Fluorescence Zinc BioSensing and Imaging Technology

这是一个与之竞争的续订申请。该项目的总体主旨是开发新的荧光传感和成像技术，以了解锌的生物学，以期开发新的和改进的人类疾病诊断和治疗方法。最近出现了锌在中风、阿尔茨海默病、癫痫、长期记忆、胃肠道感染和2型糖尿病中的作用的证据(S)，但在所有这些病例中，都有必要更详细地了解锌的参与。以前，我们已经开发了基于蛋白质的荧光指示剂，具有出色的灵敏度、选择性和可调的动力学；使用这种基本方法，我们第一次能够测量(在静止细胞中)细胞质中的游离锌，并通过使用可表达的指示剂，在静止的线粒体中。对于下一个周期，我们建议开发一种新的比率、可表达的锌指示剂调色板，在许多应用中提供快速、灵敏和可靠的锌定量。我们还提议应用我们的新传感技术来回答一个至关重要的问题，这个问题到目前为止还没有被其他研究人员所抵制：在大脑的海马结构中，突触后树突棘是否会吸收突触锌？这个问题的答案将有助于阐明锌在学习和记忆形成中的作用。

This is a competing renewal application. The overall thrust of the project is to develop novel fluorescent sensing and imaging technology for understanding the biology of zinc, with a view to developing novel and improved diagnostics and therapies for human diseases. Evidence of zinc’s role(s) in stroke, Alzheimer’s disease, epilepsy, long term memory, gastrointestinal infectious disease, and type 2 diabetes has recently emerged, but a much more detailed understanding of zinc’s involvement will be necessary in all these cases. Previously, we had developed protein-based fluorescence indicators with outstanding sensitivity, selectivity, and tunable kinetics; using this basic approach we were able for the first time to measure (in a resting cell) free zinc in the cytoplasm, and by use of an expressible indicator, in the resting mitochondrion. For the next cycle we propose to develop a palette of new ratiometric, expressible zinc indicators that will provide rapid, sensitive, and reliable zinc quantitation in many applications. We also are proposing to apply our new sensing technology to answer a question of central importance that so far has resisted other investigators: In the hippocampal formation in the brain, is synaptic zinc taken up by the post-synaptic dendritic spines? An answer to this question will help elucidate zinc’s role in learning and memory formation.