We will document and investigate the role of changes in intracellular free calcium during mitosis to determine what role they play in controlling cell structure. We have found that intracellular free calcium increases at nuclear envelope breakdown and at the onset of anaphase during mitosis both during the rapid cleavage stage of the sea urchin embryo and during cell division in animal cells in tissues culture. To extend this work we will make visual observations of the cell at the same time the calcium measurements are taken to pin-point the exact relationship to the structural events, both in time and in two- dimensional images of free calcium distribution in the cell. We will attempt to block progression of the cell cycle at nuclear envelope breakdown and at the metaphaseanaphase transition in animal cells by controlling calcium entry from outside or by lowering intracellular free calcium with microinjected calcium buffers. Our preliminary results with anti-bodies have implicated Ca2+/calmodulin-dependent protein kinase in normal progression through nuclear envelop breakdown. We will extend these these studies to include the meta-anaphase transition and try to reinitiate mitosis at both points with calcium-independent forms of this enzyme when mitoses has been arrested by low calcium buffers.
