Regulation and mechanism of clathrin coated vesicle formation

Cells continuously need to internalise material from the extracellular environment, to deliver material to that environment and to move material between the membrane-bound subcompartments inside the cell. Broadly the way that they do this is by packaging material into specialised areas of one membrane (the ?donor?). These specialised areas often have a protein ?coat? and they become increasingly curved or ?invaginated? and cargo molecules become concentrated in these invaginations. Eventually they pinch off from the donor membrane, forming small spherical structures called coated vesicles. The vesicle is then sent to a target ?acceptor? membrane where it fuses and delivers its contents. The whole process of forming coated vesicles is highly controlled to ensure that the selection of cargo and direction of transport is properly maintained.
Our lab has focussed its efforts on the formation of coated vesicles at the cell surface. A molecule, clathrin, is the major protein component of these coats, giving the vesicles their name. However many other molecules are also involved in forming the coat and selecting the cargo that will be included in the vesicle. Our lab uses a variety of approaches to understand the molecules involved in forming coated vesicles. In particular we use assays that allow us to reconstitute coated vesicle formation in the test-tube. These assays allow us to ask which molecules are required and how their assembly into the coat is regulated. The aim of this proposal is to understand more fully how clathrin coated vesicles are formed.
Clathrin coatedvesicles are responsible for the internalisation of many different molecules including nutrients, growth factors and hormones. They are also often hijacked by pathogens such as bacteria and viruses to gain entry into the cell. They thus play very important roles in the response of cells to their environment. If we can understand the molecular basis of this fundamental process and how it is modified depending on where a cell is located, we hope in the longer term to apply this knowledge to understanding defects in this process that contribute to disease.

Clathrin coated pits and coated vesicles are the major ports of entry for a huge variety of cargo, including pathogens, and they represent the paradigm for membrane-based sorting events in higher cells. Clathrin and the AP2 adaptor complex are the major components of the coat but, by a variety of approaches, a host of other proteins has been implicated in the clathrin coated vesicle cycle. The challenge for the future lies in understanding how the interactions of all of these components are regulated in space and time to efficiently sequester and internalise cargo. The aim of this proposal is to elucidate the mechanism of coated vesicle formation. Using a multi-disciplinary approach, including reconstitution assays in permeabilised cells, intact cell assays, protein chemistry and light and electron microscopy, we aim to elucidate the molecular mechanism of coated vesicle formation. We will build on our previous findings and focus in the first instance on the role of rab5 at the plasma membrane, the regulation of cargo/adaptor interactions, the temporal requirements for individual coated pit components and the regulation of these interactions by phosphorylation.