm5NM1 and Tm5NM-34kd; Fig. 8). Very strong staining either side of the Z-line can be seen in soleus longitudinal sections with the
9d antibody (Fig. 8, A and C). Ectopic Tm3 was mainly restricted to the cytoplasmic region of the myofiber (Fig. 8, G, K, and L). This staining is specific for ectopic Tm3 as although this antibody detects a number of different endogenous isoforms in the soleus muscle (Fig. 7 A) none of these are present in the cytoplasmic sarcomeric regions of the muscle (Fig. 8, D and J). In longitudinal and cross sections, ectopic Tm3 colocalized with the
-actin antibody (Fig. 8, I and N, respectively). This is consistent with an association of Tm3 with a Z-line adjacent
-actin cytoskeleton.
|
|
The dystrophic features were associated with muscle weakness that is shown by the inability of the Tm3 mice to extend their limbs when held by the tail (compare WT and Tm3/66 mice in Fig. 9, K and L, respectively). This type of muscle dysfunction is similar to that observed in SJL mice, a natural mouse model for limb girdle muscular dystrophy 2B (Bittner et al., 1999). The Tm3 mice also have altered gait when running on a treadmill; they run with their hindlimbs closer to the body and with an arched back. Further evidence of dystrophy in the Tm3 mice is the raised levels of serum creatine kinase (1300 ± 150 U/L [mean ± SEM] and 300 ± 50 U/L for Tm3 [n = 5] and WT [n = 4] mice, respectively; P = 0.023, ANOVA). Elevated plasma creatine kinase is a characteristic feature of muscular dystrophies and is thought to be an indicator of loss of sarcolemma integrity. Therefore, we conclude that incorporation of Tm3 into the novel Z-lineassociated actinTm filament network leads to mice with features of muscular dystrophy and ragged-red fiber myopathy.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previously, we found that during muscle differentiation there is a switch in Tm expression from NS to sarcomeric isoforms (Gunning et al., 1990). However, expression of a number of NS isoforms, particularly from the - and
-TM genes, persisted in adult muscle. The more detailed analysis shown here using isoform specific antibodies demonstrates that the level of NS Tm expression is highly muscle specific. In addition to the known cytoskeletal Tm (Tm5NM1) that was expressed in all muscles, skeletal muscle also expresses a novel 34-kD product recognized by CG3 that was abundant in a very restricted set of muscles, the EOM, the diaphragm, and the soleus muscle. There is no known EST corresponding to the novel
-TM 34-kD product, although we have previously reported a novel, slow muscle specific transcript from this gene (Dufour et al., 1998b). The carboxy terminus of this product has not been characterized but may correspond to this novel 34-kD product.
The novel Tm5NM-34-kD protein, a product of the -TM gene, is abundant in slow fibers, but appears not to be restricted to slow fibers because it is abundant in muscles that contain few slow fibers (EOM and diaphragm). This unusual pattern of expression suggests this isoform has some specialized function in specific myofibers in contrast to Tm5NM1, which is expressed in all muscle fibers. The muscles that contain high amounts of the novel Tm are characterized by chronic or frequent contractions. This suggests that Tm5NM-34kd may be associated with a structure unique to these fibers.
Tropomyosin isoforms define a novel Z-lineassociated element in skeletal muscle
Using antibodies to -TM gene products (
9d and CG3) and an antibody to a
-actin, a novel structural compartment in muscle has been identified. This compartment is present at a very restricted area adjacent to the Z-line. Analysis of muscle transverse sections suggests that within this compartment there are at least two separate structures located in the intermyofibrillar space. One, labeled by
9d, containing Tm5NM1, appears to be associated with a
-actin filament network, and the other, defined by CG3 (Tm5NM-34kd), appears to be part of a separate filament system. The localization of Tm5NM1 and Tm5NM-34kd in the intermyofibrillar space aligned adjacent to the Z-line suggests there are filament networks that laterally interconnects the Z-line adjacent region of individual myofibrils (Fig. 10 A). This system would be analogous to the desmin intermediate filament network that laterally interlinks the Z-disks of each myofibril (Capetanaki, 2002).
|
There is increasing evidence that many signaling molecules are present in a region adjacent to the Z-line in skeletal muscle (Ervasti, 2003). Chisel, STARS, Arpp, myopalladin, enigma, FHL3, and myopodin locate to this region in skeletal muscle (Guy et al., 1999; Bang et al., 2001; Palmer et al., 2001; Arai et al., 2002; Tsukamoto et al., 2002; Coghill et al., 2003). The colocalization of these molecules suggests that there must be a scaffold that provides the structure to organize these molecules. This provides evidence for a structural network of signaling molecules that links the sarcomere to costameres and the ECM. The NS Tmactin filament network described in the present investigation may be such a scaffold structure.
