RIDGWAY, R.L.*; CHEUNG, M.B.; BARLAM, I.: Hemocyte Activation in Molluscs is Inhibited in the Presence of a Ca²⁺-chelating Buffer

In the snail Lymnaea stagnalis, hemocyte activation is correlated with a rapid shift in cytoskeletal organization that facilitates amoeboid movement and cell aggregation. Activation involves characteristic stages: 1) substrate attachment, 2) spreading/flattening out, 3) generation of lamellipodia/filipodia, 4) random motility, 5) directed motility/phagocytosis, and 6) hemocyte aggregation/fibroblast-like morphology. Previously, a chelating buffer (containing 2 mM Na²-EDTA) was shown to prevent hemocyte aggregation (Adema et al., Dev. Comp. Immunol. 18: 25-31, 1994). We employed this buffer to examine the effects of lowering extracellular Ca²⁺ on the time course of hemocyte activation and the shift in organization of microfilaments/microtubules. In the presence of the chelating buffer hemocytes were slower to attach to the substrate, but reached stage 3 in roughly the same time (5-10 min) as control preparations. After 15 min of exposure to EDTA, however, most hemocytes began to round up (reverting to stage 1) and their degree of microfilament/microtubule organization was significantly decreased. These results support our hypothesis that Ca²⁺ concentration, perhaps mediated by Ca²⁺-binding proteins, underlie the cytoskeletal changes associated with hemocyte activation.