Developing super-resolution imaging based biophysical methods to study protein aggregate induced membrane disruption at the single-molecule level

该提案旨在开发一种数字化、超灵敏的技术，能够以最高的灵敏度检测和量化蛋白质诱导的膜渗透和破坏。膜完整性的破坏是一种普遍存在的机制，通过这种机制，蛋白质聚集体在各种人类疾病中赋予毒性，包括阿尔茨海默病和帕金森病，2型糖尿病，镰状细胞病，甚至特定类型的癌症。有毒聚集体破坏和渗透细胞膜的脂质双分子层，并允许细胞外钙离子流入，导致钙稳态被破坏和细胞死亡。在这项工作中，我们将制造纳米探针来模拟天然的双层细胞膜，并确定蛋白质聚集如何破坏膜的完整性。通过利用基于单分子定位的超分辨率成像，我们将开发一种定量方法，通过计算进入离子的数量，以高通量的方式直接观察单个蛋白质聚集体诱导的脂质膜渗透。这种新颖的超灵敏方法将为蛋白质错误折叠疾病的膜渗透机制提供新的见解，使我们能够识别和表征复杂人体样品中毒性最大的蛋白质聚集体，并筛选潜在的药物，可以预防或减少与阿尔茨海默病和帕金森病相关的蛋白质聚集体诱导的膜损伤。

This proposal aims to develop a digital, ultrasensitive technique that can detect and quantify protein-induced membrane permeabilisation and disruption at the highest level of sensitivity. Disruption of membrane integrity is a ubiquitous mechanism by which protein aggregates confer toxicity in various human diseases, including Alzheimer's and Parkinson's disease, Type II diabetes, Sickle cell disease, and even specific type of Cancer. Toxic aggregates disrupt and permeabilise lipid bilayer of the cellular membrane and allow the influx of extracellular calcium ions, which leads to disrupted calcium homeostasis and cell death. In this work, we will fabricate nanosized probe to mimic the natural bilayer of cellular membrane and determine how protein aggregate damage membrane integrity. By utilising single-molecule localisation based super-resolution imaging, we will develop a quantitative method which will enable direct observation of individual protein aggregates induced lipid membrane permeabilisation in a high-throughput manner by counting the number of entering ions. This novel ultra-sensitive methodology will provide new insights into the mechanism of membrane permeabilisation in protein-misfolding disease, allowing us to identify and characterise the most toxic protein aggregates in complex human samples and screen the potential drug which can prevent or reduce protein aggregates induced membrane damage which is related to Alzheimer's and Parkinson's disease.