Dynamics of SIN asymmetry establishment

Timing of cell division is coordinated by the Septation Initiation Network (SIN) in fission yeast. SIN activation is initiated at the two spindle pole bodies (SPB) of the cell in metaphase, but only one of these SPBs contains an active SIN in anaphase, while SIN is inactivated in the other by the Cdc16-Byr4 GAP complex. Most of the factors that are needed for such asymmetry establishment have been already characterized, but we lack the molecular details that drive such quick asymmetric distribution of molecules at the two SPBs. Here we investigate the problem by computational modeling and, after establishing a minimal system with two antagonists that can drive reliable asymmetry establishment, we incorporate the current knowledge on the basic SIN regulators into an extended model with molecular details of the key regulators. The model can capture several peculiar earlier experimental findings and also predicts the behavior of double and triple SIN mutants. We experimentally tested one prediction, that phosphorylation of the scaffold protein Cdc11 by a SIN kinase and the core cell cycle regulatory Cyclin dependent kinase (Cdk) can compensate for mutations in the SIN inhibitor Cdc16 with different efficiencies. One aspect of the prediction failed, highlighting a potential hole in our current knowledge. Further experimental tests revealed that SIN induced Cdc11 phosphorylation might have two separate effects. We conclude that SIN asymmetry is established by the antagonistic interactions between SIN and its inhibitor Cdc16-Byr4, partially through the regulation of Cdc11 phosphorylation state

Ran GTPase Cycle and Importins α and β Are Essential for Spindle Formation and Nuclear Envelope Assembly in Living Caenorhabditis elegans Embryos

The small GTPase Ran has been found to play pivotal roles in several aspects of cell function. We have investigated the role of the Ran GTPase cycle in spindle formation and nuclear envelope assembly in dividing Caenorhabditis elegans embryos in real time. We found that Ran and its cofactors RanBP2, RanGAP, and RCC1 are all essential for reformation of the nuclear envelope after cell division. Reducing the expression of any of these components of the Ran GTPase cycle by RNAi leads to strong extranuclear clustering of integral nuclear envelope proteins and nucleoporins. Ran, RanBP2, and RanGAP are also required for building a mitotic spindle, whereas astral microtubules are normal in the absence of these proteins. RCC1(RNAi) embryos have similar abnormalities in the initial phase of spindle formation but eventually recover to form a bipolar spindle. Irregular chromatin structures and chromatin bridges due to spindle failure were frequently observed in embryos where the Ran cycle was perturbed. In addition, connection between the centrosomes and the male pronucleus, and thus centrosome positioning, depends upon the Ran cycle components. Finally, we have demonstrated that both IMA-2 and IMB-1, the homologues of vertebrate importin α and β, are essential for both spindle assembly and nuclear formation in early embryos