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Fig. 4. Effect of ECM on cell-cell interactions, E-cadherin and actin subcellular
localization. (A) Caco-2 cells were plated at 20,000 cells/cm2 on
glass (a) or on native LN5-rich ECM (b), processed for immunofluorescence with
anti E-cadherin at confluence and analysed by confocal microscopy. Note that
E-cadherin is mostly located at the cell-cell junction membrane on domes
formed by cells grown on ECM. 3D confocal reconstruction (XZ) shows an
E-cadherin signal at the basal side of the Caco-2 cells grown on glass (c),
which disappears in cells grown on ECM (d) where the localization of
E-cadherin aggregation (i.e. 25 µm from the basal side out of a 30 µm
total height) is compatible with the position adherens junction (bar, 10
µm). (B) Caco-2 cells were plated at 50,000 cells/cm2 on glass
(a,a'), polylysine (b,b'), native LN5-rich ECM (c,c') type IV collagen (d,d'),
or LN2 (e,e'), processed for immunofluorescence with anti-E-cadherin and FITC
(green) and TRITC-conjugated phalloidin (red) after 72 hours of culture and
analysed by confocal microscopy. Note that ECM increases E-cadherin-actin
colocalization (yellow merge signal) and allows the formation of cortical
actin cytoskeleton (bar, 10 µm). (C) Actin labelling in cells grown (72
hours) on glass in presence of the ß1-integrin-activating antibody (bar,
10 µm). The results are representative of three independent
experiments.
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) or
on native LN5-rich ECM (
) deposited by HT29-MTX cells. (A) Phase
contrast micrography (X 25; bar, 200 µm) shows that domes appear on
cultures grown on ECM 2 days earlier than on plastic support. (B) shows the
confluence rates observed by phase microscopy. (C) At the indicated times,
domes were counted. Values represent mean±s.m.d. from triplicate wells
from three independent cultures. (D) shows a 3D confocal reconstruction of the
diffusion of FITC-conjugated biotin added at the apical side of Caco-2
cultures grown on plastic (a) or on LN5-rich ECM (b). (bar, 10 µm). Note
that the presence of ECM restricts biotin diffusion at the apical compartment,
which indicates a reinforcement of cell-cell interactions as compared to
cultures grown on plastic without ECM. The result is representative of three
independent experiments.
), 10 µg/ml (
) or 2.5µg/ml
(
6-integrin blocking antibodies.
Cells were then processed for immunofluorescence with anti-E-cadherin and FITC
(green) or TRITC-conjugated phalloidin (red) and analysed by confocal
microscopy. Colocalization (yellow merge signal) of E-cadherin and actin at
the apical-lateral side of cells was estimated by computer analysis of
confocal stack series. (A) shows that ECM components increase the
colocalization of E-cadherin and actin as compared to plastic without ECM, as
expressed in arbitrary units. (B) 3D reconstruction of cells grown on type IV
collagen in the absence (a) or in the presence (b) of anti-ß1-integrin
blocking antibody (bar, 10µm). The result is representative of three
independent experiments. (C) Effect of anti-ß1-integrin blocking antibody
on the colocalization of E-cadherin and actin in cells grown on LN2. (D)
Effect of anti-