High-resolution imaging of mammalian cells by confocal microscopy

In animals, cells need to move during development and in response to infection or tissue damage. Cell movement is driven by the cytoskeleton, principally by filaments made of actin molecules, and also requires changes in the way cells attach to other cells or to molecules around them. We want to understand more about how proteins inside cells drive or inhibit cell movement, and therefore be able to more effectively use these proteins as targets for drug therapy against several diseases including chronic inflammatory disorders, heart disease, Alzheimer's and some cancers.

The control of transmembrane proteins and associated cytoskeletal architecture at the plasma membrane is crucial to the regulation of numerous cellular functions including cell adhesion to the extracellular matrix, cell-cell adhesion in epithelia and endothelia, transendothelial migration of leukocytes, synaptogenesis and axon guidance. The spatial and temporal regulation of these events is tightly co-ordinated and can only be accurately studied using microscopy approaches, ideally in living specimens. Laser-scanning confocal microscopy allows the targeted analysis of events occurring at specific focal planes in the cell. This enables precise movements of molecules to be studied both spatially and temporally. Recent advances in technology have resulted in the development of laser scanning systems, that allow functional assays such as photo activation, FRAP (Fluorescence Recovery After Photobleaching), FRET and FLAP (Fluorescence Localisation After Photobleaching) using fluorophore-tagged proteins to analyse the dynamics of protein interactions and movement within cells. This application proposes the purchase of a state-of-the-art four-laser line confocal microscope to allow tracking of proteins in 3 dimensions over time in a living cell. We aim to employ this system to study a range of diverse membrane receptors and cytoskeletal proteins and their relative dynamics during cell adhesion, migration, synaptogenesis in brain slices and axon guidance of neuronal growth cones.