Small Grants for Exploratory Research: Isolation of Myosin-like Organelle Motor

这项SGER建议的目的是扩展对轴浆提取液中基于肌动蛋白的运动性的真正新的观察。该项目要求通过免疫荧光和免疫金标记法对运动系统进行组织化学表征。在矢量粒子运动的背景下，将使用S1修饰来确定肌动蛋白细丝的极性。此外，该项目的目标是对马达进行生化纯化，并随后根据其分子结构和酶性质进行表征，以建立其在完全表征的系统中沿着肌动蛋白细丝移动颗粒的能力。这个项目有可能极大地改变我们对轴浆颗粒运动的看法。在过去的十年里，越来越多的文献支持以微管为基础的轴浆运输运动系统。单向马达与微管相互作用，以顺行或逆行的粒子运动方式运行，主导了我们对这一过程的思考。在这一过程中，基于肌动蛋白的运动性受到的关注要少得多，尽管人们很早就知道微丝存在于作为颗粒运动分析的起始材料的轴浆提取物中。现在很清楚，微丝也可能在颗粒运动中起作用。清楚地证明基于肌动蛋白的运动是矢量的，并且在生理相关的条件下依赖于肌动蛋白细丝的极性，这将对我们理解细胞运动做出重大贡献。

The aim of this SGER proposal is to extend a truly novel observation of actin-based motility in axoplasmic extracts. The project calls for the histochemical characterization of the movement system via immunofluorescence and immunogold labelling. S1 decoration will be used to determine actin filament polarity in the context of vectoral particle movement. In addition, the project aims toward the biochemical purification of the motor and its subsequent characterization in terms of its molecular structure, and enzymatic properties in a longer-term aim of establishing its ability to move particles along actin filaments in a totally- characterized system. %%% This project has the potential to alter our perception of axoplasmic particle movement significantly. During the last decade, a growing literature has developed in favor of a microtubule-based motility system for axoplasmic transport. Unidirectional motors that interact with microtubules and operate in anterograde or retrograde particle movement have dominated our thinking about the process. Actin-based motility has received far less attention in this process, though microfilaments have long been known to be present in the axoplasmic extracts that have served as the starting material for the particle movement assays. It is now clear that microfilaments may also serve in particle movement. The clear demonstration that actin-based motility is vectorial, and dependent on the polarity of the actin filaments under physiologically- relevant conditions would be a major contribution to our understanding of cell motility.