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

用于驱动蛋白 13 与微管蛋白环相互作用的 2D EM 自动化平台

• 批准号: 8169689
• 负责人: RONALD A MILLIGAN
• 金额: $ 1.29万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169689
• 关键词: ATP Hydrolysis; Behavior; Binding; Cell physiology; Cells; Chemicals; Computer Retrieval of Information on Scientific Projects Database; Data Collection; Dolastatin Compound; Family; Funding; Grant; Human; Institutes; Institution; Kinesin; Length; Microtubule Depolymerization; Microtubule-Associated Proteins; Microtubules; Mitosis; Molecular; Motion; Motor; Nature; Phase; Plasmodium falciparum; Process; Proteins; Research; Research Personnel; Research Project Grants; Resources; Source; Techniques; Tubulin; United States National Institutes of Health; Walking; Work; beta Tubulin; cell motility; depolymerization; insight; member; particle

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 该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。 运动蛋白的驱动蛋白家族利用源自 ATP 水解的能量来结合微管并在微管上起作用（Vale 和 Fletterick，1997.Annu.Rev.Cell Dev.Biol.13：745-777）。 微管的动态性质对于基本细胞过程非常重要，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，正是与微管相关蛋白 (MAPS)、运动蛋白（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和功能（Walczack，2000. Curr. Opin. Cell.）。生物。12：52-56）。 驱动蛋白最著名的是它们能够沿着微管轨道行走，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（Kinesin-13 是其中的一个成员）尚未被证明能够参与这种行走运动。 相反，这个驱动蛋白家族利用化学能来解聚微管末端。 先前的研究表明，在微管末端解聚的过程中，形成微管蛋白环和螺旋(Moores et. al. 2002. Mol. Cell. 9: 903-909; Desai et. al. 1999 Cell. 96: 69- 78）；重要的是，将多拉司他汀添加到α和β微管蛋白中会导致自发形成环，这些环与解聚过程中形成的环无法区分（未发表的结果）。 人们认为这些微管蛋白环代表了微管末端的微管蛋白构型，并且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，我们将利用恶性疟原虫的 KinI 运动域 (pKinI) 建立一个自动化平台，通过 LEGINON 和单粒子 2D 平均技术进行数据收集和处理。 一旦建立了自动化平台，我们将扩大我们的研究范围，包括全长人类驱动蛋白 13 和该蛋白质的各种截短版本。 希望此类研究将有助于我们对微管解聚的分子理解。