Genetic analysis of myosin II assembly and organization in model organisms.

Myosins are a large family of actin-based motor proteins that are involved in a variety of cellular processes. Class II, or conventional, myosins are organized into a number of multi-component structures such as muscle thick filaments, non-muscle filaments and the actomyosin ring during cell division. A number of conditions must be met for the proper assembly and organization of myosin II-containing structures, including the correct stoichiometry of myosin and its associated proteins, and the conformation and regulation of the myosin molecule itself by molecular chaperones and protein kinases. In this review we discuss the use of model organisms in the genetic analysis of the assembly and organization of myosin-containing structures.

Myotonic dystrophy protein kinase phosphorylates the myosin phosphatase targeting subunit and inhibits myosin phosphatase activity.

Myotonic dystrophy protein kinase (DMPK) and Rho-kinase are related. An important function of Rho-kinase is to phosphorylate the myosin-binding subunit of myosin phosphatase (MYPT1) and inhibit phosphatase activity. Experiments were carried out to determine if DMPK could function similarly. MYPT1 was phosphorylated by DMPK. The phosphorylation site(s) was in the C-terminal part of the molecule. DMPK was not inhibited by the Rho-kinase inhibitors, Y-27632 and HA-1077. Several approaches were taken to determine that a major site of phosphorylation was T654. Phosphorylation at T654 inhibited phosphatase activity. Thus both DMPK and Rho-kinase may regulate myosin II phosphorylation.

STEM Analysis of Caenorhabditis elegans muscle thick filaments: evidence for microdifferentiated substructures.

In the thick filaments of body muscle in Caenorhabditis elegans, myosin A and myosin B isoforms and a subpopulation of paramyosin, a homologue of myosin heavy chain rods, are organized about a tubular core. As determined by scanning transmission electron microscopy, the thick filaments show a continuous decrease in mass-per-length (MPL) from their central zones to their polar regions. This is consistent with previously reported morphological studies and suggests that both their content and structural organization are microdifferentiated as a function of position. The cores are composed of a second distinct subpopulation of paramyosin in association with the alpha, beta, and gamma-filagenins. MPL measurements suggest that cores are formed from seven subfilaments containing four strands of paramyosin molecules, rather than the two originally proposed. The periodic locations of the filagenins within different regions and the presence of a central zone where myosin A is located, implies that the cores are also microdifferentiated with respect to molecular content and structure. This differentiation may result from a novel "induced strain" assembly mechanism based upon the interaction of the filagenins, paramyosin and myosin A. The cores may then serve as "differentiated templates" for the assembly of myosin B and paramyosin in the tapering, microdifferentiated polar regions of the thick filaments.

Differential assembly of alpha- and gamma-filagenins into thick filaments in Caenorhabditis elegans.

Muscle thick filaments are highly organized supramolecular assemblies of myosin and associated proteins with lengths, diameters and flexural rigidities characteristic of their source. The cores of body wall muscle thick filaments of the nematode Caenorhabditis elegans are tubular structures of paramyosin sub-filaments coupled by filagenins and have been proposed to serve as templates for the assembly of native thick filaments. We have characterized alpha- and gamma-filagenins, two novel proteins of the cores with calculated molecular masses of 30,043 and 19,601 and isoelectric points of 10.52 and 11.49, respectively. Western blot and immunoelectron microscopy using affinity-purified antibodies confirmed that the two proteins are core components. Immunoelectron microscopy of the cores revealed that they assemble with different periodicities. Immunofluorescence microscopy showed that alpha-filagenin is localized in the medial regions of the A-bands of body wall muscle cells whereas gamma-filagenin is localized in the flanking regions, and that alpha-filagenin is expressed in 1.5-twofold embryos while gamma-filagenin becomes detectable only in late vermiform embryos. The expression of both proteins continues throughout later stages of development. C. elegans body wall muscle thick filaments of these developmental stages have distinct lengths. Our results suggest that the differential assembly of alpha- and gamma-filagenins into thick filaments of distinct lengths may be developmentally regulated.

Rac-1 and Raf-1 kinases, components of distinct signaling pathways, activate myotonic dystrophy protein kinase.

