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

'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.

Endocytosis is a highly regulated and essential process in most eukaryotic cells. It is required for recycling of lipids and trafficking proteins, and for uptake or down-regulation of cell-surface receptors. During endocytosis the plasma membrane invaginates into the cell resulting in the production of a vesicle that then fuses with endosomes and enters the endolysosomal membrane system. This process involves at least 30 proteins that assemble transiently at sites on the plasma membrane. Work in the model organism S. cerevisiae has led to significant advances in our understanding of the distinct stages that take place during endocytosis in vivo. It is now widely believed that the broad stages of coat assembly (early), invagination (mid) and scission/inward movement (late) are conserved across evolution, and that in many cases direct homologues of proteins are responsible for carrying out equivalent steps in the process. A notable difference between yeast and vertebrate cells is that dynamins are considered central to the endocytic process in vertebrates while these proteins have been considered peripheral to endocytic function in yeast. We believe that the role for dynamins in yeast endocytosis has been underestimated due to their involvement in other processes in the cell. In our preliminary data we use electron microscopy to analyse invaginations that form at the plasma membrane. We observe very marked differences in these structures when a single yeast dynamin is deleted from cells. We also observe differences in the dynamic behaviour of other endocytosis-associated proteins in live cells. This gives the first unequivocal evidence for a function for the yeast dynamin Vps1 in endocytosis. We now aim to extend these studies using complementary live cell and ultrastructual electron microscopy approaches to address long-standing questions in the endocytic field concerning the role of dynamins and their interplay with another family of proteins the amphiphysins.