Allostery in myosins studied at the molecular level

描述（由申请人提供）：所有生物的一项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，它们会施加惊人的力量。考虑到细胞中运动蛋白的多样性，一个关键问题是运动蛋白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“专门化”马达的概念，或微调以执行特定功能的马达。尽管这些运动蛋白很重要，但人们对它们的个体适应性以及它们与细胞中发现的运动模式的关系知之甚少。在之前的研究中，我们发现了两种肌凝蛋白，它们在肌动蛋白上具有新的和不同的过程步进模式。肌凝蛋白X沿着筋膜蛋白-肌动蛋白束中的多条细丝行走。另一方面，非肌球蛋白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，包括转移性肿瘤细胞中起关键作用。肌凝蛋白X将整合素、钙粘蛋白和网状蛋白受体等必需物质输送到细胞前沿的丝状足；NMIIB出现在细胞的后部，在那里它维持细胞的极性和内部组织。因此，我们必须了解这两种肌凝蛋白是如何运作的，这样我们才能通过这些参与者来控制细胞运动。对于这两种肌凝蛋白，我们认为它们的两个运动域通过应变敏感门控机制同步。对于NMIIB和myosin X，门控被调整以适应它们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，肌凝蛋白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的、渐进的步骤并维持细胞骨架张力。目的2：我们将确定肌动蛋白丝束环境中肌凝蛋白X是如何被门控的。目的3：我们将确定两种肌凝蛋白如何扭曲肌动蛋白丝。

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.