Live imaging of apoptotic signaling flow using tunable combinatorial FRET-based bioprobes for cell population analysis of caspase cascades

Miho Suzuki, Yutaka Shindo, Ryu Yamanaka, Kotaro Oka

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)

Abstract

Understanding cellular signaling flow is required to comprehend living organisms. Various live cell imaging tools have been developed but challenges remain due to complex cross-talk between pathways and response heterogeneities among cells. We have focused on multiplex live cell imaging for statistical analysis to address the difficulties and developed simple multiple fluorescence imaging system to quantify cell signaling at single-cell resolution using Förster Resonance Energy Transfer (FRET)-based chimeric molecular sensors comprised of fluorescent proteins and dyes. The dye-fluorescent protein conjugate is robust for a wide selection of combinations, facilitating rearrangement for coordinating emission profile of molecular sensors to adjust for visualization conditions, target phenomena, and simultaneous use. As the molecular sensor could exhibit highly sensitive in detection for protease activity, we customized molecular sensor of caspase-9 and combine the established sensor for caspase-3 to validate the system by observation of caspase-9 and -3 dynamics simultaneously, key signaling flow of apoptosis. We found cumulative caspase-9 activity rather than reaction rate inversely regulated caspase-3 execution times for apoptotic cell death. Imaging-derived statistics were thus applied to discern the dominating aspects of apoptotic signaling unavailable by common live cell imaging and proteomics protein analysis. Adopted to various visualization targets, the technique can discriminate between rivalling explanations and should help unravel other protease involved signaling pathways.

Original languageEnglish
Article number21160
JournalScientific reports
Volume12
Issue number1
DOIs
Publication statusPublished - 2022 Dec

ASJC Scopus subject areas

  • General

Fingerprint

Dive into the research topics of 'Live imaging of apoptotic signaling flow using tunable combinatorial FRET-based bioprobes for cell population analysis of caspase cascades'. Together they form a unique fingerprint.

Cite this