X chromosome drive in a widespread Palearctic woodland fly, Drosophila testacea.

Selfish genes that bias their own transmission during meiosis can spread rapidly in populations, even if they contribute negatively to the fitness of their host. Driving X chromosomes provide a clear example of this type of selfish propagation. These chromosomes have important evolutionary and ecological consequences, and can be found in a broad range of taxa including plants, mammals and insects. Here, we report a new case of X chromosome drive (X drive) in a widespread woodland fly, Drosophila testacea. We show that males carrying the driving X (SR males) sire 80-100% female offspring and possess a diagnostic X chromosome haplotype that is perfectly associated with the sex ratio distortion phenotype. We find that the majority of sons produced by SR males are sterile and appear to lack a Y chromosome, suggesting that meiotic defects involving the Y chromosome may underlie X drive in this species. Abnormalities in sperm cysts of SR males reflect that some spermatids are failing to develop properly, confirming that drive is acting during gametogenesis. By screening wild-caught flies using progeny sex ratios and a diagnostic marker, we demonstrate that the driving X is present in wild populations at a frequency of ~ 10% and that suppressors of drive are segregating in the same population. The testacea species group appears to be a hot spot for X drive, and D. testacea is a promising model to compare driving X chromosomes in closely related species, some of which may even be younger than the chromosomes themselves.

The core of the mammalian centriole contains gamma-tubulin.

BACKGROUND: The microtubule network, upon which transport occurs in higher cells, is formed by the polymerization of alpha and beta tubulin. The third major tubulin isoform, gamma tubulin, is believed to serve a role in organizing this network by nucleating microtubule growth on microtubule-organizing centers, such as the centrosome. Research in vitro has shown that gamma tubulin must be restored to stripped centrioles to regenerate the centrosomal functions of duplication and microtubule nucleation. RESULTS: We have re-examined the localization of gamma tubulin in isolated and in situ mammalian centrosomes using a novel immunocytochemical technique that preserves antigenicity and morphology while allowing increased accessibility. As expected, alpha tubulin was localized in cytoplasmic and centriolar barrel microtubules and in the associated pericentriolar material. Foci of gamma tubulin were observed at the periphery of the organized pericentriolar material, as reported previously, often near the termini of microtubules. A further and major location of gamma tubulin was a structure within the proximal end of the centriolar barrel. The distributions were complementary, in that alpha tubulin was excluded from the core of the centriole, and gamma tubulin was excluded from the microtubule barrel. CONCLUSIONS: We have shown that gamma tubulin is localized both in the pericentriolar material and in the core of the mammalian centriole. This result suggests that gamma tubulin has a role in the centriolar duplication process, perhaps as a template for growth of the centriolar microtubules, in addition to its established role in the nucleation of astral microtubules.

Cryo-electron microscopy reveals ordered domains in the immature HIV-1 particle.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) is the causative agent of AIDS and the subject of intense study. The immature HIV-1 particle is traditionally described as having a well ordered, icosahedral structure made up of uncleaved Gag protein surrounded by a lipid bilayer containing envelope proteins. Expression of the Gag protein in eukaryotic cells leads to the budding of membranous virus-like particles (VLPs). RESULTS: We have used cryo-electron microscopy of VLPs from insect cells and lightly fixed, immature HIV-1 particles from human lymphocytes to determine their organization. Both types of particle were heterogeneous in size, varying in diameter from 1200-2600 A. Larger particles appeared to be broken into semi-spherical sectors, each having a radius of curvature of approximately 750 A. No evidence of icosahedral symmetry was found, but local order was evidenced by small arrays of Gag protein that formed facets within the curved sectors. A consistent 270 A radial density was seen, which included a 70 A wide low density feature corresponding to the carboxy-terminal portion of the membrane attached matrix protein and the amino-terminal portion of the capsid protein. CONCLUSIONS: Immature HIV-1 particles and VLPs both have a multi-sector structure characterized, not by an icosahedral organization, but by local order in which the structures of the matrix and capsid regions of Gag change upon cleavage. We propose a model in which lateral interactions between Gag protein molecules yields arrays that are organized into sectors for budding by RNA.

