Cell membranes can be thought of as a composite material comprising proteins and lipids. Many different lipids form the mammalian cell plasma membrane and the overall composition (the lipidome) of the membrane dictates its biophysical properties such as fluidity, rigidity and viscosity. These in turn influence its function such as bending, fusion, signalling and so on. Fine regulation of signalling is especially important in T cells, which must detect and activate in response to foreign pathogens while not reacting to self. However, T cells frequently inhabit hypoxic environments, such as tumour microenvironments. This is problematic because molecular oxygen is required to synthesise certain critical membrane lipids such as unsaturated phospholipids and sterols. Without these, membrane properties, and therefore function, cannot be maintained. The question is therefore, how does hypoxia influence T cell membrane lipidomes, membrane properties and membrane function and how do T cells maintain functionality in hypoxic conditions?
We will begin by culturing T cells conventionally and in hypoxic environments. We will use microscopy and membrane stains to document morphological changes to the cells and use mass-spectrometry to quantify the membrane lipidome. We will develop fluorescent markers for specific lipids (e.g. sterols) and use imaging to quantify and map their distribution in the cells. Then, we will use advanced fluorescence microscopy together with environmentally-sensitive membrane dyes. These allow us to map membrane physical properties, such as viscosity, across the cell. These studies will be performed in conventional/hypoxic resting T cells and at the T cell immunological synapse where T cell receptor (TCR) signalling occurs. We will then map key membrane proteins within the membrane to determine how they are affected by the altered lipidomes. To do this we will again use microscopy including the latest state-of-the art super-resolution imaging to map protein distributions on the nanoscale. To discover causal relationships between hypoxia and lipidomes, we will introduce mutations and use drugs, that affect the lipid synthesis pathways in order to recapitulate the changes we see in hypoxia. Finally, we will examine the effect of altered lipidomes on T cell signalling including signal transduction, gene expression and cell proliferation.
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