在过去的几十年里,膜接触位点(MCSs)已成为细胞生理学和病理学不同领域的深入研究对象,它们对细胞正确功能的重要性现已得到广泛认可。任何已知的细胞间细胞器之间的MCS,包括内质网(ER),线粒体,高尔基,内体,过氧化物酶体,溶酶体,脂滴,和质膜(PM),已被大量记录,在某些情况下,还确定了负责系链的分子。它们代表特定的膜集线器,其中紧密协调的离子交换,脂质,营养素,和维持适当的细胞稳态所需的因素发生。他们的精致,动态,有时难以捉摸的性质阻止和/或延迟了在生理条件下和在活生物体中容易成像细胞器间接近的工具的开发。如今,由于发现MCSs的失调与几种病理状况有关,因此这方面得到了巨大的发展。我们最近开发了模块化,基于分裂GFP的接触位点传感器(SPLICS)被设计成当ER和线粒体之间的同型和异型并置发生在特定距离范围内时发出荧光。在这里我们详细描述,通过突出优势和劣势,这些新型基因编码的SPLICS传感器的使用和应用,以及如何正确量化短程和远程ER-线粒体相互作用。
In the last decades, membrane contact sites (MCSs) have been the object of intense investigation in different fields of cell physiology and pathology and their importance for the correct functioning of the cell is now widely recognized. MCS between any known intercellular organelles, including endoplasmic reticulum (ER), mitochondria, Golgi, endosomes, peroxisomes, lysosomes, lipid droplets, and the plasma membrane (PM), have been largely documented and in some cases the molecules responsible for the tethering also identified. They represent specific membrane hubs where a tightly coordinated exchange of ions, lipids, nutrients, and factors required to maintain proper cellular homeostasis takes place. Their delicate, dynamic, and sometimes elusive nature prevented and/or delayed the development of tools to easily image interorganelle proximity under physiological conditions and in living organisms. Nowadays, this aspect received great momentum due to the finding that MCSs\' dysregulation is involved in several pathological conditions. We have recently developed modular, split-GFP-based contact site sensors (SPLICS) engineered to fluoresce when homo- and heterotypic juxtapositions between ER and mitochondria occur over a range of specific distances. Here we describe in detail, by highlighting strengths and weaknesses, the use and the application of these novel genetically encoded SPLICS sensors and how to properly quantify short- and long-range ER-mitochondria interactions.