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 movie is a z-stack (0.3 µm) of images of a FRAP experiment on MDCK cell junctions expressing E-cadherin-GFP before photobleaching. Also available is a z-stack of images immediately following photobleaching (CIL# 24067) and the time-lapse video of FRAP experiment (CIL# 24065). Photobleached regions are indicated by arrows. Scale bar: 10 µm.
MDCK E-cadherin-GFP cells were observed 3 days after seeding on glass coverslips, and ?12â24 h after confluency, in DMEMâFCS supplemented with 10 mM Hepes, in a 37 °C observation chamber (POCmini-chamber-system, Tempcontrol 37â2 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 (this movie) and after (CIL# 24067) photobleaching. The FRAP time-lapse video, with a time interval of 27 s, is CIL #24065. All three movies are from supporting information for Proc Natl Acad Sci. (209) 106: 7010-7015.
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