Allostery in myosins studied at the molecular level

描述（由申请人提供）：所有生物体的一个关键功能是在需要时移动的能力。这些运动--细胞内运输、细胞分裂、肌肉收缩和细胞运动--是由分子机器驱动的，考虑到它们只有几纳米宽，这些分子机器施加了惊人的力量。鉴于细胞中马达蛋白的多样性，一个关键问题是马达在移动货物和施加力时如何合作和竞争。一个新兴的范例是“专门”马达的概念，或被微调以执行特定功能的马达。尽管这些运动蛋白的重要性，相对较少的是知道他们的个人适应性，以及如何这些涉及到运动模式中发现的细胞。在先前的研究中，我们发现了两个肌球蛋白与新的和独特的进行性步进模式的肌动蛋白。肌球蛋白X沿着多条肌丝在肌成束蛋白-肌动蛋白束中行走。另一方面，非肌肉肌球蛋白IIB（NMIIB）沿着单个肌动蛋白丝的长螺距螺旋行走。这两种肌球蛋白在迁移细胞中起关键作用，包括转移肿瘤细胞。肌球蛋白X将必需的货物如整联蛋白、钙粘蛋白和netrin受体递送到细胞前缘的丝状伪足; NMIIB出现在细胞后部，在那里它维持细胞极性和内部组织。因此，我们必须了解这两种肌球蛋白是如何运作的，这样我们才能通过这些参与者控制细胞运动。对于这两个肌球蛋白，我们建议，他们的两个电机域同步通过应变敏感的门控机制。对于NMIIB和肌球蛋白X，门控被调整以适应它们沿着沿着肌动蛋白轨道的独特步进模式。我们假设NMIIB具有张力维持的适应性，肌球蛋白X具有竞争选择的适应性，并且两者都可能具有扭曲单个肌动蛋白丝的适应性。为了测试这些门控机制，我们将追求以下具体目标：目标1：我们将确定NMIIB如何采取短，进行步骤沿着肌动蛋白和维持细胞骨架张力。目的2：我们将确定肌球蛋白X是如何门控的环境中的肌动蛋白丝束。目的3：我们将确定两种肌球蛋白如何扭曲肌动蛋白丝。 公共卫生相关性：运动细胞利用非肌肉肌球蛋白IIB和肌球蛋白X的运动活性来组织和重新定位其内容物。我们将使用一组先进的单分子方法，以及分子遗传学的工具，以确定这些肌球蛋白是如何协调其不同类型的肌动蛋白轨道。这项工作将阐明 癌细胞如何从原发肿瘤中逃逸、迁移和侵入周围组织。

DESCRIPTION (provided by applicant): A critical function for all living organisms is the ability to move when needed. These movements-- intracellular trafficking, cell division, muscle contraction, and cell motility-- are driven by molecular machines that exert an amazing amount of force considering that they are only a few nanometers across. Given the variety of motor proteins in the cell, a key question is how motors cooperate and compete while moving cargoes and applying forces. An emerging paradigm is the notion of "specialized" motors, or motors that are fine-tuned to perform a specific function. Despite the importance of these motor proteins, relatively little is known about their individual adaptations and how these relate to the motility patterns found in the cell. In prior studies, we discovered two myosins with new and distinct processive stepping patterns on actin. Myosin X walks along multiple filaments in a fascin-actin bundle. Nonmuscle myosin IIB (NMIIB), on the other hand, walks along the long-pitch helix of a single actin filament. Both myosins play pivotal roles in migrating cells, including metastasizing tumor cells. Myosin X delivers essential cargoes such as integrins, cadherins and netrin receptors to filopodia at the leading edge of the cell; NMIIB appears in the rear of the cell, wher it maintains cell polarity and internal organization. Thus, it is essential that we understand how both myosins operate so that we can control cell motility through these players. For both myosins, we propose that their two motor domains are synchronized through strain-sensitive gating mechanisms. For both NMIIB and myosin X, gating is tuned to accommodate their unique stepping patterns along actin tracks. We hypothesize that NMIIB has adaptations for tension maintenance, myosin X has adaptations for bundle-selection, and both may have adaptations for twisting single actin filaments. To test these gating mechanisms, we will pursue the following specific aims: Aim 1: We will determine how NMIIB takes short, processive steps along actin and maintains cytoskeletal tension. Aim 2: We will determine how myosin X is gated in the environment of an actin filament bundle. Aim 3: We will determine how both myosins twist actin filaments. PUBLIC HEALTH RELEVANCE: Motile cells use the motor activity of nonmuscle myosin IIB and myosin X to organize and reposition their contents. We will use a battery of advanced single-molecule methods, as well as the tools of molecular genetics, to determine how these myosins are coordinated on their distinct types of actin tracks. Together this work will illuminate how cancer cells escape from the primary tumor, migrate, and invade surrounding tissues.