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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这个研究项目的目的是可视化Kinesin-13和微管蛋白环之间的相互作用。马达蛋白的动蛋白家族利用三磷酸腺苷水解产生的能量结合微管并在其上做功(Vale和Fletterick，1997)。安努。牧师单元格开发比奥尔。13：745-777)。微管的动态性质对于基本的细胞过程很重要，例如有丝分裂期间的纺锤体组装、细胞迁移和细胞运输；事实上，它是与微管相关蛋白(MAP)、运动蛋白等马达蛋白和其他细胞因子的相互作用，调节微管的动态行为和功能(Walczack，2000)。货币。奥平。牢房。比奥尔。12：52-56)。动蛋白最为人所知的是其沿着微管轨道行走的能力，同时将货物从细胞中的一个点运送到另一个点；然而，尚未有证据表明中间的动蛋白(kinesin-13)参与了这种行走运动。相反，这个动蛋白家族利用化学能量来解聚微管的末端。以前的研究表明，在解聚微管末端的过程中，会形成微管蛋白环和螺旋(Moore et.艾尔2002年。摩尔。牢房。9：903-909；Desai et.艾尔1999年牢房。96：69-78)；重要的是，在α和β微管蛋白中加入托伐他汀会导致自发形成的环与解聚过程中形成的环没有区别(未发表的结果)。这些微管蛋白环被认为是微管末端微管蛋白构型的代表，对这些环上的马达的研究将对微管解聚的分子机制提供重要的见解。在这个项目的第一阶段，我们将利用恶性疟原虫Kini的运动域(PKinI)建立一个通过LEGINON和单粒子2D平均技术进行数据收集和处理的自动化平台。一旦建立了自动化平台，我们将扩大我们的研究范围，包括全长人类Kinesin-13和各种截短版本的蛋白质。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.