Structural changes induced in Ca2+-regulated myosin filaments by Ca2+ and ATP.

We have used electron microscopy and proteolytic susceptibility to study the structural basis of myosin-linked regulation in synthetic filaments of scallop striated muscle myosin. Using papain as a probe of the structure of the head-rod junction, we find that this region of myosin is approximately five times more susceptible to proteolytic attack under activating (ATP/high Ca2+) or rigor (no ATP) conditions than under relaxing conditions (ATP/low Ca2+). A similar result was obtained with native myosin filaments in a crude homogenate of scallop muscle. Proteolytic susceptibility under conditions in which ADP or adenosine 5'-(beta, gamma-imidotriphosphate) (AMPPNP) replaced ATP was similar to that in the absence of nucleotide. Synthetic myosin filaments negatively stained under relaxing conditions showed a compact structure, in which the myosin cross-bridges were close to the filament backbone and well ordered, with a clear 14.5-nm axial repeat. Under activating or rigor conditions, the cross-bridges became clumped and disordered and frequently projected further from the filament backbone, as has been found with native filaments; when ADP or AMPPNP replaced ATP, the cross-bridges were also disordered. We conclude (a) that Ca2+ and ATP affect the affinity of the myosin cross-bridges for the filament backbone or for each other; (b) that the changes observed in the myosin filaments reflect a property of the myosin molecules alone, and are unlikely to be an artifact of negative staining; and (c) that the ordered structure occurs only in the relaxed state, requiring both the presence of hydrolyzed ATP on the myosin heads and the absence of Ca2+.

The higher-order structure of chromatin: evidence for a helical ribbon arrangement.

Both intact and nuclease-isolated chromatin fibers have been examined at different degrees of salt-induced compaction, using a variety of preparation techniques. The results suggest that the initial folding step in nucleosome packing involves the formation of a zig-zag ribbon as has been proposed by others (Thoma F., T. Koller, and A. Klug, 1979, J. Cell Biol., 83:403-427; Worcel A., S. Strogartz, and D. Riley, 1981, Proc. Natl. Acad. Sci. USA, 78:1461-1465), and that subsequent compaction occurs by coiling of the ribbon to form a double helical structure. This type of folding generates a fiber in which the nucleosome-nucleosome contacts established in the zig-zag ribbon are maintained and in which the histone H1 molecules occupy equivalent sites. The diameter of the fiber is not dependent upon the nucleosome repeat length. Direct mass values for individual isolated fibers obtained from electron scattering measurements showed that the mass per length was dependent on ionic strength, and ranged from 6.0 X 10(4) daltons/nm at 10 mM NaCl to 27 X 10(4) daltons/nm at 150 mM salt. These values are equivalent to 2.5 nucleosomes/11 nm at 10 mM NaCl and to 11.6 nucleosomes/11 nm at 150 mM salt and are consistent with the range of packing ratios for the proposed helical ribbon.

Differing accessibility in chromatin of the antigenic sites of regions 1-58 and 63-125 of histone H2B.

Experiments with antibodies induced by separated fragments 1-58 and 63-125 of H2B histone indicated that the 1-58 portion of the molecule is much more accessible in chromatin than is the 63-125 region. In immunoabsorption and immunoelectron microscopic assays with bovine and chicken chromatins, anti-1-58 antibodies reacted with sheared or unsheared chromatin both at low ionic strength (1 mM Tris-HCl) and in 0.14 M NaCl. Anti-63-125 antibodies were bound only weakly by chromatin at low ionic strength and not at all in 0.14 M NaCl. Antibodies to whole H2B showed intermediate reactivity with chromatin in both assays. In tests of immunofluorescence with unfixed calf liver nuclei in suspension, anti-1-58 caused nucleolar as well as nucleoplasmic fluorescence, whereas anti-63-125 did not lead to detectable fluorescence; anti-H2B showed intermediate staining intensity. In control experiments, anti-H1 antibody was bound by chromatin at low ionic strength but not in 0.14 M NaCl; anti-H3 antibody was bound poorly under either condition.

Structural repeating units in chromatin. III. A comparison of chromatin subunits from vertebrate, cilliate and angiosperm species.

A comparison was made of the chromatin subunit ("nu"body) structure present in nuclei from chicken erythrocytes, Tetrahymena cells, and Pisum sativum (pea) buds. All three types of chromatin yielded spherical subunits upon nuclease digestion which were indistinguishable in the electron microscope, and contained approximately the same amount of DNA. There were, however, consistent and significant differences in the digestion patterns of chromatin from the three organisms.

Electron microscopy of the actin-myosin head complex in the presence of ATP.

The structure of the actin-myosin head complex during the ATPase cycle has been studied by electron microscopy of negatively stained acto-heavy-meromyosin. In the absence of ATP, heavy meromyosin molecules generally showed a regular, angled appearance, with both heads attached to the actin filament. In the presence of ATP, attached molecules showed a less ordered structure, often with only one head attached. We conclude that configurations other than the rigor structure occur during the actomyosin cross-bridge cycle.

Composition of native and reconstituted chromatin particles: direct mass determination by scanning transmission electron microscopy.

