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First published online 4 December 2002
doi: 10.1242/jcs.00231

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5ß1 integrin mediates strong tissue cohesion

Department of Surgery, University of Medicine and Dentistry-Robert Wood Johnson Medical School, CAB Room 7070, New Brunswick, NJ 08648, USA

View larger version (9K): [in a new window]   Fig. 1. (A) Parallel plate compression device. The apparatus (not drawn to scale) contains inner and outer rectangular Plexiglas chambers. The outer chamber (OC) is connected to a thermostatted circulating water pump and serves to regulate the temperature of the inner chamber (IC). The lower assembly (LA) screws into the base of the inner chamber. The position of its central core (CC), whose tip is the lower compression plate (LCP), can be adjusted vertically by a screw thread to set the distance between the two plates. The upper compression plate (UCP) is a cylinder about 15 mm long suspended from the arm of a Cahn recording electrobalance, labeled as B, by a 0.15 mm diameter nickel-chromium wire (NCW). Its position can be adjusted horizontally to place the UCP directly above the LCP. Both plates are coated with poly-HEMA before each use. During an experiment, a spheroidal cell aggregate, labeled as A, is positioned on the lower plate and raised until it contacts the upper plate. Compression of the aggregate reduces the load measured by the balance by an amount equal to the force acting upon the cell aggregate. (B) Diagram of a liquid droplet compressed between two parallel plates at shape equilibrium. R1 and R2 are the two primary radii of curvature, at the droplet's equator and in a plane through its axis of symmetry, respectively. R3 is the radius of the droplet's circular area of contact with either compression plate. H is the distance between the upper and lower compression plates. Because R1, R2 and H can all be directly measured with greater accuracy than R3, the latter parameter was calculated using Eqn. 2.

View larger version (24K): [in a new window]   Fig. 2. (A) Flow cytometric analysis of CHO-Ncad and CHO-A5 transfectants. Note the significant shift in mean channel fluorescence of positive cells, representing approximately a 100-fold increase in protein expression. (B) Western blot analysis of CHO-Ncad and CHO-A5 transfectants. 25 µg of protein from cell lysates of CHO-Ncad and CHO-A5 cells were separated by SDS-PAGE, blotted to PVDF and subjected to immunoblot analysis using appropriate antibodies. Enhanced chemiluminescence detected a weak 130 kDa band corresponding approximately to the known average molecular weight for N-cadherin in the CHO-B2 parent line. A much stronger 130 kDa band is evident in the CHO-Ncad transfectant. 5 integrin was undetectable in CHO-B2 cells but was strongly expressed by the CHO-A5 cell line as represented by a 120 kDa band.

View larger version (30K): [in a new window]   Fig. 3. Aggregate formation of CHO-B2, CHO-A5 and CHO-Ncad cell lines. Hanging drop cultures containing 2.5x106 cells/ml were incubated for 2-3 days then transferred to shaker flasks and incubated for another 2-3 days. Note that CHO-B2 (A) aggregates only formed thick, flat sheets, whereas aggregates of CHO-A5 (B) and CHO-Ncad (C) formed spheres. Bar, 1.67 mm.

View larger version (11K): [in a new window]   Fig. 4. Linear regression analysis of surface tension versus volume for CHO-Ncad and CHO-A5 aggregates. Aggregates of CHO-Ncad () ranging in volume from 0.1 to 0.3mm3 were subjected to TST. Linear regression analysis of the data generated a correlation coefficient (r2) of 0.099, indicating that no statistically significant correlation exists between and volume. A similar analysis of CHO-A5 (O) aggregates produced an r2 value of 0.402, suggesting a possible weak relationship between and volume.

View larger version (10K): [in a new window]   Fig. 5. Surface tension versus days in culture for CHO-Ncad and CHO-A5 aggregates. Surface tension of CHO-Ncad aggregates (white bars) remained relatively constant between 4 and 6 days in culture. The surface tension of CHO-A5 aggregates (shaded bars), however, increased between 4 and 6 days in culture. On each day, the surface tension of the CHO-A5 aggregates was greater than that of the CHO-Ncad aggregates.

View larger version (10K): [in a new window]   Fig. 6. Linear regression analysis of surface tension versus volume of CHO-A5 aggregates after 5 () and 6 () days in culture. The surface tension of CHO-A5 aggregates cultured for 5 days remained relatively constant over a threefold range in volume. Linear regression analysis generated a correlation coefficient (r2) value of 0.164, indicating that surface tension at 5 days is size independent. At 6 days in culture, however, a greater degree of scatter in the data gave rise to an r2 value of 0.550, indicating size dependence.

View larger version (37K): [in a new window]   Fig. 7. (Top panel) Western blot analysis of N-cadherin expression of CHO-B2 and 5 integrin transfected cell lines grown as 2D or 3D cultures. Cell lysates were prepared from cells grown on tissue culture plastic and cells grown as 3D spheroids. N-cadherin was detected by immunoblot analysis. Note the presence of a 130 kDa band, corresponding to the published molecular weight of N-cadherin. Note also that transfection of CHO cells with 5 integrin did not result in increased N-cadherin expression irrespective of whether cells were grown in conventional tissue culture or as spheroids. (Bottom panel) Assessment of cadherin function by fast aggregation assay. Cells from near-confluent plates of CHO-B2 (A,B), CHO-P3 (C,D), CHO-A5 (E,F) and CHO-Ncad (G,H) were detached by trypsin/calcium (0.05% trypsin/2 mM CaCl2) treatment. Cells were stained with the membrane intercalating dye PKH-2 and resuspended at a concentration of 1x106 cells/ml in 3 ml of either calcium/magnesium-free HBSS (A,C,E,G) or HBSS with 2 mM Ca2+ (B,D,F,H), transferred to shaking flasks and placed on a gyratory shaker at 37°C and 120 rpm. Aggregation was monitored 1 hour later and imaged by fluorescence microscopy. Note that only the CHO-Ncad cell line aggregated in a calcium-dependent manner (G,H).

View larger version (54K): [in a new window]   Fig. 8. FN-dependent aggregation of CHO-A5 cells. CHO-A5 cells secrete low levels of endogenous FN. When cultured in hanging drops in FN-depleted tissue culture medium, cells formed loose sheets (A). Addition of 300 µg/ml of exogenous FN resulted in compact spheroid formation (B). Bar, 1.0 mm.

View larger version (44K): [in a new window]   Fig. 9. Dose-dependent aggregation of FN-null CHO-5 cells. CHO-5 cells express high levels of 5 integrin but do not express N-cadherin or secrete FN. Cells were cultured in hanging drops either in the absence of FN (A) or in 3 (B), 30 (C), 100 (D) or 300 (E) µg/ml rat plasma FN. Note the dose-dependent aggregation and compaction of CHO-5 cells in response to the addition of exogenous FN. Bar, 0.5 mm.

View larger version (22K): [in a new window]   Fig. 10. Aggregate formation and compaction of CHO-A5 cells in the presence of RGD peptide. CHO-A5 cells secrete low levels of endogenous FN. Culturing these cells in FN-depleted medium resulted in formation of cellular sheets (A). With the addition of 100 µM RGD peptide CHO-A5 cells failed to form aggregates (B). Bar, 1.0 mm.

View larger version (27K): [in a new window]   Fig. 11. Aggregate formation and compaction of CHO-5 cells in the presence of recombinant monomeric FN. CHO-5 cells were cultured in FN-depleted medium with 100 µg/ml of either monomeric (A) or dimeric (B) rat plasma FN. Note that aggregates failed to form in the presence of FN monomers (A). Bar, 1.0 mm.