Micropatterned Surfaces As Tools For The Study Of The Rapid Non-Genomic Actions Of Steroid Receptors

Steroid hormones control several developmental and physiological processes by binding to intracellular receptors that, in turn, interact with DNA to alter gene expression. These processes typically take at least 30 to 60 min for an increase in mRNA expression to be observed. In contrast, other regulatory actions of steroid hormones such as increases in activity of mitogen activated protein kinases are manifested within seconds to a few minutes and are far too rapid to be due to changes at the genomic level. Because these effects are not impaired by inhibitors of mRNA transcription, they are referred to non-genomic or rapid actions to distinguish them from the classical genomic effects at the transcriptional level. The non-genomic effects are thought to occur at the plasma membrane but have proven difficult to analyse in detail because of technical problems arising from capturing the receptors at the membrane due to their dynamic behaviour, subcellular sizes and complexity of action. Here we describe a novel technique for studying the non-genomic action of steroid hormones making use of dip-pen nanolithography (DPN) for patterning supported lipid bilayers containing haptenated lipids onto glass surfaces. Mast cells have been chosen for these studies because of the crucial role they play in allergic reactions and because the non-genomic action of steroid hormones have been reported as one of the means whereby allergy is regulated in these cells. Since mast cells express IgE receptors on their surfaces, they are treated with an anti-IgE antibody and allowed to settle on the patterned surfaces. The IgE receptor is then cross-linked through interaction with the haptenated lipids and this leads to the recruitment of different signalling molecules including steroid receptors to the patterned lipids. The DPN approach allows a nano-scale characterisation of the activating events afforded by the lipid bilayer. The patterns enable quantitative evaluation of co-localised cellular components and the steroid receptors to be assessed. This assay also allows visualisation and analysis of the interacting proteins to be made on a single cell level as well as receptor-proximal events triggered by allergens and regulation by steroid receptors to be measured. This method could be adapted for studying the rapid action of steroid hormones in other cell types.