Regulation of myosin V interaction with cargo

项目摘要/摘要 分子马达是细胞组织所必需的，并驱动细胞类型的特定结构。马达 在适当的时间，将细胞器、信使核糖核酸和其他细胞成分分配到正确的位置，并 调节多种途径，包括分泌和细胞内交通。因此，对分子的适当控制 马达对于动物生理学来说是必不可少的。 肌球蛋白V马达(MyoV)存在于大多数真核生物中，提供相对较长距离的运动 在肌动蛋白缆索上的货物。人类表达三个MyoV。每个MyoV的突变都与严重的 疾病。在许多情况下，这些缺陷可能是由于MyoV无法正确地与货物相关联。 我们揭示了货物附着和脱离MyoV所需的一些机制。然而， 在如何控制货物从MyoV上附着和拆卸方面，知识存在很大差距。 在酿酒酵母(以下简称酵母)中，许多货物的长距离运输仅发生在 肌动蛋白通过MyoV。这与大多数生物不同，在大多数生物体中，运输是通过运动蛋白在微管上启动的， 随后移交给MyoV。因此，酵母MyoV，Myo2的调节是一个更简单的系统。 引人注目的是，精选的酵母Myo2有不同的轨迹。这导致我们发现 MyO2从其货物中脱离是细胞器运输的一个关键性质。重要的是，由于他们的 序列相似性，酵母Myo2的机制已经为人类MyoV的功能提供了洞察力。 这一建议的重点是液泡/溶酶体遗传，它通过Myo2发生，并受到两者的控制 在空间上和时间上。我们确定了Vac17-Vac8空泡适配器，但Myo2如何与Vac17- Vac8和调节复合体形成和解离的机制(S)知之甚少。 我们最近在生化和基因分析方面取得的进展导致了以下两个目标。 1)确定Myo2-Vac17-Vac8复合体的组织结构。这个目标将使用遗传学、生物化学、 结构方法和AlphaFold多聚体预测确定Vac17如何与Myo2和Myo2相关联 与支架蛋白Vac8结合。2)确定Vps41和KYK3在调节拆卸中的作用 Myo2-Vac17-Vac8复合体。我们发现Vps41和Yck 3对Myo2- Vac17-Vac8复合体，但它们在这一途径中的作用与它们在液泡融合中的已知作用不同。至 确定它们的Myo2特定角色，我们将识别更多与Vps41和Yck 3在 Myo2-Vac17-Vac8复合体的上下文。我们将使用下拉式方法，高含量显微镜 屏幕和邻近标签。鉴于人们对分子马达是如何附着或脱离的知之甚少 货物，拟议的研究可能会揭示对细胞功能至关重要的新途径。

PROJECT SUMMARY/ABSTRACT Molecular motors are essential for cellular organization, and drive cell-type specific architectures. Motors distribute organelles, mRNA, and other cellular components to the correct place, at the proper time, and regulate multiple pathways including secretion, and intracellular traffic. Thus, the proper control of molecular motors is essential for animal physiology. Myosin V motors (MyoV) are present in most eukaryotes and provide relatively long-range movement of cargoes on actin cables. Humans express three MyoV. Mutations in each MyoV have been linked to serious diseases. In many cases, the defects may be due to an inability of the MyoV to properly associate with cargo. We uncovered some of the mechanisms required for cargo attachment and detachment from MyoV. However, there are large gaps in knowledge of how cargo attachment and detachment from MyoV is controlled. In S. cerevisiae (hereafter referred to as yeast), long-range transport of many cargoes occurs solely on actin via MyoV. This contrasts with most organisms, where transport initiates on microtubules via kinesins with a subsequent handoff to MyoV. Thus, regulation of yeast MyoV, Myo2, is a simpler system. Strikingly, selected cargoes of yeast Myo2 have distinct trajectories. This led to our discovery that disengagement of Myo2 from its cargoes is a key property of organelle transport. Importantly, due to their sequence similarity, mechanisms discovered for yeast Myo2 have provided insights into human MyoV function. This proposal is focused on vacuole/lysosome inheritance which occurs via Myo2 and is controlled both spatially and temporally. We identified the Vac17-Vac8 vacuole adaptors, yet how Myo2 interacts with Vac17- Vac8 and the mechanisms(s) that regulate formation and disassociation of the complex are poorly understood. Our recent inroads in biochemical and genetic analyses have led to the following two aims. 1) Determine the organization of the Myo2-Vac17-Vac8 complex. This aim will use genetics, biochemistry, structural approaches and AlphaFold multimer predictions to determine how Vac17 associates with Myo2 and with the scaffold protein Vac8. 2) Determine roles of Vps41 and the kinase Yck3 in the regulated disassembly of the Myo2-Vac17-Vac8 complex. We discovered that Vps41 and Yck3 are critical for disruption of the Myo2- Vac17-Vac8 complex, yet their roles in this pathway are distinct from their known roles in vacuole fusion. To determine their Myo2-specific roles, we will identify additional proteins that act with Vps41 and Yck3 in the context of the Myo2-Vac17-Vac8 complex. We will use pull-down approaches, high-content microscopy screens and proximity labeling. Given that little is known about how molecular motors attach or detach from cargoes, the proposed studies will likely reveal new pathways that are crucial for cell function.