Two genetically independent Chlamydomonas mutants, ocal and oca2, that display abnormal cell division were isolated by DNA insertional mutagenesis. The culture of these mutants contained large abnormally-shaped cells with multiple pairs of flagella. DAPI staining showed that those aberrant cells carried the same number of nuclei as that of flagella pairs. Time-lapse video microscopy revealed the following characteristics of the cell division process in the mutants: i) although the mutants, like wild-type cells, had a potential to divide into eight daughter cells by successive three rounds of division cycle, they frequently failed to cleave and formed a fused cell in the course of division; ii) a cell often grew into an extremely large cell with many pairs of flagella; iii) the large cell was suddenly divided into a number of daughter cells by simultaneously forming multiple cleavage furrows; iv) alternatively, an extremely large cell stopped dividing although many cleavage furrows were formed on its surface. These observations suggest that these mutants are partially deficient in the progression of furrowing, and that Chlamydomonas is capable of undergoing cytokinesis between many pairs of nuclei simultaneously, as in the cellularization process of insect eggs.

View full abstract

When a clone of Chinese hamster ovary (CHO) cells transfected with a plasmid containing a luciferase reporter gene under the control of the human heat shock protein (hsp) 70 gene promoter was treated with cycloheximide during heat exposure at 42 and 43°C for 15 to 100 minutes and then incubated at 37°C after removal of cycloheximide, reporter gene expression was suppressed by the protein synthesis inhibitor only at small heat shock doses (i.e., heat shock of less than 40 minutes at 42°C and 15 minutes at 43°C). A similar stress dose-dependent suppression of reporter gene expression by cycloheximide was also demonstrated by treatment with sodium arsenite at 37°C. However, dexamethasone-dependent reporter gene expression in a different CHO clone was not inhibited after the inducer treatment for different times in the presence of cycloheximide. In addition, synthesis of most cellular proteins (except for hsp) was not affected after heat shock treatment with cycloheximide. The results suggested that the cycloheximide inhibition of gene expression is specific to hsp gene expression induced by limited stress doses. Furthermore, a prior 42°C heat shock treatment for 30 minutes induced a decreased responsiveness (tolerance) to a second 42°C heat treatment for hsp gene expression, but tolerance did not develop in cells exposed to the first heat shock in the presence of cycloheximide. These results confirm previous findings that induction of hsp gene expression by stress is balanced by the severity of stress and rate of protein synthesis. They also support the proposed model of autoregulation of hsp gene expression by levels of free hsp70.

View full abstract

Use of 2-D gel and imaging plate analysis enabled biosynthetically radiolabeled immunoprecipitates to be quantitated at the very low level of gene products during processing from RER inside cells to cell surface. We used this efficient and sensitive measurement to analyse expression of HLA-DR molecules in human eosinophilic leukaemia cell lines. We found that they synthesized a constitutive amount of DRA gene products and differential amounts of DRB1 gene products. Thus, the incompletely inducible expression of DRB1 gene products was responsible for the limited accumulation of normally assembled molecules for cell surface expression and the lack of serological determination.

View full abstract

The foot-like structure of pecten (mollusk) cross striated muscle cells was studied from structural and biochemical standpoints, and compared with foot structures of vertebrate skeletal muscle cells. In vertebrate muscles, foot structures have been observed at the interspace between T-tubules and sarcoplasmic reticula (SR). In pecten muscles, T-tubules were not observed, but SR were found situated in the outer portions of the cell contacting the cell membrane, and foot-like structures were recognized at the interspace between the SR and cell membrane. We could isolate the SR fraction from these muscles in which vesicles of SR/cell membranes were included. In the SR fraction, foot-like structures were observed ultrastructurally by thin sectioning. The size and shape of the foot-like structure, whether observed in intact cells or SR fractions, appear smaller than foot structures of vertebrates. However, when calculated by SDS-PAGE, the molecular weight of the structure is similar to that of vertebrates. These findings are discussed and compared to characteristics of foot structures and foot proteins of vertebrate skeletal muscles reported in previous studies.

