Advanced fluorescence spectroscopy to unravel functional oligomerization of a newly discovered Ca2+ channel and its role in heart failure.

内质网（内质网）稳态是心力衰竭中越来越受关注的靶点，因为错误折叠或未折叠的蛋白质的积累容易导致内质网应激，而内质网应激又在心血管疾病的发展中起重要作用。ER跨膜蛋白可以发挥影响细胞死亡或存活的重要信号作用。Mitsugumin 23（MG 23）是一种新发现的内质网/肌浆网（ER/SR）跨膜蛋白，被认为是寡聚体。我们认为MG 23的动态寡聚化行为控制其功能作用。我们的最新数据表明MG 23在心脏纤维化、SR Ca 2+渗漏和心力衰竭早期阶段中的作用。这使得MG 23成为进一步深入研究的新的和及时的目标，以揭示其报告的寡聚化所发挥的分子作用，并更好地了解其潜在的可药用性。虽然我们研究质膜整合蛋白寡聚化的能力在过去几年中有了很大的进步，但这些方法依赖于极端光学切片，这是不服从内部细胞器，如ER/SR。这个博士项目的目的是开发和应用空间-使用共聚焦显微镜的时间分子亮度方法来解决病理生理学中MG 23的寡聚化动力学问题条件荧光波动光谱的使用提供了承诺，研究同质寡聚化拥挤的系统中，其中单个分子的可视化受到蛋白质密度和/或样品的局部几何形状的挑战。使用分子生物学，电生理学，心力衰竭模型和先进的显微镜技术相结合，我将可视化心肌细胞中的MG 23功能性寡聚化，解开MG 23在心力衰竭条件下的寡聚化，并将MG 23寡聚化与心脏细胞病理学，特别是细胞凋亡和纤维化相关联。该项目将开发一个工具箱来研究ER膜蛋白寡聚化，并对心力衰竭中未折叠蛋白反应途径的更广泛研究产生影响。

Endoplasmic reticulum (ER) homeostasis is becoming an increasingly interesting target in heart failure, since accumulation of misfolded or unfolded proteins can easily lead to ER stress, which in turn can play an important role in the development of cardiovascular diseases. ER transmembrane proteins can play prominent signalling roles that influence cell death or survival. Mitsugumin 23 (MG23), is a newly discovered endoplasmic/sarcoplasmic reticulum (ER/SR) transmembrane protein thought to be oligomeric in nature. We suggest that the dynamic oligomerization behaviour of MG23 controls its functional role. Our latest data suggest a role for MG23 in cardiac fibrosis, SR Ca2+ leak and the early stages of heart failure. This makes MG23 a novel and timely target for further in-depth study to unravel the molecular role played by its reported oligomerization and provide a better understanding of its potential druggability. Although our ability to study oligomerization of plasma membrane integral proteins has greatly advanced over the last few years, these approaches rely on extreme optical sectioning, which is not amenable to internal organelles such as the ER/SR. The aim of this PhD project is to develop and apply spatial-temporal molecular brightness approaches using confocal microscopy to address the problem of the oligomerization dynamics of MG23 in pathophysiological conditions. The use of fluorescence fluctuation spectroscopy offers promise to study homo-oligomerization in crowded systems, where the visualization of individual molecules is challenged either by protein density and/or by the local geometry of the sample. Using a combination of molecular biology, electrophysiology, heart failure models and advanced microscopy techniques, I will visualize MG23 functional oligomerization in cardiac cells, unravel MG23 oligomerization in heart failure conditions, and correlate MG23 oligomerization to cardiac cell pathology, specifically apoptosis and fibrosis. This project will develop a toolbox to study ER membrane protein oligomerization, with ramifications to the broader study of the unfolded protein response pathway in heart failure.