Physical Sciences of Imaging in the Biomedical Sciences CDT
Thesis project - "Single molecule imaging of the myelin proteins"
Dr Daniel Fulton, Institute of Inflammation and Ageing
Dr Rob Neely, School of Chemistry
Dr Iain Styles, School of Computer Science
This project aims at elucidating the organisation and molecular dynamics of proteins within the myelin sheath using single molecule imaging. The myelin sheath is a fatty cover used in the nervous system to insulate the nerve fibres, allowing faster, and more efficient signalling. Conditions where the myelin sheath improperly develops, or is destroyed, causes neurological impairments that impact on functions such as motor control, vision, thought, and regulation of bodily functions. Information on this sheath has previously mainly been derived from static electron microscope images, which lack information on the dynamics and temporal organisation of events which occur within it. This project intends to use single molecule imaging to expand on the knowledge of the field, by tracking individual molecules to allow information on the spread of behaviour within the system to be derived, rather than looking at the bulk behaviour within the system. This will allow information on aspects of myelin behaviour such as protein trafficking, and changes in composition of the membrane to be identified, whilst the cell is in its natural environment. The dynamic behaviours of these proteins can also be expounded to allow us to understand how the biophysical behaviours of these proteins change during processes such as repair, maintenance and formation of the sheath. This shall be achieved using the aid of genetically-encoded fusions of the myelin proteins to fluorescent proteins. We will produce novel photoswitchable and photoconvertible fluorescent protein fusions that will allow us to explore different aspects of the myelin at the single-molecule level and using super-resolution microscopy.
We will carry out a range of studies on the fusion protein fusions we produce as control experiments and in order to study their diffusional properties within a model lipid bilayer. These tests will be carried out in order to establish the optimal wavelength for fluorescence imaging, to examine photostability of the fluorescent proteins in our imaging set-up, to test the photoswitchable nature of the proteins produced, and to allow us to study the oligomerisation of the protein fusions in an adaptable model lipid bilayer. This model will be produced using the Langmuir Blodgett technique to produce the lipid bilayer within which we can seed a known concentration of selected myelin proteins. Following this the physical characteristics of these fluorescent proteins will be compared with that of standard organic fluorophores in order to determine whether or not the fluorescent protein fusions impact on the physical behaviour of the myelin proteins. .
Using the information derived from the studies within this model system, computational analysis procedures shall be developed to help analyse the behaviour of the system and gain valuable information for understanding the molecular dynamics of the proteins. This will involve developing on cell tracking and molecular identification algorithms, which would account for the interactions which occur within the system such as oligomerisation, and diffusion of the various molecules within the system. This will be developed on further when looking at the behaviours of these proteins within actual brain tissue, as the system contains other proteins, such as MHC proteins used in the immune system, and the various basic metabolic ions channels, which will have to be ignored in the analysis of the data, as well as including mechanisms to compensate for the increased complexity of trafficking dynamics present in actual brain tissue. The acquisition of information on the molecular dynamics of the various individual proteins would allow comparison of the biophysical properties of the myelin proteins in the model system compared to the real brain situation, which would allow us to further understand the various chemical interplays which are involved in myelination.