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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 该研究项目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。 运动蛋白的动力素家族利用源自ATP-Hydrolsys的能量在微管上结合并起作用（Vale and Fletterick，1997。Annu。Annu。Rev.CellDev。Biol。13：745-777）。 微管的动态性质对于有丝分裂，细胞迁移和细胞运输过程中的纺锤体组装等基本细胞过程很重要。实际上，正是与微管相关蛋白（MAP），运动蛋白（例如驱动蛋白）和其他细胞因子的相互作用，是调节动态行为的，以及微管的功能（Walczack，2000。Curr。Cell。Cell。Cell。Biol。Biol。Biol。12：52-56）。 驱动蛋白以其沿着微管轨道行走的能力而闻名，同时将货物从细胞中的一个点携带到另一个点。但是，尚未证明中间动力素（其中的动力素13是成员）从事这种行走运动。 取而代之的是，这种驱动剂家族利用化学能将微管的末端解散。 先前的研究表明，在解聚微管末端，形成微管蛋白环和螺旋（Moores等，2002。Mol。Cell。9：903-909; Desaiet。Al。1999Cell。96：69-78）;重要的是，在α和β微管蛋白中添加dolastatin会导致与在解聚过程中形成的环的自发形成（未发表的结果）。 人们认为，这些微管蛋白环是微管末端的微管蛋白构型的代表，并且对与此环的电动机进行的研究将提供有关微管解聚化分子机制的重要见解。 在该项目的第一阶段，我们将利用来自恶性疟原虫（PKINI）的Kini的运动结构域来建立一个通过Leginon和单个粒子2D平均技术进行数据收集和处理的自动化平台。 一旦建立了一个自动化平台，我们将扩展研究以包括全长的人类动力蛋白13和蛋白质的各种截短版本。 希望这样的研究将有助于我们对微管解聚的分子理解。