AUTOMATED PLATFORM FOR 2D EM OF KINESIN-13 INTERACTIONS WITH TUBULIN RINGS

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 本研究的目的是可视化驱动蛋白-13和微管蛋白环之间的相互作用。 马达蛋白的驱动蛋白家族利用来源于ATP水解的能量来结合微管并在微管上工作（Vale和Fletterick，1997. Annu.修订版细胞开发13：745-777）。 微管的动态性质对于基本细胞过程如有丝分裂期间的纺锤体组装、细胞迁移和细胞运输是重要的;事实上，它与微管相关蛋白（MAPS）、马达蛋白如驱动蛋白和其他细胞因子的相互作用调节微管的动态行为，从而调节微管的功能（Walczack，2000. Curr. Opin. Cell. 12：52-56）。 驱动蛋白最为人所知的是它们能够沿着沿着微管轨道行走，同时将货物从细胞中的一个点运送到另一个点;然而，中间驱动蛋白（驱动蛋白-13是其中的一员）尚未显示参与这种行走运动。 相反，这个家族的驱动蛋白利用化学能去激活微管的末端。 先前的研究已经表明，在微管末端解聚的过程中，形成微管蛋白环和螺旋（Moores et.等人，2002年。摩尔Cell. 9：903-909; Desai et.等人，1999 Cell.九十六：（第69-78段）;重要的是，向α和β微管蛋白中加入多拉司他汀导致自发形成环，这些环与解聚过程中形成的环是不可分离的（未发表的结果）。 人们认为，这些微管蛋白环是微管末端微管蛋白构型的代表，对与这些环结合的马达的研究将为微管解聚的分子机制提供重要的见解。 在该项目的第一阶段，我们将利用来自恶性疟原虫的KinI（pKinI）的运动域，通过LEGINON和单粒子2D平均技术建立一个自动化的数据收集和处理平台。 一旦建立了自动化平台，我们将扩展我们的研究，包括全长人类驱动蛋白-13和各种截短版本的蛋白质。 我们希望这些研究将有助于我们对微管解聚的分子理解。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The objective of this research project is to visualize the interactions between Kinesin-13 and tubulin rings. The kinesin family of motor proteins utilize energy derived from ATP-hydrolysis to bind, and do work, on microtubules (Vale and Fletterick, 1997. Annu. Rev. Cell Dev. Biol. 13: 745-777). The dynamic nature of microtubules is important for fundamental cellular processes such as spindle assembly during mitosis, cell migration, and cellular transport; in fact, it is the interactions with microtubule associated proteins (MAPS), motor proteins such as kinesin, and other cellular factors, that regulate the dynamic behavior, and so the function, of microtubules (Walczack, 2000. Curr. Opin. Cell. Biol. 12: 52-56). Kinesins are most known for their ability to walk along a microtubular track while carrying cargo from one point in the cell to another; however the intermediate kinesins, of which Kinesin-13 is a member, have not been shown to engage in such a walking motion. Instead, this family of kinesins utilize chemical energy to depolymerize the ends of microtubules. Previous studies have shown that, in the process of depolymerizing microtubule ends, tubulin rings and spirals are formed (Moores et. al. 2002. Mol. Cell. 9: 903-909; Desai et. al. 1999 Cell. 96: 69-78); importantly, addition of Dolastatin to alpha and beta tubulin results in the spontaneous formation of rings indistinguisable from those formed during the depolymerization process (unpublished results). It is thought that these tubulin rings are representative of tubulin configuration at the ends of microtubules, and that studies of motors bound to such rings will give important insights regarding the molecular mechanism of microtubule depolymerization. In the first phase of this project, we will utilize the motor domain of KinI from Plasmodium falciparum (pKinI) to institute an automated platform for data collection and processing via LEGINON and single particle 2D averaging techniques. Once, an automated platform has been established, we will extend our studies to include full-length human kinesin-13 and various truncated versions of the protein. It is the hope that such studies will contribute to our molecular understanding of microtubule depolymerization.