Robert W. Kensler

Myosin light chain phosphorylation affects the structure of rabbit skeletal muscle thick filaments

To identify the structural basis for the observed physiological effects of myosin regulatory light chain phosphorylation in skinned rabbit skeletal muscle fibers (potentiation of force development at low calcium), thick filaments separated from the muscle in the relaxed state, with unphoshorylated light chains, were incubated with specific, intact, myosin light chain kinase at moderate (pCa 5.0) and low (pCa 5.8) calcium and with calcium-independent enzyme in the absence of calcium, then examined as negatively stained preparations, by electron microscopy and optical diffraction. All such experimental filaments became disordered (lost the near-helical array of surface myosin heads typical of the relaxed state). Filaments incubated in control media, including intact enzyme in the absence of calcium, moderate calcium (pCa 5.0) without enzyme, and bovine serum albumin substituting for calcium-independent myosin light chain kinase, all retained their relaxed structure. Finally, filaments disordered by phosphorylation regained their relaxed structure after incubation with a protein phosphatase catalytic subunit. We suggest that the observed disorder is due to phosphorylation-induced increased mobility and/or changed conformation of myosin heads, which places an increased population of them close to thin filaments, thereby potentiating actin-myosin interaction at low calcium levels.

The myosin-binding protein C motif binds to F-actin in a phosphorylation-sensitive manner

Cardiac myosin-binding protein C (cMyBP-C) is a regulatory protein expressed in cardiac sarcomeres that is known to interact with myosin, titin, and actin. cMyBP-C modulates actomyosin interactions in a phosphorylation-dependent way, but it is unclear whether interactions with myosin, titin, or actin are required for these effects. Here we show using cosedimentation binding assays, that the 4 N-terminal domains of murine cMyBP-C (i.e. C0-C1-m-C2) bind to F-actin with a dissociation constant (Kd) of ∼10 μm and a molar binding ratio (Bmax) near 1.0, indicating 1:1 (mol/mol) binding to actin. Electron microscopy and light scattering analyses show that these domains cross-link F-actin filaments, implying multiple sites of interaction with actin. Phosphorylation of the MyBP-C regulatory motif, or m-domain, reduced binding to actin (reduced Bmax) and eliminated actin cross-linking. These results suggest that the N terminus of cMyBP-C interacts with F-actin through multiple distinct binding sites and that binding at one or more sites is reduced by phosphorylation. Reversible interactions with actin could contribute to effects of cMyBP-C to increase cross-bridge cycling.

Structure of Limulus telson muscle thick filaments

Abstract Computer analysis of electron micrographs of negatively stained thick filaments isolated from the telson levator muscle of the horseshoe crab ( Limulus polyphemus ) has shown that they have a four-stranded helical structure. The repeating units along each helix have a bent extended shape (measuring approximately 20 nm × 8 nm × 8 nm) and are inclined at an angle of about 30 ° to the helical path. At the resolution of this study, it was difficult to establish the exact size of the surface subunits, but our results are probably more consistent with each unit representing the two heads of a single myosin molecule rather than larger aggregates.

Atomic model of the human cardiac muscle myosin filament

Of all the myosin filaments in muscle, the most important in terms of human health, and so far the least studied, are those in the human heart. Here we report a 3D single-particle analysis of electron micrograph images of negatively stained myosin filaments isolated from human cardiac muscle in the normal (undiseased) relaxed state. The resulting 28-Å resolution 3D reconstruction shows axial and azimuthal (no radial) myosin head perturbations within the 429-Å axial repeat, with rotations between successive 132 Å-, 148 Å-, and 149 Å-spaced crowns of heads close to 60°, 35°, and 25° (all would be 40° in an unperturbed three-stranded helix). We have defined the myosin head atomic arrangements within the three crown levels and have modeled the organization of myosin subfragment 2 and the possible locations of the 39 Å-spaced domains of titin and the cardiac isoform of myosin-binding protein-C on the surface of the myosin filament backbone. Best fits were obtained with head conformations on all crowns close to the structure of the two-headed myosin molecule of vertebrate chicken smooth muscle in the dephosphorylated relaxed state. Individual crowns show differences in head-pair tilts and subfragment 2 orientations, which, together with the observed perturbations, result in different intercrown head interactions, including one not reported before. Analysis of the interactions between the myosin heads, the cardiac isoform of myosin-binding protein-C, and titin will aid in understanding of the structural effects of mutations in these proteins known to be associated with human cardiomyopathies.

Binding of the N-terminal Fragment C0–C2 of Cardiac MyBP-C to Cardiac F-actin

Cardiac myosin-binding protein C (cMyBP-C), a major accessory protein of cardiac thick filaments, is thought to play a key role in the regulation of myocardial contraction. Although current models for the function of the protein focus on its binding to myosin S2, other evidence suggests that it may also bind to F-actin. We have previously shown that the N-terminal fragment C0-C2 of cardiac myosin-binding protein-C (cMyBP-C) bundles actin, providing evidence for interaction of cMyBP-C and actin. In this paper we directly examined the interaction between C0-C2 and F-actin at physiological ionic strength and pH by negative staining and electron microscopy. We incubated C0-C2 (5-30μM, in a buffer containing in mM: 180 KCl, 1 MgCl(2), 1 EDTA, 1 DTT, 20 imidazole, at pH 7.4) with F-actin (5μM) for 30min and examined negatively-stained samples of the solution by electron microscopy (EM). Examination of EM images revealed that C0-C2 bound to F-actin to form long helically-ordered complexes. Fourier transforms indicated that C0-C2 binds with the helical periodicity of actin with strong 1st and 6th layer lines. The results provide direct evidence that the N-terminus of cMyBP-C can bind to F-actin in a periodic complex. This interaction of cMyBP-C with F-actin supports the possibility that binding of cMyBP-C to F-actin may play a role in the regulation of cardiac contraction.

