Dissecting the in vivo role of glycogen synthase kinase-3 beta (GSK3b) in the function of kinesin-1 using CRISPR/cas-1

尽管25年前就发现分子马达是磷酸化的，但关于分子马达的基本问题仍然存在。 蛋白激酶或特定磷酸化位点的身份，以及它们如何控制马达 仍然没有答案。由于激酶级联显示出相当大的串扰，并在细胞归巢中发挥多种作用， 然而，在特定过程中涉及哪种激酶的破译一直是困难的。此外，还有一些 关于磷酸化在多大程度上抑制或刺激细胞内转运，调节磷酸化的程度， 机制在物种之间是保守的，以及体外机制如何转化为体内系统。因此，在本发明中， 缺乏的是一个连贯的策略，以成功地阐明磷酸化如何有助于空间和 控制体内细胞内转运的时间调节机制，没有这些机制，靶向有效治疗- 在疾病如癌症或神经变性的早期可能被破坏的途径的治疗是不可能实现的。 长期目标是确定参与细胞内蛋白质表达调控的细胞/分子机制。 体内运输总体目标是开发一个体内平台，以梳理出特定激酶如何控制 运动功能，通过确定精确的功能位点，并通过分离的监管步骤，从一个 复杂的机制网络。核心假设是糖原合成酶激酶-3 β （GSK3b）差异磷酸化驱动蛋白-1上的特定位点以控制体内细胞内转运。的 提出的研究的基本原理是，一旦GSK3b如何参与驱动蛋白的体内机制- 介导的运输是已知的，该领域将更接近于确定复杂的机制， MT上的许多细胞货物的运动性跟踪它们向远端位点的递送。以强有力的指导， 目前尚无数据，这一假设将通过追求特定目标来检验;确定GSK 3b调节驱动蛋白-1 在体内细胞内转运过程中发挥作用。将追求两个目标：产生可遗传的GSK3b磷酸盐， 使用CRISPR/Cas系统检测磷酸化缺陷/活性KHC/KLC飞系（目的1），并鉴定其中的磷酸化缺陷/活性KHC/KLC飞系。 细胞内GSK3b介导的磷酸化如何控制驱动蛋白-1功能的体内机制 运输（目标2）。所使用的实验策略采用已经证实的体内方法， 与果蝇遗传学，结合生物化学分析和生物物理范例。这一方法 在申请人看来是创新的，因为它通过分析特定的 体内驱动蛋白1亚基上的GSK3b磷酸化事件，这将导致更好地理解 驱动蛋白-1如何发挥作用的机制细节;这似乎与目前的研究有很大不同。 从体外研究中得知。该研究具有重要意义，因为它有望纵向推进 并在生理条件下，在整个生物体环境中转化目前已知的东西。的 所获得的知识将极大地推动精确的药理学/遗传修饰剂的发展 这将有助于癌症和神经变性的治疗。

Despite the discovery that molecular motors are phosphorylated 25 years ago, fundamental questions on the identity of the protein kinase(s) or the particular phosphorylation sites, and how they function to control motors remain unanswered. Since kinase cascades display considerable crosstalk and play multiple roles in cell home- ostasis, deciphering which kinase is involved in a particular process has been difficult. Further, there is some debate as to the extent to which phosphorylation inhibits or stimulates intracellular transport, the extent regulatory mechanisms are conserved between species, and how in vitro mechanisms translate to in vivo systems. Thus, what is lacking is a cohesive strategy to successfully unravel how phosphorylation contributes to the spatial and temporal regulatory mechanisms that control intracellular transport in vivo, without which targeting effective treat- ments to a pathway that is likely disrupted early in disease such as cancer or neurodegeneration is unattainable. The long-term goal is to identify the cellular/molecular mechanisms involved in the regulation of intracellular transport in vivo. The overall objective is to develop an in vivo platform to tease out how a specific kinase controls motor function by identifying the precise functional sites involved, and by isolating the regulatory steps from a complex network of mechanisms. The central hypothesis is that the kinase glycogen synthase kinase-3beta (GSK3b) differentially phosphorylates particular sites on kinesin-1 to control intracellular transport in vivo. The rationale for the proposed research is that once the in vivo mechanisms of how GSK3b is involved in kinesin- mediated transport are known, the field will be a step closer to identifying the complex mechanisms that govern the motility of numerous cellular cargoes on MT tracks for their delivery to distal sites. Guided by strong prelimi- nary data, this hypothesis will be tested by pursuing the specific aim; identify that GSK3b-regulates kinesin-1 function during intracellular transport in vivo. Two objectives will be pursued; generate heritable GSK3b phos- phorylation defective/active KHC/KLC fly lines using the CRISPR/Cas system (Objective 1), and identify the in vivo mechanisms of how GSK3b-mediated phosphorylation controls kinesin-1 function during intracellular transport (Objective 2). The experimental strategy used employs an already proven in vivo approach, coupled with Drosophila genetics, integrated with biochemical analysis and biophysical paradigms. This methodology is innovative in the applicant’s opinion, because it departs from the status quo by enabling the analysis of particular GSK3b-phosphorylation events on kinesin-1 subunits in vivo, which will lead to a better understanding of the mechanistic details of how kinesin-1 functions; which appear to be considerably different from what is currently known from in vitro studies. The proposed research is significant, because it is expected to vertically advance and transform what is currently known, under physiological conditions, in a whole organism setting. The knowledge acquired will dramatically propel the development of precise pharmacological/genetic modifiers against defects in this pathway which will benefit the treatment of cancer and neurodegeneration.