Monitoring Integrin Activation by Fluorescence Resonance Energy Transfer

Article (PDF Available)inMethods in molecular biology (Clifton, N.J.) 757:205-14 · January 2012with48 Reads
DOI: 10.1007/978-1-61779-166-6_14 · Source: PubMed
Abstract
Aberrant integrin activation is associated with several immune pathologies. In leukocyte adhesion deficiency (LAD), the absence or inability of β(2) integrins to undergo affinity upregulation contributes to recurrent infectious episodes and impaired wound healing, while excessive integrin activity leads to an exaggerated inflammatory response with associated tissue damage. Therefore, integrin activation is an attractive target for immunotherapies, and monitoring the effect of agents on integrin activation is necessary during preclinical drug development. The activation of integrins involves the structural rearrangement of both the extracellular and cytoplasmic domains. Here, we describe methods for monitoring integrin conformational activation using fluorescence resonance energy transfer (FRET).
Metadata of the Book that will be visualized online
Book Title Integrin and Cell Adhesion Molecules
Book SubTitle Methods and Protocols
Copyright Year 2011
Copyright Holder Springer Science+Business Media, LLC
Family Name
Kim
Particle
Given Name
Minsoo
Corresponding Author
Suffix
Division Department of Microbiology and Immunology, David H. Smith Center for
Vaccine Biology and Immunology
Organization University of Rochester
Address 601 Elmwood Avenue, Box 609, 14642, Rochester, NY, USA
Email Minsoo_Kim@urmc.rochester.edu
Family Name
Lefort
Particle
Given Name
Craig T.
Author
Suffix
Division
Organization
Address
Email
Family Name
Hyun
Particle
Given Name
Young-Min
Author
Suffix
Division
Organization
Address
Email
Motomu Shimaoka (ed.), Integrin and Cell Adhesion Molecules: Methods and Protocols,
Methods in Molecular Biology, vol. 757, DOI 10.1007/978-1-61779-166-6_14, © Springer Science+Business Media, LLC 2011
Chapter 14
Monitoring Integrin Activation by Fluorescence Resonance
Energy Transfer
Craig T. Lefort, Young-Min Hyun, and Minsoo Kim
Abstract
Aberrant integrin activation is associated with several immune pathologies. In leukocyte adhesion defi-
ciency (LAD), the absence or inability of b
2
integrins to undergo affinity upregulation contributes to
recurrent infectious episodes and impaired wound healing, while excessive integrin activity leads to an
exaggerated inflammatory response with associated tissue damage. Therefore, integrin activation is an
attractive target for immunotherapies, and monitoring the effect of agents on integrin activation is necessary
during preclinical drug development. The activation of integrins involves the structural rearrangement of
both the extracellular and cytoplasmic domains. Here, we describe methods for monitoring integrin
conformational activation using fluorescence resonance energy transfer (FRET).
Key words: FRET, Integrin, Cell adhesion, LFA-1, Mac-1, VLA-4, Fluorescence microscopy, Flow
cytometry
Integrins are heterodimeric transmembrane receptors that mediate
cell–cell and cell–extracellular matrix interactions (1). Integrins play
critical roles in a wide range of processes, including embryonic devel-
opment and the coordinated immune response (1, 2). The activation
state of an integrin is regulated by its conformation, similar to many
other membrane proteins and proteins involved in signaling (3).
Integrin receptors are not fixed in a particular conformation; rather,
they equilibrate between a compact, bent structure with low affinity
for ligand, and an extended, high-affinity conformation, with several
apparent intermediate conformational states (3). In contrast to the
low-affinity integrin, the activated integrin exhibits spatially separated
cytoplasmic tails and an extracellular headpiece that is extended away
from the plasma membrane (Fig. 1).