View full abstract

We have recently established a human osteoblastic cell line (SV-HFO) in a culture system, in which the cells are mineralized by treatment with dexamethasone (Dex). Using this system, we examined the effects of all trans-retinoic acid (RA) on the mineralization of the cells. RA inhibited the mineralization, coincident with the inhibition of alkaline phosphatase (ALP). On the other hand, RA induced osteocalcin secretion and had no effect on the expression of the other osteoblastic markers such as type I collagen and osteonectin.
To further clarify the mechanism of inhibition of mineralization by RA, we used the retinoic acid receptor (RAR)α-selective (Am80), β-selective (CD2019) and Υ-selective (CD437) agonists instead of RA. RARα- and RARβ-selective agonists inhibited the mineralization and ALP activity of the cells, while the RARΥ-selective agonist had no such effects. On the other hand, the RARΥ-selective agonist induced osteocalcin secretion, but RARα- and RARβ-selective agonists had no effect on osteocalcin secretion.
These results suggested that the inhibitory effect of RA on the mineralization of human osteoblasts is mediated by the activation of RARα and/or RAR β and that RARΥ preferentially regulates the expression of osteocalcin without influence on mineralization.

View full abstract

To investigate the cardiac differentiation and the initial stages of myoflbrillogenesis, chick precardiac mesoderm explants were cultured and spatiotemporal expressions of various sarcomeric cytoskeletal proteins were examined by immunofluorescence microscope. The precardiac cells differentiated and formed well organized myofibrils in culture. The myofibrillogenesis was similar to the reorganizing process of myofibrils in cultured cardiomyocytes from older embryos.

View full abstract

Myofibril assembly requires the cell to join diverse components, correctly oriented to the rest of the cell. Titin, a huge elastic protein with a role in myogenesis, assembles during translation in vivo and may require spatially organized mRNA to allow assembly. By immunofluorescence, we examined titin and myosin protein organization early in skeletal muscle development in vitro; titin was the first organized, initially as spots, then as periodically spaced lines, and later as doublets. Titin mRNA organization during development was detected by fluorescent in situ hybridization. Only titin mRNA was seen in mononucleated myoblasts. Shortly after fusion, both titin protein and mRNA were diffuse. Titin mRNA remained diffuse when titin protein formed cables. Where titin protein formed linear arrays of spots, titin mRNA showed a colinear, continuous array. Titin mRNA remained in arrays colinear with young myofibrils until several slender myofibrils aligned laterally; then, titin mRNA formed periodic arrays. The titin probe encodes peptide sequence in the A band, where this region of titin mRNA is detected in the most organized cells. Nebulin undergoes a similar progression slightly later in development. This pattern, of narrowly spaced stripes, is too closely spaced to function in the soluble phase. Titin mRNA is the earliest mRNA to become so highly organized in muscle; it does so earlier and at a different location than do mRNAs for costamere proteins. These results, taken with earlier ones, suggest mRNA localization may be as key to somatic cell differentiation as it is to embryonic development.

View full abstract

The exchangeability of actin in cardiac myofibrils and fibroblast stress fibers was investigated using fluorescent analogue cytochemistry in combination with fluorescence recovery (FR) after photobleaching. Living embryonic chicken cardiac myocytes and fibroblasts were microinjected with rhodamine (rh)-labeled muscle and nonmuscle actins. After incorporation of the fluorescent actin analogue into cellular structures, small areas of labeled structures were photobleached with a laser pulse. In cardiac myofibrils, FR in their proximal striated portions occurred at a slower rate than that in their proximal nonstriated and distal terminal portions with each rh-isoactin injected. Thus, nascent myofibrils at different developmental stages display different actin exchangeabilities. Further, in all portions of myofibrils, FR of rh-muscle actin was faster than that of rh-nonmuscle actin. This indicates that actin molecules in cardiac myofibrils cannot be readily exchanged by heterotypic nonmuscle actin. In fibroblasts, photobleaching of stress fibers yielded similar results in both their proximal midpoints and distal terminal portions, and the FR rate was consistently faster than that observed in any part of the myofibrils. This result seems to be related to the dynamic properties of actin filaments in stress fibers at all portions. Further, the fact that stress fibers possessed a similar exchange rate with muscle and nonmuscle actins appears to be related to a more primitive nature of stress fibers than myofibrils.

