engleski

Cryoelectron Tomography: Zooming Into Intact Cells

Cryoelectron tomography has unique potential for three-dimensional visualization of intact macromolecular complexes in their natural environment. This approach is based on reconstructing three-dimensional volumes from tilt series of electron micrographs of cells preserved in their native state by vitrification.The basic idea of tomography is first to record a series of two-dimensional projection images of the object of interest as viewed from different directions, and then to synthesize these projections into a three-dimensional density map. Prerequisite for preservation of a cell and its contents in their native state suitable for electron microscopy is vitrification, i.e. embedding in amorphous ice as a result of rapid freezing. Using these techniques, resolutions of 5– 8 nm have already been achieved and the prospects for further improvement are good. Since many intracellular activities are conducted by complexes in the megadalton range with dimensions of 20– 50 nm, current resolutions should suffice to identify many of them in tomograms. However, residual noise and the dense packing of cellular constituents hamper interpretation. In cryoelectron tomography there are two conflicting requirements, which are difficult to reconcile. On the one hand, resolution and quality of a tomogram are directly dependent on spacing of the projections and on the angular range covered. Therefore, data must be collected over a tilt range as wide as possible, with increments as small as possible. On the other hand, the exposure to the electron beam must be minimized to prevent radiation damage. The development of elaborate automated data-acquisition procedures enables a dramatic reduction of the beam exposure and makes it possible to take advantage of the principle of dose fractionation. Recently, tomographic data have been collected on vitrified eukaryotic Dictyostelium cells, focusing on the actin cytoskeleton. In actin networks reconstructed without prior removal of membranes or extraction of soluble proteins, the cross-linking of individual microfilaments, their branching angles, and membrane attachment sites were analyzed. At a resolution of 5 to 6 nanometers, single macromolecules with distinct shapes, such as the 26S proteasome, were identified in an unperturbed cellular environment.

Tomography; Cytoskeleton; Cryofixation

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