1. Introduction
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The structural changes that occur in the integrin molecule
during conversion from the inactive to the active form can be
observed and quantified using fluorescence resonance energy
transfer (FRET). FRET is a spectroscopic phenomenon in which
energy emitted from a fluorophore (donor) is transferred to a
second fluorophore (acceptor) in close proximity (<10 nm) and
with overlapping spectra, resulting in the emission of energy from
the acceptor. While initial studies using electron microscopy
demonstrated the changes in conformation between the resting
and activated integrin (4), fluorescence microscopy and FRET
provide a means to study the structure, and therefore function, of
integrins on the living cell surface. In addition, FRET is a powerful
method because it has the sensitivity for measuring dynamic inte-
grin activation with nanometer resolution. There are several
modalities for measuring FRET signals, including acceptor
photobleaching FRET, sensitized emission FRET, and ratiomet-
ric FRET. Here, we describe three different FRET methods for
studying integrin activation (Fig. 1).
1. K562 human leukemia cell line (ATCC).
2. RPMI 1640 media supplemented with 10% fetal bovine serum
(FBS), 100 mg/ml streptomycin, and 100 U/ml penicillin.
2. Materials
2.1. Acceptor
Photobleaching FRET
Fig. 1. Experimental systems used for FRET analysis of integrin activation. (a) Loss of
FRET between CFP- and YFP-tagged integrin cytosolic tails indicates spatial separation
during integrin activation. (b) Loss of FRET between FITC-conjugated anti-integrin
I domain antibodies and the membrane dye ORB indicates extracellular domain extension
during integrin activation.
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14 Monitoring Integrin Activation by Fluorescence Resonance Energy Transfer
3. a
M
-mCFP and b
2
-mYFP cDNA: Generated using the mam-
malian expression vectors pECFP-N1 and pEYFP-N1
(ClonTech), respectively (see Note 1).
4. G418 sulfate.
1. L-15 media (Gibco) containing 2 mg/ml
d-glucose.
2. Nikon Eclipse TE2000-E microscope coupled to a
QuantEM:512SC CCD camera (Photometrics).
3. Imaging filters (Chroma): YFP (HQ500/20×, Q515LP,
HQ535/30M), CFP (D436/20×, 455DCLP, D480/40M),
and YFP photobleach (D535/50×, Dichroic Full Mirror,
Metal Slug).
1. NIS-Elements software (Nikon).
1. GD25 cells (5).
2. DMEM supplemented with 10% FBS, 100 mg/ml streptomy-
cin, and 100 U/ml penicillin.
3. a
4
-mCFP and b
1
-mYFP, generated using the mammalian
expression vectors pECFP-N1 and pEYFP-N1 (ClonTech),
respectively (see Note 1).
4. Amaxa transfection system, including nucleofector kit V
(Lonza).
1. Trypsin-EDTA.
2. L-15 media containing 2 mg/ml
d-glucose.
3. Delta T dish, 0.17 mm.
4. Human VCAM-1-Ig recombinant fusion protein (R&D
Systems).
5. Nikon Eclipse TE2000-E microscope coupled to a
QuantEM:512SC CCD camera (Photometrics) with a dual-
view image splitter (Photometrics).
6. CFP/YFP dual-band filter set (Chroma).
7. Filter wheel with excitation filters (431–441 nm/490–510 nm).
8. White-light TIRF illuminator (Nikon).
9. Perfect focus unit (Nikon) and vibration isolation system
(Technical Manufacturing Corp.).
1. AutoQuant and AutoDeblur software (Media Cybernetics).
1. 1-Step Polymorphs cell separation media (Accurate Chemical).
2. Neutrophil isolation buffer (NIB): Hanks’ balanced salt
solution (HBSS) without calcium chloride or magnesium
chloride buffered with 10 mM HEPES, pH 7.4, and 0.1%
bovine serum albumin (BSA).
2.1.2. K562 Cell Treatment
and FRET Measurement
2.1.3. Data Analysis
2.2. Sensitized
Emission FRET
2.2.1. GD25 Cell Culture
and Transfection
2.2.2. GD25 Cell Treatment
and FRET Measurement
2.2.3. Data Analysis
2.3. Ratiometric FRET
2.3.1. Neutrophil Isolation
2.1.1. K562 Cell Culture
and Transfection
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3. Hypotonic erythrocyte lysis buffer (HELB): 10× phosphate-
buffered saline (PBS) without calcium chloride or magnesium
chloride, pH 7.4, diluted 1:100 in water to make a final
concentration of 0.1× PBS.
