Loop L5 Acts as a Conformational Latch in the Mitotic Kinesin Eg5
Loop L5 Acts as a Conformational Latch in the Mitotic Kinesin Eg5
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Loop L5 作为有丝分裂驱动蛋白 Eg5 中的构象锁存器
DOI：
10.1016/j.bpj.2010.12.3096
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发表时间：
2011
影响因子：
3.4
通讯作者：
Behnke-Parks W
中科院分区：
文献类型：
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作者：
Behnke-Parks W
有丝分裂染色体的分离是由有丝分裂纺锤体介导的，这是一种高度动态的基于微管的结构，在每个有丝分裂周期中经历一系列不同的形态变化。影响纺锤体形态发生的主要因素是微管(MT)+末端动力学和来自Kinesin-5家族的分子马达的功能。Kinesin-5家族成员是保守的同源四聚体发动机，在活性复合体的两侧有两个催化结构域。这种特殊的结构使这些马达能够交联化和滑动来自相反纺锤体两极的反平行的MT，从而在有丝分裂纺锤体的形态发生中发挥它们的基本功能。最近的研究表明，Kinesin-5马达影响后期纺锤体对称性和中区组织[1]。酿酒酵母细胞表达两个Kinesin-5同源物Cin8p和Kip1p，它们在纺锤体组装过程中功能重叠，中期和后期B，至少其中一个需要表达才能存活。到目前为止，这两个Kinesin-5蛋白之间的冗余程度以及它们在体外的运动特性还没有得到彻底的研究。在本研究中，我们使用高时间和空间分辨率成像和FRAP来表征表达微管蛋白-GFP(2)的酿酒酵母细胞纺锤体形态发生过程中极间MT(IMT)+端的动态变化。这一方法使我们能够研究主要的中区组织蛋白Ase1(2)在控制IMT+端动态中的作用，并比较Cin8和Kip1在后期对这些动态的影响。此外，为了了解Kinesin-5 Kip1在体内的功能，我们用单分子荧光动力学实验对其运动特性进行了表征。这个测试的结果将会被公布。
Mitotic chromosome segregation is mediated by mitotic spindle, a highly dynamic microtubule-based structure, which undergoes a distinct set of morphological changes in each mitotic cycle. Major factors that contribute to spindle morphogenesis are microtubule (MT) plus-end dynamics and function of molecular motors from the Kinesin-5 family. Kinesin-5 family members are conserved, homotetrameric motors with two catalytic domains located on opposite sides of the active complex. This special architecture enables these motors to crosslink and slide anti-parallel MTs originating from opposite spindle poles and thereby perform their essential functions in mitotic spindle morphogenesis. It was recently shown that Kinesin-5 motors affect anaphase spindle symmetry and midzone organization (1). S. cerevisiae cells express two Kinesin-5 homologues, Cin8p and Kip1p that overlap in function during spindle assembly, metaphase and anaphase B and at least one of them need to be expressed for viability. So far, the extent of redundancy between these two Kinesin-5 proteins and their motile properties in vitro have not been thoroughly investigated. In the present study, we use high temporal and spatial resolution imaging and FRAP to characterize interpolar MT (iMT) plus-end dynamics during spindle morphogenesis in S. cerevisiae cells expressing tubulin-GFP (2). This approach allowed us to study the role of the major midzone organizing protein Ase1 (2) in controlling iMT plus-end dynamics and to compare between the effects of Cin8 and Kip1 on these dynamics during anaphase. In addition, in order to understand in vivo functions of the Kinesin-5 Kip1, we characterized its motile properties in single-molecule fluorescence motility assay. Results from this assay will be presented.