Myotonic dystrophy protein kinase (DMPK) is a serine-threonine protein kinase encoded by the myotonic dystrophy (DM) locus on human chromosome 19q13.3. It is a close relative of other kinases that interact with members of the Rho family of small GTPases. We show here that the actin cytoskeleton-linked GTPase Rac-1 binds to DMPK, and coexpression of Rac-1 and DMPK activates its transphosphorylation activity in a GTP-sensitive manner. DMPK can also bind Raf-1 kinase, the Ras-activated molecule of the MAP kinase pathway. Purified Raf-1 kinase phosphorylates and activates DMPK. The interaction of DMPK with these distinct signals suggests that it may play a role as a nexus for cross-talk between their respective pathways and may partially explain the remarkable pleiotropy of DM.

Myotonic dystrophy protein kinase (DMPK) induces actin cytoskeletal reorganization and apoptotic-like blebbing in lens cells.

DMPK, the product of the DM locus, is a member of the same family of serine-threonine protein kinases as the Rho-associated enzymes. In DM, membrane inclusions accumulate in lens fiber cells producing cataracts. Overexpression of DMPK in cultured lens epithelial cells led to apoptotic-like blebbing of the plasma membrane and reorganization of the actin cytoskeleton. Enzymatically active DMPK was necessary for both effects; inactive mutant DMPK protein did not produce either effect. Active RhoA but not constitutive GDP-state mutant protein produced similar effects as DMPK. The similar actions of DMPK and RhoA suggest that they may function in the same regulatory network. The observed effects of DMPK may be relevant to the removal of membrane organelles during normal lens differentiation and the retention of intracellular membranes in DM lenses.

Protein machines and self assembly in muscle organization.

The remarkable order of striated muscle is the result of a complex series of protein interactions at different levels of organization. Within muscle, the thick filament and its major protein myosin are classical examples of functioning protein machines. Our understanding of the structure and assembly of thick filaments and their organization into the regular arrays of the A-band has recently been enhanced by the application of biochemical, genetic, and structural approaches. Detailed studies of the thick filament backbone have shown that the myosins are organized into a tubular structure. Additional protein machines and specific myosin rod sequences have been identified that play significant roles in thick filament structure, assembly, and organization. These include intrinsic filament components, cross-linking molecules of the M-band and constituents of the membrane-cytoskeleton system. Muscle organization is directed by the multistep actions of protein machines that take advantage of well-established self-assembly relationships.

Unc-45 mutations in Caenorhabditis elegans implicate a CRO1/She4p-like domain in myosin assembly.

The Caenorhabditis elegans unc-45 locus has been proposed to encode a protein machine for myosin assembly. The UNC-45 protein is predicted to contain an NH2-terminal domain with three tetratricopeptide repeat motifs, a unique central region, and a COOH-terminal domain homologous to CRO1 and She4p. CRO1 and She4p are fungal proteins required for the segregation of other molecules in budding, endocytosis, and septation. Three mutations that lead to temperature-sensitive (ts) alleles have been localized to conserved residues within the CRO1/She4p-like domain, and two lethal alleles were found to result from stop codon mutations in the central region that would prevent translation of the COOH-terminal domain. Electron microscopy shows that thick filament accumulation in vivo is decreased by approximately 50% in the CB286 ts mutant grown at the restrictive temperature. The thick filaments that assemble have abnormal structure. Immunofluorescence and immunoelectron microscopy show that myosins A and B are scrambled, in contrast to their assembly into distinct regions at the permissive temperature and in wild type. This abnormal structure correlates with the high degree of instability of the filaments in vitro as reflected by their extremely low yields and shortened lengths upon isolation. These results implicate the UNC-45 CRO1/She4p-like region in the assembly of myosin isoforms in C. elegans and suggest a possible common mechanism for the function of this UCS (UNC-45/CRO1/She4p) protein family.

Muscle thick filaments are rigid coupled tubules, not flexible ropes.

Understanding the structures of thick filaments and their relation to muscle contraction has been an important problem in muscle biology. The flexural rigidity of natural thick filaments isolated from Caenorhabditis elegans as determined by statistical analysis of their electron microscopic images shows that they are considerably more rigid (persistence length=263 microm) than similarly analyzed synthetic actin filaments (6 microm) or duplex DNA (0.05 microm), which are known to be helical ropes. Indeed, cores of C. elegans thick filaments, having only 11% of the mass per unit length of intact thick filaments, are quite rigid (85 microm) compared with the thick filaments. Cores comprise the backbones of the thick filaments and are composed of tubules containing seven subfilaments cross-linked by non-myosin proteins. Microtubules reconstituted from tubulin and microtubule-associated proteins are nearly as rigid (55 microm) as the cores. We propose a model of coupled tubules as the structural basis for the observed rigidity of natural thick filaments and other linear structures such as microtubules. A similar model was recently presented for microtubules [Felgner et al., 1997]. This coupled tubule model may also explain the differences in flexural rigidity between natural rabbit skeletal muscle thick filaments (27 microm) or synthetic thick filaments reconstituted from myosin and myosin binding protein C (36 microm) and those reconstituted from purified myosin (9 microm). The more flexible myosin structures may be helical ropes like F-actin or DNA, whereas the more rigid muscle or synthetic thick filaments which contain myosin and myosin binding protein C may be constructed of subfilaments coupled into tubules as in C. elegans cores. The observed thick filament rigidity is necessary for the incompressibility and lack of flexure observed with thick filaments in contracting skeletal muscle.

