Elucidating the mechanism of endocytic invagination and scission

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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.