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

'内吞内陷和囊泡分裂-酵母中动力蛋白同系物和两性蛋白之间的相互作用' Ayscough和Goldberg总结（4000字符）内吞作用是大多数真核细胞中的一个重要过程。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。我们对两类蛋白质的作用特别感兴趣--发动蛋白和两性蛋白。这些蛋白质被认为参与胞吞作用，但它们发挥作用的确切步骤一直难以阐明。其原因是，已经用纯化的蛋白质对相关哺乳动物蛋白质进行了大量工作。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情况来研究，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，我们还使用电子显微镜。这允许对内吞作用的关键阶段进行更详细的分析，并使我们能够在2D横截面中或通过使用先进的方法在3D中看到内陷的实际形状和所涉及的蛋白质的位置。特别是，我们可以确定基因缺失或突变对膜弯曲和囊泡断裂过程的影响，其确定性比光学显微镜更高。然而，由于EM必须固定样品，因此它只能为我们提供过程的“快照”，而光学显微镜通过允许我们在过程发生时查看过程来补充这一点。我们的方法将为蛋白质在分子水平上的功能提供新的见解。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系统中的方法提供信息。

'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.