Myosin heavy chain kinase from developed Dictyostelium cells. Purification and characterization

We purified to homogeneity the Dictyostelium discoideum myosin heavy chain kinase that is implicated in the heavy chain phosphorylation increases that occur during chemotaxis. The kinase is initially found in the insoluble fraction of developed cells. The major purification step was achieved by affinity chromatography using a tail fragment of Dictyostelium myosin (LMM58) expressed in Escherichia coli (De Lozanne, A., Berlot, C. H., Leinwand, L. A., and Spudich, J. A. (1988) J. Cell Biol. 105, 2990-3005). The kinase has an apparent molecular weight of 84,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent native molecular weight by gel filtration is 240,000. The kinase catalyzes phosphorylation of myosin heavy chain or LMM58 with similar kinetics, and the extent of phosphorylation for both is 4 mol of phosphate/mol. With both substrates the Vmax is about 18 mumol/min/mg and the Km is 15 microM. The myosin heavy chain kinase is specific to Dictyostelium myosin heavy chain, and the phosphorylated amino acid is threonine. The kinase undergoes autophosphorylation. Each mole of kinase subunit incorporates about 20 mol of phosphates. Phosphorylation of myosin by this kinase inhibits myosin thick filament formation, suggesting that the kinase plays a role in the regulation of myosin assembly.     

Myosin heavy chain expression in embryonic cardiac cell cultures

Chick embryonic heart cell isolates and monolayer cultures were prepared from atria and ventricles at selected stages of cardiac development. The cardiac myocytes were assayed for myosin heavy chain (MHC) content using monoclonal antibodies (McAbs) specific in the heart for atrial (B-1), ventricular (ALD-19), or conductive system (ALD-58) isoforms. Using immunofluorescence microscopy or radioimmunoassay, MHC accumulation was measured before plating and at 48 hr or 7 days in culture. Reproducible changes in MHC antigenicity were observed by 7 days in both atrial and ventricular cultures. The changes were stage dependent and tissue specific but generally resulted in a decreased reactivity with the tissue specific MHC McAbs. In addition, the isoform recognized by ALD-58, characteristic of the conductive system cells in vivo, was never present in cultured myocytes. These results indicate that MHC isoforms produced in vivo may be replaced in monolayer cultures by an isoform(s) not recognized by our tissue specific MHC McAbs. This suggests that the intrinsic program of cardiac myogenesis, within cardiac myocytes, may not be sufficient to establish and maintain differential expression of tissue specific MHC in monolayer cell culture.     

Myosin heavy chain in avian muscular dystrophy corresponds to the neonatal isozyme

We have previously demonstrated, based on comparison of homologous amino acid sequences and of two-dimensional CNBr peptide gel patterns, that the myosin heavy chain in pectoralis muscles of Storrs, Connecticut dystrophic chickens is different from that of their normal controls (Huszar, G., Vigue, L., De-Lucia, J. Elzinga, M., and Haines, J. (1985) J. Biol. Chem. 260, 7429-7434). Others have shown, however, that genomic banks and mRNA complements of the control and dystrophic birds are not different. In the present studies, we have examined the hypothesis that the "dystrophic" myosin heavy chain is not a novel gene product, but is a developmental isozyme which is expressed in pectoralis muscles of adult chickens due to the dystrophic process. Two-dimensional maps of myosin heavy chain CNBr peptides were prepared from breast muscles of 17-day in ovo (embryonic), 25-day posthatch (neonatal), and adult birds of the Storrs dystrophic and of two control strains. Also, myosin and actomyosin ATPase enzymatic activities of the various preparations were determined in the pH range of 5.5 to 9.0. Analysis of the peptide maps demonstrates that the embyronic, neonatal, and control adult myosin heavy chain isozymes are distinctly different gene products with only minute variations between the respective developmental isozymes in dystrophic and control muscles. However, the pectoralis myosin heavy chain of adult dystrophic birds, which is a homogeneous isozyme population by amino acid sequences and gel patterns, corresponds to that of the neonatal-type myosin heavy chain. The ATPase properties of the embryonic, neonatal, or adult pectoralis myosins and actomyosins were not different, whether the level of specific activity or the pattern of pH activation is considered. Since the mobility of neonatal chicks (primarily neonatal-type isozymes) is not restricted, the differences in myosin heavy chain structures are part of the syndrome, but not the cause of avian muscular dystrophy.     

