Visualization and measurement of calcium transients in Amoeba proteus by fura-2 fluorescence

Abstract

A fura-2 microspectrofluorimeter was used to visualize and measure intracellular calcium transients in normal locomoting and experimentally treated Amoeba proteus. The results show that subcellular heterogeneities of cytosolic free calcium, [Ca2+]i, correlate in time and distribution with characteristic patterns of protoplasmic streaming and ameboid movement. In detail, calcium ions have a dual effect by regulating both the contractile activities of the actomyosin cortex and the rheological properties of the cytoplasmic matrix. A high resting [Ca2+]i of 1.5 to 2.0 x 10(-7) M in the uroid region or in retracting pseudopodia is associated with the transformation of rigid ectoplasmic gel into fluid endoplasmic sol, and a low [Ca2+]i of 10(-9) to 10(-8) M in the front region or in extending pseudopodia with the re-transformation of endoplasmic sol into ectoplasmic gel. Locally increased peripheral [Ca2+]i accumulations higher than 10(-7) M are also observed at places where the actomyosin cortex is known to generate motive force by contraction, i.e., in the intermediate region of orthotactic amebas or in large pseudopodia of polytactic cells. External application of 30 mM KCl abolishes the intracellular Ca2+ gradient such that [Ca2+]i attains a uniform distribution and a maximum concentration of 2 x 10(-7) M; as a consequence, cells can show a transient loss of their locomotor activity and polarity by undergoing spherulation and total contraction. On the other hand, high external Ca2+ concentrations in the range of 100 mM stabilize the bipolar cellular organization, enhance the movement velocity and induce the propagation of Ca2+ waves repeatedly running from the uroid to the front region. The significance of external ions for signal transmission and the control of dynamic activities as well as the origin and fate of calcium participating in the observed transients are discussed.