Department of Molecular Cellular and Developmental Biology,Yale University, New Haven, United States.
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, United States.
Elife. 2022 Sep 12;11:e76249. doi: 10.7554/eLife.76249.
Cytokinesis nodes are assemblies of stoichiometric ratios of proteins associated with the plasma membrane, which serve as precursors for the contractile ring during cytokinesis by fission yeast. The total number of nodes is uncertain, because of the limitations of the methods used previously. Here, we used the ~140 nm resolution of Airyscan super-resolution microscopy to measure the fluorescence intensity of small, single cytokinesis nodes marked with Blt1-mEGFP in live fission yeast cells early in mitosis. The ratio of the total Blt1-mEGFP fluorescence in the broad band of cytokinesis nodes to the average fluorescence of a single node gives about 190 single cytokinesis nodes in wild-type fission yeast cells early in mitosis. Most, but not all of these nodes condense into a contractile ring. The number of cytokinesis nodes scales with cell size in four strains tested, although large diameter mutant cells form somewhat fewer cytokinesis nodes than expected from the overall trend. The Pom1 kinase restricts cytokinesis nodes from the ends of cells, but the surface density of Pom1 on the plasma membrane around the equators of cells is similar with a wide range of node numbers, so Pom1 does not control cytokinesis node number. However, when the concentrations of either kinase Pom1 or kinase Cdr2 were varied with the promoter, the numbers of cytokinesis nodes increased above a baseline of about ~190 with the total cellular concentration of either kinase.
有丝分裂胞器是与质膜相关的化学计量比例的蛋白质的集合，在裂殖酵母的胞质分裂过程中，这些蛋白质作为收缩环的前体。由于之前使用的方法的限制，有丝分裂胞器的总数是不确定的。在这里，我们使用 Airyscan 超分辨率显微镜的约 140nm 分辨率来测量带有 Blt1-mEGFP 标记的小的、单个的有丝分裂早期胞质分裂节点的荧光强度，该标记位于活裂殖酵母细胞中。在有丝分裂早期的野生型裂殖酵母细胞中，整个宽带有丝分裂节点的总 Blt1-mEGFP 荧光与单个节点的平均荧光之比约为 190 个单个有丝分裂节点。大多数（但不是全部）这些节点浓缩成一个收缩环。在四个受测试的菌株中，有丝分裂胞器的数量与细胞大小成正比，尽管直径较大的突变体细胞形成的有丝分裂胞器数量比总体趋势预期的要少一些。Pom1 激酶将有丝分裂胞器限制在细胞的末端，但细胞赤道周围质膜上 Pom1 的表面密度与广泛的节点数量相似，因此 Pom1 不控制有丝分裂胞器的数量。然而，当 Pom1 激酶或 Cdr2 激酶的浓度随着 启动子而变化时，有丝分裂胞器的数量会增加到约 190 个左右的基线以上，此时两种激酶的总细胞浓度都会增加。