Incorporation of Tm3 into Z-LAC results in muscular dystrophy and ragged-red fibers
Transgenic mice that express Tm3 in their skeletal muscle display two significant pathologies. Because the Tm3 is specifically targeted to the Z-lineassociated cytoskeleton, we propose that it is the inclusion of Tm3 into this structure which directly leads to the pathologies. The pathologies and localization were observed in two independent transgenic lines indicating that these pathologies are not due to transgene integration-site effects. The impact of Tm3 does not appear to be due to competing the endogenous Tms out of this structure because CG3 and 9d staining is unchanged (unpublished data) and the levels of Tm5NM1 and Tm5NM-34kd are unchanged. This suggests that the
-actin filaments that presumably provide the backbone for these microfilaments are not saturated with tropomyosin and can accommodate the ectopic expression of Tm3. This may also account for the specific sorting of Tm3 to this site because these
-actin filaments may be the major source of microfilaments lacking tropomyosin in skeletal muscle.
Mutations in a growing number of costameric proteins and proteins that interact with the costamere have been identified as causes of muscular dystrophies (Ervasti, 2003). The best-known example is dystrophin itself, which leads to dystrophies of differing severity depending on the specific mutations (Dalkilic and Kunkel, 2003). Recently, proteins that interact with the Z-line have also been shown to cause muscular dystrophies when mutated or absent (e.g., telethonin, calpain 3; Richard et al., 1995; Moreira et al., 2000). This suggests that it is a defect in the overall function of this complex that is responsible for the majority of dystrophies. The muscles of Tm3 transgenic mice display a phenotype characteristic of muscular dystrophy. Because Tm3 specifically localizes to the Z-line adjacent microfilaments, the data suggest that it is dysfunction of these filaments resulting from inappropriately targeted Tm3 that is leading to the dystrophic phenotype. Furthermore, this provides evidence that this microfilament network is an important structural element that directly or indirectly links the sarcomere to the sarcolemma, and that mutations in this structure may account for some currently unknown causes of muscular dystrophy.
The appearance of ragged-red fibers in the Tm3 transgenic lines suggests that the NS Tm cytoskeletal structures may influence mitochondrial function. It is notable that the desmin knockout mouse displays a similar mitochondria phenotype (Milner et al., 2000). Mitochondria are normally located beneath the sarcolemma and also near the Z-line between myofibrils. Previous studies have shown an association of -actin with subsarcolemmal mitochondria (Pardo et al., 1983; Nakata et al., 2001) and the present results are consistent with NS Tms (Tm5NM1) associated with these microfilaments. It is possible that the
-actin Z-lineassociated cytoskeleton and desmin play a role in anchoring mitochondria at this site. Disruption of this anchoring may leads to mitochondrial disruption on the one hand and migration to the cell periphery on the other.
In conclusion, the novel NS microfilament structure, together with desmin, may provide a physical link between the myofibrils, the costamere and the ECM. Alterations in the function of this novel structure may lead to muscular dystrophy and/or ragged-red fibers. The tropomyosin components of this Z-lineassociated cytoskeleton should now be included as candidate genes in searches for mutations that cause these diseases.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunohistochemistry
Standard cryosectioning.
Immediately after dissection, mouse muscles were coated in tissue freezing medium (Tissue-Tek O.C.T., ProSciTech), frozen in melting isopentane prechilled in liquid nitrogen and stored at 80°C. Muscles were sectioned in a cryostat microtome (HM500OM; Carl Zeiss MicroImaging, Inc.) at 7 µm, fixed in cold 2% PFA, and washed in PBS.
Semi-thin sectioning.
