It is easy to understand the self-assembly of particles with anisotropic shapes or interactions (for example, cobalt nanoparticles or proteins) into highly extended structures. However, there is no experimentally established strategy for creating a range of anisotropic structures from common spherical nanoparticles. We demonstrate that spherical nanoparticles uniformly grafted with macromolecules ('nanoparticle amphiphiles') robustly self-assemble into a variety of anisotropic superstructures when they are dispersed in the corresponding homopolymer matrix. Theory and simulations suggest that this self-assembly reflects a balance between the energy gain when particle cores approach and the entropy of distorting the grafted polymers. The effectively directional nature of the particle interactions is thus a many-body emergent property. Our experiments demonstrate that this approach to nanoparticle self-assembly enables considerable control for the creation of polymer nanocomposites with enhanced mechanical properties. Grafted nanoparticles are thus versatile building blocks for creating tunable and functional particle superstructures with significant practical applications.
Barth syndrome is an X-linked genetic disorder caused by mutations in the tafazzin (taz) gene and characterized by dilated cardiomyopathy, exercise intolerance, chronic fatigue, delayed growth, and neutropenia. Tafazzin is a mitochondrial transacylase required for cardiolipin remodeling. Although tafazzin function has been studied in non-mammalian model organisms, mammalian genetic loss of function approaches have not been used. We examined the consequences of tafazzin knockdown on sarcomeric mitochondria and cardiac function in mice. Tafazzin knockdown resulted in a dramatic decrease of tetralinoleoyl cardiolipin in cardiac and skeletal muscles and accumulation of monolysocardiolipins and cardiolipin molecular species with aberrant acyl groups. Electron microscopy revealed pathological changes in mitochondria, myofibrils, and mitochondrion-associated membranes in skeletal and cardiac muscles. Echocardiography and magnetic resonance imaging revealed severe cardiac abnormalities, including left ventricular dilation, left ventricular mass reduction, and depression of fractional shortening and ejection fraction in tafazzin-deficient mice. Tafazzin knockdown mice provide the first mammalian model system for Barth syndrome in which the pathophysiological relationships between altered content of mitochondrial phospholipids, ultrastructural abnormalities, myocardial and mitochondrial dysfunction, and clinical outcome can be completely investigated.
F(1)F(0) ATP synthase forms dimers that tend to assemble into large supramolecular structures. We show that the presence of cardiolipin is critical for the degree of oligomerization and the degree of order in these ATP synthase assemblies. This conclusion was drawn from the statistical analysis of cryoelectron tomograms of cristae vesicles isolated from Drosophila flight-muscle mitochondria, which are very rich in ATP synthase. Our study included a wild-type control, a cardiolipin synthase mutant with nearly complete loss of cardiolipin, and a tafazzin mutant with reduced cardiolipin levels. In the wild-type, the high-curvature edge of crista vesicles was densely populated with ATP synthase molecules that were typically organized in one or two rows of dimers. In both mutants, the density of ATP synthase was reduced at the high-curvature zone despite unchanged expression levels. Compared to the wild-type, dimer rows were less extended in the mutants and there was more scatter in the orientation of dimers. These data suggest that cardiolipin promotes the ribbonlike assembly of ATP synthase dimers and thus affects lateral organization and morphology of the crista membrane.
The apoptosome is an Apaf-1 cytochrome c complex that activates procaspase-9. The three-dimensional structure of the apoptosome has been determined at 27 A resolution, to reveal a wheel-like particle with 7-fold symmetry. Molecular modeling was used to identify the caspase recruitment and WD40 domains within the apoptosome and to infer likely positions of the CED4 homology motif and cytochrome c. This analysis suggests a plausible role for cytochrome c in apoptosome assembly. In a subsequent structure, a noncleavable mutant of procaspase-9 was localized to the central region of the apoptosome. This complex promotes the efficient activation of procaspase-3. Therefore, the cleavage of procaspase-9 is not required to form an active cell death complex.
Apaf-1 and cytochrome c coassemble in the presence of dATP to form the apoptosome. We have determined a structure of the apoptosome at 12.8 A resolution by using electron cryomicroscopy and single-particle methods. We then docked appropriate crystal structures into the map to create an accurate domain model. Thus, we found that seven caspase recruitment domains (CARDs) form a central ring within the apoptosome. At a larger radius, seven copies of the nucleotide binding and oligomerization domain (NOD) associate laterally to form the hub, which encircles the CARD ring. Finally, an arm-like helical domain (HD2) links each NOD to a pair of beta propellers, which bind a single cytochrome c. This model provides insights into the roles of dATP and cytochrome c in assembly. Our structure also reveals how a CARD ring and the central hub combine to create a platform for procaspase-9 activation.
