Characterizing the interactions between prolyl isomerase pin1 and phosphatase inhibitor-2 in living cells with FRET and FCS

Yuansheng Sun; Lifu Wang; Vinod Jyothikumar; David L. Brautigan; Ammasi Periasamy

doi:10.1117/12.915315

9 February 2012 Characterizing the interactions between prolyl isomerase pin1 and phosphatase inhibitor-2 in living cells with FRET and FCS

Yuansheng Sun, Lifu Wang, Vinod Jyothikumar, David L. Brautigan, Ammasi Periasamy

Author Affiliations +

Proceedings Volume 8226, Multiphoton Microscopy in the Biomedical Sciences XII; 82263T (2012) https://doi.org/10.1117/12.915315
Event: SPIE BiOS, 2012, San Francisco, California, United States

Abstract

Phosphatase inhibitor-2 (I2) was discovered as a regulator of protein Ser/Thr phosphatase-1 and is conserved from yeast to human. Binding between purified recombinant I2 from different species and the prolyl isomerase Pin1 has been demonstrated with pull-down assays, size exclusion chromatography and nuclear magnetic resonance spectroscopy. Despite this, questions persist as to whether these proteins associate together in living cells. In this study, we prepared fluorescent protein (FP) fusions of I2 and Pin1 and employed both Förster Resonance Energy Transfer (FRET) and Fluorescence Correlation Spectroscopy (FCS) imaging techniques to characterize their interactions in living cells. In both intensity-based and time-resolved FRET studies, we observed FRET uniformly across whole cells co-expressing I2-Cerulean and Pin1-Venus that was significantly higher than in negative controls expressing Cerulean FP (without fusing to I2) as the FRET donor and Pin1-Venus, showing a specific interaction between I2-Cerulean and Pin1-Venus in living cells. We also observed the co-diffusion of I2-Cerulean and Pin1-mCherry in Fluorescence Cross Correlation Spectroscopy (FCCS) measurements. We further showed that I2 itself as well as I2-Pin1 formed complexes in living cells (predicted from in vitro studies) via a quantitative FRET assay, and demonstrated from FCS measurements that both I2 and Pin1 (fused to Cerulean) are highly mobile in living cells.

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Yuansheng Sun, Lifu Wang, Vinod Jyothikumar, David L. Brautigan, and Ammasi Periasamy "Characterizing the interactions between prolyl isomerase pin1 and phosphatase inhibitor-2 in living cells with FRET and FCS", Proc. SPIE 8226, Multiphoton Microscopy in the Biomedical Sciences XII, 82263T (9 February 2012); https://doi.org/10.1117/12.915315

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KEYWORDS

Fluorescence resonance energy transfer

Proteins

Fluorescence correlation spectroscopy

Confocal microscopy

Luminescence

Control systems

Microscopy

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