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*CIL – Cell Image Library accession number. Please use this to reference an image.

CIL:24065*  Cite 
Description

E-cadherin local dynamics were studied in mature junctions, that is, junctions engaged in adhesion for many hours, in which cadherin expression level is stable. After stable transfection with E-cadherin-GFP, E-cadherin dynamics were studied by 2-photon single-particle tracking and fluorescence recovery after photobleaching (FRAP) combined with 3D wide-field fluorescence microscopy, which allowed the recovery process to be analyzed from series of image stacks in the entire 3D region surrounding the photobleached volume. Image analysis of fluorescence recovery indicates that most E-cadherin did not diffuse in the membrane along mature junctions, but followed a first order turn-over process that was rate-limited by endocytosis. This is a time-lapse video of a FRAP experiment on MDCK cell junctions expressing E-cadherin-GFP (each image is a projection of 14 z-frames). Also available are z-stacks (0.3 µm) of images of the FRAP experiment before (CIL# 24066) and immediately following photobleaching (CIL# 24067). Photobleached regions are indicated by arrows. Scale bar: 10 µm.

Technical Details

MDCK E-cadherin-GFP cells were observed 3 days after seeding on glass coverslips, and ≈12–24 h after confluency, in DMEM–FCS supplemented with 10 mM Hepes, in a 37 °C observation chamber (POCmini-chamber-system, Tempcontrol 37–2 Digital PeCon), on an IX71 inverted microscope (Olympus) with a high numerical aperture objective (63× oil-immersion, NA = 1.25, PlanNeofluar, Zeiss). Two-photon photobleaching was performed with a femto-second laser tuned at 878 nm, pumped by a 10W CW 532 nm laser (Mira 900 and Verdi; Coherent). The position of the beam was controlled by VM500 galvanometric mirrors (GSI Lumonics) and the photobleaching duration by a shutter LS200 (NnmLaser), driven by MetaMorph. The measured excitation Point-Spread Function (PSF) is an ellipsoid with a 0.5-μm diameter in the focal plane and a 1.5-μm extension along the optical axis. Fluorescence recovery was spatially resolved under 1-photon excitation by fast 3D wide field videomicroscopy, using a DG-4 monochromator set at 480 nm (Sutter), a Coolsnap HQ CCD camera (Roper Scientific), and a PiFoc piezo-driven objective actuator (Physik Instrumente). The FRAP experiment was performed as follows. One point in a junction was photobleached for 200 ms with a 30 mW average power excitation intensity as measured at the objective back pupil. A stack of 14 images with a 0.3-μm vertical spacing were acquired before (CIL# 24066) and after (CIL# 24067) photobleaching. This is the FRAP time-lapse video, with a time interval of 27 s. All three movies are from supporting information for Proc Natl Acad Sci. (209) 106: 7010-7015.

Biological Sources
NCBI Organism Classification
Canis lupus familiaris
Cell Type
epithelial cell
kidney
Cell Line
MDCK
Cellular Component
adherens junction
catenin complex
Attribution
Names
Simon de Beco
Charles Gueudry
Francois Amblard
Sylvie Coscoy
Published
Proc Natl Acad Sci. (209) 106: 7010-7015.
Pubmed
19372377
Link
PNAS April 28, 2009 vol. 106 no. 17 7010-7015
Citation
Digital Object Identifier (DOI)
doi:10.7295/W9CIL24065
Archival Resource Key (ARK)
ark:/b7295/w9cil24065
Grouping This image is part of a group.
Imaging
Image Type
recorded image
Image Mode
widefield illumination
fluorescence microscopy
FRAP
time lapse microscopy
Parameters Imaged
fluorescence emission
Source of Contrast
distribution of a specific protein
Visualization Methods
EGFP
Processing History
annotated with arrows
Data Qualifiers
raw, unprocessed data
Sample Preparation
Methods
living tissue
Relation To Intact Cell
whole mounted tissue
Dimensions
Spatial Axis Image Size Pixel Size
X 240px ——
Y 232px ——
Time 27 seconds 66