Dataset Information

Influence of Fluorescent Protein Maturation on FRET Measurements in Living Cells.

ABSTRACT: Förster resonance energy transfer (FRET)-based sensors are a valuable tool to quantify cell biology, yet it remains necessary to identify and prevent potential artifacts in order to exploit their full potential. We show here that artifacts arising from slow donor mCerulean3 maturation can be substantially diminished by constitutive expression in both prokaryotic and eukaryotic cells, which can also be achieved by incorporation of faster-maturing FRET donors. We developed an improved version of the donor mTurquoise2 that matures faster than the parent protein. Our analysis shows that using equal maturing fluorophores in FRET-based sensors or using constitutive low expression conditions helps to reduce maturation-induced artifacts, without the need of additional noise-inducing spectral corrections. In general, we show that monitoring and controlling the maturation of fluorescent proteins in living cells is important and should be addressed in in vivo applications of genetically encoded FRET sensors.

SUBMITTER: Liu B

PROVIDER: S-EPMC6167724 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Influence of Fluorescent Protein Maturation on FRET Measurements in Living Cells.

Liu Boqun B Mavrova Sara N SN van den Berg Jonas J Kristensen Sebastian K SK Mantovanelli Luca L Veenhoff Liesbeth M LM Poolman Bert B Boersma Arnold J AJ

ACS sensors 20180912 9

Förster resonance energy transfer (FRET)-based sensors are a valuable tool to quantify cell biology, yet it remains necessary to identify and prevent potential artifacts in order to exploit their full potential. We show here that artifacts arising from slow donor mCerulean3 maturation can be substantially diminished by constitutive expression in both prokaryotic and eukaryotic cells, which can also be achieved by incorporation of faster-maturing FRET donors. We developed an improved version of t ...[more]

PMID: 30168711

Similar Datasets

Project description:PPAR? is a pharmacological target in inflammatory and metabolic diseases. Upon agonistic treatment or following antagonism, binding of co-factors is altered, which consequently affects PPAR?-dependent transactivation as well as its DNA-independent properties. Therefore, establishing techniques to characterize these interactions is an important issue in living cells. Methods: Using the FRET pair Clover/mRuby2, we set up a flow cytometry-based FRET assay by analyzing PPAR?1 binding to its heterodimerization partner RXR?. Analyses of PPAR?-reporter and co-localization studies by laser-scanning microscopy validated this system. Refining the system, we created a new readout to distinguish strong from weak interactions, focusing on PPAR?-binding to the co-repressor N-CoR2. Results: We observed high FRET in cells expressing Clover-PPAR?1 and mRuby2-RXR?, but no FRET when cells express a mRuby2-RXR? deletion mutant, lacking the PPAR? interaction domain. Focusing on the co-repressor N-CoR2, we identified in HEK293T cells the new splice variant N-CoR2-?ID1-exon. Overexpressing this isoform tagged with mRuby2, revealed no binding to Clover-PPAR?1, nor in murine J774A.1 macrophages. In HEK293T cells, binding was even lower in comparison to N-CoR2 constructs in which domains established to mediate interaction with PPAR? binding are deleted. These data suggest a possible role of N-CoR2-?ID1-exon as a dominant negative variant. Because binding to N-CoR2-mRuby2 was not altered following activation or antagonism of Clover-PPAR?1, we determined the effect of pharmacological treatment on FRET intensity. Therefore, we calculated flow cytometry-based FRET efficiencies based on our flow cytometry data. As with PPAR? antagonism, PPAR? agonist treatment did not prevent binding of N-CoR2. Conclusion: Our system allows the close determination of protein-protein interactions with a special focus on binding intensity, allowing this system to characterize the role of protein domains as well as the effect of pharmacological agents on protein-protein interactions.

Dataset Information

Influence of Fluorescent Protein Maturation on FRET Measurements in Living Cells.

Publications

Influence of Fluorescent Protein Maturation on FRET Measurements in Living Cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets