Rod Portion Research Articles

Cross-linking of contractile proteins from skeletal muscle by treatment with microbial transglutaminase.

The action on muscle proteins of microbial transglutaminase (MTGase), which catalyzes the formation of a "zero-length" covalent cross-link between glutamine and lysine residues in peptides, was studied in order to define a basis for future application of MTGase cross-linking to the study of muscle protein interaction. We examined the cross-linking of skeletal muscle myosin, myosin subfragments, actin, and myofibrils by treatment with MTGase and the possible side-effects of the cross-linking on the enzymic activity of myosin, and found that the rod portions of myosin in myosin filaments were quickly cross-linked to each other by the action of MTGase, but myosin subfragment 1 was not cross-linked to actin. The MgATPase activities at 0.5 M KCl of myosin, heavy meromyosin, subfragment 1, and subfragment 1-actin were not significantly affected by the MTGase reaction. A very small fraction of the head portion of heavy meromyosin was cross-linked to actin in their rigor complexes by MTGase, and the ATPase activity at 0.5 M KCl of the cross-linked heavy meromyosin-actin complexes was slightly enhanced.

Neonatal and adult myosin heavy chains form homodimers during avian skeletal muscle development.

Myosin isoforms contribute to the heterogeneity and adaptability of skeletal muscle fibers. Besides the well-characterized slow and fast muscle myosins, there are those isoforms that appear transiently during the course of muscle development. At a stage of development when two different myosins are coexpressed, the possibility arises for the existence of heterodimers, molecules containing two different heavy chains, or homodimers, molecules with two identical heavy chains. The question of whether neonatal and adult myosin isoforms can associate to form a stable heterodimer was addressed by using stage-specific monoclonal antibodies in conjunction with immunological and electron microscopic techniques. We find that independent of the ratio of adult to neonatal myosin, depending on the age of the animal, the myosin heavy chains form predominantly homodimeric molecules. The small amount of hybrid species present suggests that either the rod portion of the two heavy chain isoforms differs too much in sequence to form a stable alpha-helical coiled coil, or that the biosynthesis of the heavy chains precludes the formation of heterodimeric molecules.

Rabbit skeletal muscle myosin unfolded carboxyl‐terminus and its role in molecular assembly

We have expressed in E. coli segments of the rod portion of rabbit skeletal fast muscle myosin and compared physical properties of two different species, LMM-30 and LMM-30C'. LMM-30 consists of 263 amino acids including the original C-terminus of myosin heavy chain. LMM-30C' is colinear with LMM-30, but is devoid of 17 residues at the C-terminus. 1H NMR spectroscopy indicates that the C-terminus of LMM-30, but not of LMM-30C' is unfolded and freely mobile. Furthermore, the present results show that the unfolded C-terminus is essential for molecular assembly of LMM-30; at pH 8.0 LMM-30, but not LMM-30C', formed aggregates upon decreasing the ionic strength.

Flexibility of myosin in pyrophosphate and NaCl solutions. An electric birefringence study.

The orientational relaxation time of myosin has been reported as 38 microseconds when measured in pyrophosphate media at elevated pH (Hvidt et al. 1984) and as 17 microseconds when measured in 0.3 M KCl at pH 7.3 (Bernengo and Cardinaud 1982). This discrepancy, which is reexamined in the present report, suggests that in KCl solution the rod portion of the myosin molecule is bent with an average angle close to 110 degrees, whereas in pyrophosphate at elevated pH it assumes a nearly straight and rigid conformation. Electric birefringence shows that the amount of dimeric and polymeric species in pyrophosphate media at pH's 8.0 and 8.5 is certainly greater than usually thought. In these media, relaxation times can be measured correctly at pH 9.0. A comparative analysis of the influence of protein concentration, field strength, medium composition and concentration, pH and temperature showed that a high relaxation time is associated with the presence of pyrophosphate and that the myosin tail is significantly stiffened in the presence of this anion.