Developmental changes in expression of myotonic dystrophy protein kinase in the rat central nervous system.

Myotonic dystrophy protein kinase (DMPK) is the protein product of the genetic locus associated with myotonic dystrophy, in which alterations of muscle excitability, cardiac conduction defects, mental retardation, and cognitive deficiencies are inherited as an autosomal dominant trait. DMPK belongs to a novel protein serine/threonine kinase family, but its regulation and physiological functions have not been specified. In a first step toward understanding the functions of DMPK in the central nervous system, we have characterized its localization and developmental pattern of expression in rat brain and spinal cord by using a monospecific rabbit antiserum produced against bacterially expressed DMPK. Expression of DMPK begins after birth and increases gradually to peak at postnatal day 21 with antibody labeling of neuronal cell types in many regions. After postnatal day 21 and proceeding to the adult, the pattern of expression becomes more restricted, with localization to certain regions or cell groups in the central nervous system. Electron microscopy reveals localization within adult spinal motor neurons to the endoplasmic reticulum and dendritic microtubules. The adult localizations suggest that DMPK may function in membrane trafficking and secretion within neurons associated with cognition, memory, and motor control.

beta-Filagenin, a newly identified protein coassembling with myosin and paramyosin in Caenorhabditis elegans.

Muscle thick filaments are stable assemblies of myosin and associated proteins whose dimensions are precisely regulated. The mechanisms underlying the stability and regulation of the assembly are not understood. As an approach to these problems, we have studied the core proteins that, together with paramyosin, form the core structure of the thick filament backbone in the nematode Caenorhabditis elegans. We obtained partial peptide sequences from one of the core proteins, beta-filagenin, and then identified a gene that encodes a novel protein of 201-amino acid residues from databases using these sequences. beta-Filagenin has a calculated isoelectric point at 10.61 and a high percentage of aromatic amino acids. Secondary structure algorithms predict that it consists of four beta-strands but no alpha-helices. Western blotting using an affinity-purified antibody showed that beta-filagenin was associated with the cores. beta-Filagenin was localized by immunofluorescence microscopy to the A bands of body-wall muscles, but not the pharynx. beta-filagenin assembled with the myosin homologue paramyosin into the tubular cores of wild-type nematodes at a periodicity matching the 72-nm repeats of paramyosin, as revealed by immunoelectron microscopy. In CB1214 mutants where paramyosin is absent, beta-filagenin assembled with myosin to form abnormal tubular filaments with a periodicity identical to wild type. These results verify that beta-filagenin is a core protein that coassembles with either myosin or paramyosin in C. elegans to form tubular filaments.

Assemblases and coupling proteins in thick filament assembly.

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.

Induction of glyoxylate cycle expression in Caenorhabditis elegans: a fasting response throughout larval development.

The mRNA and the bifunctional protein for the two glyoxylate cycle (GC) enzymes, isocitrate lyase and malate synthase, are expressed in a tissue- and stage-specific pattern in Caenorhabditis elegans. Since expression of the two enzymes for the carbon-conserving glyoxylate cycle is regulated by the availability of carbon sources in microorganisms, we have studied the bifunctional GCP gene expression under fasting conditions and in certain mutants of C. elegans in order to understand possible mechanisms regulating its expression during nematode development. The GCP mRNA and protein levels were elevated in early larvae which were never fed, a result consistent with previous enzyme activity measurements (Khan, F.R., & McFadden, B.A. (1982) Exp. Parasitol. 54, 48-54]. However, larvae of later stages also expressed higher levels of GCP mRNA and protein when they were shifted from normal to fasting growing conditions. The GCP expression appeared to be regulated primarily at the transcriptional level throughout development. Although the expression of both the GCP gene and lin-14 peaks at about the same time during development and are induced by fasting, the regulation of the GCP gene is independent of the heterochronic lin-14 control mechanism of postembryonic lineages, as demonstrated by the fact that there was no significant change of the GCP at both mRNA and protein levels in the heterochronic lin-4 (lf) and lin-14 (gf) mutants compared to the wild type.