Greater resolution of Z-lineassociated structures was achieved with semi-thin (0.51.0 µm) longitudinal sections of maximally stretched muscle (50% greater than the resting muscle length). Immediately after dissection, the muscle was stretched and fixed in 4% formaldehyde for 30 min at 4°C and processed for cryoultramicrotomy as described by Griffiths et al. (1984) with modification. In brief, after fixation, muscles were cut into small strips (45-mm long x 1-mm wide) and transferred to 1.8 M sucrose/17% polyvinylpyrrolidone for overnight infusion. Muscle strips were trimmed further, mounted on cryopins, and snap-frozen in liquid nitrogen. Semi-thin (0.51.0 µm) sections were cut at 60°C using an Ultracut UCT ultramicrotome (Leica) equipped with an EM FCS cryochamber (Leica). Sections were suspended in 2.3 M sucrose, allowed to thaw, and placed on poly-O-lysine coated slides (Starfrost).
Immunostaining.
Both thick (7 µm) and semi-thin (0.51.0 µm) sections were blocked overnight (4°C) in PBS Triton X-100 (0.05%) containing 0.2% fish gelatin and 2% BSA. The primary antibodies were applied for 6090 min at RT, slides were washed with PBS, and secondary antibody was applied (6090 min at RT). Double staining was performed by applying primary/secondary antibody pairs sequentially, i.e., the first primary and its secondary and then the second primary and its secondary. After washing to remove unbound secondary antibody, slides were mounted with Vectashield (Vector Laboratories) and viewed using a confocal laser scanning microscope (model TCS SP2; Leica) with an oil immersion 63x objective and analyzed with confocal software (Leica).
Antibodies
The polyclonal antibodies 9d and
9d were directed against the exon 9d of the
- and
-TM genes, respectively. The
9d antibody is a polyclonal sheep antibody and detects isoforms Tm5NM1 and Tm5NM2 (Percival et al., 2004; Fig. 1 C), whereas
9d (originally WS
/9d) is a polyclonal antibody that detects Tm2, Tm3, Tm5a, Tm5b, and Tm6 from the
-TM gene and Tm1 from the ß-TM gene (Schevzov et al., 1997; Fig. 1, A and B). Either a polyclonal rabbit or sheep
9d antibody was used depending on the species of the costaining antibody. CG3 is a mouse monoclonal that recognizes exon 1b of the
-TM gene and detects all NS products (Fig. 1 C; Lin et al., 1988). The affinity purified
-actin antibody is a polyclonal sheep antibody raised against the first 14 aa of cytoplasmic
-actin (EEEIAALVIDNGSG). The commercially available mouse monoclonal 311 Tm antibody (Sigma-Aldrich), which recognizes Tms containing exon 1a sequence from the
-, ß-, and
-TM genes, was used. The
-actinin-2 antibody was a rabbit polyclonal antiserum was provided by A. Beggs (Children's Hospital, Boston, MA; North and Beggs, 1996). A rabbit polyclonal antibody recognizing the rod domain of dystrophin (Dys6-10) was provided by L. Kunkel (Children's Hospital). Slow fibers were detected with mAbs to the slow MHC isoform (undiluted BA-F8; Sigma-Aldrich) obtained from supernatants of hybridoma cultures (Borrione et al., 1988). Double staining with TRITC-labeled phalloidin (Sigma-Aldrich) was performed after the application of the primary antibody.
The following commercially available secondary antibodies were used: donkey antimouse HRP (Amersham Biosciences); donkey antirabbit HRP (Amersham Biosciences); donkey antisheep HRP (Jackson ImmunoResearch Laboratories); goat antirabbit FITC-labeled IgG (Sigma-Aldrich); goat antimouse Cy3 (Jackson ImmunoResearch Laboratories); and goat antirabbit Cy3 (Jackson ImmunoResearch Laboratories). The following Alexa-conjugated antibodies were also used (Molecular Probes): donkey antimouse IgG (L + H) Alexa 488; donkey antisheep IgG Alexa 488 and 555; goat antimouse IgM Alexa 488; goat antimouse IgG Alexa 488; and goat antirabbit IgG Alexa 488 and 555. All secondary antibodies were used at a 1:1,000 dilution.
Histopathological analysis
Mouse muscles were frozen as described above for immunohistochemistry. Sections were placed on poly-L-lysine precoated glass microscope slides, air dried, and stained with H&E or the modified Gomori-Trichrome method (Engel and Cunningham, 1963).