Localization of myosin in the cytoskeleton of brush border cells using monoclonal antibodies and confocal laser-beam scanning microscopy

Monoclonal antibodies binding to the rod portion of brush border myosin were used to localize myosin in chicken intestinal brush border cells by indirect immunofluorescence. Isolated cells, or cells still attached in a sheet, were analyzed by conventional epifluorescence microscopy, which showed that most of the immunoreactive myosin is localized in the apical brush border (terminal web), and in a basal region. In addition, a weak, diffuse granular and rod-like labeling was detected throughout the cell body. Using the laser-scanning confocal microscope (White et al., 1987), a more precise localization of the myosin within the terminal web and the cell body was obtained. In the terminal web, most of the myosin was concentrated in a circumferential ring, below the plasma membrane, and the remaining myosin was found in the inter-rootlet area. These two populations of myosin were topologically strictly related, since they were found in the same optical sections. In the cell body, as well as in the basal region, the myosin was found to be associated with the outer limiting membrane of the cell, in a cortical location, whereas essentially no myosin was detected in the cytoplasm.

Analysis of myosins from lobster muscles: Fast and slow isozymes differ in heavy‐chain composition

AbstractThe contractile speed of striated muscle is correlated with myosin adenosine triphosphatase (ATPase) activity. In crustaceans, ATPase activity in fast muscle is about four‐fold greater than that in slow muscle. Slow and fast myosins were purified from the crusher claw closer and the deep abdominal muscles, respectively, of teh American lobster, Homarus americanus, and analyzed by peptide mapping, chymotryptic digestion, and two‐dimensional polyacrylamide gel electrophoresis (PAGE). Peptide mapping of myosin heavy chains with Staphylococcus aureus V8 protease produced seven peptides unique to slow‐muscle myosin and 11 peptides unique to fastmuscle myosin. Heavy‐chain fragments produced by chymotryptic digestion of native myosin differed in molecular weight and isoelectric point between the two muscle types, suggesting possible differences in the flexible hinge region in the rod portion of the molecule. In contrast, twodimensional PAGE showed that the corresponding light chains of both fast and slow myosins were identical in size and net charge. Therefore, the molecular basis of the difference in ATPase activities between fast‐ and slow‐muscle myosins can be attributed solely to heavy‐chain isoform composition.

Ultrastructural localization of dystropin in human muscle by using gold immunolabelling

Immunolabelling with a 5 nm gold probe was used to localize dystrophin at the ultrastructural level in human muscle. The primary antibody was monoclonal, raised against a segment (amino acids 1181-1388) from the rod domain of dystrophin. The antibody (Dy4/6D3) is specific for dystrophin and shows no immunoreactivity with any protein from mdx mouse muscle or from patients with a gene deletion spanning part of the molecule recognized by the antibody (Nicholson et al. 1989 a; England et al. 1990). Using this antibody, labelling was almost entirely confined to a narrow 75 nm rim at the periphery of the muscle fibres. Histograms of the distance from the gold probe to the cytoplasmic face of the plasma membrane and of the distance between gold probes (nearest neighbour in a plane parallel with the plasma membrane) displayed modes at approximately 15 nm and 120 nm, respectively. The distribution of the probe was the same in longitudinal and transverse sections of the muscle. These observations suggest that the rod portion of the dystrophin molecule is normally arranged close to the cytoplasmic face of the plasma membrane and that the molecules form an interconnecting network. Labelling was not associated with the transverse tubular system.

Generation of a full-length human perinatal myosin heavy-chain-encoding cDNA

Vertebrate sarcomeric myosin heavy chains (MHC) are encoded by multigene families whose members show tissue-specific and developmentally-regulated patterns of expression. Molecular genetic studies have allowed the cloning of a small number of complete genes or cDNAs encoding MHC isoforms [see Warrick and Spudich, Annu. Rev. Cell Biol. 3 (1987) 379-421]. Reported here is the isolation and sequence of a 2.6-kb cDNA that encodes the subfragment 1 or head of a human perinatal skeletal MHC. A cDNA sequence encoding the rod portion of this isoform has been previously reported [Feghali and Leinwand, J. Cell Biol. 108 (1989) 1791-1797]. Polymerase chain reaction with fetal skeletal muscle RNA was used to join the two nonoverlapping cDNA sequences to construct a full-length sequence. The gene encoding the perinatal skeletal MHC has been localized to the cluster of skeletal MHC-encoding genes on chromosome 17.

