Detail of Fig5A_R-GECO



Project
Title
Live cell imaging of R-GECO in response to ionomycin stimulation in live HeLa cells.
Description
Live cell imaging of R-GECO in response to ionomycin stimulation in live HeLa cells.
Release, Updated
2022-11-23
License
CC BY
Kind
Image data
File Formats
.tif
Data size
10.5 MB

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

Datatype
-
Molecular Function (MF)
Biological Process (BP)
response to ionomycin ( GO:1904636 ) cellular response to ionomycin ( GO:1904637 )
Cellular Component (CC)
Biological Imaging Method
confocal microscopy ( Fbbi:00000251 )
X scale
433 nanometer/pixel
Y scale
433 nanometer/pixel
Z scale
-
T scale
30 seconds per frame

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

Summary of Methods
See details in Vu CQ, et. al. (2021) Sci Rep, Aug 13;11(1):16519.
Related paper(s)

Cong Quang Vu, Shun-Ichi Fukushima, Tetsuichi Wazawa, Takeharu Nagai (2021) A highly-sensitive genetically encoded temperature indicator exploiting a temperature-responsive elastin-like polypeptide., Scientific reports, Volume 11, Number 1, pp. 16519

Published in 2021 Aug 13 (Electronic publication in Aug. 13, 2021, midnight )

(Abstract) Genetically encoded temperature indicators (GETIs) allow for real-time measurement of subcellular temperature dynamics in live cells. However, GETIs have suffered from poor temperature sensitivity, which may not be sufficient to resolve small heat production from a biological process. Here, we develop a highly-sensitive GETI, denoted as ELP-TEMP, comprised of a temperature-responsive elastin-like polypeptide (ELP) fused with a cyan fluorescent protein (FP), mTurquoise2 (mT), and a yellow FP, mVenus (mV), as the donor and acceptor, respectively, of Forster resonance energy transfer (FRET). At elevated temperatures, the ELP moiety in ELP-TEMP undergoes a phase transition leading to an increase in the FRET efficiency. In HeLa cells, ELP-TEMP responded to the temperature from 33 to 40 degrees C with a maximum temperature sensitivity of 45.1 +/- 8.1%/ degrees C, which was the highest ever temperature sensitivity among hitherto-developed fluorescent nanothermometers. Although ELP-TEMP showed sensitivity not only to temperature but also to macromolecular crowding and self-concentration, we were able to correct the output of ELP-TEMP to achieve accurate temperature measurements at a subcellular resolution. We successfully applied ELP-TEMP to accurately measure temperature changes in cells induced by a local heat spot, even if the temperature difference was as small as < 1 degrees C, and to visualize heat production from stimulated Ca(2+) influx in live HeLa cells induced by a chemical stimulation. Furthermore, we investigated temperatures in the nucleus and cytoplasm of live HeLa cells and found that their temperatures were almost the same within the temperature resolution of our measurement. Our study would contribute to better understanding of cellular temperature dynamics, and ELP-TEMP would be a useful GETI for the investigation of cell thermobiology.
(MeSH Terms)

Contact
Takeharu Nagai , Osaka University , SANKEN , Nagai Laboratory
Contributors

OMERO Dataset
OMERO Project
Source