EM
Muscles were removed immediately after euthanasia and cut into very thin slices while immersed in modified Karnovski's fixative (2.5% glutaraldehyde/4% PFA in 1 M cacodylate buffer, pH 7.4). Samples were further fixed overnight in the same fixative and fixed after with 2% osmium tetroxide, dehydrated through an ascending series of ethanol, and embedded in Spurr's epoxy resin. Ultrathin sections were cut with a Reichert-Jung Ultracut ultramicrotome, double contrasted with uranyl acetate and lead citrate, and viewed and photographed with a BioTwin transmission electron microscope (model CM120; Philips).
Western blot analysis
Skeletal muscle tissue was homogenized in 20 vol of 10 mM Tris, pH 7.6/2% SDS/20 mM DTT by boiling followed by crushing with a pestle in a microtube. The samples were then solubilized by gentle sonication, boiled again, and spun at 12,000 rpm to remove insoluble particles. An equal volume of SDS-PAGE sample buffer (4% SDS, 12% glycerol, 2% mercaptoethanol, 0.01% Coomassie G-250, 50 mM Tris-Cl, pH 6.8) was added and the sample stored at 20°C. Equal volumes of protein (10 µg) were analyzed on 15% SDS-PAGE (with low 0.9% bis-acrylamide) gels with reference to a standard known amount of brain protein previously determined using a BCA protein assay kit (Pierce Chemical Co.) and volumes adjusted where necessary. Coomassie-stained gels were used to verify equal loading. Protein was transferred onto PVDF membranes (Millipore) at 80 V at 4°C. Blots were blocked in 5% skim milk at 4°C, washed in TBS, and incubated with the antibodies for 2 h at RT followed by three washes with TBS 0.05% Tween 20. HRP-labeled secondary antibody was added at 1:10,000 dilution for 1 h at RT. Excess antibody was removed with 4 x 20 min washes. Detection was performed using the Western lightning chemiluminescence detection system (PerkinElmer) on Biomax X-ray film (Kodak) for varying times (260 min).
Serum creatine kinase measurement
Blood was taken from 6-mo-old anesthetized (halothane) WT and Tm3 mice by cardiac puncture. Serum creatine kinase was measured using a commercial kit and a serum quality control sample (Trace/DMA; Thermo Electron).
![]() |
Acknowledgments |
---|
This work was supported by National Health and Medical Research Council (NHMRC) grants awarded to E.C. Hardeman and P.W. Gunning. P.W. Gunning is a Principal Research Fellow of the NHMRC.
Submitted: 30 June 2004
Accepted: 19 July 2004
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arai, A., J.A. Spencer, and E.N. Olson. 2002. STARS, a striated muscle activator of Rho signaling and serum response factor-dependent transcription. J. Biol. Chem. 277:2445324459.
Bang, M.L., R.E. Mudry, A.S. McElhinny, K. Trombitas, A.J. Geach, R. Yamasaki, H. Sorimachi, H. Granzier, C.C. Gregorio, and S. Labeit. 2001. Myopalladin, a novel 145-kilodalton sarcomeric protein with multiple roles in Z-disc and I-band protein assemblies. J. Cell Biol. 153:413427.
Banker, B.Q., and A.G. Engel. 1994. Basic Reactions of Muscle. Myology. A.G. Engel and Franzini-Armstrong C., editors. McGraw-Hill, Inc., New York. 832888.
Bittner, R.E., L.V. Anderson, E. Burkhardt, R. Bashir, E. Vafiadaki, S. Ivanova, T. Raffelsberger, I. Maerk, H. Hoger, M. Jung, et al. 1999. Dysferlin deletion in SJL mice (SJL-Dysf) defines a natural model for limb girdle muscular dystrophy 2B. Nat. Genet. 23:141142.[CrossRef][Medline]
Borrione, A.C., A.M. Zanellato, L. Saggin, M. Mazzoli, G. Azzarello, and S. Sartore. 1988. Neonatal myosin heavy chains are not expressed in Ni-induced rat rhabdomyosarcoma. Differentiation. 38:4959.[Medline]
Bottinelli, R., D.A. Coviello, C.S. Redwood, M.A. Pellegrino, B.J. Maron, P. Spirito, H. Watkins, and C. Reggiani. 1998. A mutant tropomyosin that causes hypertrophic cardiomyopathy is expressed in vivo and associated with an increased calcium sensitivity. Circ. Res. 82:106115.
