Mulvihill Daniel P, Hyams Jeremy S
Department of Biology, University College London, Gower Street, London WC1E 6BT, UK.
J Cell Sci. 2002 Sep 15;115(Pt 18):3575-86. doi: 10.1242/jcs.00031.
In dividing cells, the assembly and contraction of the cytokinetic actomyosin ring (CAR) is precisely coordinated with spindle formation and chromosome segregation. Despite having a cell wall, the fission yeast Schizosaccharomyces pombe forms a CAR reminiscent of the structure responsible for the cleavage of cells with flexible boundaries. We used the myo2-gc fission yeast strain in which the chromosomal copy of the type II myosin gene, myo2(+), is fused to the gene encoding green fluorescent protein (GFP) to investigate the dynamics of Myo2 recruitment to the cytokinetic actomyosin ring in living cells. Analysis of CAR formation in relation to spindle pole body (SPB) and centromere separation enabled us to pinpoint the timing of Myo2 recruitment into a stable CAR structure to the onset of anaphase A. Depolymerisation of actin with latrunculin B did not affect the timing of Myo2 accumulation at the cell equator (although Myo2 no longer formed a ring), whereas depolymerisation of microtubules with either thiabendazole (TBZ) or methyl 2-benzimidazolecarbamate (MBC) resulted in a delay of up to 90 minutes in CAR formation. Microtubule depolymerisation also delayed the localisation of other CAR components such as actin and Mid1/Dmf1. The delay of cytokinesis in response to loss of microtubule integrity was abolished in cells lacking the spindle assembly checkpoint protein Mad2 or containing non-functional Cdc16, a component of the fission yeast septation initiation network (SIN). The delay was also abolished in cells lacking Zfs1, a component of the previously described S. pombe cytokinesis checkpoint. Recruitment of the polo-related kinase, Plo1, a key regulator of CAR formation, to the SPBs was substantially reduced in TBZ in a Mad2-dependent manner. Loading of Cdc7, a component of the SIN and downstream of Plo1 in the cytokinesis pathway, onto the the SPBs was also delayed in TBZ to the same extent as CAR formation. We conclude that CAR formation is subject to regulation by the spindle assembly checkpoint via the loading of Plo1 onto the SPBs and the consequent activation of the SIN.
在分裂细胞中，胞质分裂肌动球蛋白环（CAR）的组装和收缩与纺锤体形成及染色体分离精确协调。尽管裂殖酵母有细胞壁，但粟酒裂殖酵母仍会形成一种CAR，类似于负责分裂具有灵活边界细胞的结构。我们使用了myo2-gc裂殖酵母菌株，其中II型肌球蛋白基因myo2(+)的染色体拷贝与编码绿色荧光蛋白（GFP）的基因融合，以研究活细胞中Myo2募集到胞质分裂肌动球蛋白环的动力学。分析CAR形成与纺锤极体（SPB）和着丝粒分离的关系，使我们能够确定Myo2募集到稳定CAR结构的时间为后期A开始时。用Latrunculin B使肌动蛋白解聚并不影响Myo2在细胞赤道面积累的时间（尽管Myo2不再形成环），而用噻苯咪唑（TBZ）或甲基2-苯并咪唑氨基甲酸酯（MBC）使微管解聚会导致CAR形成延迟长达90分钟。微管解聚还延迟了其他CAR成分如肌动蛋白和Mid1/Dmf1的定位。在缺乏纺锤体组装检查点蛋白Mad2或含有无功能Cdc16（裂殖酵母隔膜起始网络（SIN）的一个成分）的细胞中，因微管完整性丧失而导致的胞质分裂延迟被消除。在缺乏Zfs1（先前描述的粟酒裂殖酵母胞质分裂检查点的一个成分）的细胞中，延迟也被消除。在TBZ中，polo相关激酶Plo1（CAR形成的关键调节因子）募集到SPBs的过程以Mad2依赖的方式大幅减少。在TBZ中，SIN成分且在胞质分裂途径中位于Plo1下游的Cdc7加载到SPBs上的过程也与CAR形成延迟到相同程度。我们得出结论，CAR形成受纺锤体组装检查点通过将Plo1加载到SPBs上以及随后激活SIN的调控。