Molecular cloning of a splice variant of Caenorhabditis elegans YNK1, a putative element in signal transduction.

YNK1 is a 98.3-kDa protein whose sequence was originally deduced from a genomic sequence in Caenorhabditis elegans. It was recently found that YNK1 is homologous to three different proteins implicated in signal transduction, suggesting that YNK1 is a signal transduction protein. In this report we describe the isolation of a full-length cDNA that encodes a splice variant of YNK1, designated YNK1a. We also present evidence that both YNK1 and YNK1a transcripts exist in vivo. Furthermore, using an antibody raised against a YNK1a recombinant protein, we demonstrate that the YNK1 protein is expressed in vivo throughout development.

Myotonic protein kinase expression in human and bovine lenses.

Myotonic dystrophy (DM) is an autosomal dominant trait closely associated with CGT repeat expansions in the same locus on human chromosome 19q13.3. The expansions occur in the 3'untranslated region of a transcription unit encoding a serine-threonine kinase (DM kinase) of a new class based upon structure and function. Lens cataracts are a prominent finding in myotonic dystrophy. DM kinase was shown to be expressed in human and bovine lenses at the RNA level and in human lenses at the protein level. Sequencing of PCR products of RNA extracted from normal human lenses demonstrated an exact match to published genomic and cDNA 3' UTR sequences. Northern blots of bovine lens RNA showed that the transcript is similar in size to the transcript detected in other tissues that are affected in myotonic dystrophy. A polyclonal antibody (DM-2) was produced against recombinant DM protein kinase in rabbits. Development of Western blots with DM-2 showed a single reactive band of 67 kDa. Immunofluorescent studies of formalin-fixed human lens sections detected the DM kinase in the perinuclear cytoplasm of normal human lens epithelial cells and more diffusely in superficial subcapsular cortical fibers. In contrast, the same antibody labeled the nucleus most prominently in a single DM lens.

Localization of myotonic dystrophy protein kinase in skeletal muscle and its alteration with disease.

Myotonic dystrophy (DM) is an autosomal dominant disorder which affects skeletal muscle, heart, eye lens, brain, and endocrine functions. The disease-causing mutations are expansions of the triplet repeat CTG in the 3' untranslated region of a locus which encodes a serine/threonine protein kinase that represents a new family of protein kinases. A monoclonal antibody to a recombinant DM protein kinase (mAb DM-1) reacts specifically with the 64 kDa isoform of DM protein kinase in type I fibers in skeletal muscle, the fiber type which characteristically atrophies in the disease. Within type I fibers of normal muscle the isoform may be localized with mAb DM-1 to the triad region. In the DM disease state, the enzyme is redistributed to the pathologically characteristic peripheral sarcoplasmic masses. In markedly affected human distal myotonic muscle, the levels of the 64 kDa DM kinase isoform are elevated relative to slow skeletal myosin heavy chain. These results suggest that, consistent with the dominant clinical phenotype, the localization and accumulation of the 64 kDa isoform are altered in the heterozygous disease state.

Evidence that apoB-100 of low-density lipoproteins is a novel Src-related protein kinase.

Protein-tyrosine kinases of signal transduction pathways occur and function intracellularly. In contrast, the low-density lipoprotein (LDL) particle circulates in plasma, where its function is to solubilize and transport lipid. Recently, several reports showed that LDL may have a role in signal transduction. We have identified a region in the apoB-100 primary structure which shows similarity to Src-homology-1 (SH1) domains, the kinase region of protein-tyrosine kinases. Results obtained in protein kinase assays of highly purified LDL showed that only the apoB-100 was phosphorylated, suggesting that apoB-100 has the capacity to undergo autophosphorylation like known protein-tyrosine kinases. Phosphorylation was not observed for any other apolipoprotein in LDL or for any component of high-density lipoprotein and lipoprotein [a]. Our results suggest that apoB-100 may be a novel and functional member of the src protein kinase family.

Preliminary three-dimensional model for nematode thick filament core.

