The purpose of the experiments outlined in this application is to provide, from a cell biology point of view, new information concerning the factors controlling the progression of cells through the cell cycle. The many forms of cancer have as one of their common characteristics uncontrolled division of the cancerous cells. Thus the cells divide repeatedly, in amounts and place where they are not required, causing much damage. The experiment proposed here will continue a biochemical and molecular characterization of one protein that is involved in microtubule stability during mitosis and thus may be a key regulator of cell division. Specifically, the experiments proposed are designed to define the characteristics of a recently identified protein (Mrel = 62 kD) that is the substrate for calcium dependent phosphorylation at the start of anaphase. In living cells, a pulse of calcium is released at the metaphase-anaphase transition which is thought to trigger continued transit through the cell cycle. To understand the nature of this trigger more completely, the 62 kD protein will be characterized. To do this, the abundance and distribution of the protein antibodies essential for these experiments have been purified and characteristics will be determined in vitro and in vivo. The former series of experiments will determine the ability of the protein to interact with microtubules in an in vitro assembly system, and test how the protein is involved in microtubule disassembly. For the in vivo experiments, the purified protein will be microinfected into living cells at various times during the cell cycle to test the affect of the protein on normal cell cycle progression. Finally, clones encoding the 62 kD protein will be isolated from a sea urchin expression library to begin molecular analysis of the protein to compare it to other known proteins involved in cell cycle control. The experiments proposed in this application are important because they will provide new and definitive information concerning the control of cell division by beginning an identification of the factors involved in the cascade of events that signal the start of anaphase of mitosis, and, ultimately, the completion of cell division.
| Ye, X; Sloboda, R D (1995) A 62-kDa mitotic apparatus protein required for mitotic progression is sequestered to the interphase nucleus by associating with the chromosomes during anaphase. Cell Motil Cytoskeleton 30:310-23 |
| Johnston, J A; Sloboda, R D; Silver, R B (1994) Phosphoprotein phosphatase 1 (PP1) is a component of the isolated sea urchin mitotic apparatus. Cell Motil Cytoskeleton 29:280-90 |
| Johnston, J A; Sloboda, R D (1992) A 62-kD protein required for mitotic progression is associated with the mitotic apparatus during M-phase and with the nucleus during interphase. J Cell Biol 119:843-54 |