Identification of a Putative Binding Site for a Sea Urchin 53‐kD Myosin Binding Proteina

The actomyosin cytoskeleton provides the structural framework for an array of directed cellular movements such as cytokinesis and morphogenesis. Myosin is possibly the major mechanochemical transducer in such movements. The exact structural state of myosin in nonmuscle cells is unknown and may involve cycles of assembly and disassembly.’ Therefore, the regulation of myosin structure and distribution in cells may have a fundamental physiologic role. In a number of cells the redistribution of myosin has been observed to correlate with changes in cell state.’,’ Myosin’s catalytic activity resides in the head of the molecule, and phosphorylation of myosin light chains in this region mediates conformational changes necessary for the regulation of activity. The formation of bipolar filaments is a function of the rod portions of myosin heavy chain. The head-rod junction of myosin heavy chain contains the binding sites for the light chains and plays a role in conformational changes;’ This head-rod junctional region retains a large degree of flexibility. Thus, the neck region determines myosin’s shape and, consequently, filament assembly and enzymatic activity?’ We have identified a myosin-binding protein in sea urchin eggs that mediates myosin’s low ionic strength solubility. This protein has the subunit molecular weight of 53 kD and binds to myosin in a nucleotide-dependent manner. In this study the 53K binding site on myosin was examined to determine the mechanism of interaction that gives rise to a change in myosin’s properties. We used several methods to identify the 53K binding site on the myosin molecule (summarized in FIGURE 1). Myosin and 53K were isolated from soluble sea urchin extracts as described by Yabkowitz and Burgess6 Papain and chymotrypsin digests were performed on myosin and the insoluble and soluble fragments were separated according to Barylko et al.’ Papain gives rise to 165, 130, 107, 80, 62 and 24-kD fragments. The 80, 62, and 24kD fragments are head fragments as defined by actin binding and ATPase activity

Covalent cross-linking of single fibers from rabbit psoas increases oscillatory power

Single fibers from chemically skinned rabbit psoas muscle were treated with 1-ethyl-3-[3-dimethyl-amino)proyl]-carbodiimide (EDC) at 20 degrees C after rigor was induced. A 22-min treatment resulted in 18% covalent cross-linking between myosin heads and the thin filament as determined by stiffness measurements. This treatment also results in covalent cross-linking among rod portions of myosin molecules in the backbone of the thick filament. The fibers thus prepared are stable and do not dissolve in solutions at ionic strengths as high as 1,000 mM. The preparation was subjected to sinusoidal analysis, and the resulting complex modulus data were analyzed in terms of three exponential processes, (A), (B), and (C). Oscillatory work (process B) was much greater in the cross-linked fibers than in untreated ones in activating solutions of physiological ionic strength (200 mM); this difference was attributed to the decline of process (A) with EDC treatment. Consequently, the Nyquist plot of the EDC-treated preparation exhibited an insect-type response. We conclude that, under these conditions, both cross-linked and non-cross-linked myosin heads contribute to the production of oscillatory power. The cross-linked preparations also exhibited oscillatory work in high ionic strength (500-1,000 mM) solutions, indicating that cross-linked myosin heads are capable of utilizing ATP to produce work. We conclude that process (A) does not relate to an elementary step in a cross-bridge cycle, but it may relate to dynamics outside the cross-bridge such as filament sliding or sarcomere rearrangement.

Differential distribution of myosin isoforms among the myofibrils of individual developing muscle fibers.

