Combining atomic-resolution structure with high-resolution tracking in cells to dissect regulation and mechanism of the MKlp2 kinesin

将原子分辨率结构与细胞内高分辨率跟踪相结合，剖析 MKlp2 驱动蛋白的调控和机制

• 批准号: 283958999
• 负责人: Professor Dr. Jörg Enderlein, since 6/2019
• 金额: --
• 依托单位: Department of Physics
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/283958999?language=en
• 关键词: Combining; atomic; resolution; structure; tracking

Mitosis involves a complex coordination of cytoskeletal rearrangements for which molecular motors are temporally and spatially controlled to precisely divide the cell and distribute the chromosomes. The most burning open questions in this area concern the collective regulation of motors, which is also central to drug development. The kinesin 6 MKlp2 plays critical roles for the metaphase to anaphase transition and for cytokinesis. Inhibition of MKlp2 function in pancreatic adenocarcinoma cells reduced cell growth and MKlp2 inhibitors can kill tumor stem cells. The MKlp2 motor domain is 60% larger than that of other kinesins, due to several unique inserts that are believed to be involved in the regulation of this kinesin. MKlp2 interacts with the kinases polo-kinase 1 (Plk1) and AuroraB and controls their spatial and temporal action during cell division. MKlp2 also interacts with myosin II which is critical to bring kinases to the proper place for furrow ingression. Whether the motor activity of Mklp2 is important at this location and whether Myosin II activity is influenced by MKlp2 is unknown. The motor properties of MKlp2 and its precise cellular functions, as well as its regulation remain elusive. MKlp2 is a N-terminal kinesin with unique features, including an N-terminal extension, a short insertion in loop2 (important for MT binding), a long insertion in loop6 (L6) adjacent to the N-terminal extension in the tertiary structure, and a neck linker that is about four times longer than that of other kinesins. Because the large insertions are near the structural elements important for force generation, this motor is likely to have a different mechanism for generating its powerstroke. Whether the insertions contribute to and/or regulate its role in cytokinesis is unclear. Our preliminary data on how the motor interacts with microtubules confirms that this kinesin is unusual. The interaction with Plk1 located close to the mechanical element of this kinesin is likely to modulate how this motor produces force and how such force is used to allow transport of cargos or to organize microtubules during mitosis. To understand how MKlp2 functions, we propose to combine high-resolution structure determination with a functional characterization in vitro and in vivo. For single-molecule studies in vivo we will use a novel approach using single-walled carbon nanotubes (SWNTs) as precisely targetable and uniquely stable near-infrared fluorescent markers. We further have established multidisciplinary international collaborations to add transient kinetics and cryo-electron microscopy (cryoEM) experiments to study basic properties of this motor. The project is ambitious and innovative by combining atomic structure determination with functional single-molecule studies in live cells to define the properties of the motor and we will break new ground by tracking the motors in live cells using novel super-resolution live-cell imaging technology.

有丝分裂涉及细胞骨架重排的复杂协调，其中分子马达在时间和空间上受到控制，以精确地分裂细胞和分配染色体。在这一领域，最紧迫的公开问题涉及电机的集体调节，这也是药物开发的核心。驱动蛋白6 MKlp 2在细胞分裂中期到后期的转换和胞质分裂中起关键作用。在胰腺癌细胞中抑制MKlp 2功能会降低细胞生长，并且MKlp 2抑制剂可以杀死肿瘤干细胞。MKlp 2马达结构域比其他驱动蛋白的马达结构域大60%，这是由于据信参与该驱动蛋白的调节的几个独特的插入物。MKlp 2与激酶polo激酶1（Plk 1）和AuroraB相互作用，并控制它们在细胞分裂期间的空间和时间作用。MKlp 2还与肌球蛋白II相互作用，肌球蛋白II对于将激酶带到沟内陷的适当位置至关重要。Mklp 2的运动活动在该位置是否重要以及肌球蛋白II活性是否受到Mklp 2的影响尚不清楚。MKlp 2的运动特性及其精确的细胞功能，以及其调节仍然难以捉摸。MKlp 2是具有独特特征的N-末端驱动蛋白，包括N-末端延伸、环2中的短插入（对于MT结合很重要）、三级结构中与N-末端延伸相邻的环6（L 6）中的长插入以及比其他驱动蛋白长约四倍的颈接头。由于大的插入物靠近对力产生重要的结构元件，因此该马达可能具有用于产生其动力冲程的不同机制。插入是否有助于和/或调节其在胞质分裂中的作用尚不清楚。我们关于马达如何与微管相互作用的初步数据证实了这种驱动蛋白是不寻常的。与Plk 1的相互作用位于靠近该驱动蛋白的机械元件，可能会调节该电机如何产生力，以及如何使用这种力来允许货物运输或在有丝分裂期间组织微管。为了了解MKlp 2的功能，我们建议将联合收割机高分辨率结构测定与体外和体内功能表征相结合。对于体内单分子研究，我们将使用一种新的方法，使用单壁碳纳米管（SWNTs）作为精确的靶向和独特稳定的近红外荧光标记物。我们还建立了多学科的国际合作，增加瞬态动力学和低温电子显微镜（cryoEM）实验，以研究这种电机的基本特性。该项目是雄心勃勃和创新的，通过将原子结构测定与活细胞中的功能性单分子研究相结合来定义马达的特性，我们将通过使用新型超分辨率活细胞成像技术跟踪活细胞中的马达来开辟新天地。