Department of Biomedical Sciences, University of Padua, Padua, Italy.
Department of Biochemistry, Molecular Biology and Physiology, Faculty of Medicine, Unidad de Excelencia Instituto de Biología y Genética Molecular (IBGM), University of Valladolid and CSIC, Valladolid, Spain.
Nat Commun. 2024 Nov 12;15(1):9775. doi: 10.1038/s41467-024-52985-0.
Membrane contact sites (MCSs) are hubs allowing various cell organelles to coordinate their activities. The dynamic nature of these sites and their small size hinder analysis by current imaging techniques. To overcome these limitations, we here design a series of reversible chemogenetic reporters incorporating improved, low-affinity variants of splitFAST, and study the dynamics of different MCSs at high spatiotemporal resolution, both in vitro and in vivo. We demonstrate that these versatile reporters suit different experimental setups well, allowing one to address challenging biological questions. Using these probes, we identify a pathway in which calcium (Ca) signalling dynamically regulates endoplasmic reticulum-mitochondria juxtaposition, characterizing the underlying mechanism. Finally, by integrating Ca-sensing capabilities into the splitFAST technology, we introduce PRINCESS (PRobe for INterorganelle Ca-Exchange Sites based on SplitFAST), a class of reporters to simultaneously detect MCSs and measure the associated Ca dynamics using a single biosensor.
膜接触位点（MCSs）是各种细胞细胞器协调其活动的枢纽。这些位点的动态性质及其较小的尺寸阻碍了当前成像技术的分析。为了克服这些限制，我们在这里设计了一系列包含改良的、低亲和力的 FAST 分割变体的可逆化学遗传报告器，并在体外和体内以高时空分辨率研究不同 MCSs 的动态，我们证明这些多功能报告器非常适合不同的实验设置，允许人们解决具有挑战性的生物学问题。使用这些探针，我们确定了一条途径，其中钙（Ca）信号动态调节内质网-线粒体并列，表征了潜在的机制。最后，通过将 Ca 感应能力集成到 FAST 分割技术中，我们引入了 PRINCESS（基于 FAST 分割的细胞器间 Ca 交换位点探针），这是一类报告器，可以使用单个生物传感器同时检测 MCSs 并测量相关的 Ca 动力学。
