Department of Pathology, Cedars-Sinai Medical Center.
Department of Pathology, Cedars-Sinai Medical Center; Department of Biomedical Science Education, College of Medicine, Charles R. Drew University of Medicine and Science;
J Vis Exp. 2024 Oct 11(212). doi: 10.3791/66862.
Endoplasmic reticulum (ER)-mitochondria contact sites play a critical role in cell health and homeostasis, such as the regulation of Ca and lipid homeostasis, mitochondrial dynamics, autophagosome and mitophagosome biogenesis, and apoptosis. Failure to maintain normal ER-mitochondrial coupling is implicated in many neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and hereditary spastic paraplegia. It is of considerable significance to explore how the dysregulation of ER-mitochondrial contacts could lead to cell death and whether repairing these contacts to the normal level could ameliorate neurodegenerative conditions. Thus, improved assays that measure the level of these contacts could help to illuminate the pathogenic mechanisms of these diseases. Ultimately, establishing simple and reliable assays will facilitate the development of new therapeutic strategies. Here we describe a split-luciferase assay to quantitatively measure the level of ER-mitochondria contacts in live cells. This assay can be used to study the pathophysiological role of these contacts as well as to identify their modulators in high-throughput screening.
内质网（ER）-线粒体接触位点在细胞健康和稳态中起着关键作用，例如调节 Ca 和脂质稳态、线粒体动力学、自噬体和噬线粒体体的发生以及细胞凋亡。内质网-线粒体偶联不能维持正常与许多神经退行性疾病有关，如阿尔茨海默病、帕金森病、肌萎缩侧索硬化症和遗传性痉挛性截瘫。探讨内质网-线粒体接触的失调如何导致细胞死亡，以及修复这些接触到正常水平是否可以改善神经退行性疾病状态，这具有重要意义。因此，改进测量这些接触水平的检测方法可以帮助阐明这些疾病的发病机制。最终，建立简单可靠的检测方法将有助于开发新的治疗策略。在这里，我们描述了一种分裂荧光素酶检测方法，用于定量测量活细胞中内质网-线粒体接触的水平。该检测方法可用于研究这些接触的病理生理作用，以及在高通量筛选中鉴定它们的调节剂。