Myosin was localized in situ in the posthatch chicken pectoralis using isoform-specific mAbs. The distribution among myofibrils was demonstrated by immunofluorescence and by immunogold EM. Fluorescein- or rhodamine-labeled antibody (12C5) specific for the head region (S1) of myosin was used as a marker to identify "embryonic" myosin. In longitudinal semithin frozen sections, a minority population of myofibrils stained intensely with 12C5. All other myofibrils in the same cell stained only weakly. Similarly, in Lowicryl-embedded ultrathin sections prepared for EM, a minority population reacted preferentially with gold-labeled 12C5. An antibody (5B4) specific for the rod portion of "neonatal" myosin reacted strongly with nearly all myofibrils, and this was evident by light and electron microscopy. A few of the fibrils that reacted strongly with 12C5 reacted weakly with 5B4. These observations demonstrate that an epitope reacting with 12C5 is more abundant in some myofibrils than in others within the same cell. Three categories of myofibrils can be identified by their relative proportions of embryonic and neonatal forms of myosin: in nearly all fibrils, a neonatal isoform predominates; in a minority population, embryonic and neonatal isoforms are both abundant; and in a few fibrils, an embryonic isoform predominates. It is concluded that there are distinct populations of myofibrils in which specific isoforms are segregated within an individual cell.

Radio-immunolocalization in Nude Mice Using Anticytokeratin Monoclonal Antibodies

Four monoclonal antibodies reactive with the rod portion of human cytokeratin No. 8 were evaluated using experimental tumour radio-immunolocalization. Nude mice carrying human HeLa cell tumours were injected with 125I-labelled F(ab')2 fragments or intact IgG antibodies. Positive immunolocalization was observed with all the specific antibodies, the intact antibodies being superior to their analogous F(ab')2 fragments. High localization efficiency was observed despite the fact that the targets are intracellular filaments. The presence of necrotic areas in the tumours may favour the high uptake in the tumours. By computerized gamma-scintigraphic evaluation of the antibody distribution in the animals, several kinetic parameters of the antibody behaviour in vivo could be determined. The biological half-lives in the tumours were determined to be greater than 600 h for some of the antibodies compared to a half-life of 40-80 h in non-tumour tissues. Optimal imaging was obtained as late as 10-14 days after administration of the antibody. Up to 10% of the injected antibody could be identified in the tumours, indicating a potential value in radio-immunolocalization and radio-immunotherapy using anticytokeratin antibodies.

Yeast myosin heavy chain mutant: Maintenance of the cell type specific budding pattern and the normal deposition of chitin and cell wall components requires an intact myosin heavy chain gene

Recent studies with myosin heavy chain mutants in the slime mold Dictyostelium discoideum and the yeast Saccharomyces cerevisiae indicate that the myosin heavy chain gene is not essential for cell survival under laboratory growth conditions. However, cells lacking a normal myosin heavy chain gene demonstrate substantial alterations in growth and cell division. In this study, we report that a disruption mutant in the rod portion of the yeast myosin heavy chain gene, MYO1, produces abnormal chitin distribution and cell wall organization at the mother-bud neck in a high proportion of dividing cells. It is suggested that this phenotype is the cause of the cell division defect and the osmotic sensitivity of yeast MYO1 mutants. In the absence of a normal MYO1 polypeptide, yeast cells alter their cell type specific budding pattern. It is concluded that an intact myosin heavy chain gene is required to maintain the cell type specific budding pattern and the correct localization and deposition of chitin and cell wall components during cell growth and division.

Diversity in immunoreactivity of tumor-derived cytokeratin monoclonal antibodies.

The cytokeratins 8, 18, and 19, expressed in many normal and malignant epithelial cells, were purified from human gastrointestinal tumors and used as immunogen for hybridoma generation. The reactivity pattern of five of the generated ten monoclonal antibodies (MAb) was characterized biochemically and immunohistochemically. All of the generated MAb were reactive with the central rod portion of the cytokeratins, as determined after partial enzymatic degradation, and displayed characteristic reactivity patterns. MAb TS 4 exhibits pan-epithelial immunohistochemical reactivity staining of all epithelial structures, including all layers of epidermis and non-keratinizing squamous epithelium and myoepithelial cells. The determinant involved is present on several different cytokeratins, i.e., nos. 1, 5, 7, 8, and 15, as determined by immunoblotting experiments from different tissues and cell lines. MAb TS 1, TS 3, and TS 7 reveal pluri-epithelial reactivity pattern immunohistochemically, similar to TS 4, but they are unreactive with whole epidermis and with superficial cell layers of non-keratinizing squamous cells. MAb TS 1 was found to be highly specific and reactive only with cytokeratin 8. Furthermore, the TS 1 MAb alone can precipitate the antigen, indicating reactivity with repetitive epitopes on cytokeratin 8. MAb TS 3 and 7 bind to cytokeratins 7 and 8. Finally, MAb TS 8 was found to be immunohistologically the most restricted, in general lacking reactivity to hepatocytes, pancreatic and salivary gland acinar cells, proximal renal tubules, and luminal cells of the epididymis. TS 8 was mainly reactive with cytokeratin 19 and showed weak binding to cytokeratin 8 and 14.

