Fig. 5. PLC in the presence of Ca²⁺ results in the loss of filamentous actin from rat ectoplasmic specializations. Spermatids with attached ectoplasmic specializations were incubated in the presence or absence of either PLC in the presence and absence of Ca²⁺. Shown in panels A,A' to D,D' are paired phase and fluorescence micrographs of spermatids with attached adhesion complexes fixed and labeled with fluorescent phalloidin for filamentous actin immediately after isolation (A,A') or after incubation with or without PLC in the presence or absence of a calculated 11 µM free Ca²⁺ (B,B'-D,D') The least amount of filamentous actin is associated with cells treated with PLC in the presence of Ca²⁺. (E) Treatment of spermatid/adhesion complexes with PLC in the presence of Ca²⁺ resulted in increased levels of actin and gelsolin in supernatants, relative to controls, as assessed by immunoblot. In these experiments, spermatids with attached junction complexes were incubated in buffers with or without PLC in the presence or absence of a calculated 1.5 mM (upper two blots) or 11 µM free Ca²⁺ (lower two blots). Cells were removed from solution by centrifugation and equivalent volumes of supernatants assessed for actin and gelsolin on immunoblots. The amount of actin and gelsolin are greatest in supernatants from spermatid/junction complexes treated with both PLC and Ca²⁺. Significantly, treatment with PLC in the absence of Ca²⁺ results in increased gelsolin in supernatants, but not in increased actin.