Program in Cell and Developmental Dynamics, Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, MA 01655 Laboratory of Cellular and Structural Biology, Rockefeller University, New York, NY 10065 Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller University, New York, NY 10065 Proteomics and Mass Spectrometry Facility, University of Massachusetts Medical School, Shrewsbury, MA 01545.
Mol Biol Cell. 2013 Dec;24(23):3603-19. doi: 10.1091/mbc.E12-10-0714. Epub 2013 Oct 2.
Cytokinesis, the process by which cytoplasm is apportioned between dividing daughter cells, requires coordination of myosin II function, membrane trafficking, and central spindle organization. Most known regulators act during late cytokinesis; a few, including the myosin II-binding proteins anillin and supervillin, act earlier. Anillin's role in scaffolding the membrane cortex with the central spindle is well established, but the mechanism of supervillin action is relatively uncharacterized. We show here that two regions within supervillin affect cell division: residues 831-1281, which bind central spindle proteins, and residues 1-170, which bind the myosin II heavy chain (MHC) and the long form of myosin light-chain kinase. MHC binding is required to rescue supervillin deficiency, and mutagenesis of this site creates a dominant-negative phenotype. Supervillin concentrates activated and total myosin II at the furrow, and simultaneous knockdown of supervillin and anillin additively increases cell division failure. Knockdown of either protein causes mislocalization of the other, and endogenous anillin increases upon supervillin knockdown. Proteomic identification of interaction partners recovered using a high-affinity green fluorescent protein nanobody suggests that supervillin and anillin regulate the myosin II and actin cortical cytoskeletons through separate pathways. We conclude that supervillin and anillin play complementary roles during vertebrate cytokinesis.
胞质分裂是将细胞质分配给分裂的子细胞的过程，需要肌球蛋白 II 功能、膜运输和中心纺锤体组织的协调。大多数已知的调节剂在后期胞质分裂中起作用；少数调节剂，包括肌球蛋白 II 结合蛋白 anillin 和 supervillin，在早期起作用。Anillin 在与中心纺锤体支架膜皮质方面的作用已得到充分证实，但 supervillin 的作用机制相对尚未确定。我们在这里表明，supervillin 中的两个区域影响细胞分裂：残基 831-1281 与中心纺锤体蛋白结合，残基 1-170 与肌球蛋白 II 重链 (MHC) 和肌球蛋白轻链激酶的长形式结合。MHC 结合是挽救 supervillin 缺陷所必需的，并且该位点的突变会产生显性负表型。Supervillin 将激活的和总肌球蛋白募集到皱襞处，同时敲低 supervillin 和 anillin 会附加地增加细胞分裂失败。敲低任一种蛋白都会导致另一种蛋白的定位错误，并且内源性 anillin 在 supervillin 敲低时会增加。使用高亲和力绿色荧光蛋白纳米抗体回收相互作用伙伴的蛋白质组学鉴定表明，supervillin 和 anillin 通过独立的途径调节肌球蛋白 II 和肌动蛋白皮质细胞骨架。我们得出结论，supervillin 和 anillin 在脊椎动物胞质分裂中发挥互补作用。
