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

数以百万计的分子对活细胞细胞核中的基因转录做出了贡献。到目前为止，对基因转录的研究主要局限于对一个细胞内所有这些分子的集成分析。这种集成分析假设特定转录因子(Tf)的所有个体拷贝都具有相同的功能，并作为一个同源群体运行。然而，在一个蛋白质群体中，具有不同生物物理性质的Tf分子的亚群体可能存在，并在同一过程中发挥不同的作用。这需要单分子水平的分析，这是一项刚刚开始在细胞生物学研究中起飞的技术。在这一建议中，我们在单分子水平上分析了核基因转录的动态过程。我们关注SRF(血清反应因子)，它是一种调节大脑中的生理过程，如神经元生长和突触功能的因子。在包括轴突损伤、神经元变性和癫痫等神经疾病的小鼠模型中，SRF功能受损。SRF通过转录即刻早期基因(IEGs)，如c-Fos和Egr1，介导神经元活性介导的基因转录。此外，SRF调节肌动蛋白细胞骨架基因，从而影响神经纤维生长和轴突再生。我们使用SRF和Halo标签的融合蛋白(SRF-Halo)来分析活神经元中的单个SRF分子，使用光片照明的单粒子跟踪。此外，我们将在固定细胞中进行单分子定位显微镜(SMLM)。这些技术将被用来确定核SRF转录动力学的几个参数：i)未与DNA结合的SRF的比例)SRFII的DNA停留时间)亚核SRF的定位，例如与转录枢纽的共定位到目前为止，只有少数转录因子在单分子水平上被分析，研究集中在永生化细胞系上。在这里，我们将使用原代细胞，即受生长因子(如脑源性神经营养因子)刺激的原代小鼠海马神经元。在第一步中，我们将在神经元中获得的结果与从NIH3T3细胞中获得的数据进行比较，这些数据是在为这一提议而进行的工作中获得的。这提供了关于SRF在增殖细胞和分化细胞之间的结合动力学是保守的还是分化的信息。我们进一步分析了SRF的DNA结合动力学是如何随着神经元极化的时间进程而改变的，例如在第一个神经突起突出期间或后来的突触形成过程中。最后，我们将研究激光切断损伤的初级神经元，以解决轴突退化和再生对SRF活动动力学的潜在调制。由于SRF在所有被研究的器官中都具有基本的生理和病理功能，而不仅仅是大脑，我们的数据将为SRF的调节和与丰富组织相关的一般TF活性提供重要的洞察力。

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.