Faculty Bibliography

BACKGROUND: Barth syndrome is a rare, multisystem disorder caused by mutations in tafazzin that lead to cardiolipin deficiency and mitochondrial abnormalities. Patients most commonly develop an early-onset cardiomyopathy in infancy or fetal life. METHODS AND RESULTS: Knockdown of tafazzin (TAZKD) in a mouse model was induced from the start of gestation via a doxycycline-inducible shRNA transgenic approach. All liveborn TAZKD mice died within the neonatal period, and in vivo echocardiography revealed prenatal loss of TAZKD embryos at E12.5-14.5. TAZKD E13.5 embryos and newborn mice demonstrated significant tafazzin knockdown, and mass spectrometry analysis of hearts revealed abnormal cardiolipin profiles typical of Barth syndrome. Electron microscopy of TAZKD hearts demonstrated ultrastructural abnormalities in mitochondria at both E13.5 and newborn stages. Newborn TAZKD mice exhibited a significant reduction in total mitochondrial area, smaller size of individual mitochondria, reduced cristae density, and disruption of the normal parallel orientation between mitochondria and sarcomeres. Echocardiography of E13.5 and newborn TAZKD mice showed good systolic function, but early diastolic dysfunction was evident from an abnormal flow pattern in the dorsal aorta. Strikingly, histology of E13.5 and newborn TAZKD hearts showed myocardial thinning, hypertrabeculation and noncompaction, and defective ventricular septation. Altered cellular proliferation occurring within a narrow developmental window accompanied the myocardial hypertrabeculation-noncompaction. CONCLUSIONS: In this murine model, tafazzin deficiency leads to a unique developmental cardiomyopathy characterized by ventricular myocardial hypertrabeculation-noncompaction and early lethality. A central role of cardiolipin and mitochondrial functioning is strongly implicated in cardiomyocyte differentiation and myocardial patterning required for heart development. (J Am Heart Assoc. 2012;1:jah3-e000455 doi: 10.1161/JAHA.111.000455.).

This Meeting Review describes the proceedings and conclusions from the inaugural meeting of the Electron Microscopy Validation Task Force organized by the Unified Data Resource for 3DEM (http://www.emdatabank.org) and held at Rutgers University in New Brunswick, NJ on September 28 and 29, 2010. At the workshop, a group of scientists involved in collecting electron microscopy data, using the data to determine three-dimensional electron microscopy (3DEM) density maps, and building molecular models into the maps explored how to assess maps, models, and other data that are deposited into the Electron Microscopy Data Bank and Protein Data Bank public data archives. The specific recommendations resulting from the workshop aim to increase the impact of 3DEM in biology and medicine.

We present a major revision of the iterative helical real-space refinement (IHRSR) procedure and its implementation in the SPARX single particle image processing environment. We built on over a decade of experience with IHRSR helical structure determination and we took advantage of the flexible SPARX infrastructure to arrive at an implementation that offers ease of use, flexibility in designing helical structure determination strategy, and high computational efficiency. We introduced the 3D projection matching code which now is able to work with non-cubic volumes, the geometry better suited for long helical filaments, we enhanced procedures for establishing helical symmetry parameters, and we parallelized the code using distributed memory paradigm. Additional features include a graphical user interface that facilitates entering and editing of parameters controlling the structure determination strategy of the program. In addition, we present a novel approach to detect and evaluate structural heterogeneity due to conformer mixtures that takes advantage of helical structure redundancy.

Erythrocytes possess a spectrin-based cytoskeleton that provides elasticity and mechanical stability necessary to survive the shear forces within the microvasculature. The architecture of this membrane skeleton and the nature of its intermolecular contacts determine the mechanical properties of the skeleton and confer the characteristic biconcave shape of red cells. We have used cryo-electron tomography to evaluate the three-dimensional topology in intact, unexpanded membrane skeletons from mouse erythrocytes frozen in physiological buffer. The tomograms reveal a complex network of spectrin filaments converging at actin-based nodes and a gradual decrease in both the density and the thickness of the network from the center to the edge of the cell. The average contour length of spectrin filaments connecting junctional complexes is 46 +/- 15 nm, indicating that the spectrin heterotetramer in the native membrane skeleton is a fraction of its fully extended length ( approximately 190 nm). Higher-order oligomers of spectrin were prevalent, with hexamers and octamers seen between virtually every junctional complex in the network. Based on comparisons with expanded skeletons, we propose that the oligomeric state of spectrin is in a dynamic equilibrium that facilitates remodeling of the network as the cell changes shape in response to shear stress

CopA uses ATP to pump Cu(+) across cell membranes. X-ray crystallography has defined atomic structures of several related P-type ATPases. We have determined a structure of CopA at 10 A resolution by cryo-electron microscopy of a new crystal form and used computational molecular docking to study the interactions between the N-terminal metal-binding domain (NMBD) and other elements of the molecule. We found that the shorter-chain lipids used to produce these crystals are associated with movements of the cytoplasmic domains, with a novel dimer interface and with disordering of the NMBD, thus offering evidence for the transience of its interaction with the other cytoplasmic domains. Docking identified a binding site that matched the location of the NMBD in our previous structure by cryo-electron microscopy, allowing a more detailed view of its binding configuration and further support for its role in autoinhibition

Cryo-electron tomography and subtomogram averaging are utilized to determine that the bacteriophage varphi12, a member of the Cystoviridae family, contains surface complexes that are toroidal in shape, are composed of six globular domains with six-fold symmetry, and have a discrete density connecting them to the virus membrane-envelope surface. The lack of this kind of spike in a reassortant of varphi12 demonstrates that the gene for the hexameric spike is located in varphi12's medium length genome segment, likely to the P3 open reading frames which are the proteins involved in viral-host cell attachment. Based on this and on protein mass estimates derived from the obtained averaged structure, it is suggested that each of the globular domains is most likely composed of a total of four copies of P3a and/or P3c proteins. Our findings may have implications in the study of the evolution of the cystovirus species in regard to their host specificity

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 polarized domains of myelinated axons are specifically organized to maximize the efficiency of saltatory conduction. The paranodal region is directly adjacent to the node of Ranvier and contains specialized septate-like junctions that provide adhesion between axons and glial cells and that constitute a lateral diffusion barrier for nodal components. To complement and extend earlier studies on the peripheral nervous system, electron tomography was used to image paranodal regions from the central nervous system (CNS). Our three-dimensional reconstructions revealed short filamentous linkers running directly from the septate-like junctions to neurofilaments, microfilaments, and organelles within the axon. The intercellular spacing between axons and glia was measured to be 7.4 +/- 0.6 nm, over twice the value previously reported in the literature (2.5-3.0 nm). Averaging of individual junctions revealed a bifurcated structure in the intercellular space that is consistent with a dimeric complex of cell adhesion molecules composing the septate-like junction. Taken together, these findings provide new insight into the structural organization of CNS paranodes and suggest that, in addition to providing axo-glial adhesion, cytoskeletal linkage to the septate-like junctions may be required to maintain axonal domains and to regulate organelle transport in myelinated axons. (c) 2010 Wiley-Liss, Inc