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，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布染色体。这一领域最紧迫的悬而未决的问题涉及对马达的集体监管，这也是药物开发的核心。运动蛋白6mklp2在中期到后期转变和细胞分裂中起关键作用。抑制MKlp2在胰腺腺癌细胞中的功能可以降低细胞生长，MKlp2抑制剂可以杀死肿瘤干细胞。MKlp2马达结构域比其他激酶大60%，这是由于几个独特的插入物被认为参与了该激酶的调控。MKlp2与polo-kinase 1 （Plk1）和AuroraB激酶相互作用，并控制它们在细胞分裂过程中的空间和时间作用。MKlp2还与肌凝蛋白II相互作用，肌凝蛋白II对于将激酶带到沟侵入的适当位置至关重要。Mklp2的运动活性在这个位置是否重要以及Myosin II的活性是否受到Mklp2的影响尚不清楚。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n端激酶蛋白，具有独特的特征，包括n端延伸，在环2中有一个短插入（对MT结合很重要），在环6中有一个长插入（L6），在第三结构中与n端延伸相邻，并且有一个比其他激酶蛋白长约4倍的颈连接子。因为大的插入是接近的结构元素重要的力量产生，这种电机很可能有一个不同的机制，以产生其动力冲程。插入是否有助于和/或调节其在细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，这种运动蛋白是不寻常的。与靠近该驱动蛋白机械元件的Plk1的相互作用可能会调节该马达如何产生力，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表征相结合。对于体内单分子研究，我们将使用一种新颖的方法，使用单壁碳纳米管（SWNTs）作为精确靶向和独特稳定的近红外荧光标记物。我们进一步建立了多学科的国际合作，以增加瞬态动力学和低温电子显微镜（cryoEM）实验来研究该电机的基本特性。该项目雄心勃勃，具有创新性，将原子结构测定与活细胞中的功能单分子研究相结合，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来开辟新天地。

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.