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Electron Microscopy in Cell Biology: Integrating Structure and Function

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Electron Microscopy in Cell Biology: Integrating Structure and Function REVIEWS applied routinely3,7. CryoEM of isolated REVIEW molecules is a rapidly emerging and powerful approach for obtaining structural models of macromolecular complexes in the intermedi- Electron microscopy in cell biology: ate resolution range (0.5–2.0 nm). CryoEM can be applied to those complexes that are not integrating structure and function suitable for X-ray crystallography because of their size, instability, or the fact that they are Abraham J. Koster* and Judith Klumperman‡ available only in insufficient amounts (for a recent overview, see REF.8). The resolution of Electron microscopy (EM) is at the highest-resolution limit of a spectrum of cryoEM usually allows the relative positions complementary morphological techniques. When combined with molecular and orientations of individual components detection methods, EM is the only technique with sufficient resolution to of a macromolecular complex to be deter- mined to within a few angströms. For example, localize proteins to small membrane subdomains in the context of the cell. recently, single-particle cryoEM of the Recent procedural and technical developments have increasingly improved ribosome-bound class I release factor RF2 the power of EM as a cell-biological tool. indicated that binding of RF2 to a stop codon in the decoding centre of the 30S ribosomal subunit induces a conformational change in RF2 that allows it to interact simultaneously With the sequencing of the human genome at high resolution is combined with methods with a stop codon and the peptidyl-transferase nearly complete, characterization of the that specifically localize the functional com- centre of the 50S ribosomal subunit9,10. functions of the gene products will be an ponents of these membranes — that is, when important next challenge in cell biology. The cellular architecture is integrated with molec- Chemical fixation. Although there are routine rapidly growing number of human disor- ular information. For maximal output, we protocols for the high-pressure-freezing fixa- ders that are identified as an intracellular need to strive for the best possible structural tion of biological samples, precise experimen- membrane-trafficking defect unequivocally preservation and to use the most sensitive tal conditions must be established for each shows how protein function is directly protein-detection methods. sample to prevent the formation of damaging linked to intracellular localization and ice crystals, and not all material is suitable for emphasizes the importance of understanding Cryofixation. The best available approach so cryofixation. This is why many studies still membrane-mediated protein-trafficking far to immobilize and preserve cellular archi- make use of the more conventional chemical- pathways. Electron microscopy (EM) is the tecture is probably CRYOFIXATION3–5. HIGH-PRESSURE fixation methods. Chemical fixation, how- only technique that can combine sensitive FREEZING is the only way to freeze biological ever, induces structural artefacts and might protein-detection methods with detailed infor- samples of 10–200-µm thickness without cause the redistribution of particularly small mation on the substructure of intracellular generating artefacts as a result of ice-crystal soluble proteins. To weigh up the pros and compartments. This combined resolution formation, and is therefore the method of cons of the two preparation methods for power has given EM a central position in choice for cells and tissues6. In most applica- future EM studies, a careful analysis of the defining intracellular protein-distribution tions, high-pressure freezing is followed by possible artefacts that are induced by chemi- patterns and predicts a continuing and FREEZE SUBSTITUTION. These preparations are cal fixation is much needed. The more labo- increasing demand for EM in cell biology1–3. notable because of their sharp membrane rious high-pressure-freezing procedures Although EM is an established technology, delineation and the clear visibility of their might be advantageous for some, but not all, new methodologies have recently been devel- cytoskeletal elements. In particular, cellular cell-biological questions. oped to assess the fine-structure identity of compartments that are susceptible to shrink- intracellular membrane-enclosed compart- age as a result of chemical fixation — for Assessing molecular information. The most ments. At the same time, the methods to example, endosomes — retain their turgid widely used approach to detect proteins in localize molecules of interest to within these shape after high-pressure-freezing fixation cells is IMMUNOEM. Many protein-sorting compartments have been broadened