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
* Author for correspondence (e-mail: anat015{at}mail.uni-wuerzburg.de)
Accepted 13 December 2002
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Summary |
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Key words: VE-cadherin, Biophysics, Permeability, Actin
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Introduction |
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Regulation of adhesion is necessary to allow cells to adapt to changing
environmental conditions occurring during morphogenetic cell rearrangement,
migration, wound repair and several steps of tumour invasion and metastasis
(Christofori and Semb, 1999).
A well studied example of rapid agonist-induced modulation of cadherin-based
adhesion is the inflammatory response of the vascular endothelium, which is
characterised by circumscribed separation of intercellular junctions to form
large paracellular gaps through which plasma proteins and blood cells can
leave the blood compartment to fulfil a variety of important functions such as
attacking invading pathogens (Allport et
al., 1997
; Dejana,
1997
; Michel and Curry,
1999
; Hordijk et al.,
1999
; Petzelbauer et al.,
2000
; Vestweber,
2000
).
A fundamental challenge is to understand how cells can actively regulate
adhesive strength in a wide dynamic range. The presently favoured model
postulates regulation of adhesive strength by alteration of catenin-mediated
tethering of the cytodomains to the cytoskeleton
(Angres et al., 1996;
Yap et al., 1997a
;
Hordijk et al., 1999
;
Angst et al., 2001
;
Baumgartner and Drenckhahn,
2002a
; Vasioukhin and Fuchs,
2001
). Direct evidence for this hypothesis has been difficult to
obtain because correlation between catenin modification (e.g. by
phosphorylation) and concomitant cytoskeletal disconnection is mostly
indirect. Moreover, inhibition of adhesive contact formation in the presence
of cytochalasins that inhibit actin polymerisation and induce fragmentation of
F-actin (Theodoropoulos et al.,
1994
; Urbanik and Ware,
1989
; Sampath and Pollard,
1991
) does not distinguish between direct cytoskeletal effects on
cadherin function and more general cellular consequences of cytochalasins
(from Greek cytos: cell; chalasis: collapse, relaxation)
including destabilisation of plasma membrane structure, cell shape and
contractile tonus (Kolega et al.,
1991
; Van Deurs et al.,
1996
). Even if these experiments are taken as evidence for
adhesive strengthening by cytoskeletal tethering of cadherins, it still
remains to be shown whether this effect is caused by cytoskeleton- and
catenin-induced inside-out modulation of the affinity of cadherins [similar to
the mechanism involved in affinity regulation of some integrins
(Calderwood et al., 2000
)] or
by other more indirect effects, such as cytoskeleton-mediated clustering of
cadherins at sites of cumulative adhesive strength
(Angres et al., 1996
;
Yap et al., 1997a
;
Yap. et al., 1997b
) and
damping of the lateral mobility of cadherins. In view of the apparent low
adhesive affinity of purified trans-interacting cadherin ectodomains
(millimolar range determined for VE-cadherin)
(Baumgartner et al., 2000a
;
Baumgartner et al., 2000b
)
linkage of cadherins to the cytoskeleton should facilitate rapid rebinding of
cadherins after dissociation, thereby increasing the number of bonds and
overall adhesive strength between the interacting cell surface [for
theoretical evaluation of this aspect, see Baumgartner and Drenckhahn
(Baumgartner and Drenckhahn,
2002a
)].
In the present study we provide direct evidence that two
permeability-increasing compounds known to cause gradual opening of
endothelial junctions, that is, the inhibitor of actin polymerisation,
cytochalasin D, and the Ca2+-ionophore A23187
(Suttorp et al., 1989;
Schnittler et al., 1990
;
Kuhne et al., 1993
;
Drenckhahn and Ness, 1997
;
Dejana, 1997
;
Hordijk et al., 1999
;
Michel and Curry, 1999
), have
a strong negative impact on the adhesion of VE-cadherin-coated microbeads to
the surface of cultured endothelial cells. Drug-induced reduction of adhesion
was not caused by changes of the affinity for trans-interaction but depended
on the decay of F-actin, and correlated with the increase of the lateral
mobility of cadherins and dispersal of cadherin-enriched plasmalemmal
microdomains. Reduction of cytoskeletal tethering is assumed to be the main
mechanism responsible for drug-induced weakening of cadherin-mediated
intercellular adhesion.
