Pauline M. Bennett

Interaction of C-protein with myosin, myosin rod and light meromyosin*

C-protein, a component of vertebrate skeletal muscle myofibrils, is known to be located at specific positions along the thick myosin-containingc myofilaments. We have investigated its effects on the assembly of myosin into filaments in vitro and its interaction with low ionic strength aggregates of myosin rod and light meromyosin, the α-helical fragments of myosin. C-protein is not required for the formation of myosin filaments in vitro . Purified myosin, free of C-protein, can form long filaments with a demonstrable 14 nm longitudinal repeat. The presence of C-protein disrupts the regularity of the myosin filament structure, resulting in a reduced and more variable diameter and a loss of longitudinal order. However, C-protein does not appear to have any length-regulating role. Binding measurements at low ionic strength reveal a strong affinity of C-protein for myosin and also for rod and light meromyosin. The limiting stoichiometry of binding is about one mole C-protein per mole for myosin and somewhat less for rod and light meromyosin. The interaction of C-protein with rod and light meromyosin has also been investigated by electron microscopy. In the absence of C-protein, myosin rod forms large sheet-like paracrystals with a 14 nm longitudinal repeat. In the presence of C-protein the formation of these paracrystals is disrupted and in their place we find irregular narrow filaments. Formation of paracrystals of light meromyosin is not disrupted by C-protein. The C-protein forms a series of transverse stripes on the paracrystal with a longitudinal spacing identical to the principal repeat of about 40 nm which characterizes the light meromyosin assembly. We conclude that the C-protein in native thick filaments is probably bound to the shaft of the filament with a periodicity determined by the underlying myosin assembly.

The ultrastructural location of C-protein, X-protein and H-protein in rabbit muscle

SummaryPurified antibodies to the thick filament accessory proteins, C-protein, X-protein and H-protein, have been used to label fibres of three rabbit muscles, psoas (containing mainly fast white fibres), soleus (containing mainly slow red fibres) and plantaris (a muscle of mixed fibre type) and their location has been examined by electron microscopy.These accessory proteins are present on one or more of a set of eleven transverse stripes about 43 nm apart that have been observed previously in each half A-band. Each protein has a limited set of characteristic distributions. H-protein is present on stripe 3 (counting from the M-line) in the majority of psoas fibres but is absent in soleus and plantaris muscle. C-protein can occur (1) on stripes 4–11 (the commonest pattern seen in psoas); (2) on stripes 5–11 (in psoas and plantaris); (3) on stripe 3 together with stripes 5–11 (in plantaris); or (4) on none (in red fibres of all three muscles). X-protein can occur (1) on stripes 3–11 in the red fibres of all three muscles; (2) on stripe 4 only (in psoas and plantaris); (3) on stripes 3 and 4 (in psoas and plantaris) or (4) on none. Stripes labelled with anti-X are wider than those labelled with anti-C and consist of a doublet with an internal spacing of 16 nm. The patterns for the three accessory proteins, while overlapping, are in no case identical; this suggests the proteins do not simply substitute for one another.The precise axial positions of the anti-C labelled stripes differ from those of the anti-X stripes; the anti-X stripes lie about 8–9 nm further from the M-line than the corresponding anti-C stripes. This implies that the inner member of an X-protein doublet lies in a very similar position to a C-protein stripe. The anti-H labelled stripe seen in most psoas fibres lies 14 nm nearer the M-line than stripe 3 of the anti-X labelled array in psoas red fibres and is staggered from a continuation of the C-protein array by about 4 nm.The labelling patterns were constant within a fibre and suggest a very precise assembly mechanism. The number of classes of fibre, as defined by the accessory proteins present and their arrangement, exceeds the number of fibre types presently recognized.

Structure of straight flagella from a mutant Salmonella.

