Agar-overlay immunofluorescence: high-resolution studies of cytoskeletal components and their changes during...

Cells that are flattened by overlaying with a thin sheet of agarose can be instantaneously fixed with freezing absolute methanol containing 1% formalin. This procedure results in good preservation of the cytoskeleton. Use of this technique ("agar-overlay immunofluorescence") clarified that (1) Dictyostelium myosin exists in situ as thick filaments (Yumura and Fukui, 1985), (2) the thick filaments are arranged in a meshwork at the posterior cortex of a polarized cell performing directed locomotion, at the constricting portion of a dividing cell forming a contractile ring, and at the outermost lateral periphery of a cell engaging in spiral aggregation (Fig. 1f; Yumura et al., 1984; Yumura and Fukui, 1985), and (3) the distribution of thick filaments changes dramatically in response to the chemoattractant cAMP in 1 minute (Fig. 3; Yumura and Fukui, 1985). This technique can provide valuable information on the dynamic features as well as the detailed organization of cytoskeletal elements which, otherwise, cannot be visualized with sufficient resolution.