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Materials and Methods |
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Coating of polystyrene beads
After extensive vortexing, 10 µl solution of protein A-coated
superparamagnetic polystyrene microbeads (Dynabeads, diameter 2.8 µm,
Dynal, Oslo) containing 2x109 beads/ml were washed three
times using 100 µl of buffer A (100 mM Na-phosphate, pH 8.1). Washing was
performed by sedimenting the beads via application of a magnetic field for
1 minute using a magnetic tube holder (MPC-E-1, Dynal) and reuptake in
the corresponding buffer. The washed beads were suspended in 100 µl buffer
A containing 10 µg of either VE-cadherin-Fc or of the Fc-portion of human
IgG (for control experiments) and allowed to react for 30 minutes at room
temperature (RT) under permanent slow overhead rotation of the reaction tube
to avoid sedimentation and aggregation of the beads. After washing three times
in 100 µl of buffer B (200 mM triethanolamine, pH 9.0) beads were incubated
for 45 minutes in 100 µl buffer B containing 0.54 mg dimethyl pimelimidate
dihydrochloride (DMP, Pierce, Rockford, USA) at RT to covalently crosslink
protein A and bound Fc-portions. Free DMP was blocked by two washes for 30
minutes at 37°C in 100 µl 100 mM Tris pH 8.0. Finally, the beads were
washed three times in Hanks Balanced Salt Solution (HBSS, Gibco, Karlsruhe,
Germany) and stored in HBSS at 4°C for up to 5 days under permanent slow
overhead rotation. Concentration of VE-cadherin-Fc bound to the bead surface
(24.6 µm2/bead) was determined by ELISA as described previously
(Baumgartner and Drenckhahn, 2000a).
Cell culture
The microvascular endothelial cell line used (MyEnd) was generated from
mouse myocardium by immortalisation with polyoma middle T oncogene (PymT) as
described and characterised previously
(Golenhofen et al., 2002).
Cells were grown in Dulbecco's modified Eagle's medium (DMEM; Life
Technologies, Eggenstein, Germany) supplemented with 50 U/ml
penicillin-G/streptomycin and 10% fetal calf serum (Biochrom) in a humidified
atmosphere (95% O2/5% CO2) at 37°C. For experiments
cells were grown on coverslips coated with gelatine crosslinked with
glutaraldehyde (Schnittler et al.,
1993
). The culture was split once a week and used for experiments
between passages 5 and 20. MyEnd cells formed monolayers of highly elongated
cells frequently organised into whirl-like formations. The overall cell shape
and growth pattern resembled primary cultures of microvascular endothelial
cells from brain and skin (Karasek,
1989
; Rubin et al.,
1991
) and differed significantly from the typical cobblestone
pattern formed by macrovascular endothelial cells from various sources
(Schnittler et al., 1997
).
MyEnd cells were immunopositive for three endothelial marker proteins tested:
von Willebrand factor (Fig.
1A), VE-cadherin (Fig.
1B) and PECAM-1 (Fig.
1C).
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Laser tweezer
The home-built laser tweezer setup consisted of a Nd:YAG laser (1064 nm)
the beam of which was expanded to fill the back aperture of a high
NA-objective (63x1.2 oil, Zeiss), coupled through the epiillumination
port of an Axiovert 135 microscope (Zeiss, Oberkochen, Germany) and reflected
to the objective by a dicroic mirror (FT510, Zeiss). The laser intensity was
adjusted to 20 mW up to 200 mW in the focal plane. Beads resisting
displacement at 20 mW also resisted detachment at higher laser intensities.
This all-or-nothing behaviour was also observed under various drug conditions.
Therefore we decided to perform all tweezer experiments at 20 mW.
Protein-coated polystyrene microbeads were allowed to interact with the cell
surface of MyEnd monolayers for 15 minutes. Then 100-300 beads were probed
during the following 2-10 minutes by the laser tweezer.
Cytochalasin D, colchicine, the Ca2+-ionophore A23187 (all purchased from Sigma, St Louis, MO) and jasplakinolide (Calbiochem, Bad Soden, Germany) were used at 10 µM in DMEM. Jasplakinolide was applied for 60 minutes, whereas the other drugs were applied for 30 minutes under cell culture conditions as described above. Ca2+-free conditions were achieved by addition of 5 mM EGTA to the culture medium.
Cytochemistry
For visualisation of endogenous VE-cadherin, ß-catenin, and F-actin in
association with adhering VE-cadherin-Fc-coated beads, Dynabeads could not be
used because of strong autofluorescence. Therefore, immunolocalisation studies
were performed with latex-sulfate beads (Interfacial Dynamics, Portland, OR).