The structure of straight flagella from a mutant of Salmonella typhimurium has been studied by electron microscopy and optical diffraction and filtering. In the optical diffraction pattern, the axial and radial co-ordinates and the signs of the real and imaginary parts of the phases of the principal maxima on each layer-line were measured. The phase-determination required the preparation of centrosymmetric combinations of two copies of the same area of the flagellum, together with two circular apertures to provide reference fringes across the diffraction pattern. For the imaginary parts, pieces of mica were used to retard the light passing through one of the copies by π radians relative to that passing through the other. Optically filtered images of the upper and lower sides of the flagellum separately showed the surface lattice clearly, with a maximum of six long-pitch helical rows crossing any line drawn perpendicular to the helix axis. The “twosided” filtered image showed the superposition pattern characteristic of an odd number of long-pitch helices and demonstrated the polarized nature of the flagellum, in which superposed helical features form “chevrons” pointing towards the proximal (hook) end. The flagellar structure may be regarded as a single, genetic helix with just under eleven subunits in two turns. The selection rule is l = 15n + 82m. There are eleven long-pitch helical rows, running at an angle of about 7 ° to the helix axis. This angle is in the range predicted by Asakura (1970) from theoretical calculations based on observations on low-resolution electron micrographs of flagellar polymorphs of differing over-all helical waveform. A drawing of the structure with non-spherical subunits demonstrates the polarized appearance and suggests a simple model for the V-shaped breaks sometimes observed in fragmented flagella. Comparison of straight with normal flagella indicates that their structures are basically similar and that the transition between them occurs by a small change in the orientation of the subunit.

Understanding the Organisation and Role of Myosin Binding Protein C in Normal Striated Muscle by Comparison with MyBP-C Knockout Cardiac Muscle

Myosin binding protein C (MyBP-C) is a component of the thick filament of striated muscle. The importance of this protein is revealed by recent evidence that mutations in the cardiac gene are a major cause of familial hypertrophic cardiomyopathy. Here we investigate the distribution of MyBP-C in the A-bands of cardiac and skeletal muscles and compare this to the A-band structure in cardiac muscle of MyBP-C-deficient mice. We have used a novel averaging technique to obtain the axial density distribution of A-bands in electron micrographs of well-preserved specimens. We show that cardiac and skeletal A-bands are very similar, with a length of 1.58 ± 0.01 μm. In normal cardiac and skeletal muscle, the distributions are very similar, showing clearly the series of 11 prominent accessory protein stripes in each half of the A-band spaced axially at 43-nm intervals and starting at the edge of the bare zone. We show by antibody labelling that in cardiac muscle the distal nine stripes are the location of MyBP-C. These stripes are considerably suppressed in the knockout mouse hearts as expected. Myosin heads on the surface of the thick filament in relaxed muscle are thought to be arranged in a three-stranded quasi-helix with a mean 14.3-nm axial cross bridge spacing and a 43 nm helix repeat. Extra “forbidden” meridional reflections, at orders of 43 nm, in X-ray diffraction patterns of muscle have been interpreted as due to an axial perturbation of some levels of myosin heads. However, in the MyBP-C-deficient hearts these extra meridional reflections are weak or absent, suggesting that they are due to MyBP-C itself or to MyBP-C in combination with a head perturbation brought about by the presence of MyBP-C.

Structure of the myosin-containing filament assembly (A-segment) separated from frog skeletal muscle

Abstract A- and I-segments, which are the naturally occurring assemblies of the myosin-containing and actin-containing filaments, respectively, have been released by mechanical disruption of frog skeletal myofibrils in a relaxing medium. In the electron microscope, negatively stained A-segments (length ~1.6 μm) show periodic structure in much more detail than is observed in isolated filaments or sectioned fibrils. The structure is bipolar. The central M-band (width ~495 A) is flanked on either side by a heavily stained “bare” zone (width ~395 A), following which is a series of ten bands of equal width (420 A ± 15 A) showing a polarised substructure; the end of the segment has a less regular structure. Bands 1, 2 and 3 differ from bands 4 to 10, which all look alike. Optical diffraction patterns from the region of the ten bands resemble, in certain respects, the patterns from micrographs of light meromyosin paracrystals. The arrangement of the heavy meromyosin projections is not apparent; probably they have been damaged by the stain. No evidence was found of a 442 A periodicity. It is concluded that the band pattern observed represents mainly the bipolar structure of the light meromyosin filament backbone.

