Non-fitting FLIM-FRET facilitates analysis of protein interactions in live zebrafish embryos.

Molecular interactions are key to all cellular processes, and particularly interesting to investigate in the context of gene regulation. Protein-protein interactions are challenging to examine in vivo as they are dynamic, and require spatially and temporally resolved studies to interrogate them. Foerster Resonance Energy Transfer (FRET) is a highly sensitive imaging method, which can interrogate molecular interactions. FRET can be detected by Fluorescence Lifetime Imaging Microscopy (FLIM-FRET), which is more robust to concentration variations and photobleaching than intensity-based FRET but typically needs long acquisition times to achieve high photon counts. New variants of non-fitting lifetime-based FRET perform well in samples with lower signal and require less intensive instrument calibration and analysis, making these methods ideal for probing protein-protein interactions in more complex live 3D samples. Here we show that a non-fitting FLIM-FRET variant, based on the Average Arrival Time of photons per pixel (AAT- FRET), is a sensitive and simple way to detect and measure protein-protein interactions in live early stage zebrafish embryos.

Of numbers and movement - understanding transcription factor pathogenesis by advanced microscopy.

Transcription factors (TFs) are life-sustaining and, therefore, the subject of intensive research. By regulating gene expression, TFs control a plethora of developmental and physiological processes, and their abnormal function commonly leads to various developmental defects and diseases in humans. Normal TF function often depends on gene dosage, which can be altered by copy-number variation or loss-of-function mutations. This explains why TF haploinsufficiency (HI) can lead to disease. Since aberrant TF numbers frequently result in pathogenic abnormalities of gene expression, quantitative analyses of TFs are a priority in the field. In vitro single-molecule methodologies have significantly aided the identification of links between TF gene dosage and transcriptional outcomes. Additionally, advances in quantitative microscopy have contributed mechanistic insights into normal and aberrant TF function. However, to understand TF biology, TF-chromatin interactions must be characterised in vivo, in a tissue-specific manner and in the context of both normal and altered TF numbers. Here, we summarise the advanced microscopy methodologies most frequently used to link TF abundance to function and dissect the molecular mechanisms underlying TF HIs. Increased application of advanced single-molecule and super-resolution microscopy modalities will improve our understanding of how TF HIs drive disease.