and (J. L. Murk, M. Kleijmeer and B. Humbel, events occur in 40–60-nm, often coated, perfected. Furthermore, techniques are now personal communication). FIGURE 1b shows membrane subdomains, which are below the evolving that literally add an extra dimension an example of a Golgi complex that was pre- detection level of the light microscope and to EM images — electron tomography (ET) pared using this protocol. are distinguishable in the EM only under the for three-dimensional (3D) imaging and When freeze substitution is omitted and most favourable conditions. The TOKUYASU CORRELATIVE MICROSCOPY for integrating the imag- frozen cells are viewed directly in the electron CRYOSECTIONING TECHNIQUE11, which was intro- ing of live cells and EM. Here, without the microscope, we refer to this method as CRYOEM duced in 1973, has been improved and perfect- pretension of being complete, we highlight — a technique that was pioneered by ed so that it now generally surpasses other some of the most recent and developing EM Dubochet and colleagues (for more informa- techniques with respect to membrane visibility techniques, which we believe will have an tion, see REF.4). CryoEM provides the exciting and labelling sensitivity12–14. FIGURE 2f and 2g important role in the future of cell biology. possibility of viewing the cell in what is prob- show examples of a multivesicular endosome ably its most natural state. The full procedure, and small transport carriers, respectively, as ImmunoEM: crucial considerations from high-pressure freezing to visualizing they appear in ultrathin cryosections. The pre- EM is exploited to its full capacity when its slices of frozen material by EM, however, ferred tag for the visualization of antibodies by power to visualize membrane compartments needs further development before it can be EM is colloidal gold (FIG. 2d–g), which can be SS6 | SEPTEMBER 2003 www.nature.com/focus/cellbioimaging REVIEWS a b determination of the subcellular localization of secretory proteins and even cholesterol19,20. Moreover, because section fixation occurs at low temperature and is rapid, it will probably solve at least some of the alleged penetration problems of chemical fixation. Alternatively, a combination of high-pressure freezing/freeze GGsubstitution and subsequent rehydration of the LLcells or tissue is presently under development. This ‘rehydration’ procedure will allow high- pressure-frozen material to be subjected to the Tokuyasu cryosectioning technique with a minimized chance of artefacts that are caused by chemical fixation, and therefore com- bines the strongest points of the two methods c d (J. W. Slot, unpublished observations). Localizing lipids. Protein-trafficking events crucially depend on membranes, but remark- ably little is known about the in situ intracellu- lar distribution of lipids. A significant problem for lipid localization by EM is that routine fixation procedures immobilize lipids insuffi- ciently and cause their extraction and redis- tribution. The best results so far were obtained with chemically fixed and freeze-substituted cells21,22. More recently, with some simple modifications, the Tokuyasu approach was adapted to localize cholesterol19 and success- Figure 1 | The electron-tomography process on a 250-nm thick section of a high-pressure frozen, fully provided the first EM double-labelling of freeze-substituted and Epon-embedded dendritic cell. a | A digital image at 0º tilt, which was taken from two lipids — cholesterol and bis(monoacyl- a tilt series of 131 images that ranged from –65º to +65º. Because of the thickness of the section, the glycero)phosphate20. An increasing number of membranes near the Golgi complex (G) are poorly distinguished. The non-specific 10-nm colloidal gold labels (black dots) were deposited on the surface of the section to allow image alignment during the lipid-binding toxins are becoming available for reconstruction process27. L, lysosome. b | A 3.2-nm thin digital slice from the tomogram that was computed use in EM detection. Combined with the from the above-mentioned tilt series. The limited thickness of the digital slice allows the membrane methodological innovations, this gives us hope morphology to be seen more clearly. c | Computer-assisted contour drawing on the digital slice of the that in the near future the in situ distribution of tomogram that is shown in part b. The scale bars in a, b and c represent 200 nm. d | A contour model of some more types of lipids will be assessed at high res- of the structures that are visible in the tomogram shown in part b. This figure was kindly provided by W. J. C. olution. Moreover, it opens up the possibility of Geerts (Department of Molecular Cell Biology, Faculty of Biology, Utrecht, The Netherlands) and J. L. Murk (Department of Cell Biology, University Medical Center Utrecht, The Netherlands). studying
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