Endocytosis is a fundamental cellular process which facilitates the uptake of lipids and proteins from the plasma membrane. During endocytosis, the plasma membrane is internalised through the formation of an invagination, which is then pinched off to form a vesicle. Endocytosed vesicles are then able to fuse with endosomal compartments allowing cargo to be sorted within the endo-lysosomal system. One of the most well characterised forms of endocytosis is clathrin mediated, which takes place in distinct stages. These include coat and clathrin recruitment, membrane invagination and finally scission which releases the vesicle into the cytoplasm. Cells require many different molecules to orchestrate each stage including the GTPase dynamin and cytoskeletal protein actin. Dynamin facilitates the scission stage of endocytosis where it oligomerises around the vesicle neck and, through the hydrolysis of GTP, enables scission. Furthermore dynamin-1 has been reported to bind directly to actin. In Saccharomyces cerevisiae (yeast) actin is essential to endocytic invagination in order to overcome turgor pressure. Yeast contain a dynamin-like protein Vps1 which acts during endocytic scission and has orthology with dynamin-1. In this thesis the direct interaction between Vps1 and actin was explored to elucidate how this interaction may be required during endocytosis. It is understood that both dynamin and actin are involved in other cellular processes and therefore the knowledge gained from investigating this interaction during endocytosis may well provide new insight into how these two molecules work together in other molecular systems. Thus, mutations found to perturb the Vps1-actin interaction were created in human dynamin-1 and preliminary results suggest this could have an effect on mammalian cell endocytosis and cell migration.
This project has identified a novel Vps1-actin interaction which is required for the scission stage of yeast endocytosis. It also describes a point mutation in Vps1 E461K, which has been found to cause an early stage endocytic defect
