Freeze-Etch Electron Microscopy
|Pujals Riatos, Silvia||Research Associate|
|Tenkova-Heuser, Tatyana||Research Associate|
The key goal of this laboratory has long been to develop advanced new procedures for preserving the living appearance of the meso-scaled molecular machinery found inside cells. Our basic procedure is the "quick-freeze/deep-etch" method of electron microscopy, which we originally developed to visualize the mechanisms involved in the quantal release of neural transmitter substances from brain synapses and neuromuscular junctions. This we found involved secretion of the meso-scaled entities called "synaptic vesicles". Subsequently, our freeze-etch techniques were disseminated and reproduced all around the world, as other electron microscopists sought to visualize the structures and living dynamics of many different meso-machines found inside cells, including receptor and signaling complexes, cytoskeletal actomyosin networks, and a whole variety of cell-membrane differentiations, including clathrin-coated pits, caveolae, and endocytotic organelles of all sorts.
Overall, our "quick-freeze/deep-etch" techniques have been used to capture, visualize, and understand several important cellular processes that occur far too rapidly, and on too small a scale, to visualize in any other way - not only neural transmission, but also muscular contraction, viral infection, immune-cell synapse formation, vesicular transport, and cell migration during neurogenesis.
Additionally, we have modified the "quick-freeze/deep-etch" technique so that we can visualize isolated and purified protein and DNA macromolecules, in order to better understand the molecular mechanisms that underlie cellular functioning on the meso-scale. In all of our studies of macromolecules, as well as our studies of cell organelles, our TEM and SEM-imaging techniques have provided exceedingly true-to-life views that retain the full meso-architecture of cells and organelles, and thus are best viewed by modern methods of 3D-imaging including tomography and stereology.
At the present, we are well along in a further development of cryo-scanning electron microscopy for directly visualizing frozen cells without any further manipulation. In this way, we intend to make our EM laboratory in the iCeMS the world leader in 3D electron microscopy at the meso-scale.
The cross-disciplinary projects that we have already initiated with other iCeMS researchers include the following:
- EM visualization of the pathological meso-scale entities that form in and around nerve and glial cells in various neurodegenerative diseases, including the "plaques and tangles" that develop in Alzheimer's disease, as well as the various other intracellular-fibril "amyloid" aggregates that form in Parkinson's disease, Huntington's disease, ALS, etc. Here we are working closely with the Nakatsuji Lab to develop and analyze various ES and iPS cell-lines that are genetically engineered to recapitulate these diseases by forming intracellular fibril-aggregates, with the goal of determining what can be done to prevent their formation or assist the affected cells in ridding themselves of them.
- The above project also involves close collaboration with the Kusumi Lab, in order to correlate our EM observations with their high-speed single-molecule imaging of fibril-formation, in a further effort to determine the effects this has on membrane and organellar dynamics in living cells. Indeed, we are seeking to determine the EM-equivalents of many different aspects of the advanced high-speed single-molecule imaging that is always being done, on many different fronts, in the Kusumi Lab.
- Finally, we are seeking to provide EM support for a number of other multidisciplinary research projects going on within the iCeMS, including the development of "smart nanoporus materials" with the Takano and Kitagawa Labs, the development of new imaging methods to visualize lipid transport and the formation of mesoscale lipid-assemblies with the Ueda and Kusumi Labs, and the spatial and temporal organization of organelles (everything from the mundane mitochondria to the most mysterious bit of 'nuage'), which the Hiiragi, Kengaku, and Nakatsuji Labs are studying to determine the special roles they play during embryonic and neural development.
- Heuser J E. The origins and evolution of freeze-etch electron microscopy. J Electron Microsc. 60, S3-29 (2011). PMID: 22449129
- Numata T, Murakami T, Kawashima F, Morone N, Heuser JE, Takano Y, Ohkubo K, Fukuzumi S, Mori Y, Imahori H. Utilization of photoinduced charge-separated state of donor-acceptor-linked molecules for regulation of cell membrane potential and ion transport. J Am Chem Soc. 134, 6092-6095 (2012). PMID: 22449129
- Tanaka T, Takahashi K, Yamane M, Tomida S, Nakamura S, Oshima K, Niwa A, Nishikomori R, Kambe N, Hara H, Mitsuyama M, Morone N, Heuser JE, Yamamoto T, Watanabe A, Sato-Otsubo A, Ogawa S, Asaka I, Heike T, Yamanaka S, Nakahata T, Saito MK. Induced pluripotent stem cells from CINCA syndrome patients as a model for dissecting somatic mosaicism and drug discovery. Blood. 120, 1299-1308. (2012) PMID: 22723549
- Minami I, Yamada K, Otsuji TG, Yamamoto T, Shen Y, Otsuka S, Kadota S, Morone N, Barve M, Asai Y, Tenkova-Heuser T, Heuser JE, Uesugi M, Aiba K, Nakatsuji N. A small molecule that promotes cardiac differentiation of human pluripotent stem cells under defined, cytokine- and xeno-free conditions. Cell Rep. 2, 1448-1460 (2012).PMID: 23103164
- Kadota S, Minami I, Morone N, Heuser JE, Agladze K, Nakatsuji N. Development of a reentrant arrhythmia model in human pluripotent stem cell-derived cardiac cell sheets. Eur Heart J. 34, 1147-1156 (2012). PMID: 23201623
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