Mechanism by Which the Bicaudal D2-Nuclear Pore Protein 358 Interaction Activates Microtubule-based Cargo Transport

项目摘要/摘要 有许多物质，如分泌囊泡、自噬小体和液滴 胞浆动力蛋白和胞质动力蛋白沿微管双向运输 激动素。然而，目前仍不清楚这两个生物马达是如何合作定位 并实现蜂窝功能。双向运输受损导致多种 神经退行性疾病，包括阿尔茨海默病。微管相关蛋白 MAP7和dynein辅因子Lis1是在货物中起关键作用的两种重要蛋白质 运输。虽然MAP7增强了Kinesin的前进运动，但Lis1缓解了这种自发性运动。 动力蛋白抑制。在这个建议中，我们的目标是重建DDBE-K(动力蛋白，动力肌动蛋白，BicD， EGL，kinesin)复合体，并观察其在两种MAP7存在时在微管上的运动 用全内反射荧光(TIRF)显微镜观察。鉴于MAP7具有 能够延长动蛋白与微管的附着，并且Lis1更喜欢结合两个动力蛋白， 我们预测，MAP7和Lis1将在决定细胞运动方向方面发挥关键作用 微管上的复合体。使用单分子分析，我们将调查是否 适配蛋白BicD不仅作为货物和马达之间的连接物，而且还扮演着 在检测货物和马达捆绑方面的重要作用。这些拟议的研究将提供新的 深入了解动力蛋白和货物适配器蛋白的作用，有助于我们理解 胞质动力蛋白和动蛋白如何共同作用，双向运输货物 微管。

Project Summary/Abstract There are many cargo such as secretory vesicles, autophagosomes, and liquid droplets that are bi-directionally transported along microtubules by the opposing motors cytoplasmic dynein and kinesin. However, it is still unclear how these two biological motors collaborate to position the cargo and achieve cellular functions. Impaired bidirectional transport leads to a variety of neurodegenerative diseases, including Alzheimer's disease. Microtubule-associated protein MAP7 and dynein cofactor Lis1 are two important proteins that play critical roles in cargo transport. While MAP7 enhances the processive motion of kinesin, Lis1 relieves the auto- inhibition of dynein. In this proposal, we aim to reconstitute the DDBE-K (dynein, dynactin, BicD, Egl, kinesin) complex and observe its motion on microtubule tracks in the presence of both MAP7 and Lis1 using total internal reflection fluorescence (TIRF) microscopy. Given that MAP7 has the ability to prolong the attachment of kinesin to microtubules and Lis1 prefers binding two dyneins, we predict that MAP7 and Lis1 will play key roles in determining the direction of motion of the complexes on microtubules. Using single-molecule assays, we will investigate whether the adapter protein BicD not only functions as a linker between cargo and motor but also plays an essential role in sensing cargo and motor binding. These proposed studies will provide novel insights into the roles of dynein and cargo adapter proteins and contribute to our understanding of how cytoplasmic dynein and kinesin work together to transport cargo bidirectionally on microtubules.