View full abstract

Recently we described a protein, smoothelin, that has been exclusively found in smooth muscle cells (SMC). The human cDNA has been cloned from a colon cDNA library and the putative protein sequence was deduced. Smoothelin does not belong to a known protein family but shows a partial homology with members of the spectrin family. Transfection studies revealed that smoothelin has an affinity for actin and is either capable of forming filamentous structures or colocalizes with such structures. The protein is expressed in visceral as well as vascular tissues of all vertebrate classes. A study on the distribution of smoothelin in the vascular and placental system showed that smoothelin expression was largely restricted to the muscular pulsating blood vessels. Therefore, we hypothesized that smoothelin is expressed in contractile SMC only (36, 37).
No expression of smoothelin was observed in established cell lines of SMC. In tissue explants smoothelin mRNA concentration decreases to undetectable levels within 12 hours after dissection as was in general the case in primary cell cultures. Here we report on continued smoothelin expression for several passages observed in a human prostate primary cell culture system. Smoothelin was demonstrated to colocalize with actin stress fibers but not with desmin filaments. This culture system offers opportunities to study the cytological localization of smoothelin, interactions with other proteins and should provide a system to test the promoter of the smoothelin gene. On immunoblots the molecular weight of smoothelin differed between visceral and vascular smooth muscle tissue with apparent molecular weights of respectively 59 kDa and 94 kDa. There is no evidence for the existence of another gene coding for the 94 kDa smoothelin. Thus, posttranslational modification, alternative splicing and dual promoter control are the alternatives for the expression of two isoforms of smoothelin.

View full abstract

Originally, zeugmatin was identified as a 600-800 kD muscle specific protein in Z-bands of cardiac and skeletal muscles by Maher et al. (1985). In this presentation we review our work on myofibrillogenesis and present evidence that zeugmatin is actually part of the Z-Band region of titin and that this region of titin plays an important role in the assembly of the Z-bands and niyofibrils. Rhee et al. (1994) reported that during myofibrillogenesis, zeugmatin antibody localization is detected in fully formed Z-bands in the mature niyofibrils, in the Z-bodies of the nascent myofibrils, but not in the Z-bodies of the premyofibrils. These observations lead to the suggestion that zeugmatin might be responsible for the fusion of the Z-bodies to form the solid Z-bands of the mature myofibrils (Rhee et al. 1994). As part of a study to test aspects of this model of myofibrillogenesis, we isolated a 1.8 kb cDNA from a chicken cardiac expression library using an anti-zeugmatin antibody (Turnacioglu et al., 1996). We found this chicken cDNA to be 60% identical at the ami no acid level to a segment of the Z-band region of human cardiac titin (connectin) sequenced by Labeit and Kolmerer (1995). This homology along with Western blot analysis with purified titin, suggested that zeugmatin is in fact part of the N-terminal region of chicken titin. When expressed in non-muscle cells, Z1.1 product colocalized with the alpha-actinin in stress fiber dense bodies and focal adhesions. Cultures of non-muscle cells, skeletal myotubes and cardiomyocytes were also transfected with a fusion construct (Z1.1GFP) consisting of the Z1.1 kb cDNA linked to the cDNA for green fluorescent protein (GFP). The Z1.1 kb cDNA encodes only 362 of the approximately 2, 000 amino acids which comprise the Z-band region of titin; neverthelss, the Z1.1GFP fusion protein targets in vivo to the alpha-actinin rich Z-bands of contracting myofibrils. A dominant negative phenotype was observed in living cells expressing highlevels of this Z1.1GFP fusion protein with inhibition of myofibrillogenesis as well as the disassembly of preexisting myofibrils in these cells. These data indicate that the Z-band region of titin (connectin) plays an important role in organizing and maintaining the structure of the myofibril.