Myosin heavy chain composition of single cells from avian slow skeletal muscle is strongly correlated with velocity of shortening during development

We have determined the myosin heavy chain (MHC) composition (using a sensitive sodium dodecyl sulfate-polyacrylamide gel electrophoresis system) and the maximal velocity of shortening (Vmax) of single cells from neonatal and adult chicken anterior latissimus dorsi (ALD) muscles. In addition, the MHC, myosin light chain, and regulatory protein (i.e., troponin and tropomyosin subunits) compositions of bundles of ALD fibers were determined at late embryonic, neonatal, and adult ages. At young ages, there are two MHCs in ALD muscle, SM1 and SM2, with SM1 decreasing in relative amount with increasing age, as shown previously by others. The mean Vmax of single fibers also decreases from neonatal to adult ages. A strong quantitative correlation is demonstrated between the specific MHC composition and Vmax among individual cells of the ALD muscle at several ages. Since virtually no changes occur in the regulatory protein and myosin light chain compositions of the ALD muscle between late embryonic and adult ages, it appears that the MHC composition of an individual cell in this muscle is the primary determinant of the maximal shortening velocity. These results are the first to illustrate the functional significance of the developmental transition in myosin heavy chain composition of an avian slow skeletal muscle, consistent with our previous findings on mammalian muscle.     

Myosin heavy chain isoforms in histochemically defined fiber types of rat muscle

Summary Combined histochemical and biochemical analyses were performed on rat skeletal muscles in order to determine the myosin heavy chain patterns in specific fiber types. Four myosin heavy chain isoforms were separated by gradient polyacrylamide gel electrophoresis of extracts from single fibers and whole muscle homogenates. Their electrophoretic mobility increased in the order HCIIa, HCIIb, and HCI. HCIIa, HCIIb and HCI were present as unique isoforms in histochemically defined fiber types IIA, IIB and I, respectively. The isoforms HCI and HCIIa coexisted at variable ratios in type IC and IIC fibers. An additional fast myosin heavy chain isoform with an electrophoretic mobility between HCIIa and HCIIb was designated as HCIId because of its abundance in fast fibers of large diameter in the diaphragm. With the exception of slight differences in mATPase staining intensity after acid preincubation, these fibers were almost indistinguishable from type IIB fibers. In view of their specific myosin heavy chain composition (HCIId), these fibers were named type IID. In the extensor digitorum longus muscle, type IID fibers were of smaller size than type IIB and differed from the latter by higher NADH tetrazolium reductase activities. Circumstantial evidence suggests that type IID fibers are identical with the 2X fibers, previously described by Schiaffino et al. (1986).     

Myosin heavy chain gene amplification as a suppressor mutation in Caenorhabditis elegans

Summary In the nematode, Caenorhabditis elegans, the body wall muscles contain paramyosin and two different types of myosin heavy chain, MHC A and MHC B. In mutants that do not express MHC B or that express defective paramyosin, muscle structure is disrupted and movement is impaired. Second site mutations in the sup-3 locus partially reverse these defects and are correlated with a 2- to 3-fold increase in the accumulation of the MHC A isoform. The sup-3 mutations occur at a high frequency (10^–4) after ethyl methanesulfonate (EMS) mutagenesis. This is comparable to the average EMS-induced mutation rate per gene in C. elegans. In this paper we show that the sup-3 mutation is an amplification of the structural gene for the MHC A protein, myo-3. We employed genomic Southern hybridization with MHC gene-specific probes in order to measure the copy number of the myo-3 gene relative to that of the MHC B gene, unc-54. We have identified the putative amplification junctions for these sup-3 alleles using a set of cosmid clones which encompass myo-3 region. Although it has been suggested that gene amplification plays an important role in evolution, there are few known cases of gene amplification in the germ line cells of multicellular organisms. The results shown here provide a clear example of a heritable gene amplification event that occurs at a high frequency in the germ line. Similar events may thus represent the initial event in the evolution of new function and in the formation of multigene families.     

