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

项目概要/摘要 有许多货物，如分泌囊泡，自噬体和液滴， 通过相反的马达细胞质动力蛋白沿微管沿着双向运输， 驱动蛋白然而，目前还不清楚这两种生物马达如何合作定位细胞。 货物和实现细胞功能。双向运输受损导致各种 神经退行性疾病，包括阿尔茨海默病。微管相关蛋白 MAP 7和动力蛋白辅因子Lis 1是两种重要的蛋白质，在货物运输中起关键作用 运输MAP 7增强了驱动蛋白的进行性运动，而Lis 1则减轻了自激运动。 动力蛋白的抑制。在这项提议中，我们的目标是重建DDBE-K（动力蛋白，动力蛋白，BicD， Egl，驱动蛋白）复合物，并观察其在两种MAP 7存在下在微管轨道上的运动 和Lis 1使用全内反射荧光（TIRF）显微镜。由于MAP 7具有 能够延长驱动蛋白与微管的附着，并且Lis 1偏好结合两个动力蛋白， 我们预测，MAP 7和Lis 1将在决定运动方向的关键作用， 微管上的复合物。使用单分子测定，我们将研究是否 衔接蛋白BicD不仅作为货物和马达之间的接头， 在感知货物和马达绑定中起重要作用。这些研究将提供新的 深入了解动力蛋白和货物衔接蛋白的作用，并有助于我们理解 细胞质动力蛋白和驱动蛋白如何协同工作， 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.