Detail of Fig3A_hRasWT_GDP_3h



Project
Title
Images of the HeLa cells 3 h after transfection of the Alexa 488 labeled H-Ras wild-type (WT) with GDP.
Description
Images of the HeLa cells 3 h after transfection of the Alexa 488 labeled H-Ras wild-type (WT) with GDP. channel1; nucleus staining image, chennel2; Alexa 488 image, channel3; bright field image.
Release, Updated
2024-11-25
License
CC-BY
Kind
Image data
File Formats
.oif
Data size
2.3 MB

Organism
Homo sapiens ( NCBITaxon:9606 )
Strain(s)
-
Cell Line
HeLa Cell ( CLO_0003684 )

Datatype
-
Molecular Function (MF)
Biological Process (BP)
protein targeting ( GO:0006605 )
Cellular Component (CC)
Biological Imaging Method
fluorescence microscopy ( Fbbi:00000246 )
confocal microscopy ( Fbbi:00000251 )
X scale
0.172 micrometer/pixel
Y scale
0.172 micrometer/pixel
Z scale
-
T scale
-

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

Summary of Methods
See details in Ikari M, et. al. JACS Au. 2023 Jun 1;3(6):1658-1669.
Related paper(s)

Masaomi Ikari, Hiromasa Yagi, Takuma Kasai, Kohsuke Inomata, Masahiro Ito, Kae Higuchi, Natsuko Matsuda, Yutaka Ito, Takanori Kigawa (2023) Direct Observation of Membrane-Associated H-Ras in the Native Cellular Environment by In-Cell (19)F-NMR Spectroscopy., JACS Au, Volume 3, Number 6, pp. 1658-1669

Published in 2023 Jun 26 (Electronic publication in June 1, 2023, midnight )

(Abstract) Ras acts as a molecular switch to control intracellular signaling on the plasma membrane (PM). Elucidating how Ras associates with PM in the native cellular environment is crucial for understanding its control mechanism. Here, we used in-cell nuclear magnetic resonance (NMR) spectroscopy combined with site-specific (19)F-labeling to explore the membrane-associated states of H-Ras in living cells. The site-specific incorporation of p-trifluoromethoxyphenylalanine (OCF(3)Phe) at three different sites of H-Ras, i.e., Tyr32 in switch I, Tyr96 interacting with switch II, and Tyr157 on helix alpha5, allowed the characterization of their conformational states depending on the nucleotide-bound states and an oncogenic mutational state. Exogenously delivered (19)F-labeled H-Ras protein containing a C-terminal hypervariable region was assimilated via endogenous membrane-trafficking, enabling proper association with the cell membrane compartments. Despite poor sensitivity of the in-cell NMR spectra of membrane-associated H-Ras, the Bayesian spectral deconvolution identified distinct signal components on three (19)F-labeled sites, thus offering the conformational multiplicity of H-Ras on the PM. Our study may be helpful in elucidating the atomic-scale picture of membrane-associated proteins in living cells.

Contact
Takanori Kigawa , RIKEN , Center for Biosystems Dynamics Research
Contributors
Masaomi Ikari, Hiromasa Yagi

OMERO Dataset
OMERO Project
Source