Myosin heavy chain composition of muscle spindles in human biceps brachii.

Data on the myosin heavy chain (MyHC) composition of human muscle spindles are scarce in spite of the well-known correlation between MyHC composition and functional properties of skeletal muscle fibers. The MyHC composition of intrafusal fibers from 36 spindles of human biceps brachii muscle was studied in detail by immunocytochemistry with a large battery of antibodies. The MyHC content of isolated muscle spindles was assessed with SDS-PAGE and immunoblots. Four major MyHC isoforms (MyHCI, IIa, embryonic, and intrafusal) were detected with SDS-PAGE. Immunocytochemistry revealed very complex staining patterns for each intrafusal fiber type. The bag(1) fibers contained slow tonic MyHC along their entire fiber length and MyHCI, alpha-cardiac, embryonic, and fetal isoforms along a variable part of their length. The bag(2) fibers contained MyHC slow tonic, I, alpha-cardiac, embryonic, and fetal isoforms with regional variations. Chain fibers contained MyHCIIa, embryonic, and fetal isoforms throughout the fiber, and MyHCIIx at least in the juxtaequatorial region. Virtually each muscle spindle had a different allotment of numbers of bag(1), bag(2) and chain fibers. Taken together, the complexity in intrafusal fiber content and MyHC composition observed indicate that each muscle spindle in the human biceps has a unique identity.     

Myosin heavy chain isoform mRNA and protein levels after long-term paralysis

To assess the long-term influence of paralysis on muscle phenotypic mRNA and protein expression, the effects of spinal cord transection (ST) on myosin heavy chain (MyHC) isoform mRNA and protein levels in the soleus and medial gastrocnemius (MG) muscles of rats were analyzed. Control soleus contained predominantly MyHC-I with low amounts of MyHC-IIa and IIx mRNAs. After ST, MyHC-I mRNA decreased to approximately 15%, MyHC-IIa was increased by 75-200%, and MyHC-IIx was elevated by 8-10x. Low level expression of MyHC-IIb was observed post-ST, suggesting that reduced activity is not a primary stimulus for MyHC-IIb expression. Adaptations in mRNA preceded protein adaptations in the soleus. Although MyHC-I protein in the MG was reduced post-ST, no other consistent changes occurred. The relative lack of adaptation to ST by the MG suggests that the reduced activity and load bearing encountered by the MG were insufficient to induce a change in muscle phenotype.     

Myosin heavy chain kinase inactivated by Ca2+/calmodulin from aggregating cells of Dictyostelium discoideum.

Soluble myosin heavy chain kinases (MHC kinases) were partially purified from growth phase and aggregation-competent cells of Dictyostelium discoideum. In the aggregation-competent cells, two MHC kinases were distinguishable. One of these enzymes, called MHC kinase II, was inactivated by Ca2+ and calmodulin in a highly temperature-dependent reaction. A MHC kinase found in growth phase cells did not have these regulatory properties. Substrate specificities were analysed for MHC kinase II and for the MHC kinase from growth phase cells. Both enzymes phosphorylated threonine residues of the myosin heavy chains of D. discoideum and Physarum polycephalum. Phosphopeptide mapping of D. discoideum myosin and determination of the stoichiometry of its phosphorylation suggested the presence of two phosphorylation sites per heavy chain. Both sites were contained within a 38-kd chymotryptic fragment. The inactivation of MHC kinase II by Ca2+ plus calmodulin suggests this enzyme has a role in the regulation of myosin functions during the chemotactic response of a cell. The phosphorylated myosin had about one third the actin-activated Mg2+-ATPase activity of the non-phosphorylated myosin. Previous findings indicated that stimulation of D. discoideum cells with the chemo-attractant cAMP increases the cytoplasmic Ca2+ concentration. Under these conditions MHC kinase II might be inhibited and the dephosphorylated, more active form of myosin would accumulate.     