The Escherichia coli large ribosomal subunit at 7.5 A resolution.

BACKGROUND: In recent years, the three-dimensional structure of the ribosome has been visualised in different functional states by single-particle cryo-electron microscopy (cryo-EM) at 13-25 A resolution. Even more recently, X-ray crystallography has achieved resolution levels better than 10 A for the ribosomal structures of thermophilic and halophilic organisms. We present here the 7.5 A solution structure of the 50S large subunit of the Escherichia coli ribosome, as determined by cryo-EM and angular reconstitution. RESULTS: The reconstruction reveals a host of new details including the long alpha helix connecting the N- and C-terminal domains of the L9 protein, which is found wrapped like a collar around the base of the L1 stalk. A second L7/L12 dimer is now visible below the classical L7/L12 'stalk', thus revealing the position of the entire L8 complex. Extensive conformational changes occur in the 50S subunit upon 30S binding; for example, the L9 protein moves by some 50 A. Various rRNA stem-loops are found to be involved in subunit binding: helix h38, located in the A-site finger; h69, on the rim of the peptidyl transferase centre cleft; and h34, in the principal interface protrusion. CONCLUSIONS: Single-particle cryo-EM is rapidly evolving towards the resolution levels required for the direct atomic interpretation of the structure of the ribosome. Structural details such as the minor and major grooves in rRNA double helices and alpha helices of the ribosomal proteins can already be visualised directly in cryo-EM reconstructions of ribosomes frozen in different functional states.

Atrial structure and plasma ANF levels in rats with chronic diabetes mellitus.

The effect of streptozocin-induced diabetes (STZ-D) on right atrial structure was investigated in male Wistar rats. STZ (55 mg/kg) or saline (1 ml/kg) was administered by intravenous injection 12 wk before the experimental studies. Tissue was sampled from four regions of the atrium, processed, and embedded in plastic. Quantitative stereological analysis indicated that in STZ-D rats, there was a significant diminution in size of the musculi pectinati (muscular ridges), which form a network making up the wall of the atrium. In addition, within the muscular ridges, there was a significant reduction in the relative proportion of cardiocytes within the cardiac tissue. The rest of the cardiac tissue consisted of interstitial regions, connective tissue, and blood vessels, which correspondingly increased. This suggests there was some form of cardiomyopathy. When atrial granularity was determined relative to cardiocyte volume density, a significant decrease (54%) was found in tissue from STZ-D rats. The blood pressure of conscious STZ-D rats was significantly lower than control rats, whereas right atrial pressure was not different. The level of resting plasma immunoreactive atrial natriuretic factor (ANF) in conscious STZ-D rats (98 +/- 5 pg/ml) was significantly higher than in control rats (52 +/- 7 pg/ml). The decreased atrial granularity could be related to the higher resting plasma ANF levels, suggesting a more rapid turnover or increased synthesis bypassing storage in the granular form.

Three-dimensional structure of an invertebrate rhodopsin and basis for ordered alignment in the photoreceptor membrane.

Invertebrate rhodopsins activate a G-protein signalling pathway in microvillar photoreceptors. In contrast to the transducin-cyclic GMP phosphodiesterase pathway found in vertebrate rods and cones, visual transduction in cephalopod (squid, octopus, cuttlefish) invertebrates is signalled via Gq and phospholipase C. Squid rhodopsin contains the conserved residues of the G-protein coupled receptor (GPCR) family, but has only 35% identity with mammalian rhodopsins. Unlike vertebrate rhodopsins, cephalopod rhodopsin is arranged in an ordered lattice in the photoreceptor membranes. This organization confers sensitivity to the plane of polarized light and also provides the optimal orientation of the linear retinal chromophores in the cylindrical microvillar membranes for light capture. Two-dimensional crystals of squid rhodopsin show a rectilinear arrangement that is likely to be related to the alignment of rhodopsins in vivo.Here, we present a three-dimensional structure of squid rhodopsin determined by cryo-electron microscopy of two-dimensional crystals. Docking the atomic structure of bovine rhodopsin into the squid density map shows that the helix packing and extracellular plug structure are conserved. In addition, there are two novel structural features revealed by our map. The linear lattice contact appears to be made by the transverse C-terminal helix lying on the cytoplasmic surface of the membrane. Also at the cytoplasmic surface, additional density may correspond to a helix 5-6 loop insertion found in most GPCRs relative to vertebrate rhodopsins. The similarity supports the conservation in structure of rhodopsins (and other G-protein-coupled receptors) from phylogenetically distant organisms. The map provides the first indication of the structural basis for rhodopsin alignment in the microvillar membrane.

