The role of CD317/tetherin in the organisation of membrane microdomains

Our bodies are composed of many, many millions of cells working together in a highly co-ordinated manner. However, cells are not simply building blocks that are linked together to create an organism; each cell comprises a dynamic network of interacting macromolecules. A barrier (the plasma membrane) encloses the contents of the cell and allows a host of processes to occur within a confined, and regulated, environment. A major component of the plasma membrane is a lipid bilayer composed of a cocktail of different lipid (fat) molecules. The plasma membrane is not, however, just lipid molecules. It has been estimated that over one third of all the proteins encoded by the human genome are membrane proteins, that is proteins associated with lipid bilayers. Many of the proteins associated with the plasma membrane actually span that membrane (integral membrane proteins), with part of their sequence inside the cell and part outside: these two parts are linked by a transmembrane section that spans the lipid bilayer. Such integral membrane proteins can be considered to float in the sea of phospholipids of the plasma membrane. They are important molecules since they link the cell with its local environment; many are transporters that move small molecules into or out of the cell, others serve to anchor the cell to other cells or to the extracellular matrix, others bind small molecules such as growth factors or hormones at the outside of the cell and transmit signals into the interior of cell. Many different types of lipid molecule are present in the plasma membrane and they appear to partition into small, highly dynamic entities termed 'lipid rafts' or 'membrane microdomains'; thus a 'lipid raft' can be considered to be floating in the sea of phospholipids too. Lipid rafts have been implicated as being important in a broad range of fundamental and essential cellular functions. These include membrane trafficking, by helping to segregate proteins for delivery to specific locations; cell signalling, by providing platforms for the transient assembly of signalling complexes; regulated exocytosis (i.e. release of molecules from the cell), by forming sites in the plasma membrane for this to occur; pathogen entry/egress (including flu and HIV) and toxin entry, by providing sites in the plasma membrane where these events take place. In addition, lipid rafts have been implicated as playing a role in several human diseases, e.g. Alzheimer's, Parkinson's, Prion, Cardiovascular and Autoimmune diseases. Thus lipid rafts are important in health and disease. It has been proposed that certain integral membrane proteins span the lipid bilayer at the periphery of lipid rafts and are linked to the actin cytoskeleton within the cell (the actin cytoskeleton provides the architectural framework, essentially the scaffolding, within the cell). These proteins are suggested to form a 'picket fence' around the edge of lipid rafts, limiting the diffusion of molecules into and out of the rafts, and thereby serving to regulate the various functions listed above. We have shown that the integral membrane protein CD317/tetherin is localised to lipid rafts and can also be attached to the actin cytoskeleton. This protein has been associated with number of diverse disease-related states (e.g. Multiple Myeloma, HIV release from cells, metastasis, drug-resistance of tumour) and a range of cellular functions (several involved with the immune response). We hypothesise that CD317/tetherin acts as a 'tethered picket fence' serving to organise lipid rafts in the plasma membrane and therefore influence the broad range of cellular functions and disease-related states that have been associated with lipid rafts. We intend to use a range of techniques to address this hypothesis. Furthering our understanding of lipid raft organisation will therefore shed light on multiple fundamental cellular processes, several of which are related to health and disease.

Lipid rafts/membrane rafts/membrane microdomains (rafts) have been defined as small (10-200nm diameter), heterogenous, highly dynamic, sterol- and sphingolipid enriched domains that compartmentalise cellular processes. It has been proposed that certain integral membrane proteins span the lipid bilayer at the periphery of rafts and are linked with the underlying actin cytoskeleton to form a 'picket fence' around the edge of the raft and limit diffusion of molecules into and out of the raft. Regulation of movement of molecules into/out of rafts is likely to be important for cellular function since they have been implicated in a broad range of fundamental and essential cellular functions (e.g. signaling, trafficking, pathogen entry/egress). In addition, rafts have been implicated in several human diseases, e.g. Alzheimer's, Parkinson's, Prion, Cardiovascular and Autoimmune diseases. Furthering our understanding of lipid raft organisation will therefore shed light on multiple fundamental cellular processes, several of which are related to health and disease. We have shown that the protein CD317/tetherin is localised to rafts and is linked to the actin cytoskeleton. It has been shown to play a role in limiting the release of HIV particles from infected cells and its elevated expression has been associated with diverse cellular phenotypes (e.g. metastasis, NFkB activation and Multiple Myeloma). We hypothesise that CD317 plays a role in organising rafts and that this explains how changes in its level of expression can have diverse cellular effects. We will use cells (in which expression of endogenous CD317 has been knocked down) expressing a range of mutated CD317 constructs and relevant reporter molecules in combination with live cell imaging techniques (FRAP, FLIP, SPT) and EM studies to address the role of CD317 in raft organisation. These imaging studies will be complemented by biochemical studies using NFkB activation as a read-out of CD317 function.