Myosin heavy chain composition of single fibres from normal human muscle.

Electrophoretic analysis in the presence of 33% glycerol of purified myosin from normal human muscle shows three distinct protein bands which are identified as type 1, 2B, and 2A myosin heavy chain (MHC) isoforms by affinity-purified polyclonal antibodies. Analysis of MHC of single human muscle fibres shows that human muscles contain a large population of fibres showing the coexistence of type 2A and 2B MHC.     

Myosin heavy chain isoforms regulate muscle function but not myofibril assembly.

Myosin heavy chain (MHC) is the motor protein of muscle thick filaments. Most organisms produce many muscle MHC isoforms with temporally and spatially regulated expression patterns. This suggests that isoforms of MHC have different characteristics necessary for defining specific muscle properties. The single Drosophila muscle Mhc gene yields various isoforms as a result of alternative RNA splicing. To determine whether this multiplicity of MHC isoforms is critical to myofibril assembly and function, we introduced a gene encoding only an embryonic MHC into Drosophila melanogaster. The embryonic transgene acts in a dominant antimorphic manner to disrupt flight muscle function. The transgene was genetically crossed into an MHC null background. Unexpectedly, transformed flies expressing only the embryonic isoform are viable. Adult muscles containing embryonic MHC assemble normally, indicating that the isoform of MHC does not determine the dramatic ultrastructural variation among different muscle types. However, transformed flies are flightless and show reduced jumping and mating ability. Their indirect flight muscle myofibrils progressively deteriorate. Our data show that the proper MHC isoform is critical for specialized muscle function and myofibril stability.     

Myosin translocation in retinal pericytes during free-radical induced apoptosis.

Vascular pathologies induced by ischemia/reperfusion involve the production of reactive oxygen species (ROS) that in part cause tissue injury. The production of ROS that occurs upon reperfusion activates specific second messenger pathways. In diabetic retinopathy there is a characteristic loss of the microvascular pericyte. Pericytes are more sensitive than endothelial cells to low concentrations of ROS, such as hydrogen peroxide (H(2)O(2)) when tested in vitro. Whether the pericyte loss is due to toxic cell death triggered by the noxious H(2)O(2) or apoptosis, due to activation of specific second messenger pathways, is unknown. During apoptosis, a cell's nucleus and cytoplasm condense, the cell becomes fragmented, and ultimately forms apoptotic bodies. It is generally assumed that apoptosis depends on nuclear signaling, but cytoplasmic morphological processes are not well described. We find that exposing cultured retinal pericytes to 100 microM H(2)O(2) for 30 min leads to myosin heavy chain translocation from the cytosol to the cytoskeleton and a significant decrease in cell surface area. Pericyte death follows within 60-120 min. Exposing cells to 150 mJ/cm(2) ultraviolet radiation, an alternate free radical generating system, also causes pericyte myosin translocation and apoptosis. Proteolytic cleavage of actin is not observed in pericyte apoptosis. 3-aminobenzamide, a pharmacological inhibitor of the cleavage and activation of the DNA-repairing enzyme poly (ADP-ribose) polymerase (PARP) inhibits pericyte apoptosis, and prevents myosin translocation. Deferoxamine, an iron chelator known to interfere with free radical generation, also inhibits pericyte myosin translocation, contractility, and cell death. Myosin translocation to the cytoskeleton may be an early step in assembly of a competent contractile apparatus, which is involved in apoptotic cell condensation. These results suggest that pericyte loss associated with increased free radical production in diabetic retina may be by an apoptotic phenomenon.     

Myosin light chain kinase expression during smooth muscle development

The expression of smooth muscle myosin light chain kinase (MLCK) was investigated during chicken gizzard development. The molecular weight and the antigenic properties of MLCK did not change during development. The use of anion exchange high performance liquid chromatography (HPLC) enabled us to distinguish between MLCKs from post-hatched and adult chickens. A partial amino acid sequence determination of 4-day-old gizzard MLCK failed to disclose differences in the primary sequences of the two proteins. The results suggest that MLCK has the same primary sequence in all sequences of the two proteins. The results suggest that MLCK has the same primary sequence in all stages of gizzard development, although charge variants due to post-translational modifications may exist.     

