QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 29 January 2003
doi: 10.1242/jcs.00322

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Baumgartner, W. Articles by Drenckhahn, D. Search for Related Content PubMed PubMed Citation Articles by Baumgartner, W. Articles by Drenckhahn, D.

Cadherin function probed by laser tweezer and single molecule fluorescence in vascular endothelial cells

¹ Institute of Anatomy and Cell Biology, University of Würzburg, Koellikerstr. 6, D-97070 Würzburg, Germany
² Institute of Biophysics, University of Linz, Altenbergerstr. 69, A-4040 Linz, Austria

View larger version (160K): [in a new window]   Fig. 1. Characterisation of MyEnd monolayers and sites of cellular adhesion of VE-cadherin-coated microbeads by immunofluorescence (A-E), Alexa-phalloidin-staining (F), phase contrast microscopy (G-I) and scanning electron microscopy (J,K). MyEnd monolayers display a typical granular immunoreactivity for von Willebrand factor (A) and junctional immunostaining for VE-cadherin (B) and PECAM-1 (C). Adhering beads (D-F) are characterised by cellular recruitment of VE-cadherin (D, localised with antibody to cytoplasmic domain), ß-catenin (E) and F-actin (F). Staining of junctions (D,E) and stress fibers (F) is blurred because the optical plane is focussed on beads at the dorsal cell surface. Beads not associated with VE-cadherin, ß-catenin and F-actin probably represent the population of 20% of beads not firmly attached to cells. Scanning electron micrographs show small cellular protrusions abutting on the bead surface (J,K). Bars, 20 µm (A-C); 10 µm (D-I); 2 µm (J,K).

View larger version (124K): [in a new window]   Fig. 2. Example of albumin-coated (A,B) and VE-cadherin-Fccoated (C,D) beads on dorsal surface of MyEnd monolayers probed with laser tweezer. Albumin-coated beads are displaced by laser tweezer, whereas VE-cadherin-Fc-coated beads resist displacement. Phase contrast images. Bar, 5 µm.

View larger version (69K): [in a new window]   Fig. 3. Determination of relative amount of surface-exposed VE-cadherin in response to cytochalasin D (10 µM) and A23187 (10 µM) assayed by trypsin treatment of MyEnd monolayers in presence and absence of Ca2+ and subsequent visualisation of VE-cadherin by western blotting. Virtually all VE-cadherin molecules are exposed on the cell surface and are sensitive to extracellular proteolysis. The total amount of VE-cadherin remains constant as judged from constant immunoblotting signal in the presence of Ca2+ that renders cadherins resistant to trypsin cleavage.

View larger version (42K): [in a new window]   Fig. 4. Determination of relative amount of VE-cadherin exposed on the dorsal surface of MyEnd monolayers assayed by the capacity of the dorsal surface of formaldehyde-fixed monolayers to bind the mAb 11D4.1 antibody specific for VE-cadherin ectodomain. In untreated monolayers (2) surface-bound IgG heavy chain signal is slightly reduced as compared to monolayers treated for 30 minutes with 10 µM cytochalasin D (3) and 10 µM A23187 (4). MyEnd not incubated with mAb 11D4.1 (1) and HUVECs incubated with mAb 11D4.1 (5) served as negative controls.

View larger version (33K): [in a new window]   Fig. 6. Video frames (10x10 µm) of MyEnd monolayers exposed to Cy3-F(ab)-labelled VE-cadherin-Fc. Individual events of trans-interaction are marked by arrows. Lifetimes of trans-interaction of the two events shown correspond to 400 milliseconds (lower arrow) and 600 milliseconds (upper arrow).

View larger version (18K): [in a new window]   Fig. 5. Determination of activation energy (E) and Arrhenius constant (A) for trans-interaction of VE-cadherin-Fc adsorbed to glass surface and Cy3-F(ab)-labelled VE-cadherin-Fc in soluble phase. Lifetime (koff) of individual trans-interacting events was determined by single molecule fluorescence.

View larger version (25K): [in a new window]   Fig. 7. Determination of lifetimes of trans-interaction of Cy3-F(ab)-labelled VE-cadherin-Fc with VE-cadherin-Fc adsorbed to glass surface and endogenous VE-cadherin exposed to cell surface of MyEnd monolayers. Average lifetime of trans-interaction at various conditions corresponds to the theoretical distribution of an average lifetime of 700 milliseconds.

View larger version (15K): [in a new window]   Fig. 8. Effect of cytochalasin D and A23187 on mean square displacement (MSD) vs. time of endogenous VE-cadherin molecules during their trans-interaction with Cy3-F(ab)-labelled VE-cadherin-Fc. The initial slopes allow determination of the diffusion coefficient D0.017 µm2/second (untreated cells), 0.17 µm2/second (cytochalasin D) and 0.35 µm2/second (A23187). The saturation value of the MSD allows determination of the barrier free areas to be BFA0.045 µm2 (untreated), 0.18 µm2 (cytochalasin D) and 0.37 µm2 (A23187). Typical trajectories of individual cadherin molecules in A23187-treated and untreated cells are shown on the right.

View larger version (90K): [in a new window]   Fig. 9. Visualisation of VE-cadherin binding sites on the dorsal cell surface of MyEnd monolayers. Note preferential sites for trans-interaction before addition of A23187 (A) and dispersal of these sites 10 minutes after addition of A23187 (B). Frames shown in A and B were taken from the same membrane area before and after A23187 application.