View full abstract

The giant muscle protein titin (connectin) is assumed to play a crucial role in the control of Z-disk assembly. Analysis of the Z-disk region of titin/connectin revealed a novel 45 residue repeat that is spliced in variable copy numbers. The repeat region is coexpressed in normal human myocardium in size variants corresponding to between 5 and 7 repeats. Smaller isoforms can be detected in muscles with thinner Z-disks like M. psoas. Sequence analysis of chicken breast muscle titin/connectin reveals that in this tissue, where Z-disks are thin, only two Z-repeats are expressed. The greatly variable thickness of Z-disks is therefore correlated to a region of variable length in titin/connectin, constructed from a novel protein building block. Transfection experiments in myogenic cell lines demonstrate that overexpression of the entire integral Z-disk region of titin leads to a disruption of sarcomere assembly. The differentially expressed titin/connectin regions, however, show no dominant-negative effects on myofibril assembly and are not targeted to Z-disks. This supports the idea that the Z-repeat region of titin/connectin is responsible for the control Z-disk thickness as an element of highly variable length, due to extensive differential splicing.

View full abstract

Desmin, the muscle-specific member of the intermediate filament (IF) family, is one of the earliest known myogenic markers in both skeletal muscle and heart. Its expression precedes that of all known muscle proteins including the members of the MyoD family of myogenic helix-loop-helix (mHLH) regulators with the exception of myt"5. In mature striated muscle, desmin IFs surround the Z-discs, interlink them together and integrate the contractile apparatus with the sarcolemma and the nucleus. In vitro studies using both antisense RNA and homologous recombination techniques in embryonic stem (ES) cells demonstrated that desmin plays a crucial role during myogenesis, as inhibition of desmin expression blocked myoblast fusion and myotube formation. Both in C2C12 cells and differentiating embryoid bodies, the absence of desmin interferes with the normal myogenic program, as manifested by the inhibition of the mHLH transcription regulators. To investigate the function of desmin in all muscle types in vivo, we generated desmin null mice through homologous recombination. Surprisingly, a considerable number of these mice are viable and fertile, potentially due to compensation by vimentin, nestin or synemin. However, desmin null mice demonstrate a multisystem disorder involving cardiac, skeletal and smooth muscle, beginning early in their postnatal life. Histological and electron microscopic analysis in both heart and skeletal muscle tissues reveals severe disruption of muscle architecture and degeneration. Structural abnormalities include loss of lateral alignment of myofibrils, perturbation of myofibril anchorage to the sarcolemma, abnormal mitochondria! number and organization, and loss of nuclear shape and positioning. Loose cell adhesion and increased intercellular space are prominent defects. The consequences of these abnormalities are most severe in the heart, which exhibits progressive degeneration and necrosis of the myocardium accompanied by extensive calcification. Abnormalities of smooth muscle included hypoplasia and degeneration. There is a direct correlation between severity of damage and muscle usage, possibly due to increased susceptibility to normal mechanical damage and/or to repair deficiency in the absence of desmin. In conclusion, the studies so far have demonstrated that though desmin is absolutely necessary for muscle differentiation in vitro, muscle development can take place in vivo in the absence of this intermediate filament protein. However, desmin seems to play an essential role in the maintenance of myofibril, myofiber and whole muscle tissue structural and functional integrity.

View full abstract

Expression of neurofilament 140K protein in C2C12 mouse skeletal muscle cells was studied. Immunofluorescence and immunoblot analyses revealed that NF140K was expressed at the proliferative stage and was colocalized with the muscle-specific intermediate protein desmin. As muscle cell differentiation proceeded, the number of NF140K-positive cells decreased whereas the number of cells expressing muscle-specific marker proteins such as sarcomeric myosin heavy chain and troponin-T increased. Down-regulation of NF140K and upregulation of a myogenic regulatory gene, the myogenin gene, started simultaneously. In differentiated muscle cell cultures, unfused cells residing between myotubes remained NF140K-positive. NF140K and desmin doublepositive cells were also found in a primary culture of adult mouse skeletal muscle cells. The results suggest that NF140K may be a unique marker for uncommitted myoblasts.

