Elucidating the molecular mechanism of Arp2/3-independent actin nucleation by WASP family proteins

细胞是生命的基本单位，所有生物体都是由一个或多个细胞组成的。包括人类细胞在内的许多细胞的功能的核心是内部骨架或细胞骨架。这种细胞骨架是细胞具有某些形状所必需的，这些形状通常是其功能的必要部分。然而，不像我们自己的身体骨架是静态的，细胞骨架能够重塑自己以改变细胞形状，或允许细胞移动。细胞骨架中最重要的蛋白质之一叫做肌动蛋白。它是一种令人惊奇的蛋白质，因为它现在几乎和数亿年前一样，早在人类甚至脊椎动物存在之前。随着时间的推移保持如此相似被称为进化保守。对细胞功能非常重要的蛋白质是最高度保守的。肌动蛋白是一种可以与其他肌动蛋白结合在一起形成长线或细丝的蛋白质。这些细丝可以被其他蛋白质组织起来，形成细胞骨架的一部分。我们感兴趣的是肌动蛋白在细胞中是如何被控制的，特别是，我们正试图确定细丝是如何从单个肌动蛋白开始的。这个过程叫做成核。我们的研究将有助于我们了解这种成核的细胞中的机制。我们认为这很重要，因为已知肌动蛋白是细胞运动所必需的，并且这种行为在已经转移的癌细胞中经常发生变化。当病原体侵入我们的细胞时，肌动蛋白也参与其中。WASP是一种已知的蛋白质，它在帮助肌动蛋白形成新的纤维中起着重要的作用，这种蛋白质在一种称为Wiskott Aldrich综合征的免疫缺陷疾病中变得有缺陷。由于肌动蛋白是一种高度保守的蛋白质（酵母和人类之间有85%的相同性），我们已经在酵母中进行了许多研究，以深入了解肌动蛋白功能的基本方面。操纵哺乳动物系统并不总是那么简单，有些实验可能需要数月才能完成。酵母提供了一个更简单的研究系统，我们可以在整个有机体的背景下研究事物，也可以单独分析不同的成分。这种方法的互补性对于深入理解一个过程是重要的。我们还旨在通过对哺乳动物蛋白质和细胞进行知情实验来利用这些发现。在酵母中有许多研究的例子，为更复杂的生物体的过程提供了新的线索。直到最近，人们才认为WASP和其他类似的蛋白质可以激活一组称为Arp 2/3成核复合物或Arp 2/3的蛋白质。我们已经证明，酵母WASP，称为Las 17，能够核肌动蛋白没有Arp 2/3存在。我们还能够证明这种成核活性对于细胞内Las 17的功能很重要。重要的是，我们还表明，两种相关的哺乳动物蛋白质可以在相似的条件下产生细丝，这表明该特性是保守的。我们现在想更多地了解这些蛋白质的肌动蛋白成核机制，因为它可能会支持对细胞特定部位成核的新理解。总的来说，这个项目是高度相关的细胞组织，膜运输和运动的关键细胞过程的理解。虽然专注于酵母WASP同源物，但我们的初步数据表明，主要发现将对WASP家族的许多蛋白质以及其他蛋白质（如在病原体上表达的那些蛋白质）具有广泛意义，这些蛋白质也驱动肌动蛋白丝形成。我们的方法与其他实验室使用的方法高度互补，但又有所不同。我们已经为这项工作开发了许多工具和试剂，这意味着我们可以朝着我们的目标快速前进，并且成果有可能在顶级国际期刊上发表，从而提高英国在科学方面的竞争力。

Cells are the basic unit of life and all organisms are composed of one or more cells. Central to the functioning of many cells, including human cells, is the internal skeleton, or cytoskeleton. This cytoskeleton is required for cells to have certain shapes that are often a necessary part of their functioning. However, unlike our own body skeleton that is static, the cytoskeleton is able to remodel itself to change cell shape, or allow a cell to move. One of the most important proteins in the cytoskeleton is called actin. It is an amazing protein because it is almost the same now as hundreds of millions of years ago, long before humans, or even vertebrates existed. Staying so similar over time is called evolutionary conservation. Proteins that are very important to cell functioning are the most highly conserved. Actin is a protein that can join together with other actin proteins to form long lines or filaments. These filaments can be organised by other proteins to form large structures that are part of the cytoskeleton. We are interested in how actin is controlled in cells and in particular, we are trying to determine how the filaments can be started from single actin proteins. This is a process called nucleation. Our study will help us understand the mechanism of this nucleation in cells. We think that it is important because actin is known to be necessary for cell movement, and this behaviour often changes in cancer cells that have become metastatic. Actin is also involved when pathogens invade our cells. One of the proteins known to be important in helping actin form new filaments is called WASP, which becomes defective in an immune-deficiency disorder called Wiskott Aldrich Syndrome. Because actin is a highly conserved protein (85% identical between yeast and humans), we have undertaken many of our studies in yeast to gain insight into fundamental aspects of actin function. Manipulating mammalian systems is not always straightforward and some experiments can take months to perform. Yeast provides a more simple system to investigate, and we can study things within the context of the whole organism as well as analysing different components individually. This complementarity of approaches is important to gain a deep understanding of a process. We also aim to exploit finding by undertaking informed experiments on mammalian proteins and cells. There are many examples of studies in yeast that have shed new light on processes in more complex organisms.Until recently it was considered that WASP and other proteins like it, work to activate a group of proteins called the Arp2/3 nucleation complex or Arp2/3. We have shown that the yeast WASP, called Las17, is able to nucleate actin without Arp2/3 present. We were also able to show that this nucleation activity is important for the function of Las17 inside cells. Importantly, we have also shown that two related mammalian proteins can generate filaments in similar conditions, suggesting that the property is conserved. We now want to learn more about the mechanism of actin nucleation by these proteins as it may underpin a new understanding of nucleation at specific sites in cells. Overall, this project is highly relevant to our understanding of key cell processes of cell organization, membrane trafficking and motility. While focused on the yeast WASP homologue our preliminary data indicates that the major findings will be of wide significance for many proteins both of the WASP family and also other proteins such as those expressed on pathogens that also drive actin filament formation. Our approach is highly complementary to, but distinct from, those used in other labs. We have already generated many tools and reagents for this work which means that we can make rapid progress towards our goals, and the outputs have the potential to be published in top international journals thus enhancing UK competitiveness in science.