Organisation of actin waves and cups by differential GTPase activity

构成所有有机体的细胞都是高度动态的，经常必须改变形状才能发挥它们的功能。这是通过细胞骨架实现的，在细胞骨架中，一种名为肌动蛋白的蛋白质的单个分子可以组装(聚合)成细丝，以产生力并将细胞表面向外推。我们感兴趣的是调控肌动蛋白何时何地聚合以产生具有特定功能的突起的机制。我们尤其感兴趣的是一个被称为大吞噬作用的过程，在这个过程中，细胞使用肌动蛋白延伸杯状结构，可以捕获并内化环境中的大量液体。细胞需要这样做的原因有很多。最基本的是进食，获取营养以维持生长，这是阿米巴等简单生物以及癌细胞都使用的。在高等动物中，大吞饮作用也被免疫细胞用来采样环境和检测异物，病毒和细菌也可以利用大吞饮作用作为进入宿主细胞的一种方式。值得注意的是，大吞饮所需的杯状突起自发地发生在细胞表面，并在没有任何外部空间信号或模板的情况下自组装。如何实现这一点是一个主要的悬而未决的问题，也是这项提议的主要焦点。细胞的底部也可以形成类似的结构。然而，由于它们受到下面表面的物理限制，它们以平坦波的形式传播，直到最终与细胞外围相撞。在那里，它们会产生一个突起，驱动细胞迁移。巨饮细胞杯和这些基波具有相似的结构和动力学，因此我们认为它们是由相同的机制形成的。通过并行研究这两种结构，我们将确定决定肌动蛋白如何被迫在特定位置聚合以形成特定结构的一般原理。以前的工作表明，CUPS的形成是通过在细胞表面产生一块特定的磷脂(PIP3)，该斑块能够将肌动蛋白聚合到其外围。这产生了一个突出环，提供了一种挤压杯形的机构。在2D中，在基波中观察到相同的组织。肌动蛋白对PIP3结构域边缘的限制是形成这两种结构的基础，但这是如何实现的完全未知。确定这一机制是我们的主要目标。我们之前的工作导致我们提出了一个新的模型来产生肌动蛋白聚合环，基于两个不同调控蛋白的相对活性：Rac是肌动蛋白聚合的激活剂，而RAS导致抑制。我们观察到，虽然RAS和RAC的活性都与细胞中观察到的PIP3结构域一致，但RAC单独的活性略有延伸。我们的主要假设是，只有RAC活跃的这个外围环定义了肌动蛋白聚合的位置，并产生杯状和基波。我们将测试这一模型，并确定RAS和/或PIP3抑制杯状中心肌动蛋白聚合的机制。要实现这一点，我们需要利用显微镜的最新进展。这将使我们能够第一次在3D中研究这些快速移动和高度动态的结构。这项新技术还需要新的分析方法，因此该项目的一个重要部分是开发新的计算工具，这些工具将对科学界普遍使用，并为杯子的形成提供重要的新见解。结合起来，这项工作将为突出物如何组织提供新的一般性见解，与巨噬细胞吞噬和细胞迁移特别相关。

The cells that make up all organisms are highly dynamic, and often must change shape to perform their function. This is achieved through their cytoskeleton, where individual molecules of a protein called actin can be assembled (polymerised) into filaments to generate force and push the cell surface outwards. We are interested in the mechanisms that regulate where and when actin is polymerised to produce protrusions with specific functions.In particular, we are interested in a process known as macropinocytosis, where cells use actin to extend cup-shaped structures that can capture and internalise large volumes of fluid from their environment. Cells need to do this for many reasons. The most fundamental is to feed, capturing nutrients to sustain growth, which is used by both simple organisms such as amoebae, as well as cancer cells. In higher animals, macropinocytosis has also been adapted by immune cells to sample their environment and detect foreign bodies and both viruses and bacteria can also exploit macropinocytosis as a way into host cells.Remarkably, the cup-shaped protrusions required for macropinocytosis occur spontaneously on the cell surface and self-assemble without any external spatial signals or template. How this is achieved is a major unanswered question and the main focus of this proposal.Similar structures can also form on the bottom surface of the cell. However, because they are physically restricted by the surface below, they propagate as flat waves until they eventually collide with the cell periphery. There, they generate a protrusion that drive cell migration. Both macropinocytic cups and these basal waves have comparable structure and dynamics, so we propose they form by the same mechanisms. By studying both structures in parallel, we will identify the general principles that dictate how actin is coerced to polymerise in specific locations to make specific structures. Previous work has suggested that cups form by generating a patch of a specific phospholipid (PIP3) on the cell surface that is able to direct actin polymerisation to its periphery. This generates a ring of protrusion, providing a mechanism to extrude a cup shape. An identical organisation in 2D is observed in basal waves. Restriction of actin to the edge of the PIP3 domain is fundamental to form both structures, but how this is achieved is completely unknown. Identifying this mechanism is our main objective.Our previous work led us to propose a new model to generate a ring of actin polymerisation, based upon the relative activities of two different regulatory proteins: Rac, which is an activator of actin polymerisation, and Ras which we propose leads to inhibition. We observed that whilst both Ras and Rac activities coincide with the PIP3 domains observed in cells, Rac alone extends slightly further. Our main hypothesis is that this peripheral ring where only Rac is active defines where actin polymerises and generates the cup shape and basal waves. We will test this model and determine the mechanisms by which Ras and/or PIP3 inhibit actin polymerisation in the centre of the cup.To achieve this, we need to take advantage of the latest advances in microscopy. This will allow us to study these rapidly moving and highly dynamic structures in 3D for the first time. This new technology also requires new analytical methods, so an important part of this project is to develop novel computational tools that will be of general use to the scientific community as well as providing important new insights into how cups form.Combined, this work will provide new general insights into how protrusions are organised with specific relevance to macropinocytosis and cell migration.