Chasing the signaling run by tri-molecular time-lapse FRET microscopy

Hsiang-Ling Kuo; Pei-Chuan Ho; Shenq-Shyang Huang; Nan-Shan Chang

doi:10.1038/s41420-018-0047-4

Chasing the signaling run by tri-molecular time-lapse FRET microscopy

Cell Death Discov. 2018 Mar 22:4:45. doi: 10.1038/s41420-018-0047-4. eCollection 2018.

Authors

Hsiang-Ling Kuo¹, Pei-Chuan Ho¹, Shenq-Shyang Huang^{1

2}, Nan-Shan Chang^{1

3

4

5

6}

Affiliations

¹ 1Institute of Molecular Medicine, National Cheng Kung University, Tainan, Taiwan Republic of China.
² 6Present Address: Graduate Program of Biotechnology in Medicine, Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan Republic of China.
³ 2Center of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan, Taiwan Republic of China.
⁴ 3Advanced Optoelectronic Technology Center, National Cheng Kung University, Tainan, Taiwan Republic of China.
⁵ 4Department of Neurochemistry, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY USA.
⁶ 5Graduate Institute of Biomedical Sciences, College of Medicine, China Medical University, Taichung, Taiwan Republic of China.

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.