Chromatin particles reconstituted from 145-base-pair lengths of DNA and either the arginine-rich histones H3 and H4 only or all four nucleosomal core histones have been compared with native nucleosomes in terms of their ultrastructure and mass distribution, as determined by scanning transmission electron microscopy (STEM). The mass of the nucleosome derived from STEM analysis was very close to that calculated for its DNA and histone components. The reconstituted particles showed a broader mass distribution, but it was clear that the majority contained at least eight histone molecules. This was to be expected for structures reconstituted from all four core histones, but in the case of H3H4-DNA complexes clearly showed that an octamer rather than tetramer of these histones was required to fold nucleosomal DNA into a stable compact particle. The significance of the H3H4 octamer complex with respect to nucleosomal structure is discussed, and the evidence that nucleosomal DNA can accept even greater numbers of histones is considered.

Inhibition of conformational change in smooth muscle myosin by a monoclonal antibody against the 17-kDa light chain.

Monoclonal antibodies against gizzard smooth muscle myosin were generated and characterized. One of these antibodies, designated MM-2, recognized the 17-kDa light chain and modulated the ATPase activities and hydrodynamic properties of smooth muscle myosin. Rotary shadowing electron microscopy showed that MM-2 binds 51 (+/- 25) A from the head-rod junction. The depression of Ca2+- and Mg2+-ATPase activities of myosin and Ca2+-ATPase activity of heavy meromyosin at low KCl concentration were abolished by MM-2. Viscosity measurement indicated that MM-2 inhibits the transition of 6 S myosin to 10 S myosin. While the rate of the production of subfragment-1 by papain proteolysis of 6 S myosin was inhibited by MM-2, the rate of proteolysis of the heavy chain of 10 S myosin was enhanced by MM-2 and reached the same rate as that of 6 S myosin plus MM-2. These results suggest that MM-2 inhibits the formation of 10 S myosin by binding to the 17-kDa light chain which is localized at the head-neck region of the myosin molecule. MM-2 increased the Vmax of actin-activated Mg2+-ATPase activities of both dephosphorylated myosin and dephosphorylated heavy meromyosin about 10- and 20-fold, respectively. MM-2 also activated the actin-activated Mg2+-ATPase activity of phosphorylated myosin at a low MgCl2 concentration and thus abolished the Mg2+-dependence of acto phosphorylated myosin ATPase activity. These results suggest that MM-2 inhibits the formation of 10 S myosin, and this results in the activation of actin-activated Mg2+-ATPase activity even in the absence of phosphorylation.

Adhesion of particulate specimens to support films for electron microscopy: a model system for assessing the surface properties of support films, and its application to chromatin particles.

Silica microspheres bearing a known surface charge were used to test the adhesive properties of support films and support film treatments commonly used in the electron microscopy of particulate specimens. Adhesion was strongly correlated with surface charge, negatively charged microspheres binding well only to positively charged support films and vice versa in solutions of low ionic strength. This charge dependency could be overcome by increasing the ionic strength to about 100 mmol with monovalent cations; under these conditions, it was not necessary to provide an oppositely charged film surface to obtain adhesion. Chromatin particles (nucleosomes) which have a net negative charge, behaved very much like the negatively charged silica with respect to adhesion, confirming that the microspheres provided an accurate indication of support film surface properties. The chromatin particles showed dramatic structural changes under conditions when adhesion was either poor, or very strong, indicating the need for careful selection of binding conditions for delicate biological specimens. A new and simple method for pretreating carbon films to improve adhesion was developed, and a preliminary account of this technique is presented.

Mapping of histone H5 sites on nucleosomes using immunoelectron microscopy.

The location of histone H5 on nucleosomes has been determined by binding anti-H5 antibodies to dinucleosomes, and recording the position of the bound IgG molecules using electron microscopy. Two types of antibody were employed, a total IgG fraction prepared from rabbits immunized with H5/RNA, which reacted to all three domains (NH2-terminal, central globular, and COOH-terminal) of H5, and an immunospecific subfraction which bound only to the central globular peptide. After reacting with dinucleosomes, both types of antibody were localized primarily in the linker DNA entry/exit region, but the whole antibody showed a much greater affinity for the linker DNA itself than did the antiglobular peptide antibody. These results provide direct support for the concept that H5, and by inference H1, is located at the linker DNA entry/exit site of the nucleosome, and further suggest that it is the central globular portion of the molecule that is most closely associated with this site. An interaction of one or both termini of H5 with the linker DNA is also indicated.

Fine structure of active ribosomal genes.

The structure of active ribosomal genes in the newt Notophthalmus viridescens has been studied at the higher resolution permitted by negative staining. Spherical particles, 70 A to 100 A in diameter are seen on the non-transcribing "spacer" regions, but were absent from certain regions of the transcribing "matrix". The matrix core often appears thicker than a double strand of DNA. These results are discussed in the light of the recently discovered subunit structure of chromatin, and in relation to the question of the disposition of histones during transcription.

Treatment with sodium butyrate inhibits the complete condensation of interphase chromatin.

The effects of histone hyperacetylation on chromatin fiber structure were studied using direct observations with the electron microscope. Histone hyperacetylation was induced in HeLa cells by treatment with sodium butyrate, and the ultrastructure of control and of acetylated chromatin fibers examined after fixation at different stages of compaction. No differences between control and acetylated chromatin were seen when the fibers were partially unfolded (10 mM NaCl, 20 mM NaCl, 50 mM NaCl), but in 100 mM NaCl, control chromatin showed further compaction to the "30 nm" fiber, while hyperacetylated chromatin failed to undergo this final compaction step. These results strongly suggest that histone acetylation causes a moderate "relaxation" rather than complete decondensation of interphase chromatin fibers. The relationship of these findings to the increased DNase I sensitivity of acetylated chromatin, and to transcription and replication, is discussed.