Akamatsu Matthew, Lin Yu, Bewersdorf Joerg, Pollard Thomas D
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520-8103.
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8103.
Mol Biol Cell. 2017 Nov 7;28(23):3203-3214. doi: 10.1091/mbc.E16-07-0522. Epub 2017 May 24.
We used quantitative confocal microscopy and FPALM superresolution microscopy of live fission yeast to investigate the structures and assembly of two types of interphase nodes-multiprotein complexes associated with the plasma membrane that merge together and mature into the precursors of the cytokinetic contractile ring. During the long G2 phase of the cell cycle, seven different interphase node proteins maintain constant concentrations as they accumulate in proportion to cell volume. During mitosis, the total numbers of type 1 node proteins (cell cycle kinases Cdr1p, Cdr2p, Wee1p, and anillin Mid1p) are constant even when the nodes disassemble. Quantitative measurements provide strong evidence that both types of nodes have defined sizes and numbers of constituent proteins, as observed for cytokinesis nodes. Type 1 nodes assemble in two phases-a burst at the end of mitosis, followed by steady increase during interphase to double the initial number. Type 2 nodes containing Blt1p, Rho-GEF Gef2p, and kinesin Klp8p remain intact throughout the cell cycle and are constituents of the contractile ring. They are released from the contractile ring as it disassembles and then associate with type 1 nodes around the equator of the cell during interphase.
我们使用定量共聚焦显微镜和活裂殖酵母的FPALM超分辨率显微镜来研究两种间期节点的结构和组装，这两种节点是与质膜相关的多蛋白复合物，它们融合在一起并成熟为细胞分裂收缩环的前体。在细胞周期漫长的G2期，七种不同的间期节点蛋白随着它们与细胞体积成比例积累而保持恒定浓度。在有丝分裂期间，即使节点解体，1型节点蛋白（细胞周期激酶Cdr1p、Cdr2p、Wee1p和膜收缩蛋白Mid1p）的总数也是恒定的。定量测量提供了有力证据，表明这两种节点都有确定的大小和组成蛋白数量，这与胞质分裂节点的情况相同。1型节点分两个阶段组装——在有丝分裂末期爆发，随后在间期稳定增加至初始数量的两倍。包含Blt1p、Rho-GEF Gef2p和驱动蛋白Klp8p的2型节点在整个细胞周期中保持完整，并且是收缩环的组成部分。当收缩环解体时，它们从收缩环释放出来，然后在间期与细胞赤道周围的1型节点结合。