Understanding the structure and the mechanism of assembly of thick filaments have been long-standing problems in the field of muscle biology. Cores which represent the backbones of thick filaments and consist of paramyosin and associated proteins were isolated from the nematode Caenorhabditis elegans. Electron microscopy of negatively stained and frozen hydrated cores was performed. The resulting images were analyzed by computing their Fourier transforms, three-dimensional reconstruction, and by modeling. A preliminary three-dimensional model is proposed in which the paramyosin constitutes an outer sheath of seven subfilaments about a set of inner 54-nm-long tubules which repeat every 72 nm. The subfilaments are not closely packed but require cross-linking by the internal tubules. Each subfilament consists of two strands of paramyosin molecules which are staggered by 72 nm with respect to one another. This stagger introduces a 22-nm gap between consecutive paramyosin molecules in each strand. An offset of the center of the inner tubules relative to the center of the gap of 6 nm was consistent with the images and their transforms. This model suggests that the nonhelical ends of paramyosin and the unpaired gap between adjacent paramyosin molecules contain sites for the interaction with the inner tubular proteins. The molecular interactions at this locus would appear to be critical in the assembly of thick filaments and their regulation.

Cardiac involvement in a large kindred with myotonic dystrophy. Quantitative assessment and relation to size of CTG repeat expansion.

OBJECTIVE: To evaluate and quantitate cardiac involvement in myotonic dystrophy and assess whether the size of the trinucleotide (cytosine-thymine-guanine [CTG]) repeat expansion is a significant predictor of cardiac abnormalities. DESIGN: Case-control study of a large kindred with myotonic dystrophy. PATIENTS: Ninety-one bloodline members of the kindred underwent clinical and cardiac evaluation with electrocardiograms, echocardiography (with Doppler in the majority of cases), and genetic and neurologic evaluations. Affected individuals were age-matched to normal family members. MAIN OUTCOME MEASURES: Electrocardiographic conduction abnormalities, wall motion abnormalities, mitral valve prolapse, and global parameters of systolic and diastolic function were determined by an observer blinded to all clinical data and genetic analysis. RESULTS: Compared with age-matched normals, patients with myotonic dystrophy (n = 25) were more likely to have conduction abnormality (52% vs 9%), mitral valve prolapse (32% vs 9%), and wall motion abnormality (28% vs 0%) (all P < .05). Left ventricular ejection fraction and stroke volume were reduced compared with normals matched for age and heart rate (P < .05), whereas Doppler indexes of diastolic function were only marginally altered. Using multivariate analysis, the number of CTG repeats (range, 69 to 1367; normal, < or = 37) was the strongest predictor of abnormalities in wall motion and electrocardiographic conduction (odds ratio of 16.5 and 5.07 per 500 repeats, respectively). The relation of mitral valve prolapse to the size of the CTG repeat was of borderline significance. Patients with more extensive neurologic findings (n = 12) had a higher incidence of wall motion and/or electrocardiographic conduction abnormalities (83% vs 43%; P = .04). CONCLUSIONS: Cardiac involvement in myotonic dystrophy affects predominantly the conduction system and myocardial function. Alterations in myocardial relaxation and diastolic properties, in contrast to skeletal muscle myotonia, are minor. In this kindred, the number of CTG repeats was a significant predictor of cardiac dysfunction in myotonic dystrophy.

Bifunctional glyoxylate cycle protein of Caenorhabditis elegans: a developmentally regulated protein of intestine and muscle.

The reaction of an abundant 106-kDa polypeptide with a specific monoclonal antibody has been localized in intestinal and muscle cells of the nematode Caenorhabditis elegans. This protein was first detected in 4-6 cells of the clonal E lineage of 100-cell embryos. This lineage is committed to the intestinal cell fate. The antigen continued to be expressed in the differentiating gut and then appeared in early differentiating body wall muscle cells of 400- to 500-cell embryos. Molecular cloning and sequencing showed that the largest cDNA clone contained 3274 bp and encoded a sequence of 1005 amino acids. The predicted polypeptide of 112,799 MW contains separate domains for the glyoxylate cycle enzymes isocitrate lyase and malate synthase. Their enzymatic activities had been shown previously to be highest in embryos and L1 larvae (Khan, F. R., and McFadden, B. A. (1980). FEBS Lett. 115, 312-314; Khan, F. R., and McFadden, B. A. (1982). Exp. Parasitol. 54, 48-54; Wadsworth, W. G., and Riddle, D. L. (1989). Dev. Biol. 132, 167-173). The domain-specific sequences were shown to be contiguous in genomic DNA and are separated by an intron of 68 bp. A single polypeptide and both enzymatic activities are precipitated by the antibody, and peptide fragments resulting from limited proteolytic digestion contained amino acid sequences which overlap the predicted junctional region. The physical localization of the gene correlates with a small region of the left arm of Linkage Group V to which multiple embryonic lethal mutations have been mapped.