Abstract
A feasible design is made to measure three protein/protein interactions to visualize signal pathways by time-lapse Förster resonance energy transfer (FRET) microscopy. When interacting proteins are in close proximity, excitation energy is provided to allow the energy flow from the first molecule to excite the second, followed by energy transfer to the third. By phorbol ester/calcium ionophore stimulation, for example, a real-time complex formation of ectopic IκBα/ERK/WWOX occurs as measured by FRET microscopy, indicative of an ongoing functional signaling. Hyaluronan induces membrane Hyal-2 signaling, which allows FRET measurement of the complex formation of ectopic Smad4/WWOX/Hyal-2 for causing bubbling cell death. If ectopic p53 is recruited to replace Hyal-2, the resulting ectopic Smad4/WWOX/p53 complex induces membrane blebbing without cell death. Together, in this perspective review article, we demonstrate the utilization of time-lapse FRET microscopy to visualize the signaling event via the tri-molecular protein complex formation and their biological outcomes. We show an initial two-protein binding to form the driving force to jumpstart the tri-molecular execution for the signal pathway.
Highlights
To understand how a molecular signaling path goes and whether the signaling works properly in cells, it is important to measure the protein/protein interactions in vivo in a real-time mode[1,2,3]
To further demonstrate the validity of time-lapse Förster resonance energy transfer (FRET) for signaling, here we show hyaluronan (HA)-mediated cell death caused by transiently overexpressed proteins[14]
If the driver signal is too strong in the beginning, the driver phase is directly merged with the execution phase such as in the Smad4/Hyal-2(-sp)/WWOX-mediated bubbling cell death (Figs. 1e, 3e; Video S4)
Summary
To understand how a molecular signaling path goes and whether the signaling works properly in cells, it is important to measure the protein/protein interactions in vivo in a real-time mode[1,2,3]. Supporting evidence as shown below reveals that time-lapse trimolecular FRET microscopy allows determination of the complex formation of the first two molecules as a “driver” leading to the tri-molecular interactions for signaling “execution” and eventual biological outcome. Hyaluronan initiates Hyal-2/WWOX/Smad[4] signaling for bubbling cell death
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