125 µl bead solution (5 µl packed beads) were washed twice in 1 ml MES
buffer (2-morpholino-ethane sulfonic acid, 25 mM, pH 6.0) by resuspension and
centrifugation at 3000 g for 10 minutes. Beads were
resuspended in 500 µl MES including 2.5 µg VE-cadherin-Fc and incubated
overnight at RT under permanent slow overhead rotation. After centrifugation
at 3000 g for 10 minutes beads were washed three times in 1 ml
PBS containing 0.1% glycine. Beads were stored for up to 5 days under
permanent overhead rotation at 4°C. Monolayers with adhering beads were
fixed at RT with 2% formaldehyde in phosphate buffered saline (PBS, pH 7.4)
for 5 minutes and permeabilised using 0.1% (v/v) Triton X-100 (Sigma) in PBS
for 3 minutes. After preincubation of the cells with 10% normal goat serum
(NGS) and 1% bovine serum albumin (BSA) (both from Sigma) in PBS for 30
minutes at RT, monolayers were incubated overnight at 4°C with rabbit
polyclonal VE-cadherin antibody directed against the cytoplasmic domain of
mouse VE-cadherin (kindly provided by D. Vestweber, Münster, Germany) or
mouse monoclonal antibody against ß-catenin (Transduction Laboratories,
San Diego, CA) (dilution 1:100 and 1:300, respectively). For visualisation of
F-actin coverslips were incubated with Alexa-phalloidin (1 U/µl, Molecular
Probes, Eugene, OR) for 1 hour at 37°C. For characterisation of MyEnd
cells immunostaining was performed with rabbit polyclonal antibodies against
von Willebrand factor (Sigma, dilution 1:200) or rat monoclonal antibody
against PECAM-1 (kindly provided by B. Nieswandt, Würzburg, Germany;
dilution 1:100). After washing with PBS (3x5 minutes) cells were
incubated with one of the following: Cy3-labelled goat anti-rabbit, goat
anti-rat or goat anti-mouse IgG (Dianova, Hamburg, Germany) at RT for 30
minutes. After final rinsing with PBS cells were mounted in 60% glycerol in
PBS containing 1.5% N-propyl gallate as an antifading compound.
For scanning electron microscopy endothelial cell monolayers with adhering VE-cadherin-Fc coated beads were fixed for 24 hours with 4% glutaraldehyde in HBSS. After dehydration with graded aceton series, critical point drying and sputtercoating with palladium-gold (CPD 030, Bal-Tec, Schalksmühle, Germany) cells were examined with a DSM-962 scanning electron microscope (Zeiss, Germany).
Quantification of F-actin and VE-cadherin
For determination of the relative F-actin contents of endothelial cells,
monolayers were fixed at RT with 3% formaldehyde in PBS for 15 minutes and
then permeabilised with 0.1% (v/v) Triton X-100 in PBS for 5 minutes.
Afterwards each coverslip was incubated with 500 µl (1 µg/ml) phalloidin
covalently labelled with tetramethyl-rhodamine isothiocyanate (TRITC) for 1
hour at 37°C (Faulstich et al.,
1983; Franke et al.,
1984
). Series of experiments with changing concentrations of
TRITC-phalloidin (0.1-10 µM) were conducted to show that the conditions
chosen allow saturation of binding. After washing three times for 5 minutes in
PBS, TRITC-phalloidin was extracted from the cells by two subsequent 1 hour
incubation steps with 1 ml of methanol at 37°C. Methanol supernatants were
pooled, centrifuged at 100,000 g for 20 minutes and quantified
in a fluorescence spectrometer at an excitation wavelength of 540 nm and an
emission wavelength of 563 nm.
The relative amount of endogenous VE-cadherin exposed on the cell surface
of MyEnd cells was determined by western blotting of cell cultures treated for
7 minutes with 0.05% trypsin (Serva) in DMEM in either the presence or the
absence of 5 mM EGTA at 37°C. After removal of the trpysin-containing
supernatant, monolayers were washed three times with PBS containing a mixture
of protease inhibitors (leupeptin, aprotinin, pepstatin, 20 µg/ml each,
Sigma). Cells were then removed from the culture dish by a rubber policeman
and dissolved immediately in 10% sodium docecylsulfate (SDS)-containing sample
buffer at 95°C for 3 minutes. Afterwards, samples were subjected to SDS
polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent western blotting
and immunodetection with hybridoma supernatant containing rat monoclonal
VE-cadherin antibody [mAb 11D4.1, specific for mouse VE-cadherin ectodomain
(Gotsch et al., 1997)] using
horseradish-peroxidase labelled goat anti-rat IgG (Dianova, Hamburg, Germany)
and the enhanced chemilumeniscence technique (ECL, Amersham, Braunschweig,
Germany).
For determination of the amount of VE-cadherin exposed on dorsal cell
surface cells were fixed with 2% formaldehyde for 10 minutes at RT without
permeabilisation. After blocking non-specific binding sites for 1 hour with
PBS containing 10% NGS and 1% BSA, monolayers were incubated overnight with
mAb 11D4.1 at 4°C. Under these conditions antibodies have no access to
lateral and basal cell surface as assayed by immunostaining. After several
washes with PBS monolayers were subjected to SDS-PAGE and western blotting.
Bound mAb 11D4.1 (rat IgG1) was detected by horseradishperoxidase-labelled
goat anti-rat IgG and ECL-technique. Confluent monolayers of human umbilical
cord venous endothelial cells (HUVEC)
(Schnittler et al., 1997)
served as controls (mAb 11D4.1 is specific for mouse VE-cadherin).
