视觉化和量化内源性器官内蛋白相互作用在ER-线粒体接触点的内源性线粒体内蛋白相互作用,通过近距离结合测定
Vera Filipa Monteiro-Cardoso1, Romain Le Bars2, Francesca Giordano3
1Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Saclay; Inserm U1280.
Journal of visualized experiments : JoVE
|November 6, 2023
概括
这项研究引入了一种新方法,结合了近距离结合试验 (PLA),线粒体染色和3D成像,以检测内网膜-线粒体膜接触点的蛋白质. 该技术成功地识别了ORP5-ORP8蛋白质复合体,该复合体位于线粒体附近.
科学领域:
- 细胞生物学 细胞生物学
- 分子生物学分子生物学
- 生物物理学的生物物理.
背景情况:
- 膜接触部位 (MCSs) 对于器官通信至关重要,促进生物分子的交换而不是融合.
- 细胞内膜网膜 (ER) - 线粒体MCS对于细胞代谢和生物能量学至关重要.
- 在MCS中识别蛋白质需要先进的方法,因为它们的动态性质和近距离.
研究的目的:
- 开发和验证一种用于检测ER-线粒体MCS中的蛋白质的新方案.
- 为了研究特定蛋白质的空间关系,如ORP5和ORP8,在这些接触点.
- 使用先进的成像技术量化蛋白质复合体与线粒体的接近程度.
主要方法:
- 结合近距离结合试验 (PLA) 与线粒体染色和3D成像细分.
- 利用PLA检测在相邻的有机细胞膜上物理接近 (>40nm) 的蛋白质.
- 采用细胞成像软件进行空间分析和测量线粒体中的蛋白质复合体的距离.
主要成果:
- 在ER-线粒体MCS中成功检测出位于同一膜上的蛋白质.
- 量化了ORP5-ORP8复合体与线粒体表面的距离.
- 证明大约50%的ORP5-ORP8相互作用发生在线粒体附近的ER子域中.
结论:
- 开发的协议对于研究ER-线粒体MCS中的蛋白质定位和相互作用是有效的.
- 这些发现突出了ORP5-ORP8复合体在线粒体附近的特定定位.
- 这种方法促进了MCS组件及其在细胞功能中的作用的研究.
更多相关视频
08:27Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays
Published on: October 20, 2023
1.7K
09:09Author Spotlight: Regulation and Dysregulation of ER-Mitochondria Contacts — Implications for Neurodegenerative Disease Pathogenesis
Published on: October 11, 2024
2.4K
相关概念视频
01:33Enzyme-Linked Immunosorbent Assay
In 1971, Peter Perlman and Eva Engvall developed an Enzyme-linked immunosorbent assay (ELISA or EIA). ELISA differs from western blot in that the assays are conducted in microtiter plates or in vivo rather than on an absorbent membrane.
There are many different types of ELISAs, but they all involve an antibody molecule whose constant region binds an enzyme, leaving the variable region free to bind its specific antigen. Enzyme-substrate reaction allows the antigen to be visualized or quantified.
There are many different types of ELISAs, but they all involve an antibody molecule whose constant region binds an enzyme, leaving the variable region free to bind its specific antigen. Enzyme-substrate reaction allows the antigen to be visualized or quantified.
01:20Immunogold Electron Microscopy
Immunoelectron microscopy utilizes immunogold labeling of endogenous proteins with specific antibodies to detect and localize these proteins in cells and tissues. The procedure provides insights into the distribution and quantification of protein under different stimulation conditions offering clues about their functions. Conjugating highly electron-dense gold particles with primary or secondary antibodies allow antigen detection on and within cells, with high resolution and specificity.