Molecular interactions in paracrystals of a fragment corresponding to the alpha-helical coiled-coil rod portion of glial fibrillary acidic protein: evidence for an antiparallel packing of molecules and polymorphism related to intermediate filament structure.

We have expressed in Escherichia coli a fragment of c-DNA that broadly corresponds to the alpha-helical coiled-coil rod section of glial fibrillary acidic protein (GFAP) and have used the resultant protein to prepare paracrystals in which molecular interactions can be investigated. An engineered fragment of mouse GFAP c-DNA was inserted into a modified version of the E. coli expression vector pLcII, from which large quantities of a lambda cII-GFAP rod fusion protein were prepared. A protein fragment corresponding to the GFAP rod was then obtained by proteolysis with thrombin. Paracrystals of this material were produced using divalent cations (Mg, Ca, Ba) in the presence of a chaotrophic agent such as thiocyanate. These paracrystals showed a number of polymorphic patterns that were based on a fundamental pattern that had dyad symmetry and an axial repeat of 57 nm. Analysis of both positive and negative staining patterns showed that this fundamental pattern was consistent with a unit cell containing two 48-nm-long molecules in an antiparallel arrangement with their NH2 termini overlapping by approximately 34 nm. More complicated patterns were produced by stacking the fundamental pattern with staggers of approximately 1/5, 2/5, and 1/2 the axial repeat. The molecular packing the unit cell was consistent with a range of solution studies on intermediate filaments that have indicated that a molecular dimer (i.e., a tetramer containing four chains or two coiled-coil molecules) is an intermediate in filament assembly. Moreover, these paracrystals allow the molecular interactions involved in the tetramer to be investigated in some detail.

Immunochemical and immunocytochemical identification of a myosin heavy chain polypeptide in Nicotiana pollen tubes.

A myosin heavy chain polypeptide has been identified and localized in Nicotiana pollen tubes using monoclonal anti-myosin antibodies. The epitopes of these antibodies were found to reside on the myosin heavy chain head and rod portion and were, therefore, designated anti-S-1 (myosin S-1) and anti-LMM (light meromyosin). On Western blots of the total soluble pollen tube proteins, both anti-S-1 and anti-LMM label a polypeptide of approximately 175,000 Mr. Immunofluorescence microscopy shows that both antibodies yield numerous fluorescent spots throughout the whole length of the tube, often with an enrichment in the tube tip. These fluorescent spots are thought to represent vesicles and/or organelles in the pollen tubes. In addition to this common pattern, anti-S-1 stains both the generative cell and the vegetative nuclear envelope. The different staining patterns of the nucleus between anti-S-1 and anti-LMM may be caused by some organization and/or anchorage state of the myosin molecules on the nuclear surface that differs from those on the vesicles and/or organelles.

Release of flagellar filament-hook-rod complex by a Salmonella typhimurium mutant defective in the M ring of the basal body.

A Salmonella typhimurium strain possessing a mutation in the fliF gene (coding for the component protein of the M ring of the flagellar basal body) swarmed poorly on a semisolid plate. However, cells grown in liquid medium swam normally and did not show any differences from wild-type cells in terms of swimming speed or tumbling frequency. When mutant cells were grown in a viscous medium, detached bundles of flagellar filaments as long as 100 microns were formed and the cells had impaired motility. Electron microscopy and immunoelectron microscopy revealed that the filaments released from the cells had the hook and a part of the rod of the flagellar basal body still attached. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis showed that the rod portion of the released structures consisted of the 30-kilodalton FlgG protein. Double mutants containing this fliF mutation and various che mutations were constructed, and their behavior in viscous media was analyzed. When the flagellar rotation of the mutants was strongly biased to either a counterclockwise or a clockwise direction, detached bundles were not formed. The formation of large bundles was most extreme in mutants weakly biased to clockwise rotation.

