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

CIL:24069*  Cite 

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 z-stack (0.3 µm) of images is of a FRAP experiment on MCF7 cell junctions expressing E-cadherin-GFP before photobleaching. Also available is the z-stack immediately following photobleaching (CIL# 24070) and the time-lapse video of FRAP experiment (CIL# 24068). Photobleached regions are indicated by arrows. Scale bar: 10 µm.

Technical Details

MCF7 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 12 images with a 0.3-μm vertical spacing were acquired before (this movie) and after (CIL# 24070) photobleaching. The FRAP movie, with a time interval of 7.3 s, is CIL #24068. All three movies are from supporting information for Proc Natl Acad Sci. (209) 106: 7010-7015.

Biological Sources
NCBI Organism Classification
Homo sapiens
Cell Type
epithelial cell
invasive breast ductal carcinoma
mammary gland
Cell Line
Cellular Component
adherens junction
catenin complex
Simon de Beco
Charles Gueudry
Francois Amblard
Sylvie Coscoy
Proc Natl Acad Sci. (209) 106: 7010-7015.
PNAS April 28, 2009 vol. 106 no. 17 7010-7015
Digital Object Identifier (DOI)
Archival Resource Key (ARK)
Grouping This image is part of a group.
Image Type
recorded image
Image Mode
widefield illumination
fluorescence microscopy
Parameters Imaged
fluorescence emission
Source of Contrast
distribution of a specific protein
Visualization Methods
Processing History
annotated with arrows
Data Qualifiers
raw, unprocessed data
Sample Preparation
living tissue
Relation To Intact Cell
whole mounted tissue
Spatial Axis Image Size Pixel Size
X 428px ——
Y 258px ——
Z 12px 0.3µm