Boyd, J., J.I. Risinger, R.W. Wiseman, B.A. Merrick, J.K. Selkirk, and J.C. Barrett. 1995. Regulation of microfilament organization and anchorage-independent growth by tropomyosin 1. Proc. Natl. Acad. Sci. USA. 92:1153411538.[Abstract]
Bryce, N.S., G. Schevzov, V. Ferguson, J.M. Percival, J.J. Lin, F. Matsumura, J.R. Bamburg, P.L. Jeffrey, E.C. Hardeman, P. Gunning, and R.P. Weinberger. 2003. Specification of actin filament function and molecular composition by tropomyosin isoforms. Mol. Biol. Cell. 14:10021016.
Capetanaki, Y. 2002. Desmin cytoskeleton: a potential regulator of muscle mitochondrial behavior and function. Trends Cardiovasc. Med. 12:339348.[CrossRef][Medline]
Clark, K.A., A.S. McElhinny, M.C. Beckerle, and C.C. Gregorio. 2002. Striated muscle cytoarchitecture: an intricate web of form and function. Annu. Rev. Cell Dev. Biol. 18:637706.[CrossRef][Medline]
Coghill, I.D., S. Brown, D.L. Cottle, M.J. McGrath, P.A. Robinson, H.H. Nandurkar, J.M. Dyson, and C.A. Mitchell. 2003. FHL3 is an actin-binding protein that regulates alpha-actinin-mediated actin bundling: FHL3 localizes to actin stress fibers and enhances cell spreading and stress fiber disassembly. J. Biol. Chem. 278:2413924152.
Craig, S.W., and J.V. Pardo. 1983. Gamma actin, spectrin, and intermediate filament proteins colocalize with vinculin at costameres, myofibril-to-sarcolemma attachment sites. Cell Motil. 3:449462.[Medline]
Dalby-Payne, J.R., E.V. O'Loughlin, and P. Gunning. 2003. Polarization of specific tropomyosin isoforms in gastrointestinal epithelial cells and their impact on CFTR at the apical surface. Mol. Biol. Cell. 14:43654375.
Dalkilic, I., and L.M. Kunkel. 2003. Muscular dystrophies: genes to pathogenesis. Curr. Opin. Genet. Dev. 13:231238.[CrossRef][Medline]
Dufour, C., R.P. Weinberger, and P. Gunning. 1998a. Tropomyosin isoform diversity and neuronal morphogenesis. Immunol. Cell Biol. 76:424429.[CrossRef][Medline]
Dufour, C., R.P. Weinberger, G. Schevzov, P.L. Jeffrey, and P. Gunning. 1998b. Splicing of two internal and four carboxyl-terminal alternative exons in nonmuscle tropomyosin 5 pre-mRNA is independently regulated during development. J. Biol. Chem. 273:1854718555.
Engel, W.K., and G.G. Cunningham. 1963. Rapid examination of muscle tissue. An improved trichrome method for fresh-frozen biopsy sections. Neurology. 13:919923.
Erdelyi, M., A.M. Michon, A. Guichet, J.B. Glotzer, and A. Ephrussi. 1995. Requirement for Drosophila cytoplasmic tropomyosin in oskar mRNA localization. Nature. 377:524527.[CrossRef][Medline]
Ervasti, J.M. 2003. Costameres: the Achilles' heel of Herculean muscle. J. Biol. Chem. 278:1359113594.
Fanning, A.S., J.S. Wolenski, M.S. Mooseker, and J.G. Izant. 1994. Differential regulation of skeletal muscle myosin-II and brush border myosin-I enzymology and mechanochemistry by bacterially produced tropomyosin isoforms. Cell Motil. Cytoskeleton. 29:2945.[Medline]
Flicker, P.F., G.N. Phillips Jr., and C. Cohen. 1982. Troponin and its interactions with tropomyosin. An electron microscope study. J. Mol. Biol. 162:495501.[Medline]
Gimona, M., J.A. Kazzaz, and D.M. Helfman. 1996. Forced expression of tropomyosin 2 or 3 in v-Ki-ras-transformed fibroblasts results in distinct phenotypic effects. Proc. Natl. Acad. Sci. USA. 93:96189623.