The septum of the lateral axon of the earthworm: A thin section and freeze-fracture study

SummarySepta occur between the axonal segments in the lateral giant septate axon of the nerve cord of the earthworm. This septum is demonstrated here to be permeable to fluorescein and to exhibit a negligible time delay for impulse transmission. Periodic anastomoses between the two lateral axons of the nerve cord are revealed by fluorescein. The permeability of the septum is correlated with the demonstration that nexuses occur along the septum. In thin sections, the nexuses may appear as long septilaminar or pentalaminar membrane appositions, but most frequently appear as a series of short or punctate membrane appositions. In freeze-fracture replicas, the nexuses appear as particles 10–12 nm in diameter on the PF face and as pits on the EF face. The particles and pits are arranged in plaques, in anastomosing strands, or most frequently in small plaques with strands of particles or pits emerging from the periphery. In addition to the nexuses, a junction characterized by the presence of 31 nm diameter hemispherical densities on the cytoplasmic surfaces of the septal membranes is revealed in thin sections. The densities are paired on the adjacent septal membranes, and most frequently are shown by optical diffraction to be arranged on the membrane surfaces in hexagonal or rhomboidal lattices with a centre-to-centre spacing of 34.8 nm. In freeze-fracture replicas, an array of particles and pits with a similar lattice symmetry and spacing to the arrays of hemispherical densities is demonstrated.

Structure and orientation of troponin in the thin filament

The troponin complex on the thin filament plays a crucial role in the regulation of muscle contraction. However, the precise location of troponin relative to actin and tropomyosin remains uncertain. We have developed a method of reconstructing thin filaments using single particle analysis that does not impose the helical symmetry of actin and is independent of a starting model. We present a single particle three-dimensional reconstruction of the thin filament. Atomic models of the F-actin filament were fitted into the electron density maps and troponin and tropomyosin located. The structure provides evidence that the globular head region of troponin labels the two strands of actin with a 27.5-Å axial stagger. The density attributed to troponin appears tapered with the widest point toward the barbed end. This leads us to interpret the polarity of the troponin complex in the thin filament as reversed with respect to the widely accepted model.

Myosin filament 3D structure in mammalian cardiac muscle

A number of cardiac myopathies (e.g. familial hypertrophic cardiomyopathy and dilated cardiomyopathy) are linked to mutations in cardiac muscle myosin filament proteins, including myosin and myosin binding protein C (MyBP-C). To understand the myopathies it is necessary to know the normal 3D structure of these filaments. We have carried out 3D single particle analysis of electron micrograph images of negatively stained isolated myosin filaments from rabbit cardiac muscle. Single filament images were aligned and divided into segments about 2 × 430 Å long, each of which was treated as an independent ‘particle’. The resulting 40 Å resolution 3D reconstruction showed both axial and azimuthal (no radial) myosin head perturbations within the 430 Å repeat, with successive crown rotations of approximately 60°, 60° and 0°, rather than the regular 40° for an unperturbed helix. However, it is shown that the projecting density peaks appear to start at low radius from origins closer to those expected for an unperturbed helical filament, and that the azimuthal perturbation especially increases with radius. The head arrangements in rabbit cardiac myosin filaments are very similar to those in fish skeletal muscle myosin filaments, suggesting a possible general structural theme for myosin filaments in all vertebrate striated muscles (skeletal and cardiac).

Mammalian Cardiac Muscle Thick Filaments: Their Periodicity and Interactions with Actin

Cardiac muscle has been extensively studied, but little information is available on the detailed macromolecular structure of its thick filament. To elucidate the structure of these filaments I have developed a procedure to isolate the cardiac thick filaments for study by electron microscopy and computer image analysis. This procedure uses chemical skinning with Triton X-100 to avoid contraction of the muscle that occurs using the procedures previously developed for isolation of skeletal muscle thick filaments. The negatively stained isolated filaments appear highly periodic, with a helical repeat every third cross-bridge level (43 nm). Computed Fourier transforms of the filaments show a strong set of layer lines corresponding to a 43-nm near-helical repeat out to the 6th layer line. Additional meridional reflections extend to at least the 12th layer line in averaged transforms of the filaments. The highly periodic structure of the filaments clearly suggests that the weakness of the layer lines in x-ray diffraction patterns of heart muscle is not due to an inherently more disordered cross-bridge arrangement. In addition, the isolated thick filaments are unusual in their strong tendency to remain bound to actin by anti-rigor oriented cross-bridges (state II or state III cross-bridges) under relaxing conditions.

Determination of the handedness of the crossbridge helix ofLimulus thick filaments

SummaryThick filaments, isolated in their long conformation from unstimulatedLimulus telson muscles, were shadowed with platinum or platinum-carbon and examined using electron microscopy and optical diffraction techniques. All filaments showed evidence of a right-handed surface helix, which had a major repeat at approx. 43 nm. In fortuitously oriented specimens the subunits, presumably crossbridges, which comprised the helical strands were clearly delineated. Optical transforms obtained from images of shadowed filaments confirmed the helical repeat at approx. 43 nm and could be readily interpreted as patterns expected from a one-surface view of the four-stranded filament structure we have previously reported.The striking resemblance between optically filtered images of shadowed filaments and the computed reconstruction of the one-surface filament further confirm our model for the myosin lattice of theLimulus thick filament.