The natural ebb and flow of cytoplasmic streaming inside the giant multinucleate plasmodia of Physarum polycephalum has been much studied (5, 10). Beause both actin and myosin can be extracted from Physarum (7), it is reasonable to suppose that streaming might be due to a calciumsensitive contractile system similar to that found in muscle. In order to test this hypothesis, we injected the calcium-specific pbotoprotein, aequorin, into short single strands of Physarum. The aequorin method for the detection of changes in the ionized intracellular calcium level has previously revealed calcium transients associated with contraction in barnacle muscle (9) and calcium permeability changes in squid axons (1).Our main conclusions are: first, that cyclic changes in the ionized intracellular calcium level do occur during streaming in Physarum; and second, that the polarity of the streaming is such that contraction occurs in the region of elevated Ca ++ '
METHODSPhysarum polycephalum strain M3cV, a gift of Dr. JoyceMohberg of the University of Wisconsin, Madison, Wis., was grown on an axenic medium (2) solidified with 2% agar. Small pieces of plasmodium were allowed to migrate on non-nutrient agar containing 1 mM NaCI, 1 mM CaCI2 for the experiments reported here.Microinjection of Physarum is difficult because of the efficiency of the surface precipitation, or wound-healing, response. We used beveled glass micropipets which had been washed with 20 mM EDTA then distilled water, and dried. Micropipets were filled with 0.2 #1 of aequorin solution (see reference 1 for details), mounted on a Leitz micromanipulator, and inserted gently into a large strand of plasmodium. Then, small aliquots of the aequorin solution were forced into the strand with a low air pressure, after which the organism was allowed to recover for a few minutes.After microinjection, the strand of plasmodium together with the supporting rectangular block of agar was placed on a glass slide that held Ag-AgCI electrodes (see Fig. 1 A). Next, two deep "V" cuts were made from opposite sides through the agar block, leaving only the strand of plasmodium bridging the gap at the junction of the two V cuts. The total volume of Physarum cytoplasm in our preparations was on the order of 3 5,1.The electrodes were coupled through a high-impedance differential voltage amplifier to a chart recorder. Although slightly different from the method of Kamiya (5), this set-up produced similar "electroplasmograms" (EPG) recording the differences in electrical potential between the two ends of a plasmodium. By observing the streaming movements of the aequorin-injected plasmodium with a compound microscope and simultaneously measuring the "'electroplasmogram," we were able to verify that protoplasm flows away from the end of the strand which is more negative. Fig. 1 B illustrates the experimental apparatus for recording Ca ++ -mediated light emission (9) from an aequorin-injected strand of plasmodium. Light produced during the reaction between the injected aequorin and intracellular C...
