Benhammouda Sara, Vishwakarma Anjali, Gatti Priya, Germain Marc
Groupe de Recherche en Signalisation Cellulaire and Département de Biologie Médicale, Université Du Québec à Trois-Rivières, Trois-Rivières, QC, Canada.
Centre D'Excellence en Recherche sur les Maladies Orphelines - Fondation Courtois, Université du Québec à Montréal, Montréal, QC, Canada.
Front Cell Dev Biol. 2021 Dec 3;9:789959. doi: 10.3389/fcell.2021.789959. eCollection 2021.
Organelles cooperate with each other to regulate vital cellular homoeostatic functions. This occurs through the formation of close connections through membrane contact sites. Mitochondria-Endoplasmic-Reticulum (ER) contact sites (MERCS) are one of such contact sites that regulate numerous biological processes by controlling calcium and metabolic homeostasis. However, the extent to which contact sites shape cellular biology and the underlying mechanisms remain to be fully elucidated. A number of biochemical and imaging approaches have been established to address these questions, resulting in the identification of a number of molecular tethers between mitochondria and the ER. Among these techniques, fluorescence-based imaging is widely used, including analysing signal overlap between two organelles and more selective techniques such as proximity ligation assay (PLA). While these two techniques allow the detection of endogenous proteins, preventing some problems associated with techniques relying on overexpression (FRET, split fluorescence probes), they come with their own issues. In addition, proper image analysis is required to minimise potential artefacts associated with these methods. In this review, we discuss the protocols and outline the limitations of fluorescence-based approaches used to assess MERCs using endogenous proteins.
细胞器相互协作以调节重要的细胞稳态功能。这是通过在膜接触位点形成紧密连接来实现的。线粒体-内质网（ER）接触位点（MERCS）就是这样一种接触位点，它通过控制钙和代谢稳态来调节众多生物学过程。然而，接触位点塑造细胞生物学的程度及其潜在机制仍有待充分阐明。为了解决这些问题，已经建立了许多生化和成像方法，从而鉴定出线粒体与内质网之间的一些分子连接体。在这些技术中，基于荧光的成像被广泛使用，包括分析两个细胞器之间的信号重叠以及更具选择性的技术，如邻近连接分析（PLA）。虽然这两种技术能够检测内源性蛋白质，避免了一些与依赖过表达的技术（荧光共振能量转移、分裂荧光探针）相关的问题，但它们也有自身的问题。此外，需要进行适当的图像分析，以尽量减少与这些方法相关的潜在假象。在这篇综述中，我们讨论了使用内源性蛋白质评估MERCS的基于荧光的方法的实验方案，并概述了其局限性。
