The goals of the proposed research are: 1) to determine the roles of microfilaments and microtubules in movements of the ooplasm in the medaka egg, 2) to determine the role of cytoplasmic calcium pulses, gradients, and waves in movements of the ooplasm of the medaka egg, and 3) to determine the relationship between the mechanical events of cytokinesis and microfilaments, calcium pulses, gradients and waves in the blastodisc during the first two cleavages of the medaka egg. Ooplasmic movements and cytokinesis will be studied with time-lapse video microscopy, using phase contrast, Nomarski DIC, and epifluorescence optics where appropriate. The role of cytoskeletal systems will be studied by 1) applying or microinjecting drugs that disrupt these systems, and 2) localizing microfilaments and microtubules with fluorescent phalloidins and by indirect immunofluorescence, respectively. The role of calcium will be studied by 1) looking for calcium pulses, gradients, and waves with the calcium-sensitive photoprotein, Aequorin, and 2) injecting calcium buffers into the cytoplasm of the egg. Using the fish egg as a model, these studies should provide new insights into the physiological bases of two fundamental activities of the cytoplasm of the fertilized egg: its structural rearrangement and its division into daughter cells. This research will also provide the opportunity for undergraduates to participate in an active research program using state of the art techniques of light microscopy.