Griffiths, G., A. McDowall, R. Back, and J. Dubochet. 1984. On the preparation of cryosection for immunocytochemistry. J. Ultrastruct. Res. 89:6578.[Medline]
Gunning, P., M. Gordon, R. Wade, R. Gahlmann, C.S. Lin, and E. Hardeman. 1990. Differential control of tropomyosin mRNA levels during myogenesis suggests the existence of an isoform competition-autoregulatory compensation control mechanism. Dev. Biol. 138:443453.[Medline]
Gunning, P., E. Hardeman, P. Jeffrey, and R. Weinberger. 1998a. Creating intracellular structural domains: spatial segregation of actin and tropomyosin isoforms in neurons. Bioessays. 20:892900.[CrossRef][Medline]
Gunning, P., R. Weinberger, P. Jeffrey, and E. Hardeman. 1998b. Isoform sorting and the creation of intracellular compartments. Annu. Rev. Cell Dev. Biol. 14:339372.[CrossRef][Medline]
Guy, P.M., D.A. Kenny, and G.N. Gill. 1999. The PDZ domain of the LIM protein enigma binds to beta-tropomyosin. Mol. Biol. Cell. 10:19731984.
Hall, Z.W., B.W. Lubit, and J.H. Schwartz. 1981. Cytoplasmic actin in postsynaptic structures at the neuromuscular junction. J. Cell Biol. 90:789792.[Abstract]
Hannan, A.J., P. Gunning, P.L. Jeffrey, and R.P. Weinberger. 1998. Structural compartments within neurons: developmentally regulated organization of microfilament isoform mRNA and protein. Mol. Cell. Neurosci. 11:289304.[CrossRef][Medline]
Heimann, K., J.M. Percival, R. Weinberger, P. Gunning, and J.L. Stow. 1999. Specific isoforms of actin-binding proteins on distinct populations of Golgi-derived vesicles. J. Biol. Chem. 274:1074310750.
Herman, I.M. 1993. Actin isoforms. Curr. Opin. Cell Biol. 5:4855.[Medline]
Hogan, B., R. Beddington, F. Costantini, and E. Lacy. 1994. Manipulating the Mouse Embryo. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 332 pp.
Hook, J., F. Lemckert, H. Qin, G. Schevzov, and P. Gunning. 2004. Gamma tropomyosin gene products are required for embryonic development. Mol. Cell. Biol. 24:23182323.
Ishikawa, R., S. Yamashiro, and F. Matsumura. 1989a. Annealing of gelsolin-severed actin fragments by tropomyosin in the presence of Ca2+. Potentiation of the annealing process by caldesmon. J. Biol. Chem. 264:1676416770.
Ishikawa, R., S. Yamashiro, and F. Matsumura. 1989b. Differential modulation of actin-severing activity of gelsolin by multiple isoforms of cultured rat cell tropomyosin. Potentiation of protective ability of tropomyosins by 83-kDa nonmuscle caldesmon. J. Biol. Chem. 264:74907497.
Kislauskis, E.H., X. Zhu, and R.H. Singer. 1997. ß-Actin messenger RNA localization and protein synthesis augment cell motility. J. Cell Biol. 136:12631270.
Kojima, H., A. Ishijima, and T. Yanagida. 1994. Direct measurement of stiffness of single actin filaments with and without tropomyosin by in vitro nanomanipulation. Proc. Natl. Acad. Sci. USA. 91:1296212966.
Kumar, A., K. Crawford, L. Close, M. Madison, J. Lorenz, T. Doetschman, S. Pawlowski, J. Duffy, J. Neumann, J. Robbins, et al. 1997. Rescue of cardiac alpha-actin-deficient mice by enteric smooth muscle gamma-actin. Proc. Natl. Acad. Sci. USA. 94:44064411.
