Interactions between actin and myosin filaments in skeletal muscle visualized in frozen-hydrated thin sections.

For the purpose of determining net interactions between actin and myosin filaments in muscle cells, perhaps the single most informative view of the myofilament lattice is its averaged axial projection. We have studied frozen-hydrated transverse thin sections with the goal of obtaining axial projections that are not subject to the limitations of conventional thin sectioning (suspect preservation of native structure) or of equatorial x-ray diffraction analysis (lack of experimental phases). In principle, good preservation of native structure may be achieved with fast freezing, followed by low-dose electron imaging of unstained vitrified cryosections. In practice, however, cryosections undergo large-scale distortions, including irreversible compression; furthermore, phase contrast imaging results in a nonlinear relationship between the projected density of the specimen and the optical density of the micrograph. To overcome these limitations, we have devised methods of image restoration and generalized correlation averaging, and applied them to cryosections of rabbit psoas fibers in both the relaxed and rigor states. Thus visualized, myosin filaments appear thicker than actin filaments by a much smaller margin than in conventional thin sections, and particularly so for rigor muscle. This may result from a significant fraction of the myosin S1-cross-bridges averaging out in projection and thus contributing only to the baseline of projected density. Entering rigor incurs a loss of density from an annulus around the myosin filament, with a compensating accumulation of density around the actin filament. This redistribution of mass represents attachment of the fraction of cross-bridges that are visible above background. Myosin filaments in the "nonoverlap" zone appear to broaden on entering rigor, suggesting that on deprivation of ATP, cross-bridges in situ move outwards even without actin in their immediate proximity.     

Molecular substructure of a viral receptor-recognition protein. The gp17 tail-fiber of bacteriophage T7

The bacteriophage T7 tail complex consists of a conical tail-tube surrounded by six kinked tail-fibers, which are oligomers of the viral protein gp17 (Mr 61,400). We have derived a molecular model for the tail-fiber by integrating secondary structure predictions with ultrastructural information obtained by correlation averaging of electron micrographs of negatively stained tail complexes. This model has been further refined by high-resolution scanning transmission electron microscopy of purified fibers, both negatively stained and unstained. Mass measurements made from the latter images establish that the fiber is a trimer of gp17. The proximal half-fiber is a uniform rod, about 2.0 nm in diameter and 16.4 nm long, which we infer to be a triple-stranded coiled-coil, containing three copies of an alpha-helical domain of about 117 residues, starting at Phe151. The distal half-fiber is 15.5 nm long, and is made up of four globules, 3.1 to 4.8 nm in diameter, in rigid linear array: it contains the carboxy-terminal halves (residues approximately 268 to 553) of the constituent gp17 chains, arranged with 3-fold symmetry around its long axis. The amino-terminal domains (residues 1 to 149) link the fiber to the tail-tube. We conclude that the three gp17 chains are quasi-equivalent in the proximal half-fiber, equivalent in the distal half-fiber, and non-equivalent in the kink region that separates the two half-fibers: such localized non-equivalence may represent a general mechanism for the formation of kinked joints in segmented homo-oligomeric proteins.     

Hexavalent capsomers of herpes simplex virus type 2: symmetry, shape, dimensions, and oligomeric status

The structures of the hexavalent capsomers of herpes simplex virus type 2 were analyzed by negative staining electron microscopy of capsomer patches derived from partially disrupted nucleocapsids. Optimally computer-averaged images were formed for each of the three classes of capsomer distinguished by their respective positions on the surface of the icosahedral capsid with a triangulation number of 16; in projection, each capsomer exhibited unequivocal sixfold symmetry. According to correspondence analysis of our set of capsomer images, no significant structural differences were detected among the three classes of capsomers, as visualized under these conditions. Taking into account information from images of freeze-dried, platinum-shadowed nucleocapsid fragments, it was established that each hexavalent capsomer is a hexamer of the 155-kilodalton major capsid protein. The capsomer has the form of a sixfold hollow cone approximately 12 nm in diameter and approximately 15 nm in depth, whose axial channel tapers in width from the outside towards the inner capsid surface.     

