Mitotic cell cycle control in Physarum. Unprecedented insights via flow-cytometry

High resolution flow-cytometric studies of isolated macroplasmodial nuclei of the myxomycete, Physarum polycephalum provide definite evidence for the persistence of natural synchrony at mitosis throughout the entire cell cycle, i.e. completely synchronous DNA replication and traverse of G2. Even if nuclei within a given macroplasmodium belong to two distinct genome size classes (mixoploidy), they cycle and traverse mitosis in strict synchrony. This cannot be explained by current models of regulation of division based solely upon nuclear size and/or nuclear/cytoplasm ratios. Constitutional DNA content variation was apparent among all tested strains, and loss of late-replicating, presumably AT-rich DNA accounts for this variation. A constant duration of the S phase is maintained, irrespective of DNA content, via differential slowdown of replication rates during the 2nd and 3rd hours of replication. A frequently described extension of nuclear replication into G2 could not be substantiated. Interference with DNA and protein synthesis provides the first evidence for a brief "G1 phase" equivalent of 3-4 min duration in asynchronous microplasmodial cultures, and temporally assigns a protein synthesis-dependent "transition point" for completion of mitosis and initiation of DNA synthesis at 5 min prior to actual division, nuclei which have passed this point at the time of addition of cycloheximide replicate 5% of their DNA before they become arrested. These findings provide strong experimental support for the transition point concept of cell cycle control, and additionally are commensurate with some form of the replicon-set hypothesis in Physarum.