Lehman, W., V. Hatch, V. Korman, M. Rosol, L. Thomas, R. Maytum, M.A. Geeves, J.E. Van Eyk, L.S. Tobacman, and R. Craig. 2000. Tropomyosin and actin isoforms modulate the localization of tropomyosin strands on actin filaments. J. Mol. Biol. 302:593606.[CrossRef][Medline]
Lin, J.J., T.E. Hegmann, and J.L. Lin. 1988. Differential localization of tropomyosin isoforms in cultured nonmuscle cells. J. Cell Biol. 107:563572.[Abstract]
Lin, J.J., K.S. Warren, D.D. Wamboldt, T. Wang, and J.L. Lin. 1997. Tropomyosin isoforms in nonmuscle cells. Int. Rev. Cytol. 170:138.[Medline]
Lubit, B.W. 1984. Association of beta-cytoplasmic actin with high concentrations of acetylcholine receptor (AChR) in normal and anti-AChR-treated primary rat muscle cultures. J. Histochem. Cytochem. 32:973981.[Abstract]
Matsumura, F., and S. Yamashiro-Matsumura. 1985. Purification and characterization of multiple isoforms of tropomyosin from rat cultured cells. J. Biol. Chem. 260:1385113859.
Matsumura, F., S. Yamashiro-Matsumura, and J.J. Lin. 1983. Isolation and characterization of tropomyosin-containing microfilaments from cultured cells. J. Biol. Chem. 258:66366644.
Milner, D.J., M. Mavroidis, N. Weisleder, and Y. Capetanaki. 2000. Desmin cytoskeleton linked to muscle mitochondrial distribution and respiratory function. J. Cell Biol. 150:12831298.
Miyado, K., M. Kimura, and S. Taniguchi. 1996. Decreased expression of a single tropomyosin isoform, TM5/TM30nm, results in reduction in motility of highly metastatic B16-F10 mouse melanoma cells. Biochem. Biophys. Res. Commun. 225:427435.[CrossRef][Medline]
Moreira, E.S., T.J. Wiltshire, G. Faulkner, A. Nilforoushan, M. Vainzof, O.T. Suzuki, G. Valle, R. Reeves, M. Zatz, M.R. Passos-Bueno, and D.E. Jenne. 2000. Limb-girdle muscular dystrophy type 2G is caused by mutations in the gene encoding the sarcomeric protein telethonin. Nat. Genet. 24:163166.[CrossRef][Medline]
Muthuchamy, M., K. Pieples, P. Rethinasamy, B. Hoit, I.L. Grupp, G.P. Boivin, B. Wolska, C. Evans, R.J. Solaro, and D.F. Wieczorek. 1999. Mouse model of a familial hypertrophic cardiomyopathy mutation in alpha-tropomyosin manifests cardiac dysfunction. Circ. Res. 85:4756.
Nakata, T., Y. Nishina, and H. Yorifuji. 2001. Cytoplasmic gamma actin as a Z-disc protein. Biochem. Biophys. Res. Commun. 286:156163.[CrossRef][Medline]
North, K.N., and A.H. Beggs. 1996. Deficiency of a skeletal muscle isoform of alpha-actinin (alpha-actinin-3) in merosin-positive congenital muscular dystrophy. Neuromuscul. Disord. 6:229235.[CrossRef][Medline]
North, A.J., M. Gimona, Z. Lando, and J.V. Small. 1994. Actin isoform compartments in chicken gizzard smooth muscle cells. J. Cell Sci. 107:445455.
Pardo, J.V., M.F. Pittenger, and S.W. Craig. 1983. Subcellular sorting of isoactins: selective association of gamma-actin with skeletal muscle mitochondria. Cell. 32:10931103.[CrossRef][Medline]
Palmer, S., N. Groves, A. Schindeler, T. Yeoh, C. Biben, C.C. Wang, D.B. Sparrow, L. Barnett, N.A. Jenkins, N.G. Copeland, et al. 2001. The small muscle-specific protein Csl modifies cell shape and promotes myocyte fusion in an insulin-like growth factor 1-dependent manner. J. Cell Biol. 153:985998.
Percival, J.M., G. Thomas, T.A. Cock, E.M. Gardiner, P.L. Jeffrey, J.J. Lin, R.P. Weinberger, and P. Gunning. 2000. Sorting of tropomyosin isoforms in synchronised NIH 3T3 fibroblasts: evidence for distinct microfilament populations. Cell Motil. Cytoskeleton. 47:189208.[CrossRef][Medline]
Percival, J.M., J.A.I. Hughes, D.L. Brown, G. Schevzov, K. Heimann, B. Vrhovski, N. Bryce, J.L. Stow, and P.W. Gunning. 2004. Targeting of a tropomyosin isoform to short microfilaments associated with the Golgi complex. Mol. Biol. Cell. 15:268280.