The first step: activation of the Semliki Forest virus spike protein precursor causes a localized conformational change in the trimeric spike.

The structure of the particle formed by the SFVmSQL mutant of Semliki Forest virus (SFV) has been defined by cryo-electron microscopy and image reconstruction to a resolution of 21 A. The SQL mutation blocks the cleavage of p62, the precursor of the spike proteins E2 and E3, which normally occurs in the trans-Golgi. The uncleaved spike protein is insensitive to the low pH treatment that triggers membrane fusion during entry of the wild-type virus. The conformation of the spike in the SFVmSQL particle should correspond to that of the inactive precursor found in the early stages of the secretory pathway. Comparison of this "precursor" structure with that of the mature, wild-type, virus allows visualization of the changes that lead to activation, the first step in the pathway toward fusion. We find that the conformational change in the spike is dramatic but localized. The projecting domains of the spikes are completely separated in the precursor and close to generate a cavity in the mature spike. E1, the fusion peptide-bearing protein, interacts only with the p62 in its own third of the trimer before cleavage and then collapses to form a trimer of heterotrimers (E1E2E3)3 surrounding the cavity, poised for the pH-induced conformational change that leads to fusion. The capsid, transmembrane regions and the spike skirts (thin layers of protein that link spikes above the membrane) remain unchanged by cleavage. Similarly, the interactions of the spikes with the nucleocapsid through the transmembrane domains remain constant. Hence, the interactions that lead to virus assembly are unaffected by the SFVmSQL mutation.

The collagenous domain of class A scavenger receptors is involved in macrophage adhesion to collagens.

Class A macrophage scavenger receptors (MSRs) have a remarkably broad ligand specificity and are well-known for their roles in atherogenesis and host defense. Recently, we demonstrated that these receptors also recognize and mediate adhesion to denatured forms of type I collagen. In this study, the involvement of the collagenous domain of MSRs in binding to denatured type I collagen was investigated. Transient expression of full-length, native type II MSR in COS-1 cells conferred adhesion to denatured type I collagens, whereas expression of a truncated receptor lacking the distal portion of the collagenous domain did not. Further, a synthetic peptide derived from the collagenous domain was effective in abrogating Mphi adhesion to denatured forms of type I collagen. We also addressed collagen-type specificity by examining MSR affinity for type III and type IV collagens. As with type I collagen, Mphis adhered only to denatured forms of type III collagen. Moreover, the adhesion was mediated by MSRs. In contrast, adhesion to denatured type IV collagen was not shown to be MSR-dependent, but adhesion to the native form was. MSR-mediated adhesion to types III and IV collagens was also shown to be dependent on the collagenous domain. Taken together, these data strongly suggest that the collagenous domain is involved in MSR-mediated adhesion to denatured forms of types I and III collagens and native, but not denatured, type IV collagen.

Selective adhesion of macrophages to denatured forms of type I collagen is mediated by scavenger receptors.