Single molecule optical microscopy
The experimental setup including data acquisition and automatic data
analysis has been described in detail elsewhere
(Schmidt et al., 1995). In
brief, samples were illuminated for 5 milliseconds by 514 nm light from an
Ar+ laser (C306, Coherent, CA) using a 100x objective
(PlanNeofluar, NA=1.3, Zeiss) in an epi-fluorescence microscope (Axiovert
135TV, Zeiss). The laser beam was defocused to an area of
500
µm2 at a mean intensity of 1.2 kW/cm2. Rayleigh
scattered light was effectively blocked by appropriate filter combinations
(515DRLPEXT02, Omega; 605DF50, Omega; 2xGG530; Schott, Mainz, Germany). Images
were obtained by a liquid-nitrogen-cooled, back-illuminated CCD-camera system
(Roper Scientific, Trenton, NJ; Micro Max 1300-PB) and stored on a PC.
Consecutive images were obtained at an illumination time of 5 milliseconds and
a constant delay of either 140, 170, 200, 250 or 500 milliseconds. During the
delay time, the laser was turned off by an accoustooptic modulator (1205 c-1
Isomet, Springfield, VA).
Single molecule fluorescence imaging was applied to both VE-cadherin-Fc-coated glass slides and to MyEnd cells. Glass slides (width 25 mm; Knittel, Berlin, Germany) were cleaned by sonification for 5 minutes in chloroform, subsequently dried in a stream of nitrogen and immediately incubated with 1 ml HBSS containing 0.01 mg/ml VE-cadherin-Fc and 1 mg/ml BSA (to block unspecific protein-glass interaction) for 45 minutes. Cells were treated as described above. Coated glass slides or glass slides with MyEnd cells were mounted in a home-built sample stage with temperature controller (20/20 Technology, Whimington, NC). For these experiments VE-cadherin was fluorescently labeled in solution (HBSS containing 1.8 mM Ca2+ and 1% w/v BSA) by addition of a tenfold molar excess of Cy3-labelled anti-human F(ab).
Single molecule microscopy data analysis
The position of each fluorescently labeled molecule was obtained with an
accuracy of 50 nm by an automatic analysis program
(Schmidt et al., 1995
).
Correlation of consecutive images allowed to reconstruct the 2-dimensional
trajectory of each molecule observed. The length of a trajectory is directly
related to the lifetime of a single cadherin-cadherin bond. The probability
for a trajectory of length n, i.e. n consecutive
observations given the underlying lifetime t is as follows:
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Results |
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Typically 70-80% of the beads suspended in DMEM (containing 1.8 mM
Ca2+) were tightly bound to the cell surface and resisted
displacement (detachment) by laser tweezers. Specificity of binding of
VE-cadherin-coated beads to the endothelial cell surface was confirmed by the
following control experiments: beads coated with BSA or human IgG instead of
VE-cadherin-Fc displayed strongly reduced frequency of binding
(Fig. 2, Table 1) with
9-12% of
beads resisting detachment by laser tweezer. In the presence of 5 mM EGTA
binding of beads was reduced to 23%. Preincubation of both VE-cadherin-coated
beads as well as cell cultures with hybridoma supernatant containing rat mAb
11D4.1 to external domain of mouse VE-cadherin reduced binding by
40%.
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|
Role of actin filaments
Vascular endothelial cells have been shown to respond to cytochalasin D
treatment by local dissociation of cell-to-cell junctions (intercellular gap
formation) and concomitant breakdown of the permeability barrier for
macromolecules (Drenckhahn and Ness,
1997; Michel and Curry,
1999
; Hordijk et al.,
1999
). Treatment of monolayers and microperfused capillaries with
the Ca2+-ionophore A23187 (1-10 µM) resulted in similar changes
(gap formation, increase in permeability), which was explained by
Ca2+-stimulated contraction of the cellular actomyosin system,
which was assumed to be strong enough to pull the cells apart by overcoming
cadherin-mediated adhesion (Suttorp et
al., 1989
; Schnittler et al.,
1990
; Goeckeler and
Wysolmerski, 1995
). However, another model for explaining
A23187-induced junctional dissociation implies
Ca2+-gelsolin-induced severing of F-actin as a major cause for
junctional barrier breakdown (Kuhne et
al., 1993
).
To obtain direct insight in the effect of cytochalasin D and A23187 on cadherin-mediated adhesion, we determined binding of VE-cadherin-coated beads to MyEnd-cells preincubated for 30 minutes with 10 µM cytochalasin D and 10 µM A23187, respectively. In confluent monolayers preincubated with either of these compounds, F-actin contents (assayed by TRITC-phalloidin binding) dropped to 68±7% (cytochalasin D) and 39±6% (A23187) of control cultures treated with the drug solvent ethanol (0.1%). Percentage of VE-cadherin-coated beads resisting detachment by laser tweezer dropped to 33.3±1.3% (n=3) (cytochalasin D) and to 34.4±1.9% (n=3) (A23187) of control levels (untreated cultures), whereas preincubation for 40 minutes with 0.1% ethanol or 10 µM colchicine (microtubule disrupting compound) did not attenuate adhesion (Table 1).