Structure of the cross-striated adductor muscle of the scallop

Abstract The structure of the cross-striated adductor muscle of the scallop has been studied by electron microscopy and X-ray diffraction using living relaxed, glycerol-extracted (rigor), fixed and dried muscles. The thick filaments are arranged in a hexagonal lattice whose size varies with sarcomere length so as to maintain a constant lattice volume. In the overlap region there are approximately 12 thin filaments about each thick filament and these are arranged in a partially disordered lattice similar to that found in other invertebrate muscles, giving a thin-to-thick filament ratio in this region of 6:1. The thin filaments, which contain actin and tropomyosin, are about 1 μm long and the actin subunits are arranged on a helix of pitch 2 × 38.5 nm. The thick filaments, which contain myosin and paramyosin, are about 1.76 μm long and have a backbone diameter of about 21 nm. We propose that these filaments have a core of paramyosin about 6 nm in diameter, around which the myosin molecules pack. In living relaxed muscle, the projecting myosin heads are symmetrically arranged. The data are consistent with a six-stranded helix, each strand having a pitch of 290 nm. The projections along the strands each correspond to the heads of one or two myosin molecules and occur at alternating intervals of 13 and 16 nm. In rigor muscle these projections move away from the backbone and attach to the thin filaments. In both living and dried muscle, alternate planes of thick filaments are staggered longitudinally relative to each other by about 7.2 nm. This gives rise to a body-centred orthorhombic lattice with a unit cell twice the volume of the basic filament lattice.

The CKK Domain (DUF1781) Binds Microtubules and Defines the CAMSAP/ssp4 Family of Animal Proteins

We describe a structural domain common to proteins related to human calmodulin-regulated spectrin-associated protein1 (CAMSAP1). Analysis of the sequence of CAMSAP1 identified a domain near the C-terminus common to CAMSAP1 and two other mammalian proteins KIAA1078 and KIAA1543, which we term a CKK domain. This domain was also present in invertebrate CAMSAP1 homologues and was found in all available eumetazoan genomes (including cnidaria), but not in the placozoan Trichoplax adherens, nor in any nonmetazoan organism. Analysis of codon alignments by the sitewise likelihood ratio method gave evidence for strong purifying selection on all codons of mammalian CKK domains, potentially indicating conserved function. Interestingly, the Drosophila homologue of the CAMSAP family is encoded by the ssp4 gene, which is required for normal formation of mitotic spindles. To investigate function of the CKK domain, human CAMSAP1-enhanced green fluorescent protein (EGFP) and fragments including the CKK domain were expressed in HeLa cells. Both whole CAMSAP1 and the CKK domain showed localization coincident with microtubules. In vitro, both whole CAMSAP1-glutathione-s-transferase (GST) and CKK-GST bound to microtubules. Immunofluorescence using anti-CAMSAP1 antibodies on cerebellar granule neurons revealed a microtubule pattern. Overexpression of the CKK domain in PC12 cells blocked production of neurites, a process that requires microtubule function. We conclude that the CKK domain binds microtubules and represents a domain that evolved with the metazoa.

Caldesmon binds to smooth muscle myosin and myosin rod and crosslinks thick filaments to actin filaments

SummaryIt is well established that caldesmon binds to actin (Kb–107-108m−1) and to tropomyosin (Kb106m−1) and that it is a potent inhibitor of actomyosin ATPase. Caldesmon can also bind tightly to myosin. We investigated the binding of smooth muscle and nonmuscle caldesmon isoforms (CDh and CDl respectively) to myosin using proteins from sheep aorta. Both caldesmon isoforms bind to myosin with indistinguishable affinity. The affinity is about 106m−1 in low salt buffer, but is weakened by increasing [KCl] reaching 105mM−1 in 100mm KCl. The stoichiometry of binding is about three caldesmon per myosin molecule. Stoichiometry and affinity are not dependent on whether myosin is phosphorylated nor on the presence of Mg2+ and ATP, provided the ionic strength is maintained constant. The caldesmon binding site of smooth muscle myosin is located in the S-2 region, consequently both HMM and myosin rod bind to caldesmon. Over a range of conditions myosin and myosin rod binding to caldesmon were indistinguishable. Skeletal muscle myosin has no caldesmon binding site. Smooth muscle myosin rods form side-polar filaments in low salt buffer in which the backbone packing of LMM into the filament shaft is clearly visible in negatively-stained electron microscopic images. Sometimes the S-2 portions can be seen ‘frayed’ from the filament shaft. When caldesmon is bound the filament shaft appears to be about 20% thicker and the frayed effect is dramatically increased; long filamentous ‘whiskers’ are often seen curving out from the filament shaft. Similar structures are observed with smooth muscle and with non-muscle caldesmon. Myosin also binds to caldesmon when it is incorporated into the thin filament; however, this interaction is qualitatively different. Measurements of smooth muscle HMM binding to native thin filaments in the presence of 3mm MgATP shows there is a high affinity binding (Kb=106m−1) which is independent of [Ca2+] and of the level of myosin phosphorylation. The stoichiometry is one HMM molecule per actin monomer which is equivalent to up to 14 HMM bound at high affinity per caldesmon. Negatively stained electron microscopic images of the HMM.ADP.Pi-thin filament complex have failed to show any attachment of HMM to the thin filaments. When rod filaments are added to actin plus caldesmon or to native thin filaments the rod filaments are strongly associated with the actin filament bundles. The majority of rod filaments are lined up parallel and in close proximity to actin filaments. Similar crosslinking is observed with non-muscle caldesmon. In the smooth muscle cell, caldesmon-containing thin filaments are found together with myosin filaments in the ‘contractile domain’ in parallel arrays not unlike those shown in our synthetic systems. Thus caldesmon ought to be able to crosslink thick and thin filamentsin vivo.

