Single-Molecule Analysis of the Microtubule Cross-Linking Protein MAP65-1 Reveals a Molecular Mechanism for Contact-Angle-Dependent Microtubule Bundling
Single-Molecule Analysis of the Microtubule Cross-Linking Protein MAP65-1 Reveals a Molecular Mechanism for Contact-Angle-Dependent Microtubule Bundling
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DOI：
10.1016/j.bpj.2012.01.008
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发表时间：
2012-02-22
影响因子：
3.4
通讯作者：
Dixit, Ram
中科院分区：
文献类型：
--
作者：
Tulin, Amanda;McClerklin, Sheri;Dixit, Ram
微管的成束是形成复杂微管阵列的关键。在陆地植物中，间期皮质MT形成束，特别是在它们之间的浅角相遇之后。为了研究细胞如何选择特定的MT接触角进行捆绑，我们使用了由动态MT和MT交联蛋白MAP 65 -1组成的体外重建方法。我们发现，MAP 65 -1结合到作为单体的MT，并固有地靶向反平行MT进行捆绑。停留时间分析显示，MAP 65 -1对反平行重叠MT的亲和力比其对单个MT和平行重叠MT的亲和力高约3倍。我们还发现，纯化的MAP 65 -1专门选择浅角MT遇到捆绑，表明这种活动是MAP 65 -1的内在属性。重建实验与突变的MAP 65 -1蛋白质与不同数目的血影蛋白重复内的N-末端杆域表明，杆域的长度是一个主要的决定因素的MT捆绑角的范围。杆域的长度也决定了束内MT之间的距离。总之，我们的数据表明，杆域的MAP 65 -1作为一个间隔区，并作为一个结构元素，指定MT遇到的角度，有利于捆绑。
Bundling of microtubules (MTs) is critical for the formation of complex MT arrays. In land plants, the interphase cortical MTs form bundles specifically following shallow-angle encounters between them. To investigate how cells select particular MT contact angles for bundling, we used an in vitro reconstitution approach consisting of dynamic MTs and the MT-cross-linking protein MAP65-1. We found that MAP65-1 binds to MTs as monomers and inherently targets antiparallel MTs for bundling. Dwell-time analysis showed that the affinity of MAP65-1 for antiparallel overlapping MTs is about three times higher than its affinity for single MTs and parallel overlapping MTs. We also found that purified MAP65-1 exclusively selects shallow-angle MT encounters for bundling, indicating that this activity is an intrinsic property of MAP65-1. Reconstitution experiments with mutant MAP65-1 proteins with different numbers of spectrin repeats within the N-terminal rod domain showed that the length of the rod domain is a major determinant of the range of MT bundling angles. The length of the rod domain also determined the distance between MTs within a bundle. Together, our data show that the rod domain of MAP65-1 acts both as a spacer and as a structural element that specifies the MT encounter angles that are conducive for bundling.