Tracing paternal ancestry in mice, using the Y-linked, sex-determining locus, Sry

The molecular evolution of mammalian Y-linked DNA sequences is of special interest because of their unique mode of inheritance: most Y- linked sequences are clonally inherited from father to son. Here we investigate the use of Y-linked sequences for phylogenetic inference. We describe a comparative analysis of a 515-bp region from the male sex- determining locus, Sry, in 22 murine rodents (subfamily Murinae, family Muridae), including representatives from nine species of Mus, and from two additional murine genera--Mastomys and Hylomyscus. Percent sequence divergence was < 0.01% for comparisons between populations within a species and was 0.19%-8.16% for comparisons between species. Our phylogenetic analysis of 12 murine taxa resulted in a single most- parsimonius tree that is highly concordant with phylogenies based on mitochondrial DNA and allozymes. A total evidence tree based on the combined data from Sry, mitochondrial DNA, and allozymes supports (1) the monophyly of the subgenus Mus, (2) its division into a Palearctic group (M. musculus, M. domesticus, M. spicilegus, M. Macedonicus, and M. spretus) and an Oriental group (M. cookii++, M. cervicolor, and M. caroli), and (3) sister-group relationships between M. spicilegus and M. macedonicus and between M. cookii and M. cervicolor. We argue that Y- chromosome DNA sequences represent a valuable new source of characters for phylogenetic inference.      

Hexon-only binding of VP26 reflects differences between the hexon and penton conformations of VP5, the major capsid protein of herpes simplex virus.

VP26 is a 12-kDa capsid protein of herpes simplex virus 1. Although VP26 is dispensable for assembly, the native capsid (a T=16 icosahedron) contains 900 copies: six on each of the 150 hexons of VP5 (149 kDa) but none on the 12 VP5 pentons at its vertices. We have investigated this interaction by expressing VP26 in Escherichia coli and studying the properties of the purified protein in solution and its binding to capsids. Circular dichroism spectroscopy reveals that the conformation of purified VP26 consists mainly of beta-sheets (approximately 80%), with a small alpha-helical component (approximately 15%). Its state of association was determined by analytical ultracentrifugation to be a reversible monomer-dimer equilibrium, with a dissociation constant of approximately 2 x 10(-5) M. Bacterially expressed VP26 binds to capsids in the normal amount, as determined by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cryoelectron microscopy shows that the protein occupies its usual sites on hexons but does not bind to pentons, even when available in 100-fold molar excess. Quasi-equivalence requires that penton VP5 must differ in conformation from hexon VP5: our data show that in mature capsids, this difference is sufficiently pronounced to abrogate its ability to bind VP26.     

Sequence regularities and packing of collagen molecules

Fourier analysis of sequences along edges of the type I collagen molecule constructed from two α1(I) and one α2 chains shows that the molecule is two-sided if the supercoil pitch of the α chains along the molecular axis, P, is 39 residues ($\text{D}\text{6}$, where D = 234 residues or 67 nm). One side has alternating charged and hydrophobic regions with spacings of $\text{D}\text{6}$, while the other side has an excess of hydrophobic residues with a spacing of $\text{D}\text{11}$. These characteristics arise from sequence regularities in the α chains and the geometric relationship between the chains. The pattern is marginally strongest with α2 as chain 1. The $\text{D}\text{6}$ sides could form the inside of a helical microfibril where contacts between molecules would fall P apart along the α chains. The $\text{D}\text{11}$ sides could form the outside of the microfibril where contacts between microfibrils would be spaced apart by the α chain supercoil along the microfibril axis, P′. If the microfibril is a 5₄ helix of D-staggered collagen molecules with a left-handed supercoil of pitch $\text{20D}\text{11}$, P′ is close to $\text{2D}\text{11}$ (43 residues). $\text{2D}\text{11}$ subsets in the α chains give rise to the $\text{D}\text{11}$ spacing along the molecule. The microfibril has 4₁ screw symmetry satisfying X-ray diffraction evidence that microfibrils pack in a tetragonal unit cell.This model is the same as proposed previously by us (Trus & Piez, 1976: Piez & Trus, 1977) except that P = 39 rather than 30 residues. Contrary to our earlier assumption, P = 39 residues is within the range allowed by X-ray diffraction measurements. The present results favor P = 39 since it relates regularities in the α chain sequences to helical parameters in a direct way. Furthermore, model studies show that geometric arguments which support P = 30 are equally strong at P = 39 residues.     

Image averaging of flexible fibrous macromolecules: the clathrin triskelion has an elastic proximal segment.

