A team led by Paola Pizzo and Riccardo Filadi of the University of Padova has just enabled endoplasmic reticulum-mitochondria junction studies with splitFAST of The Twinkle Factory. The interest in such membrane contact sites (MCSs) between membranous organelles has dramatically increased these years in medical research. Indeed, early disturbances in MCS structure and/or function have been described in different high-incidence disorders, such as neurodegenerative diseases and several cancers.
ER-mitochondria splitFAST – © 2023 The Authors
The endoplasmic reticulum-mitochondria junction is one example of those membrane contact sites (MCSs). They hence behave as hubs, allowing cell organelles to coordinate activities. However, the dynamic nature of these sites and their small size hinder analysis by common imaging techniques. In order to overcome these limitations, the authors designed a series of reversible chemogenetic reporters incorporating splitFAST2. They hence could monitor the dynamics of different MCSs at high spatiotemporal resolution, both in vitro and in vivo. As a result, they identified a hitherto unknown pathway involving calcium (Ca2+) signalling! Finally, by integrating Ca2+ -sensing capabilities into splitFAST, they designed PRINCESS (PRobe for INterorganelle Ca 2+ -Exchange Sites based on SplitFAST). As a result, they provide an unprecedented class of reporters detecting MCSs and measuring the associated Ca2+ dynamics using a single biosensor.
Interestingly, the authors have implemented splitFAST2, the next-gen FAST complementation system recently published in ACS Chem. Biol. 2024 by Prof. Gautier’s team. Prof. Gautier released the initial splitFAST, a breakthrough complementation reporter for protein−protein interaction (PPIs) based on the chemogenetic fluorescent FAST in 2019. Four years later, they disclosed splitFAST2, which displays higher brightness, lower self-complementation, and higher dynamic range. As a result, splitFAST2 allows higher spatial and temporal resolution, opening great prospects to decipher PPIs and empower cell-based assays for drug screening.
splitFAST2 – © 2024 American Chemical Society
The Twinkle Factory supplies a whole range of fluorogenic reporters for splitFAST, varying in colors and affinity. Initially introduced with FAST and splitFAST, they work equally well with splitFAST2.
Stain different. Tag FAST!
More reading
Garcia Casas, P., Rossini, M., Pavenius, L., Saeed, M., Arnst, N., Sonda, S., … & Filadi, R. (2023). Simultaneous detection of membrane contact dynamics and associated Ca2+ signals by reversible chemogenetic reporters. bioRxiv, 2023-12 (https://doi.org/10.1101/2023.12.28.573515)
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