Electron Microscopy Of The Cytoskeleton Of Cultured Cells T. M. Svitkina, A. B.Verkhovsky, and G.G. Borisy In: "Recent Advances in Microscopy of Cells, Tissues and Organs" (ed. P. M. Motta), pp. 93-100. Antonio Delfino Editore S.r.l., Rome. 1997.

We have developed an improved procedure appropriate for correlative light and electron microscopic study of the cytoskeleton of cultured cells. The electron microscopic component of the procedure is based on detergent extraction, chemical fixation, critical point drying and platinum/carbon coating. Minor adjustments were made at each step of the procedure which collectively produced a substantial improvement in the quality of images. Examples of cytoskeletal features and compatibility with molecular identification techniques are presented. Long and straight actin filaments were clearly seen in stress fibers and newly formed lamellipodia, and their polarity could be revealed by decoration with myosin subfragment 1. Various cytoskeletal elements could be identified by immunogold labelling. Depletion of actin from cytoskeletons by gelsolin treatment allowed for better visualization of myosin II, intermediate filaments, and microtubules. Myosin II bipolar minifilaments in the cytoskeleton associated with each other by their head-containing ends and were arranged parallel to each other or at low angles, so that myosin filament stacks of variable length were formed. These stacks corresponded to myosin spots and ribbons observed by fluorescence light microscopy as well as by immunoelectron microscopy. Intermediate filaments exposed after removal of actin exhibited numerous side projections that were made of plectin. These plectin sidearms connected intermediate filaments to microtubules and the actin-based cytoskeleton consistent with their possible role in the integration of the cytoplasm.

	Figure 1a (116k) - Lamellipodia at the leading edge of a cell contains a dense array of overlapping actin filaments
	Figure 1b (110k) - Actin filaments in a stress fiber are aligned and densely packed
	Figure 1c (138k) - Decoration of actin filaments with myosin S1 in lamellipodia
	Figure 1d (143k) - Decoration of actin filaments with myosin S1 in stress fiber

	Figure 2a (143k) - Overview of intact cytoskeleton immunolabeled for myosin II with 10 nm colloidal gold
	Figure 2b (138k) - Enlargement of intact cytoskeleton immunolabeled for myosin II with 10 nm colloidal gold
	Figure 2c (143k) - Myosin II bipolar filaments are visualized more clearly in gelsolin-treated cytoskeletons
	Figure 2d (149k) - Myosin II bipolar filaments are visualized more clearly in gelsolin-treated cytoskeletons

	Figure 3a (127k) - Gelsolin treatment of extracted cells reveals abundant millipede-like structures that consist of intermediate filaments with numerous sidearms
	Figure 3b (110k) - Immunolabeling for vimentin with 10 nm colloidal gold
	Figure 3c (99k) - Immunolabeling for plectin with 10 nm colloidal gold
	Figure 3d (132k) - Immunolabeling for tubulin with 10 nm colloidal gold confirms the identity of microtubules participating in interaction with intermediate filaments via plectin sidearms