The Protein 4.1 family: hub proteins in animals for organizing membrane proteins.

Proteins of the 4.1 family are characteristic of eumetazoan organisms. Invertebrates contain single 4.1 genes and the Drosophila model suggests that 4.1 is essential for animal life. Vertebrates have four paralogues, known as 4.1R, 4.1N, 4.1G and 4.1B, which are additionally duplicated in the ray-finned fish. Protein 4.1R was the first to be discovered: it is a major mammalian erythrocyte cytoskeletal protein, essential to the mechanochemical properties of red cell membranes because it promotes the interaction between spectrin and actin in the membrane cytoskeleton. 4.1R also binds certain phospholipids and is required for the stable cell surface accumulation of a number of erythrocyte transmembrane proteins that span multiple functional classes; these include cell adhesion molecules, transporters and a chemokine receptor. The vertebrate 4.1 proteins are expressed in most tissues, and they are required for the correct cell surface accumulation of a very wide variety of membrane proteins including G-Protein coupled receptors, voltage-gated and ligand-gated channels, as well as the classes identified in erythrocytes. Indeed, such large numbers of protein interactions have been mapped for mammalian 4.1 proteins, most especially 4.1R, that it appears that they can act as hubs for membrane protein organization. The range of critical interactions of 4.1 proteins is reflected in disease relationships that include hereditary anaemias, tumour suppression, control of heartbeat and nervous system function. The 4.1 proteins are defined by their domain structure: apart from the spectrin/actin-binding domain they have FERM and FERM-adjacent domains and a unique C-terminal domain. Both the FERM and C-terminal domains can bind transmembrane proteins, thus they have the potential to be cross-linkers for membrane proteins. The activity of the FERM domain is subject to multiple modes of regulation via binding of regulatory ligands, phosphorylation of the FERM associated domain and differential mRNA splicing. Finally, the spectrum of interactions of the 4.1 proteins overlaps with that of another membrane-cytoskeleton linker, ankyrin. Both ankyrin and 4.1 link to the actin cytoskeleton via spectrin, and we hypothesize that differential regulation of 4.1 proteins and ankyrins allows highly selective control of cell surface protein accumulation and, hence, function. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé

Cytoskeletal Protein 4.1R Affects Repolarization and Regulates Calcium Handling in the Heart

The 4.1 proteins are a family of multifunctional adaptor proteins. They promote the mechanical stability of plasma membranes by interaction with the cytoskeletal proteins spectrin and actin and are required for the cell surface expression of a number of transmembrane proteins. Protein 4.1R is expressed in heart and upregulated in deteriorating human heart failure, but its functional role in myocardium is unknown. To investigate the role of protein 4.1R on myocardial contractility and electrophysiology, we studied 4.1R-deficient (knockout) mice (4.1R KO). ECG analysis revealed reduced heart rate with prolonged Q-T interval in 4.1R KO. No changes in ejection fraction and fractional shortening, assessed by echocardiography, were found. The action potential duration in isolated ventricular myocytes was prolonged in 4.1R KO. Ca2+ transients were larger and slower to decay in 4.1R KO. The sarcoplasmic reticulum Ca2+ content and Ca2+ sparks frequency were increased. The Na+/Ca2+ exchanger current density was reduced in 4.1R KO. The transient inward current inactivation was faster and the persistent Na+ current density was increased in the 4.1R KO group, with possible effects on action potential duration. Although no major morphological changes were noted, 4.1R KO hearts showed reduced expression of NaV1.5&agr; and increased expression of protein 4.1G. Our data indicate an unexpected and novel role for the cytoskeletal protein 4.1R in modulating the functional properties of several cardiac ion transporters with consequences on cardiac electrophysiology and with possible significant roles during normal cardiac function and disease.