Direct measurement of protein–protein interactions by FLIM-FRET at UV laser-induced DNA damage sites in living cells

Abstract

Protein–protein interactions are essential to ensure timely and precise recruitment of chromatin remodellers and repair factors to DNA damage sites. Conventional analyses of protein–protein interactions at a population level may mask the complexity of interaction dynamics, highlighting the need for a method that enables quantification of DNA damage-dependent interactions at a single-cell level. To this end, we integrated a pulsed UV laser on a confocal fluorescence lifetime imaging (FLIM) microscope to induce localized DNA damage. To quantify protein–protein interactions in live cells, we measured Förster resonance energy transfer (FRET) between mEGFP- and mCherry-tagged proteins, based on the fluorescence lifetime reduction of the mEGFP donor protein. The UV-FLIM-FRET system offers a unique combination of real-time and single-cell quantification of DNA damage-dependent interactions, and can distinguish between direct protein–protein interactions, as opposed to those mediated by chromatin proximity. Using the UV-FLIM-FRET system, we show the dynamic changes in the interaction between poly(ADP-ribose) polymerase 1, amplified in liver cancer 1, X-ray repair cross-complementing protein 1 and tripartite motif containing 33 after DNA damage. This new set-up complements the toolset for studying DNA damage response by providing single-cell quantitative and dynamic information about protein–protein interactions at DNA damage sites.

PARP1 proximity proteomics reveals interaction partners at stressed replication forks

“…We identified 185 proteins that dissociated from PARP1 in the presence of olaparib. As reported before, CHD1L was identified in the proximity of PARP1 dependent on ADP-ribosylation ( 82 , 83 ). Moreover, the proximity of proteins involved in DNA replication (ATR, MCM2/3/4/6, RNASEH2A/B, and PCNA) was reduced after inhibition of PARP1 (Figure 1E ).…”

Fluorescence-based sensing of the bioenergetic and physicochemical status of the cell

“…This strategy has also been employed to study proteins and RNA turnover (Trauth et al, 2020). Interactions between macromolecules can be observed by tagging different putative interacting partners with different fluorescent reporters and subsequently measuring the FRET efficiency, which will increase as a function of the proximity of the two fluorescent molecules (Kaufmann et al, 2020). In a similar way, changes in conformation of macromolecules can be studied via FRET measurements by tagging with different fluorescent reporters different parts of the analyzed macromolecules (G€ otz et al, 2021).…”

“…Subsequently, FPs have been used to develop a great variety of sensors (Berg, Hung, & Yellen, 2009;Miyawaki et al, 1997;Nadler, Morgan, Flamholz, Kortright, & Savage, 2016) to study both molecular and global changes in the tagged macromolecule or structure of the cell. The availability of FPs emitting at different wavelengths allowed the application of the F€ orster Resonance Energy Transfer (FRET) mechanism to create sensors (Calamera et al, 2019;Miyawaki et al, 1997;Otten et al, 2019;Sadoine, Reger, Wong, & Frommer, 2021) and to determine interaction between proteins or protein domains (Ivanusic, Eschricht, & Denner, 2014;Kaufmann et al, 2020). Circularly permuted fluorescent proteins (cpFPs) variants (Topell, Hennecke, & Glockshuber, 1999) have been developed to create sensors (Kostyuk, Demidovich, Kotova, Belousov, & Bilan, 2019) to detect variations in the concentration of specific molecules.…”

Single-Cell Biochemical Multiplexing by Multidimensional Phasor Demixing and Spectral Fluorescence Lifetime Imaging Microscopy

“…However, computational frameworks for the robust and sensitive multiplexing of FRET are at their infancy, and further work is necessary to improve our multiplexing capabilities. Phasor analysis of time decays has often been applied to quantify FRET for single probes by separating the two states of a typical sensor (low/high FRET) [10,14,18,26,35,36,38,41,47,49,50,55,60,70,72,73]. Similarly, the integration of spectrally resolved FLIM (sFLIM) [34] and multidimensional phasor analysis has been successfully applied to the quantification of single FRET probes [25].…”