In a further series of experiments beads were first allowed to settle on monolayers for 15 minutes. Thereafter monolayers were treated with 10 µM cytochalasin D or 10 µM A23187 for another 30 minutes (postincubation protocol). The fraction of beads resisting displacement by laser tweezer dropped to 40-50% of control levels.
To further study the role of the actin filament system on cadherin-mediated adhesion, monolayers were preincubation with the F-actin stabilising compound jasplakinolide (10 µM), which promotes actin-polymerisation and inhibits depolymerisation-repolymerisation cycles. Preincubation for 60 minutes reduced bead adhesion by 40% of control levels showing again that polymerisation of actin is important for establishment of new adherens contacts. However, if jasplakinolide incubation was performed after beads had been allowed to settle and attach to the monolayer for 15 minutes (postincubation protocol) significant strengthening of bead adhesion was observed (20% above control values). These experiments show that stabilisation of F-actin enhances adhesion of established cadherin-cadherin contacts. Moreover these experiments allowed us to directly address the question whether A23187-mediated weakening of bead adhesion is causally related to A23187-induced depolymerisation of F-actin. In monolayers in which bead adhesion was stabilised by postincubation by jasplakinolide A23187 treatment was ineffective and did not cause significant weakening of bead adhesion. Together these experiments show the important role of actin dynamics in cadherin-mediated adhesion.
In order to rule out the possibility that reduction of adhesion induced by
cytochalasin D and A23187 is caused by loss (internalisation) of
surface-exposed cadherin molecules (Le et
al., 1999) or, alternatively, by changes of the affinity of
cadherin dimers by some kind of inside-out signalling, we determined both the
relative amount of surface-exposed cadherins and their apparent affinity in
response to treatment with cytochalasin D and A23187.
Amount of surface-exposed VE-cadherin in cytochalasin D and A23187
treated cells
The amount of VE-cadherin exposed on the total cell surface of MyEnd cells
was assayed by extracellular cleavage with trypsin. Previous experiments with
E-cadherin (Hyafil et al.,
1981; Pokutta et al.,
1994
) and VE-cadherin
(Baumgartner and Drenckhahn,
2002b
) have shown that cadherins are only sensitive to trypsin
cleavage in the absence of Ca2+, whereas in the presence of
millimolar Ca2+ cadherins completely resist cleavage. Treatment of
monolayers with 0.05% trypsin for 7 minutes in the presence and absence of
Ca2+ caused moderate dissociation but still no detachment of cells.
In the absence of Ca2+ (5 mM EGTA) trypsin treatment resulted in
complete disappearance of western blotting signals for VE-cadherin in both
control and drug-treated cultures (Fig.
3). This shows that virtually the entire VE-cadherin pool is
exposed on the cell surface and that drug treatment did not cause any
significant internalisation of VE-cadherins. The total amount of VE-cadherin
was not affected by drug treatment as judged by the western blotting signals
for VE-cadherin in corresponding cultures treated with trypsin in the presence
of 2 mM Ca2+. To determine whether these data obtained for the
whole cell surface of MyEnd cells are also valid for the dorsal cell surface
of the monolayer (site of bead interaction) we fixed control and drug-treated
monolayers with 2% formaldehyde and incubated the monolayers with monoclonal
antibody 11D4.1 that has access only to the dorsal cell surface under these
conditions as seen by diffuse immunostaining of the dorsal cell surface and
absence of any immunosignal in association with the junction-containing
lateral cell surface (not shown). The amount of bound mAb 11D4.1 (monoclonal
rat antibody) was determined by western blotting of monolayers with antibody
to rat IgG. As shown in Fig. 4,
there was only a slight increase of the immunoblotting signal in monolayers
treated for 30 minutes with A23187 and cytochalasin D. Both compounds do not
cause significant opening of intercellular junctions of MyEnd cells during
this time interval as judged from continuous non-interrupted immunostaining
for VE-cadherin and ß-catenin (not shown). Endothelial cells from human
umbilical cord (HUVEC) served as a negative control (mAb 11D4.1 is specific
for mouse VE-cadherin external domain).
|
|
These experiments exclude the possibility that significant reduction of bead adhesion on drug-treated monolayers might be caused by loss (reduction) of VE-cadherin from the dorsal cell surface.
Determination of koff and lateral mobility of VE-cadherin
by single molecule fluorescence
The experiments described above showed that significant reduction of
VE-cadherin-mediated adhesion in response to reduction of F-actin by treatment
with cytochalasin D and A23187 cannot be explained by a decrease of the
surface concentration of VE-cadherin molecules. In order to address the
question whether changes of translational entropy (lateral mobility),
reduction of affinity or a combination of both are responsible for decreased
adhesion in response to cytochalasin D or A23187, soluble VE-cadherin-Fc was
indirectly labelled by Cy3-tagged F(ab) directed against Fc-portion of human
IgG (further denoted as VE-cadherin-Fc*).
