Imaging Cytoskeleton Components by Electron Microscopy

Abstract

The cytoskeleton is a complex of detergent-insoluble components of the cytoplasm playing critical roles in cell motility, shape generation, and mechanical properties of a cell. Fibrillar polymers-actin filaments, microtubules, and intermediate filaments-are major constituents of the cytoskeleton, which constantly change their organization during cellular activities. The actin cytoskeleton is especially polymorphic, as actin filaments can form multiple higher order assemblies performing different functions. Structural information about cytoskeleton organization is critical for understanding its functions and mechanisms underlying various forms of cellular activity. Because of the nanometer-scale thickness of cytoskeletal fibers, electron microscopy (EM) is a key tool to determine the structure of the cytoskeleton. This article describes application of rotary shadowing (or metal replica) EM for visualization of the cytoskeleton. The procedure is applicable to thin cultured cells growing on glass coverslips and consists of detergent extraction of cells to expose their cytoskeleton, chemical fixation to provide stability, ethanol dehydration and critical point drying to preserve three-dimensionality, rotary shadowing with platinum to create contrast, and carbon coating to stabilize replicas. This technique provides easily interpretable three-dimensional images, in which individual cytoskeletal fibers are clearly resolved, and individual proteins can be identified by immunogold labeling. More importantly, replica EM is easily compatible with live cell imaging, so that one can correlate the dynamics of a cell or its components, e.g., expressed fluorescent proteins, with high resolution structural organization of the cytoskeleton in the same cell.

Keywords: Actin; Correlative microscopy; Critical point drying; Cytoskeleton; Electron microscopy; Immunogold; Microtubules; Rotary shadowing.

Fig. 1

Correlative phase-contrast and EM of cultured Rat-2 fibroblast combined with immunogold staining of ADF/cofilin. (a and b) Frames from time-lapse sequence showing the last live cell image (b) and an image 12 s earlier (a). Black line in (b) shows the cell edge outline from (a). (c) Low magnification EM image of the cell overlaid with the cell outline, as in (b). (d) EM of the protruding edge (asterisk in (c)) comprising a lamellipodium filled with dense actin network. Before the EM processing, the sample was immunogold labeled by cofilin antibody; inset in (d) shows gold particles as white dots

Fig. 2

Correlative fluorescence and EM of cultured B16F1 mouse melanoma cell expressing EGFP-capping protein. (a) Map showing position of the cell (number 9 in a circle) relative to the reference marks on the coverslip. (b) Fluorescence image the cell showing localization of EGFP-capping protein to the edge of lamellipodia and puncta in lamella. (c) Phase contrast image of the same cell. (d) Low magnification EM image of the same cell. Box indicates a region enlarged in (e). (e) High magnification EM of the boxed region from (d) showing actin filament bundle in a filopodium in the center and dense branched network of actin filaments in lamellipodia. Inset shows the same region by fluorescence microscopy. Bright fluorescence corresponds to lamellipodia, while the dim region (arrow) corresponds to the filopodium