Actin depolymerization and polymerization are required during apoptosis in endothelial cells

In order to understand the role of actin microfilaments in the apoptotic process, we followed their evolution during tumor necrosis factor-alpha (TNF)-induced apoptosis in bovine aortic endothelial (BAE) cells. Using Western blotting analysis and immunofluorescence microscopy, we observed that the actin microfilaments network was disrupted in apoptotic cells. Depolymerization of F-actin was concomitant with internucleosomal DNA degradation and with the morphological changes associated with apoptotic cell death. However, using the actin microfilament disrupting agent, cytochalasin, we present evidence that the formation of blebs leading to apoptotic cell fragmentation requires neopolymerization of actin. Indeed, in the presence of cyochalasin, induction of apoptosis (internucleosomal DNA degradation) in BAE cells by TNF and cycloheximide was not associated with these classical morphological markers of apoptosis. Moreover, when added to BAE cells showing incipient apoptotic fragmentation, cytochalasin E reversed this process. We also observed an accumulation of actin at the basis of the apoptotic bodies in formation in these cells. Together, these results suggest that the actin network of flattened cells is disrupted concomitantly to the morphological modifications associated to the apoptotic cell death, and that the cytochalasin-sensitive reorganisation of actin is required to the formation of apoptotic blebs.     

Myosin heavy chain isoform expression and in vivo isometric performance: a regression model

This investigation estimated the amount of variance in voluntary in vivo muscle performance that can be explained by relative myosin heavy chain (MHC) isoform expression. The role of the relative expression of these proteins in relation to in vitro force and velocity performance is well understood, but the in vivo model is less clear. Twenty-two men and women (mean +/- SD age, 27 +/- 6 years) performed isometric knee extensor actions in which peak force and rate of force development (RFD) were measured. The results of regression analysis showed that the inclusion of MHC IIb explained a significant (19.9%, p < 0.05) amount of variance in relative peak force (adjusted for muscle mass) and 14.1% of the variance in the first half of the rise phase of the force-time curve (RFD(0-50%)) (p < 0.1). The addition of MHC I into this model explained a significant (p < 0.05) amount of variance above that accounted for by MHC IIb in RFD (45.4%), RFD(0-50%) (50.8%), and RFD(50-100%) (second half of the rise phase of the force-time curve) (37.4%). Since the percentage of MHC IIb is reduced rather quickly with training, these data suggest that peak force may also be affected quickly by training. The percentage of MHC I has a longer course for change with training; therefore, it may be inferred that the greatest changes in RFD variables will likely occur during a longer course.     

Absence of the functional Myosin heavy chain 2b isoform in equine skeletal muscles

Nucleotide sequences which included the full coding region for three types of myosin heavy chain (MyHC) isoforms were determined from equine skeletal muscles. The deduced amino acid sequences were 1937, 1938, and 1935 residues for the MyHC-2a, -2x, and -slow, respectively. No MyHC-2b isoform was amplified from the equine muscle cDNA except for one pseudogene fragment. One nucleotide was inserted in the coding region of the equine pseudogene product, a minute amount of which was expressed in the skeletal muscle. The 596 bp sequence of the equine MyHC pseudogene was categorized into the MyHC-2b genes on the phylogenetic tree of the mammalian MyHC genes. These results suggest that an ancestral MyHC-2b gene had lost its function and changed to a pseudogene during the course of horse history. The MyHC genes in some ungulates were analyzed through the PCR amplifications using the MyHC isoform-specific primers to confirm the presence of the MyHC-2b and -2x genes. The exon coding the 3' untranslated region of the MyHC-2x was successfully amplified from the all ungulates examined; however, that of the MyHC-2b gene was amplified only from horses, pigs and lesser mouse deer. The PCR analyses from rhinoceros, sika deer, moose, giraffes, water buffalo, bovine, Japanese serow and sheep genes implied the absence of the MyHC-2b-specific sequence in their genomes. These results suggest that the MyHC-2b gene independently lost its function in some ungulate species.     

