Endocytic invagination and vesicle scission - interplay between dynamin homologues and amphiphysins in budding yeast

Ayscough和Goldberg摘要(4000字)内吞作用是大多数真核细胞中的一个基本过程。它涉及到细胞的少量外膜(质膜)被拉入细胞内，直到这些膜的一部分被掐断，形成一个称为囊泡的小球体。这个囊泡将包含来自细胞外的液体，在它的膜内将包含表面上的蛋白质。细胞可能想要从表面移除这些蛋白质，因为它们受到了破坏，或者因为它们可以结合或响应来自外部的信号，而细胞不再想要或需要对此做出反应。内吞作用是细胞控制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，引发内吞作用。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中被早期发现，例如阿尔茨海默氏症。艾斯考实验室的研究使用了一种简单的单细胞生物酿酒酵母(面包师酵母)作为模型系统。众所周知，在这种细胞类型和哺乳动物等更复杂的生物的细胞中，许多过程都是以相同的方式发生的。我们特别感兴趣的是两类蛋白质的作用--动力素和两栖生物素。这些蛋白质被认为与内吞作用有关，但它们的确切作用步骤一直很难阐明。这样做的原因是，许多关于相关哺乳动物蛋白质的工作都是用纯化的蛋白质进行的。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一帆风顺的，一些实验可能需要几个月的时间才能完成。酵母菌提供了一种更简单的研究情况，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到内吞作用部位，以及它们在那里停留多长时间。这种成像需要非常敏感，因为内吞部位的大小只有微米的一小部分。此外，实际的膜内陷和断裂事件发生在几秒钟的时间尺度上。使用酵母，我们可以很容易地研究只改变动力蛋白或两栖动物蛋白中的单一氨基酸的效果。除了使用活细胞成像，我们还使用电子显微镜。这使得我们能够更详细地分析内吞作用的关键阶段，并使我们能够看到内陷的实际形状和所涉及的蛋白质的位置，无论是在2D横截面上，还是通过使用先进的方法，在3D上。特别是，我们可以比光学显微镜更确定地确定基因缺失或突变对膜弯曲和囊泡破裂过程的影响。然而，由于EM的样品必须是固定的，所以它只能给我们一个过程的‘快照’，而光学显微镜则通过允许我们观察过程的发生来补充这一点。我们的方法将在分子水平上为蛋白质的功能提供新的见解。反过来，这将为在健康和疾病细胞类型的背景下研究这些蛋白质的其他系统的方法提供信息。

'Endocytic Invagination and Vesicle Scission - interplay between dynamin homologues and amphiphysins in yeast' Ayscough and Goldberg Summary (4000 characters) 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 role of two classes of proteins - the dynamins and the amphiphysins. These proteins are proposed to be involved in endocytosis but the exact step at which they function has been difficult to elucidate. The reason for this, is that much work on the relevant mammalian proteins has been performed 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. The 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 use electron microscopy. This allows a much more detailed analysis of the key stages of endocytosis and enables us to see the actual shape of the invaginations and the locations of the proteins involved, either in 2D cross sections, or, by using advanced methods, in 3D. In particular, we can determine the impact of gene deletions or mutations on the proceses of membrane curvature and vesicle scission with much more certainty than is possible with light microscopy. However, because samples have to be fixed for EM, it only gives us a 'snapshot' of the process, whereas light microscopy complements this by allowing us to view processes as they happen. Our approach will give new insights into the functioning of the proteins at the molecular level. In turn this will inform approaches in other systems studying these proteins in the context of both healthy and diseased cell types.