View full abstract

The vertebrate sarcomere is a complex structure composed of numerous proteins arranged in an exquisitely precise manner. Sarcomeric proteins are organized into interdigitating thick or thin filaments and the sliding of these filaments relative to one another constitutes muscle contraction at the sarcomere level. Consequently, an understanding of sarcomeric structure and function requires a thorough knowledge of the individual components of the thick and thin filaments, as well as their associations. Thick filaments are comprised of myosin, which provides the force required to drive muscle contraction and also plays a major structural role in thick filament formation. In addition, a family of thick filament-associated proteins plays a role in organization of the thick filament. We have used both molecular genetic and cell biological approaches to define the diversity of the myosin heavy chain gene family and to analyze the assembly of myosin and it's associated proteins into thick filaments.

View full abstract

We investigated the assembly of the first sarcomeres in chicken embryos by conf ocal microscopy of immunofluorescently stained whole mount rudiments of early chicken hearts isolated around the onset of beating. In embryos with merely 9 somites, myomesin was found to be present in a cross striated pattern, indicating that myomesin is expressed rather early during development. RNA studies confirmed these findings and RT-PCR revealed the presence of myomesin mRNA already in the 7 somite embryo. The expression of myomesin mRNA coding for the skeletal isoform preceded the heart specific transcript. In the adult heart, however, only the heart isoform was detectable.
The interaction of myomesin domains with the sarcomere was investigated by transf ection of epitope tagged constructs into cultured cardiomyocytes. The second domain of myomesin, an immunoglobulin-like domain, was found to specifically bind to the M-band region of chicken cardiomyocytes. All constructs containing this domain also showed M-band localization. Additionally, constructs consisting of either the second domain of myomesin or the myosin light chain isoform MLC 3f fused to the green fluorescent protein (eGFP) were expressed in rat cardiomyocytes and were found to be distributed in the same manner as the expression constructs tagged only with the much shorter VSV epitope. Transfected cells did not show any alteration in beating activity and no alteration of myofibrillar structure, as judged by simultaneous staining for the Z-disc protein α-actinin and other sarcomeric markers. Apparently, the addition of eGFP did not disturb the assembly properties or the function of the two proteins and therefore allowed the easy visualization of assembly and contraction processes directly in the living cardiomyocyte.

View full abstract

Muscle regeneration is a potentially useful model for denning the mechanisms responsible for nerve-dependent myosin isogene regulation in skeletal muscle. As a first step towards this goal we have characterized the pattern of expression of the four myosin heavy chain (MHC) genes, MHC-β/slow, -2A, -2X and -2B isogenes, during early stages of muscle regeneration both in the presence and in the absence of the nerve. Muscle degeneration/regeneration was induced by intramuscular injection of the myotoxic drug, bupivacaine, in the rat slow soleus muscle. MHC transcripts were identified by in situ hybridization with specific riboprobes during the period from day 3 to day 7 after muscle injury. The four genes are not detected at day 3, when the regenerating muscle contains predominantly embryonic and neonatal MHC isoforms. MHC-2X and -2B transcripts are first detected at day 4 in both innervated and denervated muscles. These transcripts remain as major transcripts in denervated muscles whereas they are down-regulated by day 5 and disappear by day 6-7 in the presence of the nerve. Innervation induces strong up-regulation of MHC-2A at day 4 and MHC-β/slow transcripts at day 5. MHC-2A transcripts are first homogeneously expressed in most fibers then become segregated in a minor population of fibers by day 6-7 while MHC-β/slow transcripts increase in most fibers. In the absence of the nerve MHC-β/slow transcripts are never expressed and MHC-2A transcripts are detected in rare fibers at days 5-7.

