The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast.
The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast.
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DOI：
10.1091/mbc.e16-01-0010
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发表时间：
2016-06-01
影响因子：
3.3
通讯作者：
Kovar DR
中科院分区：
文献类型：
--
作者：
Li Y;Christensen JR;Homa KE;Hocky GM;Fok A;Sees JA;Voth GA;Kovar DR
对裂殖酵母α-辅肌动蛋白SpAin 1的高动态成束活性进行了生化表征，并测试了其在体内收缩环形成中的重要性。调查的突变体具有较高的捆绑活动，SpAin 1（216 E），揭示了动态SpAin 1介导的捆绑是至关重要的适当的环组装和收缩。肌动球蛋白收缩环是一个交联的肌动蛋白丝网络，促进分裂细胞的胞质分裂。在裂殖酵母中，收缩环的形成已经得到很好的表征，其中交联蛋白α-辅肌动蛋白SpAin 1将肌动蛋白丝网络捆绑起来。然而，SpAin 1的具体生化特性以及它们是否适合胞质分裂尚不清楚。因此，我们纯化SpAin 1和量化的能力，动态绑定和捆绑肌动蛋白丝在体外使用的组合散装沉降测定和直接可视化的双色全内反射荧光显微镜。我们发现，虽然SpAin 1像其他α-辅肌动蛋白一样捆绑混合极性的肌动蛋白丝，但SpAin 1具有较低的捆绑活性，并且比人α-辅肌动蛋白HsACTN 4更具动态性。为了确定动态捆绑是否对裂殖酵母中的胞质分裂重要，我们创建了较少动态捆绑突变体SpAin 1（R216 E）。我们发现，动态捆绑是至关重要的胞质分裂，细胞表达SpAin 1（R216 E）显示混乱的环材料和延迟环的形成和收缩。此外，计算机模拟的初始肌动蛋白丝的伸长和对齐显示，一个中间水平的交联最有利于丝对齐。总之，我们的研究结果表明，动态捆绑SpAin 1是重要的适当的收缩环的形成和收缩。
The highly dynamic bundling activity of fission yeast α-actinin SpAin1 was biochemically characterized, and its importance for contractile ring formation in vivo was tested. Investigation of a mutant with higher bundling activity, SpAin1(216E), revealed that dynamic SpAin1-mediated bundling is crucial for proper ring assembly and constriction. The actomyosin contractile ring is a network of cross-linked actin filaments that facilitates cytokinesis in dividing cells. Contractile ring formation has been well characterized in Schizosaccharomyces pombe, in which the cross-linking protein α-actinin SpAin1 bundles the actin filament network. However, the specific biochemical properties of SpAin1 and whether they are tailored for cytokinesis are not known. Therefore we purified SpAin1 and quantified its ability to dynamically bind and bundle actin filaments in vitro using a combination of bulk sedimentation assays and direct visualization by two-color total internal reflection fluorescence microscopy. We found that, while SpAin1 bundles actin filaments of mixed polarity like other α-actinins, SpAin1 has lower bundling activity and is more dynamic than human α-actinin HsACTN4. To determine whether dynamic bundling is important for cytokinesis in fission yeast, we created the less dynamic bundling mutant SpAin1(R216E). We found that dynamic bundling is critical for cytokinesis, as cells expressing SpAin1(R216E) display disorganized ring material and delays in both ring formation and constriction. Furthermore, computer simulations of initial actin filament elongation and alignment revealed that an intermediate level of cross-linking best facilitates filament alignment. Together our results demonstrate that dynamic bundling by SpAin1 is important for proper contractile ring formation and constriction.