Yang Rui, Bentley Marvin, Huang Chung-Fang, Banker Gary
Jungers Center for Neurosciences Research, Oregon Health and Science University, Portland, OR, USA.
Methods Cell Biol. 2016;131:217-232. doi: 10.1016/bs.mcb.2015.06.021. Epub 2015 Sep 3.
Neuronal microtubules are subject to extensive posttranslational modifications and are bound by MAPs, tip-binding proteins, and other accessory proteins. All of these features, which are difficult to replicate in vitro, are likely to influence the translocation of kinesin motors. Here we describe assays for evaluating the translocation of a population of fluorescently labeled kinesin motor domains, based on their accumulation in regions of the cell enriched in microtubule plus ends. Neurons lend themselves to these experiments because of their microtubule organization. In axons, microtubules are oriented with their plus ends out; dendrites contain a mixed population of microtubules, but those near the tips are also plus end out. The assays involve the expression of constitutively active kinesins that can walk processively, but that lack the autoinhibitory domain in the tail that normally prevents their binding to microtubules until they attach to vesicles. The degree to which such motor domains accumulate at neurite tips serves as a measure of the efficiency of their translocation. Although these assays cannot provide the kind of quantitative kinetic information obtained from in vitro assays, they offer a simple way to examine kinesin translocation in living neurons. They can be used to compare the translocation efficiency of different kinesin motors and to evaluate how mutations or posttranslational modifications within the motor domain influence kinesin translocation. Changes to motor domain accumulation in these assays can also serve as readout for changes in the microtubule cytoskeleton that affect kinesin translocation.
神经元微管会经历广泛的翻译后修饰，并与微管相关蛋白（MAPs）、末端结合蛋白及其他辅助蛋白结合。所有这些在体外难以复制的特性，都可能影响驱动蛋白的转运。在此，我们描述了基于荧光标记的驱动蛋白运动结构域在富含微管正端的细胞区域中的积累情况来评估其转运的实验方法。神经元因其微管组织而适合进行这些实验。在轴突中，微管的正端向外；树突含有混合的微管群体，但靠近末端的那些微管也是正端向外。这些实验方法涉及组成型激活的驱动蛋白的表达，这些驱动蛋白能够持续移动，但尾部缺乏通常会阻止它们与微管结合直至它们附着到囊泡上的自抑制结构域。此类运动结构域在神经突末端积累的程度可作为其转运效率的一种衡量指标。尽管这些实验方法无法提供从体外实验中获得的那种定量动力学信息，但它们提供了一种在活神经元中检测驱动蛋白转运的简单方法。它们可用于比较不同驱动蛋白的转运效率，并评估运动结构域内的突变或翻译后修饰如何影响驱动蛋白的转运。在这些实验方法中，运动结构域积累的变化也可作为影响驱动蛋白转运的微管细胞骨架变化的读数。