Trans-interaction of soluble VE-cadherin-Fc* with immobilised
VE-cadherin-Fc
In a first attempt, unlabelled VE-cadherin-Fc was adsorbed to glass surface
(solid phase) and then covered with 10 µg/ml VE-cadherin-Fc* in HBSS/BSA
(soluble phase) still containing free Cy3-F(ab) directed against human Fc. In
a first step, free Cy3-F(ab) molecules were allowed to bind to Fc-portion of
adsorbed VE-cadherin-Fc under saturation (30 minutes). This allowed precise
focussing of the laser beam to the glass surface and determination of the
bleaching characteristics. Complete bleaches at 100 kW/cm2 within
10 seconds, no significant bleaching at 1 kW/cm2 within 150
illumination periods of 10 milliseconds each. Surface-bound Cy3-F(ab) was
absolutely immobile. After complete bleaching of surface-bound Cy3-F(ab)
individual trans-interaction events between soluble VE-cadherin-Fc* to
VE-cadherin-Fc on solid phase were recorded at illumination periods of 10
milliseconds. Binding events were seen as individual fluorescent signals
(fluorescence peaks) that required binding for 5 milliseconds to be
detected as specific signal against the background noise of freely diffusing
VE-cadherin-Fc* and Cy3-F(ab) in the soluble phase. An example of video frames
is shown in Fig. 6 for
trans-interaction of VE-cadherin-Fc* with endogenous VE-cadherin expressed on
endothelial cells (see paragraph below). The characteristic lifetime for
trans-interaction at 21°C between VE-cadherin-Fc* to VE-cadherin-Fc
adsorbed to glass surface was
=670±50 milliseconds, which
corresponds to an off-rate constant at 21°C of
koff=
1
1.49 second1.
No binding events (fluorescence signals) were seen if the glass surface was
coated with BSA only or if VE-cadherin-Fc-coated glass surface was covered
with Cy3-F(ab) in the absence of VE-cadherin-Fc in soluble phase. Furthermore,
trans-interaction was completely abolished by removal of Ca2+ from
the soluble phase.
|
In addition to determination of koff, this approach allowed us
for the first time to determine activation energy of cadherin
trans-interaction. Fig. 5 shows
an Arrhenius plot of the temperature dependency of koff. The
logarithm of koff is linearly dependent on 1/T (T, absolute
temperature) with a slope of 1.33x103. This allows
determination of the activation energy of E9.7 kJ/mol. This value is
typical of weak hydrophobic interactions
(Mortimer, 1987
).
|
Trans-interaction of soluble VE-cadherin-Fc* with endogeneous
VE-cadherin of endothelial cells
Endogenous VE-cadherin exposed on the cell surface of MyEnd monolayers
served as solid phase for determination of lifetime (koff) of
trans-interaction. Unlike experiments with coated glass surface there was no
unspecific binding of Cy3-F(ab) to cell surface as shown in controls in which
VE-cadherin-Fc was omitted from the Cy3-F(ab)-containing supernatant. Only in
the presence of both VE-cadherin-Fc* and Ca2+ (1.8 mM)
trans-interaction events (>5 ms) were observed
(Fig. 6). Average lifetime for
trans-interaction (duration of surface bound fluorescent spots) was =710
milliseconds (koff
1.41 second1) as calculated
by maximum likelyhood fitting of relative frequencies of consecutive
observations (Fig. 7) according
to Eqn 3 (see Materials and Methods section). After treatment with either
cytochalasin D or A23187, frequency of binding events increased 1.6-1.7 fold,
whereas the lifetime of trans-interaction events (koff) remained
unchanged (
=690 minutes, koff
1.45
second1). Slight increase of binding events (factor below 2)
corresponds to the slight increase of VE-cadherin exposed on the dorsal cell
surface in response to drug treatment (Fig.
4). This allows us to conclude that the association rate constant
(kon) and the overall affinity is not significantly altered by drug
treatment (similar level of trans-interaction events at similar surface
concentration of VE-cadherin).
|
Lateral mobility of VE-cadherin
The degree of lateral mobility of VE-cadherin in the plane of the lipid
bilayer of the dorsal surface of endothelial cells was determined by mean
square displacement (MSD) over time for individual molecules during the
lifetime of their trans-interaction with VE-cadherin-Fc* in soluble phase
(Fig. 8). Whereas cytochalasin
D and A23187 had no effect on lifetime of trans-interaction, both compounds
had a strong impact on lateral mobility of endogenous VE-cadherin during
trans-interaction with VE-cadherin-Fc*. Correlation of the position of
consecutive images allowed to reconstruct the two-dimensional trajectory of
each trans-interacting molecule. In control cells the diffusion coefficient
(calculated from initial slopes of plots shown in
Fig. 8) was D0.017
µm2 second1, whereas in cells treated with
A23187 and cytochalasin D average lateral mobility increased significantly to
D
0.17 µm2 second1 (cytochalasin D) and
D
0.35 µm2 second1 (A23187). Barrier free
diffusion occurred only at time scales of up to 500 ms. This indicates
corralling of trans-interacting complexes into restricted submicron barrier
free plasmalemmal areas (BFA), which expanded in response to actin
depolymerisation. BFA was calculated according to Edidin et al.