View full abstract

Thick filaments are stable assemblies of myosin that are characteristic of specific muscle types from both vertebrates and invertebrates. In general, their structure and assembly require remarkably precise determination of lengths and diameters, structural differentiation and nonequivalence of myosins, a high degree of inelasticity and rigidity, and dynamic regulation of assembly and disassembly in response to both extracellular and intracellular signals. Directed assembly of myosin in which additional proteins function in key roles, therefore, is more likely to be significant than the simple self assembly of myosin into thick filaments. The nematode Caenorhabditis elegans permits a wide spectrum of biochemical, genetic, molecular and structural approaches to be applied to the experimental testing of this hypothesis. Biochemical analysis of C. elegans thick filaments reveals that paramyosin, a homologue of the myosin rod that is the unique product of a single genetic locus, exists as two populations which differ by post-translational modification. The major paramyosin species interacts with the two genetically specified myosin heavy chain isoforms. The minor paramyosin species is organized within the cores of the thick filaments, where it is associated stoichiometrically with three recently identified proteins P20, P28 and P30. These proteins have now been characterized molecularly and contain unique, novel amino acid sequences. Structural analysis of the core shows that seven paramyosin subfilaments are crosslinked by additional internal proteins into a highly rigid tubule. P20, P28 and P30 are proposed to couple the paramyosin subfilaments together into the core tubule during filament assembly. Mutants that affect paramyosin assembly are being characterized for alterations in the core proteins. A fourth protein has been identified recently as the product of the unc-45 gene. Computational analysis of this gene's DNA suggests that the predicted protein may exhibit protein phosphatase and chaperone activities. Genetic analysis shows that three classes of specific unc-45 mutant proteins differentially interact with the two myosins during thick filament assembly. The unc-45 protein is proposed to be a myosin assemblase, a protein catalyst of thick filament assembly.

View full abstract

Immunochemical experiments and in vitro binding studies have revealed that titin/connectin, the elastic protein that spans the whole distance of a half-sarcomere, associates with several myosin-binding proteins of the sarcomeric A and M band. Two of these proteins, M-protein and myomesin, anchor titin in the region of the M band. A detailed molecular map describing the arrangement of titin, M-protein and myomesin in this part of the sarcomere was recently proposed. Furthermore, specific binding sites between the molecules were identified. How these polypeptides function in the assembly of the sarcomeric M band region has, however, remained unclear. Here we describe the distribution patterns of different epitopes recognized by newly developed antibodies against the extreme carboxyterminal portion of titin that is anchored in the M band, during the in vitro differentiation of human skeletal muscle cells. In contrast to a set of antibodies directed against Z band, I band and A band titin epitopes, anti-M band titin did not stain titin aggregates or titin in non-striated myofibrils (NSMF). The M band epitopes were only revealed in their characteristic sarcomeric locations, and were obviously not accessible in the non-striated part of nascent myofibrils, or during earlier developmental stages. We speculate that this phenomenon is associated with "immature" tertiary/quarternary structures of M band titin that avoid preliminary binding of M band proteins. In contrast to earlier observations on myofibrillogenesis in the mouse embryo, all the titin epitopes studied were simultaneously detected in their specific positions. Thus, sarcomere assembly in the widely used in vitro model systems seems to proceed at a much higher speed than in vivo. Similarly, myomesin and M-protein were only perceptible in striated myofibrils. While myomesin antibodies stained myofibrils at the time-point of appearance of the first titin striations, the incorporation of M-protein was found to be slightly delayed. In several myotubes no expression of M-protein was observed even during mature stages. These observations indicate its less important general role in the process of myofibrillogenesis. Furthermore, the relative number of M-protein negative myotubes varied in cultures derived from different muscles. This confirms the observation that cultured satellite cells are predestined to form a certain type of myofibers.