Macrophages (Mφs) are multifunctional immune cells which are involved in the regulation of immune and inflammatory responses, as well as in tissue repair and remodeling. In tissues, Mφs reside in areas which are rich in extracellular matrix (ECM), the structural component which also plays an essential role in regulating a variety of cellular functions. A major ECM protein encountered by Mφs is type I collagen, the most abundant of the fibril-forming collagens. In this study, the adhesion of RAW 264.7 murine Mphis to native fibrillar, monomeric, and denatured type I collagen was investigated. Using atomic force microscopy, structural differences between fibrillar and monomeric type I collagen were clearly resolved. When cultured on fibrillar type I collagen, Mphis adhered poorly. In contrast, they adhered significantly to monomeric, heat-denatured, or collagenase-modified type I collagen. Studies utilizing anti-beta1 and -beta2 integrin adhesion-blocking antibodies, RGD-containing peptides, or divalent cation-free conditions did not inhibit Mphi; adhesion to monomeric or denatured type I collagen. However, macrophage scavenger receptor (MSR) ligands and anti-MSR antibodies significantly blocked Mphi; adhesion to denatured and monomeric type I collagen strongly suggesting the involvement of the MSR as an adhesion molecule for denatured type I collagen. Further analysis by Western blot identified the MSR as the primary receptor for denatured type I collagen among Mphi; proteins purified from a heat-denatured type I collagen affinity column. These findings indicate that Mphis adhere selectively to denatured forms of type I collagen, but not the native fibrillar conformation, via their scavenger receptors.

Arterial wall and smooth muscle cell development in young Wistar rats and the effects of surgical denervation.

Development of the muscular saphenous artery and the effect of surgical denervation on normal development was investigated in young rats at 3 and 6 weeks of age. During this interval, the weight and blood pressures (systolic, diastolic, and mean) of the animals increased significantly. The tunica media of the artery and the lumen increased significantly with age, but the proportion of smooth muscle cell to paracellular matrix did not alter. Computer-assisted three-dimensional reconstructions were used to investigate the smooth muscle cells. They increased significantly in length, volume, and angle of orientation within the vessel wall with age but maintained an approximate surface area-to-volume ratio. The cells in any one vessel tended to be oriented in either a clockwise or counterclockwise direction. The size of the nucleus also increased significantly in length and volume with age, but an approximate surface area-to-volume ratio and a constant nucleocytoplasmic ratio were maintained. The nuclei tended to be eccentrically located, with less than half of all nuclei wholly within the middle third of the cell. Surgical denervation at 10 days of age resulted in abnormalities of growth in vessel dimensions, thinner tunica media at 3 weeks (denervated 11 days previously), and smaller lumen at 6 weeks (denervated 32 days previously). Elevated amounts of paracellular matrix occurred in both age groups, but denervation did not alter smooth muscle cell size. In the 3-week-old animals, denervation resulted in smooth muscle cells with hypertrophied nuclei. This may account for the increase in growth of the tunica media between 3 and 6 weeks of age in the denervated artery.(ABSTRACT TRUNCATED AT 250 WORDS)

Three-dimensional reconstruction of the mammalian centriole from cryoelectron micrographs: the use of common lines for orientation and alignment.

The microtubule organizing center of the animal cell (S. D. Fuller et al., 1992, Curr. Opin. Struct. Biol. 2, 264-274; D. M. Glover et al., 1993, Sci. Am. 268, 62-68; E. B. Wilson, 1925), (The Cell in Development and Heredity) comprises two centrioles and the pericentriolar material. We have completed several three-dimensional reconstructions of individual centrioles from tilt series of cryoelectron micrographs. The reconstruction procedure uses minimization of the common lines residual to define the orientation of the centriolar minefold symmetry axis and then uses this symmetry to generate a structure by weighted backprojection to 28-nm resolution. Many of the features of these reconstructions agree with previous, conventional transmission electron microscopy studies (M. Paintrand et al., 1992, J. Struct. Biol. 108, 107-128). The microtubule barrel of the centriole is roughly 500 nm long and 300 nm in diameter and the microtubule bundles appear to taper toward the distal end. In addition, we see a handedness to the pericentriolar material at the base (distal end) of the centriole which is opposite to the skew of the microtubule triplets. The region at which the microtubule barrel joins this base is intriguingly complex and includes an internal cylindrical feature which is a site of gamma tubulin localization.

Contractile activity of neonatal platelets.