(Edidin et al., 1994
). In
control conditions BFA was
0.045 µm2 and increased several
fold in response to cytochalasin D (0.18 µm2) and A23187 (0.37
µm2) (Fig. 8).
Overall lateral mobility of cadherins (apparent D) in response to actin
depolymerisation can be concluded from concomitant increase of both, D and
BFA.
|
Distribution of trans-interacting VE-cadherins on endothelial cell
surface
In untreated controls, trans-interaction of VE-cadherin-Fc* occurred with
high frequency at circumscribed microdomains (up to 1 µm2
in size, Fig. 9A). Surface
areas between these preferential sites for trans-interaction displayed no or
only infrequent binding events. Treatment of cells with either A23187
(Fig. 9B) or cytochalasin D
caused significant dispersal of these preformed cadherin microdomains. At the
same time overall frequency of trans-interaction increased 1.6-1.7-fold
compared with controls (see above) showing that F-actin plays an important
role in control of both the size of cadherin microdomains as well as the pool
of cadherins present within the free dorsal cell surface.
|
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Discussion |
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---|
Regulation of adhesion by cytoskeletal tethering
Cadherin-coated microbeads adhere to the free dorsal cell surface of
neuronal cells (Lambert et al.,
2000) and endothelial monolayers (this study) by recruitment of
cellular cadherins, catenins and F-actin. Adhesion of VE-cadherin-coated beads
to the endothelial cell surface could be significantly reduced by cytochalasin
D and the Ca2+-ionophore A23187. Both compounds reduced cellular
F-actin content by 30-60%. Stabilisation of F-actin by pretreatment of cells
with jasplakinolide prevented drug-induced weakening of bead adhesion
demonstrating that both, cytochalasin D and A23187 mediate anti-adhesive
activity via destabilisation of the actin filament system.
These observations are in line with studies reporting disturbance of
junctional integrity of endothelial and epithelial monolayers by both,
cytochalasin D (Hirano et al.,
1987; Stevensen and Begg, 1994; Nybom and Magnusson, 1986;
Drenckhahn and Ness, 1997
) and
A23187 (Suttorp et al., 1989
;
Kuhne et al., 1993
;
Schnittler et al., 1997
;
Michel and Curry, 1999
).
A23187-induced barrier breakdown was suggested to be caused by
Ca2+-calmodulin-mediated myosin-based contractility
(Schnittler et al., 1990
;
Goeckler and Wysolmerski, 1995) and by Ca2+-gelsolin-induced actin
depolymerisation (Kuhne et al.,
1993
). Whereas these studies could not discriminate between an
essential facilitative role of cytoskeletal activity on overall monolayer
structure [cytochalasin-treated cells undergo shape changes and display
contraction of stress fibres and of actin-myosin-based gels
(Kolega et al., 1991
)] versus
a direct modulatory action of the actin-based cytoskeleton on adhesive
strength, the laser tweezer experiments performed in the present study allow
to conclude that drug-induced depolymerisation of actin exerts a direct
inhibitory action on cadherin-mediated adhesion. Importantly, weakening of
bead adhesion associated with depolymerisation of F-actin was not caused by
reduction of extracellular affinity of VE-cadherin. Thus the possibility of
inside-out modulation of affinity can be excluded as a possible mechanism of
how cadherin adhesion might be regulated by the actin filament system.
Based on our previous theoretical evaluation of the effects between
cytoskeletal linkage and extracellular adhesion we favor a mechanism by which
adhesion (transmembrane cooperativity) is primarily controlled by the degree
of lateral mobility of cadherins in the plane of the plasma membrane. As shown
in the present study on living cells and in our previous study using single
molecule atomic force microscopy, the life time of adhesive trans-interaction
of cadherins is only 550-700ms and occurs at extremely low affinity
[KD104 M
(Baumgartner and Drenckhahn,
2002b
)]. These very weak binding properties require immobilisation
of cadherins within the cytoskeleton in order to guarantee that cadherins can
rapidly rebind after dissociation. Without tethering to the cytoskeleton,
cadherins would be driven apart by increased lateral mobility (
10-to
20-fold increase, this study) and would require a prolonged time interval for
new collision and rebinding. Inhibition of lateral mobility by any kind of
immobilisation of cadherins would increase the number of bonds per unit
surface area and hence the overall adhesive strength by a factor of 10 to 100
(Baumgartner and Drenckhahn,
2002a
). This conclusion is further supported by experiments in
which the cytoplasmic domains of cadherins were extensively crosslinked and
clustered by the FKBP/FK1012 crosslinking system. Under conditions of
crosslinking and immobilisation of cadherins a significant improvement of
adhesion to cadherin-coated substrates was observed
(Yap et al., 1997b
).