Myosin heavy chain composition in human masticatory muscles by immunohistochemistry and gel electrophoresis.

In this study we compared the immunohistochemically quantified fiber type area with the myosin heavy chain (MyHC) contents of a bundle of fibers from a human masticatory muscle. The total cross-sectional areas were determined immunohistochemically for the three major fiber types (I, IIA, and IIX) in bundles of fibers (n=42) taken from the anterior and posterior belly of the human digastric muscle (n=7). The relative MyHC contents of the same fiber bundles were determined electrophoretically (MyHC-I, -IIA, and -IIX; anterior, 32%, 35%, and 33%; posterior, 39%, 42%, and 19%) and compared with the immunohistochemical data (MyHC-I, -IIA, and -IIX; anterior, 32%, 31%, and 37%; posterior, 39%, 45%, and 15%). No significant differences were seen in the mean fiber type distribution between the two techniques; the correlation coefficient ranged from 0.71 to 0.96. The correlation coefficient was higher for MyHC type I and MyHC type IIX than for MyHC type IIA. The MyHC contents of single fibers taken from the posterior belly indicated that many fibers in this belly co-express MyHC-IIA and MyHC-IIX. Despite the presence of these hybrid fibers, the correspondence between both methods was relatively large.     

Myosin heavy chain isoform diversity in smooth muscle is produced by differential RNA processing

We have isolated and characterized two distinct myosin heavy chain cDNA clones from a neonatal rat aorta cDNA library. These clones encode part of the light meromyosin region and the carboxyl terminus of smooth muscle myosin heavy chain. The two rat aorta cDNA clones were identical in their 5' coding sequence but diverged at the 3' coding and in a portion of the 3' untranslated regions. One cDNA clone, RAMHC21, encoded 43 unique amino acids from the point of divergence of the two cDNAs. The second cDNA clone, RAMHC 15, encoded a shorter carboxyl terminus of nine unique amino acids and was the result of a 39 nucleotide insertion. This extra nucleotide sequence was not present in RAMHC21. The rest of the 3' untranslated sequences were common to both cDNA clones. Genomic cloning and DNA sequence analysis demonstrated that an exon specifying the 39 nucleotides unique to RAMHC15 mRNA was present, together with the 5' upstream common exons in the same contiguous stretch of genomic DNA. The 39 nucleotide exon is flanked on either side by two relatively large introns of approximately 2600 and 2700 bases in size. RNase protection analysis indicated that the two corresponding mRNAs were coexpressed in both vascular and non-vascular smooth muscle tissues. This is the first demonstration of alternative RNA processing in a vertebrate myosin heavy chain gene and provides a novel mechanism for generating myosin heavy chain protein diversity in smooth muscle tissues.     

Myosin II heavy chain null mutant of Dictyostelium exhibits defective intracellular particle movement

Both cellular motility and intracellular particle movement are compared between normal Dictyostelium amebae of strain AX4 and amebae of a myosin II heavy chain null mutant, HS2215, using the computer assisted Dynamic Morphology System. In AX4 cells rapidly translocating in buffer, cytoplasmic expansion is apical and the majority of intracellular particles move anteriorly, towards the site of expansion. When these cells are pulsed with 10(-6) M cAMP, the peak concentration of the natural cAMP wave, cells stop translocating and average particle velocity decreases threefold within 2-4 s after cAMP addition. After 8 s, there is a partial rebound both in cytoplasmic expansion and particle velocity, but in both cases, original apical polarity is lost. In HS2215 cells in buffer, both cellular translocation and average particle velocity are already at the depressed levels observed in normal cells immediately after cAMP addition, and no anterior bias is observed in either the direction of cytoplasmic expansion or the direction of particle movement. The addition of cAMP to myosin-minus cells results in no additional effect. The results demonstrate that myosin II is necessary for (a) the rapid rate of intracellular particle movement, (b) the biased anterior directionality of particle movement, and (c) the rapid inhibition of particle movement by cAMP.