Platelet contractile activity was evaluated by observation of tension development during isometric contraction of platelet-fibrin clots. Cylindrical clots were made with platelet-rich plasma obtained from cord blood or from adult controls. These clots were allowed to contract isometrically at 37 degrees C while attached to a transducer to record tension development. The rate of tension development was dependent on platelet concentration but was equivalent for neonatal and adult platelet clots. Although abnormalities in neonatal platelet aggregation and secretion have been well documented the platelet functions required for clot contraction such as fibrin binding and actin-myosin interaction appear to be intact in neonatal platelets.

Actin associates with the nucleocapsid domain of the human immunodeficiency virus Gag polyprotein.

Recently, it was shown that actin molecules are present in human immunodeficiency virus type 1 (HIV-1) particles. We have examined the basis for incorporation and the location of actin molecules within HIV-1 and murine retrovirus particles. Our results show that the retroviral Gag polyprotein is sufficient for actin uptake. Immunolabeling studies demonstrate that actin molecules localize to a specific radial position within the immature particle, clearly displaced from the matrix domain underneath the viral membrane but in proximity to the nucleocapsid (NC) domain of the Gag polyprotein. When virus or subviral Gag particles were disrupted with nonionic detergent, actin molecules remained associated with the disrupted particles. Actin molecules remained in a stable complex with the NC cleavage product (or an NC-RNA complex) after treatment of the disrupted HIV-1 particles with recombinant HIV-1 protease. In contrast, matrix and capsid molecules were released. The same result was obtained when mature HIV-1 particles were disrupted with detergent. Taken together, these results indicate that actin molecules are associated with the NC domain of the viral polyprotein.

Cryo-electron microscopy reveals the functional organization of an enveloped virus, Semliki Forest virus.

Semliki Forest virus serves as a paradigm for membrane fusion and assembly. Our icosahedral reconstruction combined 5276 particle images from 48 cryo-electron micrographs and determined the virion structure to 9 A resolution. The improved resolution of this map reveals an N-terminal arm linking capsid subunits and defines the spike-capsid interaction sites. It illustrates the paired helical nature of the transmembrane segments and the elongated structures connecting them to the spike projecting domains. A 10 A diameter density in the fusion protein lines the cavity at the center of the spike. These clearly visible features combine with the variation in order between the layers to provide a framework for understanding the structural changes during the life cycle of an enveloped virus.

Structures of unliganded and ATP-bound states of the Escherichia coli chaperonin GroEL by cryoelectron microscopy.

We have developed an angular refinement procedure incorporating correction for the microscope contrast transfer function, to determine cryoelectron microscopy (cryo-EM) structures of the Escherichia coli chaperonin GroEL in its apo and ATP-bound forms. This image reconstruction procedure is verified to 13-A resolution by comparison of the cryo-EM structure of unliganded GroEL with the crystal structure. Binding, encapsulation, and release of nonnative proteins by GroEL and its cochaperone GroES are controlled by the binding and hydrolysis of ATP. Seven ATP molecules bind cooperatively to one heptameric ring of GroEL. This binding causes long-range conformational changes that determine the orientations of remote substrate-binding sites, and it also determines the conformation of subunits in the opposite ring of GroEL, in a negatively cooperative mechanism. The conformation of GroEL-ATP was determined at approximately 15-A resolution. In one ring of GroEL-ATP, the apical (substrate-binding) domains are extremely disordered, consistent with the high mobility needed for them to achieve the 60 degrees elevation and 90 degrees twist of the GroES-bound state. Unexpectedly, ATP binding also increases the separation between the two rings, although the interring contacts are present in the density map.

Association of DOCA hypertension with induction of atherosclerosis in rats with short-term diabetes mellitus.