Effect of chronic lithium on sensitivity to light in male and female bipolar patients

1. Sensitivity to white light was quantified in euthymic bipolar male and female patients maintained on long term lithium therapy and age and sex matched unmedicated controls. 2. The Dark Adaptation Threshold procedure was used to assess sensitivity of both the cone and rod photoreceptors to short pulses of light. 3. Male and female controls did not differ in sensitivity to light. 4. Male patients in comparison to both controls and female patients evidenced reduced sensitivity to light during the cone and rod portion of the dark adaptation procedure. 5. Female patients did not differ from controls on sensitivity to light. 6. Using these and other published data the results were interpreted as suggesting that lithium reduces sensitivity to light during adaptation to dark.

Regulatory light chain-a myosin kinase (aMK) catalyzes phosphorylation of smooth muscle myosin heavy chains of scallop, Patinopecten yessoensis.

Regulatory light chain-a myosin kinase (aMK), which phosphorylates one of the myosin regulatory light chains, RLC-a, contained in the catch muscle of scallop, was also found to phosphorylate heavy chains of scallop myosin. After incubation of myosin isolated from the opaque portion of scallop smooth muscle (opaque myosin) with aMK in the presence of [gamma-32P]ATP, about 2 mol of 32P was incorporated per mol of the myosin. The radioactivity was mostly found in the heavy chain at 0.26 M KCl. The pH-activity curve and MgCl2 requirement for the heavy chain phosphorylation were similar to those for RLC-a phosphorylation. In contrast, the dependency of activity on KCl concentration was different from that for RLC-a. The heavy chain phosphorylation activity decreased with increase in KCl concentration up to 0.06 M, and then increased at concentrations over 0.06 M to a maximum at around 0.26 M KCl. This complicated profile probably reflects the solubility of myosin, and the phosphorylation site may be located in the rod portion insoluble at low KCl concentrations. Phosphorylation of heavy chain did not change the solubility of the opaque myosin molecule at all. The acto-opaque myosin ATPase activity in the presence of Ca2+ was found to be decreased to less than one-fourth by the heavy chain phosphorylation.

Brush border myosin filament assembly and interaction with actin investigated with monoclonal antibodies.

Monoclonal antibodies binding to epitopes in the rod portion of brush border myosin were used to study the mechanism of filament assembly and its role in myosin interaction with actin. The antibodies and their Fab fragments had specific effects on the size of the filaments assembled in vitro. Two antibodies (BM3 and BM4), directed against the tip of the myosin tail, completely inhibited myosin filament assembly. The other antibodies (BM1, BM2 and BM5), binding to other sites along the myosin rod, only partially blocked filament growth, and short filaments could be assembled. Thiophosphorylated brush border myosin filaments appeared slightly more stable to the effects of the antibodies than those composed of dephosphorylated myosin. Only one (BM3) of the antibodies which completely inhibited the assembly of new filaments was capable of disassembling preformed myosin filaments. The other antibody, BM4, partially disassembled filaments, leaving approximately 0.2-microns long 'cores', suggesting that polymerization in this myosin occurs by a biphasic mechanism, i.e. the formation of a stable nucleus of antiparallely packed molecules, followed by elongation. The antibodies BM1 and BM2 bound to myosin filaments generating a regular transverse pattern with a approximately 14-nm periodicity, and had little effect on the stability of these preformed filaments. Inhibition of filament formation and solubilization of the myosin by the antibodies appeared to be associated with inhibition of myosin interaction with actin, as measured by the actin-activated MgATPase activity. In the presence of the antibodies which completely inhibit filament assembly, we observed a decrease to approximately 20% (BM4-Fab) and to approximately 50% (BM3) of the control actin-activated myosin MgATPase activity, and this activity was kinetically different from that of the soluble myosin S1 fragment, suggesting that the rod has a profound effect on the kinetics of actomyosin interaction.