Using viruses to study kinesin-1 recruitment, regulation and function

细胞需要将货物分类并运输到细胞的不同部分，这是细胞存活和确定细胞结构和功能的基础。为此，细胞拥有由微管组成的骨架（细胞骨架）。微管与马达相关，马达以高度特异性和受调控的方式结合和运输货物。一种称为驱动蛋白-1的马达负责将货物运输到细胞内的特定位置。驱动蛋白-1转运的缺陷会导致人类疾病，从年龄相关性黄斑变性和白内障到侵袭性癌症和神经系统疾病。驱动蛋白-1转运对于使货物沿沿着神经轴突移动更长的距离非常重要，并且驱动蛋白-1的缺陷导致几种神经退行性疾病，包括帕金森氏症、亨廷顿氏症、阿尔茨海默氏症和遗传性痉挛性截瘫。驱动蛋白-1如何结合到微管上并沿着微管移动已经得到了很好的研究。相反，驱动蛋白1如何招募特定的货物，以及驱动蛋白1的活性和目标目的地如何调节仍然知之甚少。病毒在细胞内复制，并进化出劫持细胞机器的机制，以增强其复制和传播。一些病毒在复制周期中利用驱动蛋白-1，例如帮助将新形成的病毒颗粒输出细胞。一个例子是牛痘病毒（VACV），原型痘病毒和用于根除天花的疫苗。本实验室以VACV对驱动蛋白1的募集为模型，研究了驱动蛋白1与货物结合及激活马达的机制。驱动蛋白1复合体由两条驱动蛋白重链（KHC）和两条驱动蛋白轻链（KLC）组成。其活性受自身抑制调节。激活涉及货物蛋白的多个结合事件，其诱导复合物中的结构变化，从而能够与微管结合。哺乳动物细胞表达多种类型的驱动蛋白-1（同种型），每种类型具有略微不同的生化特性，其可以结合不同的货物或移动到不同的destination.VACV蛋白A36和F12/E2复合物参与募集驱动蛋白-1以将病毒颗粒运输到细胞表面。为了了解这些蛋白质中的每一种的作用，它们彼此之间以及与驱动蛋白-1复合物的组分之间的相互作用将被生物化学和结构生物学方法表征。我们已经表明，F12/E2和A36与KLC的不同结构域相关，并优先结合不同的KLC亚型。使用RNA干扰的基因敲除和使用CRISPR/Cas9介导的基因编辑的基因敲除将用于确定不同KLC亚型对病毒转运和正常细胞功能的重要性。各种KLC亚型在细胞功能中的作用在很大程度上仍然未知。将通过显微镜表征单个KLC或KLC组合已被敲除的细胞。此外，单个KLC的细胞功能将通过鉴定和比较与其相关的蛋白质来确定。还将研究发现与病毒转运复合物相关的蛋白质，以确定它们是否是病毒输出所需的。病毒蛋白，个别KLC亚型和细胞蛋白的驱动蛋白-1的激活的贡献，以及它们在其调制中可能发挥的作用将使用在体外和在细胞assays.The拟议的研究项目的组合进行测量，旨在调查所使用的VACV招募和激活驱动蛋白-1的机制，通过表征病毒和细胞蛋白参与。这项研究也将有助于我们了解正常的细胞过程，以及疾病如何加剧这些功能障碍。关于病毒与驱动蛋白-1相互作用的结构信息将有助于开发抑制这一过程并预防痘病毒引起的疾病的化合物。

Cells need to sort and transport cargo to distinct parts of the cell and this is fundamental for cell survival and for determining cell structure and function. To do this cells possess a skeleton (cytoskeleton) that is composed of microtubules. Microtubules are associated with motors that bind and transport cargo in a highly specific and regulated manner. One type of motor, called kinesin-1, is responsible for transporting cargos to specific locations within the cell. Defects in kinesin-1 transport cause human diseases, ranging from age-related macular degeneration and cataracts, to invasive cancers and neurological disorders. Kinesin-1 transport is very important for moving cargos longer distances along nerve axons and defects in kinesin-1 cause several neurodegenerative disorders, including Parkinson's, Huntington's, Alzheimer's and inherited spastic paraplegia. How kinesin-1 binds to and moves along microtubules is quite well studied. In contrast, how kinesin-1 recruits specific cargos and how kinesin-1 activity and target destination are regulated remain poorly understood.Viruses replicate inside cells and have evolved mechanisms to hijack cellular machinery to enhance their replication and spread. Several viruses exploit kinesin-1 during their replication cycle, for instance to help export newly formed virus particles out of the cell. An example is vaccinia virus (VACV), the prototype poxvirus and the vaccine used to eradicate smallpox. Our lab uses the recruitment of kinesin-1 by VACV as a model to study the mechanisms that control the binding of cargo to kinesin-1 and activation of the motor.The kinesin-1 complex is made up of two kinesin heavy chains (KHC) and two kinesin light chains (KLC). Its activity is regulated by autoinhibition. Activation involves multiple binding events by cargo proteins that induce a structural change in the complex enabling association with microtubules. Mammalian cells express multiple kinesin-1 types (isoforms), each with slightly different biochemical properties that may bind different cargos or move to different destinations.Three VACV proteins, A36 and the F12/E2 complex, are involved in recruiting kinesin-1 to transport virus particles to the cell surface. To understand the role of each of these proteins, their interaction with each other and with components of the kinesin-1 complex will be characterised biochemically and using structural biology approaches. We have shown that F12/E2 and A36 associate with different domains of KLC and preferentially bind to different KLC isoforms. Gene knock-down using RNA interference and gene knock-out using CRISPR/Cas9-mediated gene editing will be used to determine the importance of different KLC isoforms for virus transport and for normal cellular function.The roles of various KLC isoforms in cell function remain largely unknown. Cells in which individual KLCs or combinations of KLC have been knocked-out will be characterised by microscopy. Additionally, the cellular functions of individual KLCs will be determined by identifying and comparing the proteins that associate with them. Proteins that are also found to associate with the viral transport complex will be studied to determine if they are required for virus export. The contribution of viral proteins, individual KLC isoforms and cellular proteins to the activation of kinesin-1, and their possible role in its modulation will be measured using a combination of in vitro and in cell assays.The proposed research project aims to investigate the mechanisms used by VACV to recruit and activate kinesin-1 by characterising the virus and cellular proteins involved. This research will also help our understanding of the normal cellular processes and how disease ensues if these dysfunction. Structural information about the interaction of the virus with kinesin-1 will aid the development of compounds that inhibit this process and prevent disease caused by poxviruses.