Protein patterns in the naturally synchronous mitotic cycle of Physarum polycephalum : studies of the cell cycle engine

Vita.Based on molecular, cellular and genetic studies in a wide variety of organisms, a universal model of cell cycle control emerged. According to the model, mitosis is initiated by the transient activation of Mitosis Promoting Factor which is a heterodimer. It is composed of a catalytic subunit encoded by the cdc2 gene and a regulatory subunit encoded by cyclin B gene. Cyclic mitosis promoting factor activation is achieved by cyclin accumulation and destruction. The model also describes two major control points, one at entry into mitosis (G2/M) and the other at START (G1/S). The G2/M point is controlled by endogenous factors (oscillators) whereas START defines a branch point in the cell cycle where exogenous factors can influence the proliferation and differentiation of cells. However, this basic mitotic oscillator model has to be modified in order to explain the roles of numerous cyclins and several cdc2 related kinases, as well as the function of growth factors, oncogenes and tumor suppresser genes during the G1 phase of specialized cells. The experimental results presented in this dissertation offer some insight into how the model can be modified. Most notably, cyclin B does not cycle in Physarum. This was demonstrated utilizing 1) the anti PSTAIR antibody specific for the detection of p34[^cdc2], 2) suc1 beads, a specific affinity matrix to assay H1 kinase of the cdc2/cyclin B complex, 3) antibodies directed against cyclins A and B, anti phosphotyrosine antibody and standard procedure of DNA cloning and polymerase chain reaction. Major results are the demonstration of a constant level of p34[^cdc2] during the whole cell cycle, whereas the associated H1 kinase activity fluctuated dramatically during mitosis, as expected. Surprisingly, and in contrast to the current paradigm, the mitotic cyclin of Physarum did not cycle, i.e. this regulatory subunit of the H1 kinase does not degraded at the exit from mitosis. The bulk of the research is described to numerous control experiments all of which are consistent with the exceptional conclusion of a constitutive level of mitotic cyclin. Another atypical result was lack of cyclin phosphorylation at mitosis seen in many other systems. Yet another exception to the rule was the continuous presence of phosphotyrosine on cdc2 kinase subunit. In other systems, tyrosine dephosphorylation correlates with kinase activation. The only modification seen in Physarum that correlates with at least some other cell types is a mobility shift, precisely at mitosis, probably due to transient phosphorylation