Dynamics and function of the transcription factor SRF inside the nucleus of neurons revealed by single-molecule analysis and super-resolution optical microscopy

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Millions of molecules contribute to gene transcription in nuclei of living cells. So far, investigation of gene transcription is mostly restricted to ensemble analysis of all these molecules within one cell. Such ensemble analysis assumes that all individual copies of a particular transcription factor (TF) have identical function and operate as a homogenous population. However, within a protein population, sub-populations of TF molecules with distinct biophysical properties might exist and fulfil different roles in the same process. This requires analysis at the single molecule level, a technique just starting to take-off in cell biological research. In this proposal we analyse such dynamic processes of nuclear gene transcription at the single molecule level. We focus on SRF (serum response factor), a TF regulating physiological processes in the brain such as neuronal growth and synaptic function. In mouse models of neuronal disease including axon injury, neuronal degeneration and e.g. epilepsy, SRF function is impaired. SRF mediates neuronal activity mediated gene transcription through transcription of immediate early genes (IEGs) such as c-Fos and Egr1. In addition, SRF regulates actin cytoskeletal genes thereby impinging on nerve fibre growth and axonal regeneration. We employ a fusion protein of SRF and the Halo tag (SRF-Halo) to analyse single SRF molecules using single particle tracking with light sheet illumination in living neurons. In addition, we will perform single molecule localisation microscopy (SMLM) in fixed cells. These techniques will be used to determine several parameters of nuclear SRF transcriptional dynamics:i) the fraction of unbound to DNA-bound SRFii) the DNA residence time of SRFiii) the sub-nuclear SRF localization, e.g. co-localization with transcription hubsSo far, only few TFs have been analysed at the single molecule level and research focused on immortalised cell culture lines. Here, we will use primary cells, i.e. primary mouse hippocampal neurons stimulated with growth factors such as BDNF (brain derived neurotrophic factor). In a first step, we compare results obtained in neurons with data obtained from NIH3T3 cells, acquired in work building up to this proposal. This provides information as to whether SRF binding dynamics between proliferative and differentiated cells are conserved or divergent. We further analyse how DNA binding dynamics of SRF are altered along the time course of neuronal polarisation, e.g. during protrusion of the first neurites or later on in synapse formation. Finally, we will investigate injured primary neurons generated using laser axotomy to address a potential modulation of SRF activity dynamics by axonal degeneration and regeneration.Since SRF fulfils essential physiological and pathological functions in all organs investigated and not only the brain, our data will provide important insight into regulation of SRF and general TF activity relevant for a wealth of tissues.