Reactivation of a cell-free model from Physarum polycephalum: studies on cryosections indicate an inhibitory effect of Ca++ on cytoplasmic actomyosin contraction.

Cell-free models should offer "in situ conditions" to study the physiology of cytoplasmic actomyosin in its natural environment, while, if possible, still associated with its regulatory control proteins and other cytoplasmic components. Detergents and glycerol as the usual media to permeabilize the plasmalemma and to extract a portion of the cytoplasmic components, are accompanied by several disadvantages. We investigated a cell-free model consisting of cryosections of plasmodial strands that were previously enriched with "stress fibrils" and fluorescently labelled with phallotoxins and that contain the non-denatured structures that are to be reactivated in situ. The contraction reaction can be directly observed in the fluorescence microscope. This procedure allows the study of contraction conditions in the natural environment of the fibrils. The aim of these reactivation experiments was to identify the role of calcium ions. According to our results, a reactivation of cryosections is not Ca++ dependent but is partly inhibited at concentrations of 10(-4) to 5 X 10(-2)M Ca++. Complete inhibition occurs at 10 to 20 mM Ca++. Electron microscopical investigations revealed that the fluorescently labelled contracting structures were identical to actomyosin fibrils.