Dissecting the regulation and function of actin flows during cell motility

细胞迁移是一种基本的细胞行为，发生在正常生理过程中，如胚胎发生，以及病理过程中，如癌症转移。因此，了解细胞如何控制它们的运动对于许多基本的生物过程是重要的，并且具有翻译相关性。在过去的50年里，我们对细胞迁移的看法涉及亚细胞行为的逐步循环，始于细胞前部膜的延伸。然而，最近的证据表明，在一些细胞类型的前沿是即时协调与其他“步骤”的迁移周期：细胞运动，因此不是一个逐步的过程。我们现在的目标是了解这些迁移行为是如何在空间和时间上协调控制细胞运动的。我们的实验室率先使用胚胎果蝇巨噬细胞（血细胞）作为体内迁移模型，以解决控制细胞运动的基本过程。这些细胞可以在动物体内以非常高的分辨率进行实时成像，再加上果蝇的遗传可塑性，因此该系统是了解正常生理过程中细胞运动控制的强大模型。使用这个模型，我们最近发现，肌动蛋白细胞骨架网络，这是已知的细胞运动的驱动程序，经历血细胞内的恒定流动。通过计算工具的发展，我们已经发现，这种肌动蛋白流在整个细胞中是高度协调的，导致流场内稳定的汇，似乎控制肌动蛋白流穿过细胞并指导细胞运动。我们假设这种协调的肌动蛋白流是控制细胞迁移中所涉及的亚细胞行为的关键因素，这将在本提案中进一步详细剖析。由于肌动蛋白流动是所有迁移细胞的中心特征，因此这项工作的结果将与许多生物过程相关。 在第一个目标中，我们将进一步开发我们的计算工具来分析肌动蛋白流，并随后使用这些新的方法来确定正常血细胞行为期间肌动蛋白流变化的时程，例如对伤口信号的反应。我们还将研究肌动蛋白运动如何与肌球蛋白的运动相关，肌球蛋白是负责肌动蛋白网络收缩和随后流动的关键马达。这一目标的结果将形成一个基线理解，使我们能够精确地确定监管机构在后续目标中的作用。在下一个目标中，我们将研究如何肌动蛋白流的调节测试苍蝇含有突变的基因假设控制肌动蛋白网络。此外，我们将研究肌动蛋白网络的运动是如何控制和/或协调的微管网络，第二个细胞骨架网络，是重要的细胞迁移。同样，将测试包含微管网络的已知调节器中的突变的果蝇品系，以确定该网络如何调节肌动蛋白运动。在最后一个目标中，我们将研究肌动蛋白流如何直接控制负责定义细胞极性的细胞迁移调节因子的活性。膜突蛋白是一种肌动蛋白调节剂，已知定位于许多迁移细胞类型的后部，在那里它控制极化运动。初步数据显示，膜突蛋白定位于迁移血细胞的后部，膜突蛋白突变体在细胞运动和肌动蛋白流动中存在缺陷。以前的数学模型表明，任何蛋白质，结合到肌动蛋白网络具有足够的强度将进行向后的肌动蛋白流导致梯度，随后可以控制细胞的行为。我们假设，膜突蛋白是一个理想的候选人，这种直接调节肌动蛋白流，我们将研究膜突蛋白是如何控制的流量，随后反馈控制肌动蛋白网络。

Cell migration is a fundamental cellular behaviour that occurs during normal physiology, such as embryogenesis, as well as pathologies, such as cancer metastasis. Understanding how cells control their movement is therefore important for numerous fundamental biological processes and is of translational relevance. For the past 50 years our view of cell migration has involved a step-wise cycle of subcellular behaviours beginning with extension of the membrane at the front of the cell. However, recent evidence in a number of cell types has revealed that the leading edge is instantaneously coordinated with other 'steps' of the migratory cycle: cell motility is therefore not a step-wise process. Our goal now is to understand how these migratory behaviours are coordinated in space and time to control cell motion. Our laboratory has pioneered the use of embryonic Drosophila macrophages (hemocytes) as an in vivo migration model to address fundamental processes controlling cell motility. These cells can be live imaged at very high resolution within the animal, and coupled with the genetic tractability of the fruitfly, this system is therefore a powerful model to understand the control of cell motility during normal physiology. Using this model we have recently revealed that the actin cytoskeletal network, which is known to be the driver of cell motility, undergoes constant flow within hemocytes. Through the development of computational tools we have revealed that this actin flow is highly coordinated across the entire cell resulting in stable sinks within the flowfield that appear to control actin flows across the cell and direct cell motion. We hypothesise that this orchestrated actin flow is a critical factor that controls the subcellular behaviours involved in cell migration, which will be dissected in further detail in this proposal. As actin flows are a central feature of all migrating cells, the results of this work will be relevant to many biological processes. In the first Objective we will further develop our computational tools to analyse actin flows and subsequently use these novel approaches to determine the timecourse of actin flow changes during normal hemocyte behaviours, such as responses to wound signals. We will also investigate how actin motion is correlated with the movement of myosin, a critical motor responsible for contraction and subsequent flow of the actin network. The results of this objective will form a baseline understanding that will allow us to precisely determine the function of regulators in subsequent objectives. In the next Objective we will examine how actin flows are regulated by testing flies containing mutations in genes hypothesized to control the actin network. Additionally, we will examine how the motion of the actin network is controlling and/or coordinated by the microtubule network, a second cytoskeletal network that is important in cell migration. Again, fly lines containing mutations in known regulators of the microtubule network will be tested to determine how this network is regulating actin motion. In the final Objective we will examine how actin flow may be directly controlling the activity of a regulator of cell migration responsible for defining cell polarity. Moesin is an actin regulator known to localize to the rear of many migratory cell types where it controls polarised motility. Preliminary data reveals that moesin localises to the rear of migrating hemocytes and that moesin mutants have defects in cell movement and actin flow. Previous mathematical modelling has suggested that any protein that binds to the actin network with sufficient strength will be carried rearward by the actin flow resulting in a gradient that may subsequently control cellular behaviour. We hypothesise that moesin is an ideal candidate for such direct regulation by actin flow and we will examine how moesin is controlled by the flow and subsequently feeds back to control the actin network.