Detail of Figure3_Casp9_Casp3_0418-2



Project
Title
Time-course live imaging of Caspase-9 and Caspase-3 activation in apoptotic HeLa cells.
Description
Time-course live imaging of Caspase-9 and Caspase-3 activation in apoptotic HeLa cells. You can refer to the URL to get the corresponding quantitative information about the fluorescence of the leakage ratios for each chromophore-derived fluorescence into each measurement channel while re-analysis is needed: https://ssbd.riken.jp/data/279-Suzuki-CaspaseActivity/supplement/Figure3(Dual)-Signal-Separation-Matrix.xls
Release, Updated
2023-07-20
License
CC BY
Kind
Image data
File Formats
.oib
Data size
521.7 MB

Organism
Homo sapiens ( NCBI:txid9606 )
Strain(s)
-
Cell Line
HeLa cell ( CLO_0003684 )

Datatype
-
Molecular Function (MF)
cysteine-type endopeptidase activity ( GO:0004197 ) cysteine-type endopeptidase activity involved in apoptotic process ( GO:0097153 )
Biological Process (BP)
execution phase of apoptosis ( GO:0097194 )
Cellular Component (CC)
caspase complex ( GO:0008303 )
Biological Imaging Method
confocal microscopy ( Fbbi:00000251 )
fluorescence microscopy ( Fbbi:00000246 )
X scale
0.621 micrometer/pixel
Y scale
0.621 micrometer/pixel
Z scale
-
T scale
20 seconds of time interval

Image Acquisition
Experiment type
-
Microscope type
-
Acquisition mode
-
Contrast method
-
Microscope model
-
Detector model
-
Objective model
-
Filter set
-

Summary of Methods
See details in Suzuki M, et. al. Sci Rep. 2022 Dec 7;12(1):21160.
Related paper(s)

Miho Suzuki, Yutaka Shindo, Ryu Yamanaka, Kotaro Oka (2022) Live imaging of apoptotic signaling flow using tunable combinatorial FRET-based bioprobes for cell population analysis of caspase cascades., Scientific reports, Volume 12, Number 1, pp. 21160

Published in 2022 Dec 7 (Electronic publication in Dec. 7, 2022, midnight )

(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 Forster 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.

Contact
Miho Suzuki , Graduate School of Science and Engineering, Saitama University , Department of Applied Chemistry
Contributors

OMERO Dataset
OMERO Project
Source