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

有丝分裂涉及细胞骨架重排的复杂协调，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。这一领域最紧迫的悬而未决的问题与发动机的集体监管有关，这也是药物开发的核心。激动素6MKlp2在细胞中期到后期的转换和胞质分裂过程中起着关键作用。抑制胰腺癌细胞MKlp2功能抑制细胞生长，MKlp2抑制剂可杀伤肿瘤干细胞。MKlp2运动结构域比其他运动蛋白大60%，这是因为有几个独特的插入被认为参与了该运动蛋白的调节。MKlp2与Polo-kinase1(Plk1)和AuroraB相互作用，控制它们在细胞分裂过程中的空间和时间作用。MKlp2还与肌球蛋白II相互作用，而肌球蛋白II是将激酶带到适当的沟槽进入位置的关键因素。目前尚不清楚MKLp2在该部位的运动活性是否重要，以及MKlp2是否影响肌球蛋白II的活性。MKlp2的运动特性及其精确的细胞功能及其调控仍然难以捉摸。MKlp2是一种具有独特功能的N-末端动蛋白，包括N-末端延伸，在loop2中的短插入(对MT结合很重要)，在三级结构中与N-末端延伸相邻的loop6(L6)中的长插入，以及大约是其他kinesin的4倍长的颈部连接物。由于大型插入物靠近对力产生很重要的结构元素，因此这种马达很可能具有不同的产生动力冲程的机制。目前尚不清楚这些插入是否有助于和/或调节其在胞质分裂中的作用。我们关于马达如何与微管相互作用的初步数据证实，这种动蛋白是不寻常的。与Plk1的相互作用位于这种激动素的机械元件附近，可能调节这种马达如何产生力，以及这种力如何被用来允许货物的运输或在有丝分裂期间组织微管。为了了解MKlp2的功能，我们建议将高分辨率结构测定与体外和体内功能鉴定相结合。对于活体中的单分子研究，我们将使用一种新的方法，使用单壁碳纳米管(SWNTs)作为精确靶向和唯一稳定的近红外荧光标记。我们进一步建立了多学科的国际合作，增加了瞬变动力学和冷冻电子显微镜(Cryo-EM)实验，以研究这种电机的基本特性。该项目雄心勃勃，具有创新性，将原子结构测定与活细胞中功能性单分子研究相结合，以确定马达的特性，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，从而取得新的突破。

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.