Clustering of cadherins at the bead interface will probably further
strengthen adhesion by improving immobilisation of cadherins within clusters
and, in addition by increasing the local concentration of cadherins at
clustered sites. Moreover clusters stabilised by cytoskeletal linkage might
allow multivalent zipper-like binding between opposing cadherins which further
would increase the strength of adhesion
(Shapiro et al., 1995).
However such a mechanism is less likely to occur at sites of bead adhesion
because the average concentration of cadherins covering the bead surface (up
to 103 molecules/µm2) would be too low to allow
zipper-like formation (average distance between cadherins on bead surface is
about 30-60 nm).
In the present study we show that the diffusion coefficient of actively
trans-interacting endogenous VE-cadherin molecules within the free dorsal
plasma membrane (D=0.017 µm2 second1) is in
the order of the mobility determined previously for 90% of transfected
E-cadherin-GFP fusion proteins in MDCK-cells by fluorescence recovery after
photobleaching (Adams et al.,
1998
) and for the mobility of
50% of small particles (40-200
nm) coated with IgG against ectodomain of E-cadherin in transfected L-cells
(Sako et al., 1998
). Tenfold
higher values (0.3 µm2 second1) were observed
for
50% of E-cadherin-GFP fusion proteins in transfected L-cells by
single molecule fluorescence (Iino et al.,
2001
).
Whereas we have observed that lateral mobility of endogenous VE-cadherin
increased up to 10 fold in response to reduction of F-actin by 10 µM
cytochalasin D and A23187, respectively, overall mobility of cadherin
antibody-coated particles was reported by Sako et al. to decrease rather than
to increase in response to 1 µM cytochalasin D
(Sako et al., 1998). These
differences between both studies may depend on different drug concentrations
applied and on the fact that tracking of antibody-coated particles
(Sako et al., 1998
) may
primarily allow to determine bulk motion of clustered rather than of free
single molecules which were recorded in our study.
With respect to the surface distribution of actively trans-interacting
VE-cadherin molecules our observations indicate non-homogeneous distribution
of VE-cadherin. The size of microdomains enriched in actively
trans-interacting VE-cadherin was up to 1 µm2 and these hot
spots depended on an intact actin filament system as indicated by their
dispersal in response to cytochalasin D and A23187
(Fig. 9). At the same time
cytochalasin D and A23187 resulted in an enlargement of submicron domains in
which barrier free diffusion (BFA) is possible.
Cadherin microdomains located at the free cell surface may serve as preformed building blocks facilitating rapid formation of new junctions as soon as neighbouring cells approach each other for a limited time interval. The recruitment of freely diffusing single molecules into such initial sites of cell-to-cell contact may be more time consuming and less effective than the involvement of preformed microdomains with high local cadherin concentrations.
Within the cadherin microdomains VE-cadherin molecules were
compartmentalised (corralled) to submicron-sized BFA with average size of
0.045 µm2. Three fold larger BFA (0.16 µm2) was
observed by particle tracking for E-cadherin in transfected L-cells
(Sako et al., 1998) whereas
class I MHC molecules displayed BFA about twentyfold larger (1.13
µm2) (Edidin et al.,
1994
). Cytochalasin D and A23187 caused significant enlargement of
BFA for VE-cadherin (0.18-0.37 µm2) which is in the range of BFA
for E-cadherin with truncated cytodomain (0.50 µm2). Enlargement
of BFA for both cadherins can be most readily explained by loss of
cytoskeletal tethering caused either by truncation of E-cadherin cytodomain
(Sako et al., 1998
) or
inhibition of actin polymerisation (this study). Similar observations were
made with GPI-anchored membrane protein Qa-2 that appears to be indirectly
linked via transmembrane proteins to the actin cytoskeleton as the main
elastic barrier for diffusion sensitive to cytochalasin D
(Suzuki and Sheetz, 2001
).
Studies by single particle tracking and laser tweezers have shown that
barriers (tethers) determining BFA do not represent absolute obstacles because
they can be overcome by jumping events, dissociation of tethers or by pulling
forces (Kusumi and Sako, 1996
;
Sako et al., 1998
). Although
confinement to BFA is a phenomenon now documented for a variety of integral
membrane proteins and shown in this study for VE-cadherin to be sensitive to
the state of actin polymerisation, the functional significance of BFA is still
enigmatic.
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Acknowledgments |
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