View full abstract

Muscle differentiation involves a profound change in cell cytoarchitecture. This is accompanied by extensive isoform replacement in which the major non-muscle isoforms of the actin filament system are replaced by their muscle isoform counterparts. We have tested whether the sequential expression of the actin isoforms is functionally significant by precociously expressing the two striated muscle actins (α-skeletal and α-cardiac) in mouse myoblasts. The human α-skeletal and α-cardiac actin genes were transfected into mouse C2 myoblasts and clones expressing the human genes at the highest level were identified. Expression of the human α-skeletal actin gene was low with the highest mRNA level found to be 4% of that in adult human skeletal muscle. Clones expressing α-cardiac actin accumulated the mRNA up to 13% of the level of α-skeletal actin in adult human skeletal muscle. Despite the low level of α-skeletal actin expression, myoblasts transfected with this gene displayed a profound decrease in cell spreading. In contrast, α-cardiac actin had no impact on cell spreading. Neither α-skeletal actin nor α-cardiac actin had any impact on the total actin protein pool nor on the levels of the high molecular weight tropomyosins. The organisation of actin and tropomyosin into stress fibres was similar between transfected and control cells. We conclude that precocious expression of α-skeletal actin, but not acardiac actin, compromises myoblast morphology but not the ability of the cell to assemble stress-fibre-like structures.

View full abstract

We previously demonstrated that small G-actin binding proteins, cofllin, ADF and profilin, are involved in the actin dynamics during myoflbrillogenesis (OBINATA, T. (1993). Int. Rev. Cytol, 143: 153-189.). To better understand how they are responsible for the regulation of actin assembly, the amounts of the actin-binding proteins were quantified by means of quantitative immunoblotting and compared with that of G-actin pool. The sum of the amounts of cofilin, ADF and profilin was insufficient at early developmental stages but sufficient at later stages to account for the pool of G-actin in muscle cells. We detected expression of thymosin β4 at a considerable level in young embryonic but not in adult skeletal muscles. We, therefore, conclude that the G-actin pool in young embryonic skeletal muscle is mainly due to cofilin, ADF, profilin and thymosin β4. Switching from a non-muscle-type (NM-) cofilin to a muscle-type (M-) cofilin was observed during muscle development of mammals. In order to clarify cofilin-dependent regulation of actin assembly in muscle cells, cofilin tagged with fluorescence dyes was introduced into C2 myoblasts by a micro injection method. The exogeneous cofilin, but not ADF, caused quick disassembly of actin filaments and accumulated in furrow region of dividing cells. The analogs of the unphosphorylated form (A3-cofilin) and the phosphorylated form (D3-cofilin) were prepared by converting Ser3, a regulatory phosphorylation site, to Ala or Asp. When A3-cofilin and D3-cofilin were injected into living cells, the former was concentrated at the membrane ruffles and cleavage furrow, while the latter showed only diffuse distribution in the cytoplasm. These results suggest that the subcellular distribution of cofilin as well as its interaction with actin in vivo is regulated by its phosphorylation and dephosphorylation.

View full abstract

The assembly and functional characteristics of many contractile proteins are different in skeletal and cardiac muscle. Two models for thin filament assembly are consistent with observations from recent studies focused on determining the functional significance of actin filament capping in primary cultures of embryonic chick myogenic cells and cardiac myocytes. Future experiments will test the validity of the proposed models for in vivo embryonic development.

View full abstract

During development of the ascidian Halocynthia roretzi, the tadpole larva hatched from the tailbud embryo metamorphoses to the adult with a body wall muscle. Although the adult body wall muscle is morphologically nonsarcomeric smooth muscle, it contains a troponin complex consisting of three subunits (T, I, and C) as do vertebrate striated muscles. Different from vertebrate troponins, however, the smooth muscle troponin promotes actin-myosin interaction in the presence of high concentration of Ca²⁺, and this promoting property is attributable to troponin T. To address whether the embryonic/larval tail striated muscle and the adult smooth muscle utilize identical or different regulatory machinery, we cloned troponin T cDNAs from each cDNA library. The embryonic and the adult troponin Ts were encoded by distinct genes and shared only <60% identity with each other. These isoforms were specifically expressed in the embryonic/larval tail striated muscle and the adult smooth muscle, respectively. These results may imply that these isoforms regulate actin-myosin interaction in different manners. The adult troponin T under forced expression in mouse fibroblasts was unexpectedly located in the nuclei. However, a truncated protein with a deletion including a cluster of basic amino acids colocalized with tropomyosin on actin filaments. Thus, complex formation with troponin I and C immediately after the synthesis is likely to be essential for the protein to properly localize on the thin filaments.

