(Supported in part by NSF MCB 9219043 to C. Mannella) Thick sections (0.25-1.0 (m) of isolated and in situ rat liver mitochondria are conventionally fixed or high-pressure frozen and freeze substituted, and embedded in epoxy resin. The biological goals are to determine the interactions between the inner and outer mitochondrial membranes, the continuity between internal compartments, and the interactions between mitochondria and other cytoplasmic structures under various metabolic conditions. In the future, this work will be extended to frozen-hydrated material. This year we concentrated on interactions between the mitochondria and the endoplasmic reticulum, and on developing cryo preparation techniques. First, using perfusion-fixed, conventionally-embedded liver tissue, we made two double-tilt reconstructions (0.25 (m thick sections) of in situ mitochondria to compare with isolated mitochondria examined previously. The cristae were flatter, more lammelar in form, but still connected to the intramembrane space by narrow (30-40nm) channels, as we found with the isolated mitochondria. We included in these reconstructions the adjacent endoplasmic reticulum (ER), and found that fine projections (lacking ribosomes) from the ER were closely apposed to the mitochondrial outer membrane. We re-examined a reconstruction of an isolated mitochondrion which had a fragment of smooth ER attached, and found """"""""bridges"""""""" connection the ER to the outer mitochondrial membrane. We also made maps of the """"""""contacts"""""""" between the inner and outer membranes, and found them to be similar to those of the isolated mitochondria. Sterecon tracing and visualization for this project was with the assistance of Shanno n Pixley, a student of Sienna College in Albany. Then, to confirm our findings using better-preserved, cryo-prepared mitochondria, we obtained high-pressure frozen/freeze substituted rat liver tissue from the lab of Martin Maller (ETH, Zurich). We made a reconstruction from a 0.25lm thick section, but the cristae were difficult to see, due to the better retention of material. Nevertheless, the interaction between the mitochondria and ER could be clearly seen, so we made a large-area reconstruction from a 1.5lm thick section, in which several mitochondria were included. We reconstructed and visualized sheet-like cisternae of ER enveloping mitochondria and forming groups of mitochondria connected by ER. Tracing and visualization was assisted by Meghna Gupta, a Cornell University student. In hopes of better membrane visualization, and better control of the mitochondria prior to fixation, we made our own s. Material was frozen at three different time intervals, during which the tissue was kept in buffer. The first-frozen material (within 5 min) looked very much like that of Muller, with the cristae somewhat more visible than in his preparation. The other two time periods (15 and 30 min) had expanded intracristal spaces typical of degenerating mitochondria. So far, we have made a reconstruction of a mitochondrion from the middle time period, and found that the cristae were somewhat plate-like, but still connected by narrow channels to the intramembrane space. We also made a reconstruction of a mitochondrion from a cryo-section of fresh-frozen liver prepared by the Tokuyasu post-embedding procedure, which uses sugar embedding with uranyl acetate stain. The cristae looked similar to the middle-period high-pressure frozen, freeze-substituted preparation. For comparison to the liver mitochondria, we made a reconstruction of a mitochondrion in a conventionally-fixed rat motor neuron. The cristae were very twisted in the example reconstructed, and interpretation and tracing of the cristae was very difficult, so firm conclusions could not be reached. We collaborated with other labs interested in mitochondrial structure. See CS sub-projects """"""""Regulation of Steroidogenesis"""""""" by Dr. J. Strauss of U. of PA and """"""""Mitochondrial Transmembrane Processes in Human Mitochondriopathies"""""""" by Dr. W. Ruitenbeek of University Hospital, Nijmegen, The Netherlands. Every reconstruction required sophisticated visualization techniques. We were aided in this by special programs, and modifications to existing programs, written by the BMIRR Senior Programmer, Dr. A. Leith. We made attempts to segment cristae and membranes by automatic means, but found that we had to rely on interactive tracing using Sterecon for all the present work. We made many striking color views of the reconstructions, and several videotapes of animation's showing the features to be emphasized. These were presented at several meetings, and included in a publication. Mannella, C.A., Marko, M., Buttle, K. (1997) Reconsidering mitochondrial structure: New views of an old organelle. Trends in Biochemical Science 22,37-38 Mannella, C.A., Marko, M., Buttle. K., D'Archangelis, D., O'Farrell, K., Tessitore, K. (1997) Interactions of the mitochondrial outer membrane with the inner membrane and endoplasmic reticulum, Proc. 41st Ann. Meet. Biophysical Society, New Orleans, LA, Biophysical J. (in press) Mannella, C.A., Buttle, K., Tessitore, K., Pixley, S., Rath, B., D'Arcangelis, D., Marko, M. (1997) Electron tomography of rat-liver mitochondria: structure of cristae and interactions with endoplasmic reticulum. Proc. Microscopy and Microanalysis 97 Ed. G. Bailey, Jones and Begell, New York, 225-226 Frank, J., Mannella, C.A., Rieder, C. (1997) An integrated biological imaging facility: capabilities of the Biological Microscopy and Image Reconstruction Resource. Proc. Microscopy and Microanalysis 97 Ed. G. Bailey, Jones and Begell, New York, 271-272. Mannella, C. A., Buttle, K., Tessitore, K., Pixely, S., Gupta, M., Rath, B., D'Arcangelis, D., Marko, M. (1997) Workshop on New Perspectives in Mitochondrial Research, Univ of Padua, Italy, September (sponsored by IUBMB, UNESCO and Italian Society of Biochemistry).
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