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Fig. 2. Schematic drawing of negatively stained and hydrated specimens and distri-
bution of integrins in a supported bilayer. (
A
) negatively stained specimen, (
B
)
hydrated vesicle, (
C
) orientation of integrins in a quartz supported bilayer with their
extracellular domains adsorbed to the quartz or in solution.
of a negatively stained specimen. The vesicles adsorbed on the grid became
flattened and dehydrated. With this technique, only the outside of the vesicle
can be visualized.
The vesicles with reconstituted integrin were further used to generate
supported planar bilayers by vesicle fusion to quartz slides
(14)
. Using
fluorescently labeled integrin
3, the time course of vesicle fusion to quartz
slides was monitored by total internal reflection fluorescence microscopy,
TIRFM (
Fig. 3
). This method allows excitation of fluorophores, which are in
close proximity to the quartz buffer interface (distance less than 100 nm)
(15)
.
The integrins are homogeneously distributed in the plane of the supported bilayer.
Fluorescence recovery after photobleaching (FRAP) experiments with
FITC-labeled
α
IIb
β
3 incorporated into supported planar bilayers of DMPG/
DMPC were performed to determine the lateral mobility of
α
IIb
β
3 incorpo-
rated into supported planar bilayers. As a control, the mobility of the
fluorescently labeled lipid NBD-PE in the same lipid mixture was determined.
DMPG/DMPC vesicles (molar ratio 1:1) containing 0.5 mol% NBD-PE were
prepared by the extrusion method according to
(16)
. By fusion to quartz slides,
supported planar bilayers with homogeneously distributed labeled-NBD PE
were generated. FRAP experiments were performed according to Kalb and
Tamm
(17)
and for NBD-PE a diffusion coefficient
D
= (4.4
α
IIb
β
10
-8
cm
2
/s
±
0.40)
×
with a mobile fraction of
F
= (93
±
7)% was calculated. These values are in
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