View full abstract

In the higher vertebrates troponin I (TnI) is encoded by three related genes, each of which is expressed specifically in one of the three major sarcomeric muscle cell classes, i.e. cardiomyocytes or fast or slow skeletal muscle fibers. The TnIcardiac isoform contains an "extra" block of proline-rich protein sequence near the N-terminus encoded by an exon that has no counterpart in the TnIfast and TnIslow genes. All three TnI isoforms appear to be orthologously related between birds and mammals, indicating that the TnI gene family was already established in its modern form in the early reptile common ancestor to birds and mammals. Analysis of ascidian TnI suggests that early vertebrate ancestors contained a single TnI gene and that the gene duplications that established the family occured after the ascidian/vertebrate divergence. Evidence from organisms representing evolutionary intermediates between ascidians and reptiles is incomplete and does not yet delineate the exact order and timing of the TnI gene duplication events. However it does appear that early tetrapods already contained specialized TnI genes encoding long and short isoforms and that multiple differentially expressed TnI genes were present in the vertebrate lineage before the teleost/tetrapod divergence. Ascidians and the protostome invertebrate Drosophila produce long and short TnI isoforms (the longer isoforms containing a prolinerich block of extra sequence near the N-terminus) by an alternative RNA splicing mechanism from a single gene. It is likely that the alternative splicing mechanism is an ancestral feature, and that during vertebrate evolution this mechanism was abandoned in favor of transcriptional regulatory mechanisms directing tissue-specific expression of multiple genes separately encoding long and short TnI isoforms.

View full abstract

How does muscle gene mutation affect the muscle structure and function of an animal? Mutant animals of the tropomyosin and troponin C genes of Caenorhabditis elegans show Pat (paralyzed, arrested elongation at twofold) phenotypes together with abnormal muscle filament assembly. We present evidence that the mutation sites of lev-11 gene was in the tropomyosin gene, tmy-1 and that of pat-10 was in the troponin C gene, tnc-1, of the worm, respectively. The lev-11 (stSST) mutation occurred at the splice donor site of exon 1 and results in translation termination. Although the gene product from heterozygous (+ /st557) animal was not detected, our result could be the reason for the Pat phenotype of this mutation. The lev-11(xl2) mutation, isolated as an allele of levamisole resistance, occurred in exon 7 and results in amino acid substitution at 234 from Glu to Lys. This substitution give a charge change from - to + at this point which is common in three isoforms. There may be functional importance of this region for molecular interaction of the tropomyosin. Mutation site of pat-10(st575) was Asp64 to Asn and Trp153 to termination in the troponin C. The first mutation site was in the second calcium binding site and the second mutation raised the deletion of H helix in the troponin C. Both might affect the calcium binding or the retaining of the conformation for its function. Results presented here will be useful to understand the interaction site between the tropomyosin and troponin complex.

View full abstract

Skeletal muscle contraction is regulated by a complex of five polypeptides which are stably associated with the actin filament. This complex consists of two proteins: troponin with three subunits (TnC; TnI and TnT) and tropomyosin (a dimer of two chains). Using deletion mutants of TnC, TnI and TnT we determined that each of these polypeptides can be divided into at least two domains. One domain is responsible for the regulatory properties of the protein. Its interaction with the other components of the system change upon calcium binding to TnC. A second domain present in each of these proteins is responsible for the stable association of the complex to the actin filament. The interactions among this second set of domains is not influenced by calcium binding to TnC. The structural interactions are: 1) interactions between the C-domain of TnC with the N-domain of TnI; 2) interactions of the N-domain of TnI with the C-terminal domain of TnT and 3) interactions between the N-domain of TnT (T1) and actin/tropomyosin.

View full abstract