High pressure freezing to capture secretory pathway dynamics

The grant application is two fold. Firstly the proposal is for funding to continue supporting the running of a high-pressure freezing facility for the plant science community. This is BBSRC funded equipment that permits the very rapid preservation of biological material for electron microscopy by freezing under high pressure. This means that artefacts caused by chemical preservation are avoided and also ice crystal damage to tissue is avoided. A number of scientists are currently using the facility and need to continue to do so in order to complete their research programmes. Secondly, funding is requested to support the electron microscopy which is required to support the Oxford Brookes plant cell biology research into the functioning of the sub-cellular system (the secretory pathway) involved in carbohydrate production and protein processing. A new technique, electron tomography will be used to work out the three-dimensional organisation of the cell compartments involved in these processes. At the same time material preserved by high/pressure freezing will be used in combination with antibodies as stains, in order to locate, within the cell specific proteins that are involved in the organisation and functioning of the structures within the secretory pathway.

This proposal has two main objectives. Firstly funding is requested to continue running the high-pressure freezing facility at Oxford Brookes to service (principally) the plant science community. The equipment was funded by a previous BBSRC grant and a number of BBSRC funded research groups are currently using the facility or have expressed a requirement for the facility in the near future. These include Dr I Moore, location of plant Rab proteins; Dr L. Frigerio, Golgi structure and protein targeting in castor beans; Prof. H. Dickinson, radial microtubule systems in meiocytes; Prof. P. Shewry, gluten distribution in wheat endosperm; Dr D. Logan, location of proteins involved in mitochondrial distribution. Secondly, funding is requested to support the immunocytochemical and ultrastructural studies required to underpin the plant endomembrane research programmes at Oxford Brookes, most of which are BBSRC funded. Specifically high pressure freezing and freeze-substitution will be used in combination with electron tomography to analyse the structural nature of the ER/Golgi interface in a range of plant material exhibiting different cargo export programmes. This is in order to confirm our hypothesis that in plant cells, ER export sites and the Golgi exist as a unique, mobile ER associated secretory unit. The unique ability to combine good structural preservation and well preserved antigenicity though rapid freezing technology will also be used in the project to immunolocate a wide range of plant Golgi associated proteins. These include a range of matrix proteins putatively involved in vesicle tethering and cisternal organisation, plus a range of SNARE proteins, which mediate membrane fusion processes.