Axial arrangement of the myosin rod in vertebrate thick filaments: immunoelectron microscopy with a monoclonal antibody to light meromyosin.

Abstract

A monoclonal antibody, MF20, which has been shown previously to bind the myosin heavy chain of vertebrate striated muscle, has been proven to bind the light meromyosin (LMM) fragment by solid phase radioimmune assay with alpha-chymotryptic digests of purified myosin. Epitope mapping by electron microscopy of rotary-shadowed, myosinantibody complexes has localized the antibody binding site to LMM at a point ~92 nm from the C-terminus of the myosin heavy chain. Since this epitope in native thick filaments is accessible to monoclonal antibodies, we used this antibody as a high affinity ligand to analyze the packing of LMM along the backbone of the thick filament. By immunofluorescence microscopy, MF20 was shown to bind along the entire A-band of chicken pectoralis myofibrils, although the epitope accessibility was greater near the ends than at the center of the A-bands. Thin-section, transmission electron microscopy of myofibrils decorated with MF20 revealed 50 regularly spaced, cross-striations in each half A-band, with a repeat distance of ~13 nm. These were numbered consecutively, 1-50, from the A-band to the last stripe, ~68 nm from the filament tips. These same striations could be visualized by negative staining of native thick filaments labeled with MF20. All 50 striations were of a consecutive, uninterrupted repeat which approximated the 14-15-nm axial translation of cross-bridges. Each half M-region contained five MF20 striations (~13 nm apart) with a distance between stripes 1 and 1', on each half of the bare zone, of ~18 nm. This is compatible with a packing model with full, antiparallel overlap of the myosin rods in the bare zone region. Differences in the spacings measured with negatively stained myofilaments and thin-sectioned myofibrils have been shown to arise from specimen shrinkage in the fixed and embedded preparations.These observations provide strong support for Huxley's original proposal for myosin packing in thick filaments of vertebrate muscle (Huxley, H. E., 1963, J. Mol. Biol., 7:281-308) and, for the first time, directly demonstrate that the 14-15-nm axial translation of LMM in the thick filament backbone corresponds to the cross-bridge repeat detected with x-ray diffraction of living muscle.The thick myofilaments of vertebrate skeletal muscle are complex polymeric structures composed predominantly of myosin but also contain a set of minor proteins including Mprotein (32, 50), C-protein (34, 40), myomesin (16), M-creatine kinase (51), H-protein (6), AMP deaminase (1), endprotein (49), and titin or connectin (31,54).A basic model for the structure of thick filaments was first described by H. E. Huxley (22). Based on electron microscopy

Three distinct sarcomeric patterns of skeletal muscle revealed by SHG and TPEF Microscopy

“…Indeed destructive interferences are created in this region which could reduce the SHG signal at the center of the sarcomere as it has been reported [1,19,21]. The size of the M-band was found to vary between 120 nm and 200 nm in vertebrates and it is important to note that the antiparallel packing region of myosin tail molecules is always lower and has been estimated to be between 85 -130 nm [12,24]. Both the size of the antiparallel packing and the value of the optical resolution which is far greater (≈ 400 nm) explain 1/ no visible attenuation of the SHG signal due to centrosymmetry was detected at the center of the sarcomere and 2/ predominance of single-band sarcomeric SHG pattern.…”

“…Double-band sarcomeric SHG pattern has been observed in C-elegans body-wall muscles [1,3], frog heart muscles [19], mouse tibialis anterior muscle [20], mouse leg and chicken heart [21], rabbit psoas muscles [15], drosophilla flight muscles [22,23]. Such change of the sarcomeric SHG pattern is puzzling since it is well known from electron microscopy (EM) studies that the size of thick filaments is constant and that of the M-band is between (120-200 nm) in vertebrates [12][13][14]24]. In order to understand the sarcomeric SHG pattern and the conditions of its change, we undertook a quantitative study of the sarcomeric SHG signal taking into account the influence of animal species (rat versus xenopus), age (adult versus larval) and tissue preparation (fixed or fresh) using high optical resolution objectives.…”

Shortened Outflow Tract Leads to Altered Cardiac Looping After Neural Crest Ablation

“…As they differentiate the myocardial phenotype, they become positive for MF-20, which has been shown previously to bind the light meromyosin fragment of myosin heavy chain in vertebrate striated muscle. 32 HNK-1 is a glycoprotein that is expressed by migratory neural crest cells, by cardiac myocytes as they differentiate into conduction myocardium, and by neuronal processes. 33,34 It has also been described in the endocardium.…”

Dual function of the UNC-45b Chaperone with myosin and GATA4 in cardiac development

“…Note that the mouse and human UNC-45A(a) and UNC-45B(b) proteins are greater than 95% identical in sequence . Anti-sarcomeric myosin monoclonal antibody (MF20) was kindly provided by Donald Fischman, Weill Cornell Medical College, New York (Shimizu et al, 1985). Commercially available affinity purified rabbit polyclonal anti-GATA4 antibody (Santa Cruz Biotechnology) was used.…”