We have developed computational techniques that allow image averaging to be applied to electron micrographs of filamentous molecules that exhibit tight and variable curvature. These techniques, which involve straightening by cubic-spline interpolation, image classification, and statistical analysis of the molecules' curvature properties, have been applied to purified brain clathrin. This trimeric filamentous protein polymerizes, both in vivo and in vitro, into a wide range of polyhedral structures. Contrasted by low-angle rotary shadowing, dissociated clathrin molecules appear as distinctive three-legged structures, called "triskelions" (E. Ungewickell and D. Branton (1981) Nature 289, 420). We find triskelion legs to vary from 35 to 62 nm in total length, according to an approximately bell-shaped distribution (mu = 51.6 nm). Peaks in averaged curvature profiles mark hinges or sites of enhanced flexibility. Such profiles, calculated for each length class, show that triskelion legs are flexible over their entire lengths. However, three curvature peaks are observed in every case: their locations define a proximal segment of systematically increasing length (14.0-19.0 nm), a mid-segment of fixed length (approximately 12 nm), and a rather variable end-segment (11.6-19.5 nm), terminating in a hinge just before the globular terminal domain (approximately 7.3 nm diameter). Thus, two major factors contribute to the overall variability in leg length: (1) stretching of the proximal segment and (2) stretching of the end-segment and/or scrolling of the terminal domain. The observed elasticity of the proximal segment may reflect phosphorylation of the clathrin light chains.     

Le ionophore A23187

The acrosomic status of spermatozoa prepared for IVF has been evaluated by means of immunofluorescence test from Fenichel and Hsi using calcium A 23187 ionophore as inductor of acrosome reaction (AR). The spontaneous AR remains slight, even after 6 hour-incubation in Menezo B2 (6,8+2,7%). The response to ionophore, moderate before (11,2+9%), frankly increases after a 6h-capacitation (28,9+8,3%) in a group of 25 IVF couples (tubal indication, normal semen, positive fertilization). Nevertheless, it remains slight or null in 4 cases of unexplained repeated failure of fertilization. The response to ionophore A 23187 allows to explore the kinetics of capacitation of spermatozoa and their ability to perform AR. Its significance in terms of fecondance remains to be precised.     

Behavioural and chemical mechanisms behind a Mediterranean ant–ant association

1. Interspecific competition among ants is common, and so is competitive exclusion among dominant ant species. In contrast, specific associations between non‐parasitic ant species are rare, especially in the temperate zones. As an exception, the subordinate ant Camponotus lateralis frequently co‐occurs with the dominant Crematogaster scutellaris but rarely with other dominant ants. 2. This association is one of various associations between Camponotus and Crematogaster species across the world. However, the mechanisms behind these co‐occurences are largely unknown. 3. In the present study, we therefore investigated the association of Ca. lateralis and Cr. scutellaris. We studied the spatial association of the nests, interspecific aggression, both species' cuticular hydrocarbon profiles, and their propensity to follow the other species' pheromone trails. 4. Crematogaster scutellaris usually attacked and displaced the generally submissive Ca. lateralis, but was significantly less aggressive at jointly used trails. Camponotus nests were always in close proximity to Crematogaster nests. 5. The cuticular hydrocarbons of both species consisted of alkanes with chain lengths between C21 and C35. The two species had 25 hydrocarbons in common, including mono‐, di‐, and tetramethyl alkanes. Despite this qualitative similarity, however, the quantitative hydrocarbon composition differed between the two species. 6. Camponotus lateralis followed artificial trails containing trail pheromones of Cr. scutellaris, but the latter did not follow Ca. lateralis trail pheromones. Interspecific trail‐following by Camponotus, but not vice versa, has been observed in another Camponotus–Crematogaster association and may be a more general mechanism that facilitates associations between the two ant genera.     

Dynamics of herpes simplex virus capsid maturation visualized by time-lapse cryo-electron microscopy

The capsid of the herpes simplex virus initially assembles as a procapsid that matures through a massive conformational change of its 182 MDa surface shell. This transition, which stabilizes the fragile procapsid, is facilitated by the viral protease that releases the interaction between the shell and the underlying scaffold; however, protease-deficient procapsids mature slowly in vitro. To study procapsid maturation as a time-resolved process, we monitored this reaction by cryo-electron microscopy (cryo-EM). The resulting images were sorted into 17 distinct classes, and three-dimensional density maps were calculated for each. When arranged in a chronological series, these maps yielded molecular movies of procapsid maturation. A single major switching event takes place at stages 8-9, preceded by relatively subtle adjustments in the pattern of interactions and followed by similarly small 'aftershocks'. The primary mechanism underlying maturation is relative rotations of domains of VP5, the major capsid protein.