Allostery in myosins studied at the molecular level

描述(申请人提供)：所有生物的一个重要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，考虑到它们只有几个纳米宽，分子机器施加了惊人的力量。考虑到细胞中马达蛋白的多样性，一个关键问题是马达在搬运货物和施力时如何合作和竞争。一种新兴的范例是“专门的”马达的概念，即微调以执行特定功能的马达。尽管这些马达蛋白很重要，但人们对它们的个体适应以及它们与细胞中发现的运动模式的关系知之甚少。在以前的研究中，我们发现了两个肌球蛋白，它们在肌动蛋白上具有新的和不同的过程步进模式。肌球蛋白X沿着束蛋白-肌动蛋白束中的多条细丝行走。另一方面，非肌肉肌球蛋白IIB(NMIIB)沿着单个肌动蛋白细丝的长螺旋行走。这两种肌球蛋白在细胞迁移中都起着关键作用，包括转移的肿瘤细胞。肌球蛋白X将整合素类、钙粘附素和网状蛋白受体等重要物质输送到细胞前沿的丝状附着体；NMIIB出现在细胞的后部，维持细胞的极性和内部组织。因此，我们必须了解这两个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的运动。对于这两个肌球蛋白，我们认为它们的两个运动域是通过应变敏感的门控机制同步的。对于NMIIB和myosin X，门控都进行了调整，以适应它们沿着肌动蛋白轨迹的独特步进模式。我们假设NMIIB具有张力维持的适应性，肌球蛋白X具有束选择的适应性，两者都可能具有扭转单个肌动蛋白细丝的适应性。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如何沿着肌动蛋白采取短暂、渐进的步骤并保持细胞骨架的张力。目的2：我们将确定肌球蛋白X在肌动蛋白细丝束环境中是如何被门控的。目标3：我们将确定两种肌球蛋白是如何扭曲肌动蛋白细丝的。

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.