Pittenger, M.F., J.A. Kazzaz, and D.M. Helfman. 1994. Functional properties of non-muscle tropomyosin isoforms. Curr. Opin. Cell Biol. 6:96104.[Medline]
Pittenger, M.F., A. Kistler, and D.M. Helfman. 1995. Alternatively spliced exons of the beta tropomyosin gene exhibit different affinities for F-actin and effects with nonmuscle caldesmon. J. Cell Sci. 108:32533265.
Prasad, G.L., R.A. Fuldner, and H.L. Cooper. 1993. Expression of transduced tropomyosin 1 cDNA suppresses neoplastic growth of cells transformed by the ras oncogene. Proc. Natl. Acad. Sci. USA. 90:70397043.[Abstract]
Richard, I., O. Broux, V. Allamand, F. Fougerousse, N. Chiannilkulchai, N. Bourg, L. Brenguier, C. Devaud, P. Pasturaud, C. Roudaut, et al. 1995. Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A. Cell. 81:2740.[Medline]
Ronnov-Jessen, L., and O.W. Petersen. 1996. A function for filamentous -smooth muscle actin: retardation of motility in fibroblasts. J. Cell Biol. 134:6780.[Abstract]
Rybakova, I.N., J.R. Patel, and J.M. Ervasti. 2000. The dystrophin complex forms a mechanically strong link between the sarcolemma and costameric actin. J. Cell Biol. 150:12091214.
Schevzov, G., C. Lloyd, and P. Gunning. 1992. High level expression of transfected ß- and -actin genes differentially impacts on myoblast cytoarchitecture. J. Cell Biol. 117:775785.[Abstract]
Schevzov, G., P. Gunning, P.L. Jeffrey, C. Temm-Grove, D.M. Helfman, J.J. Lin, and R.P. Weinberger. 1997. Tropomyosin localization reveals distinct populations of microfilaments in neurites and growth cones. Mol. Cell. Neurosci. 8:439454.[CrossRef][Medline]
Seward, D.J., J.C. Haney, M.A. Rudnicki, and S.J. Swoap. 2001. bHLH transcription factor MyoD affects myosin heavy chain expression pattern in a muscle-specific fashion. Am. J. Physiol. Cell Physiol. 280:C408C413.
Temm-Grove, C.J., B.M. Jockusch, R.P. Weinberger, G. Schevzov, and D.M. Helfman. 1998. Distinct localizations of tropomyosin isoforms in LLC-PK1 epithelial cells suggests specialized function at cell-cell adhesions. Cell Motil. Cytoskeleton. 40:393407.[CrossRef][Medline]
Thierfelder, L., H. Watkins, C. MacRae, R. Lamas, W. McKenna, H.P. Vosberg, J.G. Seidman, and C.E. Seidman. 1994. Alpha-tropomyosin and cardiac troponin T mutations cause familial hypertrophic cardiomyopathy: a disease of the sarcomere. Cell. 77:701712.[Medline]
Tsukamoto, Y., T. Senda, T. Nakano, C. Nakada, T. Hida, N. Ishiguro, G. Kondo, T. Baba, K. Sato, M. Osaki, et al. 2002. Arpp, a new homolog of carp, is preferentially expressed in type 1 skeletal muscle fibers and is markedly induced by denervation. Lab. Invest. 82:645655.[Medline]
von Arx, P., S. Bantle, T. Soldati, and J.C. Perriard. 1995. Dominant negative effect of cytoplasmic actin isoproteins on cardiomyocyte cytoarchitecture and function. J. Cell Biol. 131:17591773.[Abstract]
Weinberger, R., G. Schevzov, P. Jeffrey, K. Gordon, M. Hill, and P. Gunning. 1996. The molecular composition of neuronal microfilaments is spatially and temporally regulated. J. Neurosci. 16:238252.[Abstract]
Wieczorek, D.F., M. Periasamy, G.S. Butler-Browne, R.G. Whalen, and B. Nadal-Ginard. 1985. Co-expression of multiple myosin heavy chain genes, in addition to a tissue-specific one, in extraocular musculature. J. Cell Biol. 101:618629.[Abstract]