This past year, we finished a seminal study on the structure of neuronal mitochondria from chick and rat using electron microscope tomography of chemically fixed tissue. Three-dimensional reconstructions of representative mitochondria were made from single-axis tilt series acquired with the intermediate voltage electron microscope (400 kV) at NCMIR. We found that the mitochondrial ultrastructure was similar across species and neuronal regions. The outer and inner membranes were each ~7 nm thick. The inner boundary membrane was found to lie close to the outer membrane with a total thickness across both membranes of ~22 nm. We discovered that the inner membrane invaginates to form cristae only through narrow, tubular openings, which we call crista junctions. Sometimes the cristae remain tubular throughout their length, but often multiple tubular cristae merge to form lamellar compartments. Punctate regions, ~14 nm in diameter, were observed in which the inner and outer membranes appeared in contact (total thickness of both membranes ~14 nm) These contact sites are known to a play a key role in the transport of proteins into the mitochondrion. It has been hypothesized that contact sites may be proximal to crista junctions to facilitate transport of proteins destined for the cristae. However, our statistical analyses indicated that contact sites are randomly located with respect to these junctions. In addition, a close association was observed between endoplasmic reticulum membranes and the outer mitochondrial membrane, consistent with the reported mechanism of transport of certain lipids into the mitochondrion. Another study that has recently been finished used electron microscope tomography on both cryofixed and chemically fixed brown adipose tissue (BAT) to examine the membrane topology of BAT mitochondria, which possess unique bioenergetics due to an uncoupling protein. The three-dimensional reconstructions of BAT mitochondria provided a view different in important biomembrane features from descriptions found in the literature. We gained new insight into BAT mitochondria architecture by identifying crista junctions, including multiple junctions connecting a crista to the same side of the intermembrane space, in a class of mitochondria that have no tubular cristae, but only lamellar cristae. We found that the cristae architecture of cryofixed mitochondria, including crista junctions, is identical to that found in chemically fixed mitochondria suggesting that this architecture is not a fixation artifact and is likely found in vivo. In cryofixed mitochondria almost all of the outer membrane was observed to be in close contact with the inner boundary membrane, which has implications for the exchange of ATP/ADP across these membranes. The stacks of lamellar cristae extended through more of the BAT mitochondrial volume than did the cristae we observed in neuronal mitochondria. Hence, the inner membrane surface area was larger in the former, which may reflect the additional surface occupied by the uncoupling protein, but may also result from a higher concentration of electron transport proteins. This is consistent with the high metabolic/thermogenic activity of BAT mitochondria. cAMP-dependent protein kinase (PKA), one of the first protein kinases discovered, mediates a variety of hormonal and neurotransmitter responses by phosphorylating different substrate proteins in the cell. Compartmentalization of PKA is achieved in part by interaction with A-kinase anchoring proteins (AKAPs). This past year, we have made significant progress in identifying the physiological partners of PKA and PKC with a selected subset of AKAPs using immunochemical methods coupled with confocal microscopy and electron microscopy Most of this work has been with a novel AKAP, called D-AKAP1 which binds both type I and type II regulatory subunits of PKA. Although PKA is a multifunctional enzyme with a broad substrate specificity, activation of this kinase permits preferential phosphorylation of specific target substrates. While the importance of PKA in regulating many cellular processes has long been apparent, the potential importance of compartmentalization for the function and regulation of PKA has only recently been recognized. Investigation of compartmentalization of PKA by fluorescently labeling the regulatory subunits and D-AKAP1 has been our major endeavor this past year and utilized both laser scanning confocal microscopy and electron microscopy to determine labeling on three levels. The first level is cellular, i.e. to determine which cell type expresses D-AKAP1. The second level is subcellular; which subcellular structures have D-AKAP1. The third level is suborganellar, e.g., does D-AKAP1 bind to the inner or outer mitochondrial membrane. Confocal microscopy is principally used at the first two levels, while electron microscopy is required for the third level. Being able to visualize these anchoring proteins and the physiological PKA and PKC partners in cells is now affording us an understanding of how these molecules function in living cells. In the coming year, we will generate high-resolution 3-D reconstructions of antibody-labeled mitochondra by electron tomography to provide a foundation for the mapping of AKAPs, PKA and PKC in those cells that show mitochondrial labeling. Another project involving the IVEM was completed in 1997. This project looked at RXRa null mutant mice which display ocular and cardiac malformations, liver developmental delay, and die from cardiac failure around embryonic day (E) 14.5 pc. To help dissect the molecular basis of the RXRa-associated cardiomyopathy, we performed ultrastructural studies on sections of embryonic heart which suggested that the density of mitochondria per myocyte was higher in the RXRa mutant compared to wild-type littermates.