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Elucidating the mechanism of endocytic invagination and scission

阐明内吞内陷和分裂的机制

基本信息

批准号：
BB/K002511/1
负责人：
Kathryn Ayscough
金额：
$ 87.87万
依托单位：
University of Sheffield
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2013
资助国家：
英国
起止时间：
2013 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FK002511%2F1
关键词：
Elucidating mechanism endocytic invagination scission

项目摘要

Cells are the basic unit of life and all organisms are composed of one or more cells. Cells need to interact with their environment to ensure that they respond correctly to signals that come from their surroundings. The majority of this interaction takes place through the proteins that lie on its surface. Endocytosis is an essential process in most eukaryotic cells. It involves a small amount of the outer (plasma) membrane of the cell being pulled inwards into the cell until some of this membrane pinches off to form a little sphere called a vesicle. This vesicle will contain fluid from outside the cell and, within its membrane it will contain proteins that were on the surface. A cell may want to remove these proteins from the surface because they are damaged, or because they can bind or respond to signals from outside that the cell no longer wants, or needs to respond to. Endocytosis is a very important way for a cell to control what is on its surface. Some pathogens or toxins can bind to proteins on the cell surface and trigger endocytosis. In this way these inappropriate substances can gain entry to the cell. Defects in the endocytic process have also been detected early in some neurological disorders such as Alzheimers. Research in the Ayscough laboratory uses a simple one-celled organism Saccharomyces cerevisiae (bakers yeast) as a model system. Many processes are known to happen in the same way in this cell-type and in cells of more complex organisms such as mammals. We are particularly interested in the interplay between three types of protein that we, and others, have shown are critical in the inward movement of the membrane and its pinching off (scission) to form a vesicle. These proteins are called, dynamins, amphiphysins and actin. They are proposed to be involved in endocytosis but the exact step at which they function has been difficult to elucidate. One reason for this, is that much work on the relevant mammalian proteins has been performed only with purified proteins. It is not always easy to then translate this data into a physiological context. Manipulating the various mammalian systems has not always been straightforward and some experiments can take months to perform. Yeast provides a more simple situation to investigate, and we can study things within the context of the whole organism. We use imaging of fluorescently tagged proteins to investigate how the proteins of interest move in the cell. We can determine when the proteins localise to sites of endocytosis and how long they stay there. This imaging needs to be very sensitive as the endocytic sites are only fractions of a micron in size. Furthermore, the actual membrane invagination and scission events occur on a seconds timescale. Using yeast we can readily investigate the effect of changing just single amino acids within the dynamin or amphiphysin proteins. As well as using live cell imaging we are trying to generate synthetic systems, using pure proteins to reproduce the events that we have studied in the cells. Understanding how to manipulate membranes might be important in the future to generate functioning synthetic cells. Our approach will give new insights into how the proteins work at the molecular level. In turn, this will inform approaches in other, more complex systems studying these proteins in the context of both healthy and diseased cell types.

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。细胞需要与环境相互作用，以确保它们对来自周围环境的信号做出正确的反应。这种相互作用的大部分通过位于其表面的蛋白质发生。内吞作用是大多数真核细胞的重要过程。它涉及到细胞的少量外（质膜）被向内拉入细胞，直到一些膜被夹断，形成一个称为囊泡的小球。这个囊泡将包含来自细胞外的液体，在其膜内，它将包含表面上的蛋白质。细胞可能想要从表面去除这些蛋白质，因为它们被损坏，或者因为它们可以结合或响应细胞不再想要或需要响应的外部信号。内吞作用是细胞控制其表面物质的一种非常重要的方式。一些病原体或毒素可以与细胞表面的蛋白质结合并触发内吞作用。通过这种方式，这些不适当的物质可以进入细胞。内吞过程中的缺陷也在一些神经系统疾病如阿尔茨海默氏症中早期发现。Ayscough实验室的研究使用简单的单细胞生物酿酒酵母（面包酵母）作为模型系统。已知许多过程以相同的方式发生在这种细胞类型和更复杂的生物体（如哺乳动物）的细胞中。我们特别感兴趣的是三种类型的蛋白质之间的相互作用，我们和其他人已经证明，这三种蛋白质在膜的向内运动和它的夹断（切断）形成囊泡中是至关重要的。这些蛋白质被称为发动蛋白、两性蛋白和肌动蛋白。它们被认为参与内吞作用，但它们发挥作用的确切步骤一直难以阐明。其中一个原因是，有关哺乳动物蛋白质的许多工作仅用纯化的蛋白质进行。然后将这些数据转化为生理背景并不总是容易的。操纵各种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环境，我们可以在整个有机体的背景下研究事物。我们使用荧光标记蛋白质的成像来研究感兴趣的蛋白质如何在细胞中移动。我们可以确定蛋白质何时定位于内吞作用的位点以及它们在那里停留多久。这种成像需要非常灵敏，因为内吞位点的尺寸只有几分之一微米。此外，实际的膜内陷和断裂事件发生在秒的时间尺度上。使用酵母，我们可以很容易地研究改变发动蛋白或两性蛋白质中的单个氨基酸的影响。除了使用活细胞成像，我们还试图产生合成系统，使用纯蛋白质来重现我们在细胞中研究的事件。了解如何操纵膜在未来产生功能性合成细胞可能很重要。我们的方法将为蛋白质如何在分子水平上工作提供新的见解。反过来，这将为在健康和患病细胞类型的背景下研究这些蛋白质的其他更复杂系统中的方法提供信息。