splitFAST2 is the next-gen complementation system just published in ACS Chem. Biol. 2024 by Prof. Gautier’s team.  Remember!  In 2019, they introduced splitFAST, a breakthrough complementation reporter for protein−protein interaction (PPIs) based on the chemogenetic fluorescent FAST.  Today, they introduce splitFAST2, displaying higher brightness, lower self-complementation, and higher dynamic range.  As a result, splitFAST2 allows high spatial and temporal resolution, opening great prospects to decipher PPIs in any biological context.


splitFAST2 – © 2024 American Chemical Society

It is hence no coincidence that a team led by Paola Pizzo and Riccardo Filadi of the University of Padova has just leveraged splitFAST2 for endoplasmic reticulum-mitochondria junction studies, their research focus.

ER-mitochondria splitFAST

ER-mitochondria splitFAST – © 2023 The Authors

Membrane contact sites (MCSs), such as the ER-mit junction, are indeed 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), an unprecedented class of reporters to simultaneously detect MCSs and measure the associated Ca2+ dynamics using a single biosensor.

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 on splitFAST2

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, doi.org/10.1101/2023.12.28.573515

Rakotoarison, L. M., Tebo, A. G., Böken, D., Board, S., El Hajji, L., & Gautier, A. (2024). Improving split reporters of protein-protein interactions through orthology-based protein engineering. ACS Chem. Biol., doi.org/10.1021/acschembio.3c00631