Isolation and Analysis of Regulators Coordinating the Nuclear Cycle with Cytokinesis in Schizosaccharomyces Pombe

During the division of a mother cell into two daughter cells the genetic material of the mother cell is replicated and segregated to the daughter cells before the cytoplasm divides into two portions in a process called cytokinesis. In order to maintain the genetic stability of the daughter cells it is crucial that the bisection of the cells only occurs once the genetic material has been faithfully partitioned. In the model organism S. pombe the timely coordination of cytokinesis is regulated by a cellular signaling cascade called the Septation Initiation Network (SIN). The SIN is required for the formation and contraction of the contractile actomyosin ring (CAR) that assembles in the cell centre and guides the centripetal deposition of septum material during its contraction. Most of the regulators of the SIN are located to the SPB at some stage of the cell cycle. Interestingly, in anaphase B, when the SIN is supposed to be fully active, positive and negative regulators of the SIN localize to opposite SPBs. This asymmetric configuration of the SIN is lost at the end of cytokinesis, when the SIN is turned off; the positive regulators dissociate from the new SPB and the negative regulators assemble on this SPB. In order to understand this process in more detail we analyzed the localization of Cdc7p, a positive regulator of the SIN. We found that the disappearance of Cdc7p from the new SPB at the end of cytokinesis correlated with the separation of the two halves of the cytoplasm. Moreover, our data suggested that the separation of the two SPBs resulting from the completion of septum formation is important for the resetting of the SIN. Interestingly, we observed that Sid2p, the downstream effector of the SIN, was important for the maintenance of asymmetry in anaphase B and for the timing of the resetting of the SIN by acting in a negative feedback. Taken together, we suggested a model where the downstream effector of the SIN may promote the loading of the negative regulators to the SPBs; in anaphase to the old SPB (due to higher affinity of the negative regulators for this SPB) and upon cell separation to both SPBs, because the old SPB cannot buffer the negative feedback. In a separate study we designed a screen to isolate new regulators of the SIN. Mutagenesis of a strain that over-expressed spg1, the core activator of the SIN, led to the isolation of 28 mutants that are dependent on high levels of Spg1p for viability. The characterization of the nine heat-sensitive mutants among them revealed that they are all new alleles of known SIN genes. Interestingly, two mutants that mapped to the SIN effector sid2, lysed at the time of cytokinesis, when shifted to the restrictive temperature. This observation points to a possible role of the SIN in regulating cell separation. Surprisingly, one of the mutants that mapped to the essential positive SIN regulator sid1 contained a premature STOP codon after 243 nucleotides. The analysis of this mutant revealed that translational read-through level of the STOP codon is sufficient for successful cytokinesis. Similarly, high levels of sid1 expression did not interfere with cytokinesis. This indicated that the regulation of the SIN by Sid1p does not depend on its levels but on a precise control of its action. Since over expression of sid1 led to the accumulation of the protein in the nucleus this may hint that Sid1p recruitment to the SPB is regulated by nuclear proteins.