We investigated the effect of deoxycorticosterone acetate (DOCA)-induced hypertension on plasma lipid and cholesterol levels and the development of vascular atherosclerotic changes in male Wistar rats injected with streptozotocin (STZ) or saline (CON). Rats given STZ alone demonstrated a mild hyperlipidemia and hypercholesterolemia without any change in blood pressure. One week of DOCA administration was without effect on blood pressure in CON and STZ groups, but at 3 and 6 wk caused a significant and similar elevation in both groups. This DOCA-induced elevation in blood pressure appeared to be associated with the increase in plasma lipid and cholesterol levels seen in both CON and STZ groups at 3 and 6 wk, although the elevation in lipid and cholesterol levels was significantly more pronounced in the STZ rats. Both CON and STZ groups injected with DOCA developed significant pathological changes in all vessels under investigation. However, the degree of atherosclerosis appeared, from a semiquantitative analysis, to be worse in the thoracic aortas and renal arteries of the STZ group. Neither normotensive group developed any atherosclerosis. It is concluded that hypertension is associated with atherosclerosis in normal rats and rats with short-term STZ-induced diabetes mellitus, although the higher plasma lipid and cholesterol levels of the latter group may potentiate the degree of vascular damage.

Postembedding alpha-tubulin immunolabelling of isolated centrosomes.

Accurate ultrastructural localization of the components of centrosomes is an important step toward the determination of their function. We have used an electron microscopy procedure to preserve centrosome-associated antigens which enables their high-resolution localization. The unique part of our procedure is the application of a post-sectioning fixation step which overcomes the poor section contrast and morphological appearance that limits the use of low-temperature processing and Lowicryl embedding. The efficacy of our approach is demonstrated by the efficient labelling of alpha-tubulin in the well-preserved and contrasted microtubule barrels of the centrides of isolated mammalian centrosomes.

Low pH induces swiveling of the glycoprotein heterodimers in the Semliki Forest virus spike complex.

Time-resolved cryoelectron microscopy reveals the first step in the conformational changes that enable membrane fusion in Semliki Forest virus. The neutral pH structure reveals a central cavity within the spike complex, plate-like extensions forming a layer above the membrane, and the paths of the paired transmembrane domains connecting the trimeric spikes and pentamer-hexamer clustered capsid subunits. Low pH treatment results in centrifugal movement of E2, the receptor-binding subunit, centripetal movement of E1 to narrow the central cavity initiating the formation of an E1 trimer, and the extension of the E1 fusion sequence toward the target membrane.

Escherichia coli RNA polymerase core and holoenzyme structures.

Multisubunit RNA polymerase is an essential enzyme for regulated gene expression. Here we report two Escherichia coli RNA polymerase structures: an 11.0 A structure of the core RNA polymerase and a 9.5 A structure of the sigma(70) holoenzyme. Both structures were obtained by cryo-electron microscopy and angular reconstitution. Core RNA polymerase exists in an open conformation. Extensive conformational changes occur between the core and the holoenzyme forms of the RNA polymerase, which are largely associated with movements in ss'. All common RNA polymerase subunits (alpha(2), ss, ss') could be localized in both structures, thus suggesting the position of sigma(70) in the holoenzyme.

Projection structure of an invertebrate rhodopsin.

Rhodopsin is the G-protein-coupled membrane receptor that initiates the visual transduction cascade in retinal photoreceptors. In the present study rhodopsin from the dark-adapted retinas of squid (Loligo forbesi) was detergent-extracted, purified, and reconstituted into native squid photoreceptor lipids following proteolytic cleavage of its prolinerich C-terminus. Two-dimensional crystals of C-terminally truncated rhodopsin reconstituted from octyl glucoside solution formed in a p222(1) lattice (a = 44 A, b = 131 A). Electron micrographs of frozen-hydrated crystals were processed and a projection structure to 8 A resolution was calculated. The projection map obtained is very similar to maps previously determined for bovine and frog rhodopsins although the crystal packing of the molecules is quite different. Comparison of the maps shows that the arrangement of alpha-helices in the proteins is very similar despite their great phylogenetic distance; this structure is likely to be present in the whole superfamily of G-protein-coupled receptors. Invertebrate rhodopsins have a large insertion in the helix 5-helix 6 loop. Assignment of an additional density in the squid rhodopsin map to this region supports a previously proposed helix assignment and identifies the end-to-end contacts as helices 1 and 5.