459-Abe-EGFRdynamics/Fig3A_PIP2_after_EGF
Author reviewedThe tracjectories of JF549–PI(4,5)P2 from colocalization of EGFR and PIP2 in living cells after EGF stimulation
Project
Dataset ID
Fig3A_PIP2_after_EGF
Kind
Image data
Title
The tracjectories of JF549–PI(4,5)P2 from colocalization of EGFR and PIP2 in living cells after EGF stimulation
Description
Time-lapse images of Halo-PI(4,5)P_{2} ( Halo-PLCdelta-PH) on HeLa cells between 1 and 5 min after EGF (epidermal growth factor) stimulation.
To simultaneously observe the dynamics of both EGFR(epidermal growth factor receptor) molecules and PI(4,5)P_{2} (phosphatidylinositol-4,5-bisphosphate) molecules upon EGF stimulation, EGFR-GFP and Halo-PI(4,5)P_{2} were imaged using dual-color total internal reflection (TIRF) microscopy. Halo-PI(4,5)P_{2} was visualized with JF549, a HaloTag ligand. This dataset includes only Halo-PI(4,5)P_{2} images.
The images of EGFR-GFP detected by GFP were deposited in another dataset in this project; Fig3A_EGFR_after_EGF.
Folder Name
Fig3A_PIP2_after_EGF
File Format
tif
File Size
104.9MB
File Separation Method
-
Figure Number
Fig3A
Dimensions
512x512x1x1x200
X Scale
33.28 micrometer
Y Scale
33.28 micrometer
Z Scale
-
T Scale
30 milliseconds
Channel
1 channel
Quantitative Data
Yes
Paper(s)
Biosample
Description
Halo-PLCdelta-PH, which serves as a reporter for PI(4,5)P_{2}, was labeled with Janelia Fluor 549 (JF549) after the expression of EGFR-GFP and Halo-PLCdelta-PH fusion proteins in EGFR-knockout (KO) HeLa cells.
Organism
Homo sapiens (NCBITaxon:9606)
Strain
-
Cell
-
Cell line
HeLa cell (CLO:0003684)
MeSH
Intrinsic Variable
The endogenous EGFR in HeLa cells was knocked out by using CRISPR/Cas9 gene editing.
Extrinsic Variable
EGFR-GFP and Halo-PLCdelta-PH were expressed exogenously. Ten nM Janelia Fluor 549 (JF549), a HaloTag ligand was added to visualize Halo–tagged PLCdelta–PH.
Ontology
Anatomical Entity
-
UBERON
-
Biological Process
response to epidermal growth factor (GO:0070849)
regulation of epidermal growth factor receptor signaling pathway (GO:0042058)
Cellular Component
plasma membrane (GO:0005886)
Molecular Function
phosphatidylinositol-4,5-bisphosphate binding (GO:0005546)
MeSH Anatomy
MeSH BP
MeSH CC
MeSH MF
Genetics
Gene
EGFR
Protein / Probe
-
Method / Genotype
gene knock out
Gene
-
Protein / Probe
Epidermal growth factor receptor
Method / Genotype
transfection
Gene
-
Protein / Probe
-Janelia Fluor 549
Method / Genotype
transfection
Treatments
Reagent / Compound
epidermal growth factor
Concentration
20 nanomolar
Fold Dilution
-
Imaging Method
Imaging Method
evanescent wave microscopy
Detection Method
complementary metal oxide semiconductor (CMOS)
Visualization Method
Janelia Fluor 549
Illumination Method
coherent illumination
Source of Contrast
distribution of a specific protein
Imaged Parameter
-
Sample Preparation Method
living cells
MeSH
Instrument
Body
Nikon TiE
Module
Hamamatsu W-view Gemini-2C
Light Source
a 488 nm laser and a 561 nm laser
Detector
Hamamatsu ORCA Fusion BT
Objective
Zido
Filter Set
Chroma ET525/50m and Semrock FF01-600/52-25
Dichroic
-