Application of automated high-content live-cell fluorescence imaging in multidisciplinary studies of epithelial neuronal and stem cell function

We request a microscope system that is capable of detecting cell fluorescence and has the capacity to analyse in a single experiment many different samples with measurement of many different functions relevant to a wide variety of cell types. Project 1: Two-way interactions between the cells that line hollow organs (gut, airways etc) and underlining cells determine the organisation of the organ. They are especially important in development, wound healing and the progression to cancer. Our experimental plan is based on the idea that responses of the lining (ie epithelial) cells to infection or damage activate sub-epithelial cells that in turn liberate growth factors which in the short term restore tissue organisation, but chronically lead to disorganised states with increased cell proliferation. We will study communication between different subsets of cells obtained from human stomach. By understanding the interactions between these cells we will be able to develop new approaches to therapeutically controlling abnormal responses to organ damage, injury or infection. Projects 2 & 3: Study of the control of neuronal function is important for an understanding of the basic function of the nervous system and defects in these processes contribute to human diseases including epilepsy. Signalling between nerve cells (ie neurotransmission) and the control of nerve cell excitability are crucial for brain activity. Neurotransmission occurs by the process of exocytosis in which small membrane bound vesicles are triggered to fuse with the cell membrane to allow their contents to be released. Similar vesicles are also used to move newly synthesis proteins through the cell to reach the cell surface. Both of these processes are tightly controlled. We intend to explore the key enzymes that play roles in the regulation of these two processes. Project 4: Food intake is controlled by signals from the digestive tract some of which are carried by specific nerve cells known as vagal afferent neurons. These respond to a variety of hormones, but their sensitivity appears to be controlled by prior ingestion of nutrients which in turn may be due to changes in the production of different signalling molecules. Using previous studies as a guide we want to explore the relevant mechanisms by studying how molecules that control gene expression change their position within cells in different circumstances known to be involved stimulation or inhibition of food intake. Project 5: Stem cells develop to provide the mature cells specific to different tissues and organs during development, and are also necessary for the replacement of mature cells in the adult to substitute for those lost by normal turnover or damage. It has recently become apparent that many cancers arise by the uncontrolled proliferation of stem cells. This project will investigate how stem cell behaviour is controlled by interactions with their environment, thereby providing us with a better understanding of some of the basic processes of development, and giving us information that will help in designing new therapies to treat cancer. Project 6: The response of the brain to damage involves many proteins that work together to salvage and protect brain function. We work on two such proteins, whose actions are reflected in their names, Neuron Restrictive Silence Factor (NRSF) and Activity Dependent Neuroprotective Protein (ADNP). The latter is now attracting interest because a short synthetic peptide derived from it, has been shown to have remarkable properties in a variety of neurological conditions ranging from head trauma to Alzheimer's disease. We believe that both these proteins may have key roles in our body, both in normal cognitive function and in our response to damage to the brain. Our aims therefore are to determine properties of both ADNP and NRSF in neuroprotection by mimicking 'at the bench' how they might act in stroke or epilepsy.

The equipment requested will be used in the first instance for the specific projects outlined below. Project 1: We will study the interactions between primary human epithelial and mesenchymal cells relevant to the maintenance of the proliferating cell niche using cells expressing fluorescent tags and focussing on novel intercellular signalling pathways emerging from our ongoing proteomic studies. Projects 2 & 3: Protein kinases modulate exocytosis in many neuronal and non-neuronal cells. The Kv4 K+ channels play crucial roles in controlling the excitability of neurons and considerable attention has been given to the control of their cell surface expression. We will modify our established assays to determine trafficking of Kv4 channels to the cell surface in HeLa cells using a high-content image-based screen with a commercially available oligo library for all of the 726 human protein kinases to determine which protein kinases modify exocytosis or Kv4 traffic. Project 4: Nuclear translocation of two key transcription factors (NF?B and STAT3), and plasma membrane translocation of PKC, will be studied in primary vagal afferent neurons using fluorescent protein tagged molecules in order to determine mechanisms underlying nutrient- regulated gene expression in these cells. Project 5: We will investigate the ability of synthetic culture substrates to control the degree of ES cell spreading thereby testing the hypotheses that ES cell shape is a crucial factor in determining behaviour, and permitting elucidation of the intracellular signalling mechanisms involved. Project 6: Molecular biological approaches will be applied to studies of the mechanisms by which NRSF and ADNP mediate CNS responses to damage, focusing on neuroprotective effects in primary culture.