Dipartimento di Scienze Fisiche ed Astronomiche, University of Palermo, Palermo, Italy.
Laboratory for Fluorescence Dynamics, University of California, Irvine, California.
Biophys J. 2011 Feb 2;100(3):774-783. doi: 10.1016/j.bpj.2010.11.089.
Prefibrillar oligomers of proteins are suspected to be the primary pathogenic agents in several neurodegenerative diseases. A key approach for elucidating the pathogenic mechanisms is to probe the existence of oligomers directly in living cells. In this work, we were able to monitor the process of aggregation of Concanavalin A in live cells. We used number and brightness analysis, two-color cross number and brightness analysis, and Raster image correlation spectroscopy to obtain the number of molecules, aggregation state, and diffusion coefficient as a function of time and cell location. We observed that binding of Concanavalin A to the membrane and the formation of small aggregates paralleled cell morphology changes, indicating progressive cell compaction and death. Upon protein aggregation, we observed increased membrane water penetration as reported by Laurdan generalized polarization imaging.
蛋白原纤维寡聚物被怀疑是几种神经退行性疾病的主要致病因子。阐明致病机制的一个关键方法是直接在活细胞中探测寡聚物的存在。在这项工作中，我们能够监测伴刀豆球蛋白 A 在活细胞中的聚集过程。我们使用了数量和亮度分析、双色交叉数量和亮度分析以及光栅图像相关光谱学，以获得分子数量、聚集状态和扩散系数随时间和细胞位置的变化。我们观察到，伴刀豆球蛋白 A 与膜的结合和小聚集体的形成与细胞形态变化平行，表明细胞逐渐紧缩和死亡。在蛋白质聚集时，我们观察到膜水渗透增加，正如 Laurdan 广义偏振成像所报道的那样。