Background & Aims Immune cells of the liver must be able to recognize and react to pathogens yet remain tolerant to food molecules and other non-pathogens. Dendritic cells (DC) are thought to contribute to hepatic tolerance. Lipids have been implicated in dysfunction of DC in cancer. Therefore, we investigated whether high lipid content in liver DC affects induction of tolerance. Methods Mouse and human hepatic non-parenchymal cells were isolated by mechanical and enzymatic digestion. DC were purified by fluorescence-activated cell sorting or with immunomagnetic beads. DC lipid content was assessed by flow cytometry, immune fluorescence, and electron microscopy and by measuring intracellular component lipids. DC activation was determined from surface phenotype and cytokine profile. DC function was assessed in T-cell, natural killer (NK)-cell, and NKT-cell co-culture assays, as well as in vivo. Results We observed 2 distinct populations of hepatic DC in mice and humans based on their lipid content and expression of markers associated with adipogensis and lipid metabolism. This lipid-based dichotomy in DC was unique to the liver and specific to DC, compared with other hepatic immune cells. However, rather than mediate tolerance, the liver DC population with high concentrations of lipid was immunogenic in multiple models—they activated T cells, NK cells, and NKT cells. Conversely, liver DC with low levels of lipid induced T-regulatory cells, anergy to cancer, and oral tolerance. The immunogenicity of lipid-rich liver DC required their secretion of tumor necrosis factor-α and was directly related to their high lipid content; blocking DC synthesis of fatty acids or inhibiting adipogenesis (by reducing endoplasmic reticular stress) reduced DC immunogenicity. Conclusions Human and mouse hepatic DC are comprised of distinct populations that contain different concentrations of lipid, which regulates immunogenic versus tolerogenic responses in the liver.
Barth syndrome (BTHS) is a mitochondrial disorder that is caused by mutations in the tafazzin gene, which affects phospholipid composition. To determine whether this defect leads to alterations in the internal three-dimensional organization of mitochondrial membranes, we applied electron microscopic tomography to lymphoblast mitochondria from BTHS patients and controls. Tomograms were formed from 50 nm and 150 nm sections of chemically fixed lymphoblasts and the data were used to manually segment volumes of relevant structural details. Normal lymphoblast mitochondria contained well aligned, lamellar cristae with slot-like junctions to the inner boundary membrane. In BTHS, mitochondrial size was more variable and the total mitochondrial volume per cell increased, mainly due to clusters of fragmented mitochondria inside nuclear invaginations. However, mitochondria showed reduced cristae density, less cristae alignment, and inhomogeneous cristae distribution. Three-dimensional reconstruction of BTHS mitochondria revealed zones of adhesion of the opposing inner membranes, causing obliteration of the intracrista space. We found small isolated patches of adhesion as well as extended adhesion zones, resulting in sheets of collapsed cristae packaged in multiple concentric layers. We also found large tubular structures (diameter 30-150 nm) that appeared to be derivatives of the adhesion zones. The data suggest that mitochondrial abnormalities of BTHS involve adhesions of inner mitochondrial membranes with subsequent collapse of the intracristae space. KeywordsCardiomyopathy; Electron microscopic tomography; Mitochondrial disease; Mitochondrial ultrastructure; Skeletal myopathy Barth syndrome (BTHS) is a mitochondrial disorder presenting with cardiomyopathy, skeletal muscle weakness, neutropenia, and growth retardation (1-3). This syndrome is caused by mutations in the tafazzin gene that has an X-linked recessive inheritance pattern (4). Although tafazzin is lacking a typical mitochondrial targeting signal, its mitochondrial localization has been demonstrated in yeast (5-7). The amino acid sequence of tafazzin contains an acyltransferase motif, suggesting its involvement in lipid metabolism (8). Indeed, altered phospholipid composition was found in heart, skeletal muscle, platelets, lymphoblasts, and fibroblasts of BTHS patients (9-11). The phospholipid most affected by the disease is cardiolipin.Cardiolipin is a phospholipid dimer with four fatty acids that is specifically localized in mitochondria. Cardiolipin is tightly bound to virtually all major proteins of the inner mitochondrial membrane, notably the solute carriers, the F 0 F 1 -ATPase, as well as respiratory complex III and IV (for a review see ref. 12). Only in the presence of cardiolipin are the respiratory complexes able to form stable oligomeric supercomplexes (13,14). Thus, cardiolipin is thought to play an essential role in the supramolecular organization of the inner mitochondrial membrane. In most cardiolipin molecular species only one or two typ...
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