Correlative Förster Resonance Electron Transfer‐Proximity Ligation Assay (FRET‐PLA) Technique for Studying Interactions Involving Membrane Proteins

Abstract

This unit provides a guide and detailed protocol for studying membrane protein-protein interactions (PPI) using the acceptor-sensitized Förster resonance electron transfer (FRET) method in combination with the proximity ligation assay (PLA). The protocol in this unit is focused on the preparation of FRET-PLA samples and the detection of correlative FRET/PLA signals as well as on the analysis of FRET-PLA data and interpretation of correlative results when using cyan fluorescent protein (CFP) as a FRET donor and yellow fluorescent protein (YFP) as a FRET acceptor. The correlative application of FRET and PLA combines two powerful tools for monitoring PPI, yielding results that are more reliable than with either technique alone. © 2016 by John Wiley & Sons, Inc.

The large extracellular loop of CD63 interacts with gp41 of HIV-1 and is essential for establishing the virological synapse

“…In the constructed vector pCMV-CD63ΔLEL, the mCherry tag was introduced by ligation of Xho I and Apa I digested mCherry fragment from the donor vector pCMV-CD63-mCherry. The vector pCMV-CD63 C145A,C146A -YFP used for FRET experiments was generated by exchange of the mCherry sequence with the YFP sequence from the vector pCMV-CD63-YFP-FLAG 40 using Xho I and Apa I restriction sites, all other vectors used were previously described 40 , 55 . All sequences of cloning sites were verified by Sanger sequencing.…”

“…Transfected HEK293T or Jurkat cells were prepared for PLA analysis as previously described 40 , 55 . We used the following antibodies for PLA experiments: mouse anti-V5 (MCA1360, Serotec), anti-FLAG (NB600–344, NovusBio) and anti-Na + , K + -ATPase (ab98787, Abcam).…”

“…Fluorescence signals were detected by Zeiss ZEN smart setup instrument settings for 488 nm and 594 nm dyes. PLA images were obtained and quantification of PLA signals was performed as previously described 40 , 55 . Pearson correlation coefficient (PCC) was calculated using cLSM images processed by using ImageJ Fiji's plugin Coloc2 for co-localization analysis.…”

The Large Extracellular Loop of CD63 Interacts with gp41 of HIV-1 and is Essential for Establishing the Virological Synapse

“…In the constructed vector pCMV-CD63ΔLEL, the mCherry tag was introduced by ligation of XhoI and ApaI digested mCherry fragment from the donor vector pCMV-CD63-mCherry. The vector pCMV-CD63 C145A,C146A -YFP used for FRET experiments was generated by exchange of the mCherry sequence with the YFP sequence from the vector pCMV-CD63-YFP-FLAG 40 using XhoI and ApaI restriction sites, all other vectors used were previously described 40,56 . All sequences of cloning sites were veri ed by Sanger sequencing.…”

Let There Be Light!

“…The different technologies described herein all have their advantages and drawbacks, so the choice of method depends on what question needs answering. There is also the possibility of combining the methods described above—for example, to utilize the superior resolution of the new types of microscopy in combination with molecular tools, or to apply different types of molecular tools in conjunction (e.g., in situ PLA and FRET) [37]. Microscopic analysis retaining the architectural information of where each analyzed cell is positioned, combined with highly multiplexed information on signaling network activity measured by mRNA-, protein expression, as well as post-translational modifications and protein interactions [38,39] will facilitate studies on cellular communication and on how these signals are interpreted in cells with different genetic and epigenetic background.…”