phosphorus-radioisotopes and diacetylmonoxime

When an unfertilized sea urchin egg was exposed to calyculin-A (CL-A), an inhibitor of protein phosphatases, for a short period and then lysed, the cortex contracted to exclude cytoplasm and became a cup-shaped mass. We call the contracted cortex "actin cup" since actin filaments were major structural components. Electron microscopic observation revealed that the cup consisted of inner electron-dense layer, middle microfilamentous layer, and outermost granular region. Microfilaments were heavily accumulated in the inner electron-dense layer. The middle layer also contained numerous microfilaments, which were determined to be actin filaments by myosin S1 decoration, and they were aligned so that their barbed ends directed toward the outermost region. Myosin II, Arp2, Arp3, and spectrin were concentrated in the actin cup. Immuno-electron microscopy revealed that myosin II was localized to the electron-dense layer. We further found that the cortical tension of the egg increased just after application of CL-A and reached maximum within 10 min. Cytochalasin B or butanedione monoxime blocked the contraction, which suggested that both actin filaments and myosin ATPase activity were required for the contraction. Myosin regulatory light chain (MRLC) in the actin cup was shown to be phosphorylated at the activation sites Ser-19 and Thr-18, by immunoblotting with anti-phosphoepitope antibodies. The phosphorylation of MRLC was also confirmed by a (32)P in vivo labeling experiment. The CL-A-induced cortical contraction may be a good model system for studying the mechanism of cytokinesis.

Topics: Actins; Animals; Cell Division; Cell Size; Cytochalasin B; Diacetyl; Enzyme Inhibitors; Marine Toxins; Microscopy, Immunoelectron; Myosins; Ovum; Oxazoles; Phosphoric Monoester Hydrolases; Phosphorus Radioisotopes; Phosphorylation; Sea Urchins; Stress, Mechanical

Topics: Action Potentials; Animals; Butanones; Calcium; Cats; Cell Membrane; Cyclic AMP; Depression, Chemical; Diacetyl; Dose-Response Relationship, Drug; Female; Heart Ventricles; In Vitro Techniques; Male; Myocardial Contraction; Oximes; Phosphorus Radioisotopes; Phosphorylation; Protein Kinases; Time Factors