4. Normalization buffer: 10× PBS without calcium chloride or
magnesium chloride, pH 7.4, diluted 1:3 in water to make a
final concentration of 4× PBS, and containing 0.4% BSA.
1. Neutrophil labeling buffer (NLB): HBSS containing calcium
chloride, magnesium chloride, and 0.1% BSA (Sigma).
2. Fluorescein isothiocyanate (FITC)-conjugated monoclonal
antibody (mAb) directed against the Mac-1 integrin head
domain (see Note 4).
1. Octadecyl rhodamine B (ORB; Sigma): Prepared as a 200 mM
stock solution in DMSO.
Acceptor photobleaching FRET represents a simple end-point
technique for analyzing the spatial relationship of donor and
acceptor fluorophores. In this method, FRET is measured by
determining the fluorescence intensity of the donor before and
after photodestruction of the acceptor. In the protocol below, we
describe a technique we have used to measure the separation of
the a- and b-subunit cytoplasmic tails of the integrin Mac-1
(a
M
b
2
) in response to activation (Fig. 1). Monomeric forms of
CFP and YFP are genetically fused to the carboxy-terminus of the
a
M
- and b
2
-subunits, respectively. Cells expressing a
M
-mCFP and
b
2
-mYFP are analyzed by fluorescence microscopy. The proximity
of the Mac-1 cytoplasmic domains is reported as FRET efficiency,
or the extent to which the acceptor fluorophore quenches donor
fluorescence. Using this technique, it has been shown that activa-
tion of both LFA-1 (6) and Mac-1 leads to the separation of the
integrin a and b cytoplasmic tails.
1. K562 cells are maintained in RPMI media containing FBS
and penicillin/streptomycin.
2. K562 cells are transfected with a
M
-mCFP and b
2
-mYFP by
electroporation and maintained under selection with 1 mg/
ml G418 sulfate.
3. Stable K562 transfectants are sorted twice by immunofluores-
cence with anti-a
M
clone ICRF44 and then seeded at a single
cell per well in 96-well plates to obtain homogeneous and
stable clones.
2.3.2. Neutrophil Treatment
and Labeling
2.3.3. Flow Cytometry Data
Acquisition
3. Methods
3.1. Acceptor
Photobleaching FRET
3.1.1. K562 Cell Culture
and Transfection
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14 Monitoring Integrin Activation by Fluorescence Resonance Energy Transfer
1. K562 cells stably expressing a
M
-mCFP and b
2
-mYFP are
maintained in RPMI media containing FBS, penicillin/strep-
tomycin, and G418 sulfate.
2. Wash K562 cells and then resuspend in L-15 media supple-
mented with 2 mg/ml
d-glucose.
3. Allow K562 cells to settle for 5–10 min on coverslips mounted
on the microscope stage.
4. Treat K562 cells with integrin activation agonists/antagonists.
5. Acquire initial CFP and YFP images using a 60× oil immer-
sion, 1.40 NA objective lens (see Note 2).
6. Expose K562 cells to YFP photobleach excitation wavelength
for 3 min, without using any neutral density filters.
7. Acquire post-photobleach CFP and YFP images, using set-
tings identical to those used for the initial image capture
(exposure time, neutral density filter, etc.).
1. For each image, analyze only the membrane region on each
cell of interest.
2. Measure the mean CFP fluorescence intensity of the region
of interest on each individual cell before and after acceptor
photobleaching using Nikon NIS-Elements (or similar)
software.
3. Repeat the analysis above for YFP to verify >90% destruction
of the initial YFP signal after photobleaching.
4. Calculate FRET efficiency (E) with the following formula:
CFP Pre CFP Post
1 ( () / () )=−E Fd Fd
(7),
where F
CFP
(d)
Pre
and F
CFP
(d)
Post
are the mean CFP emission
intensity of pre- and post-photobleach images.
In the previous FRET method, acceptor photobleaching FRET,
the donor fluorescence was monitored to determine the extent of
energy transfer and, thus, distance between donor and acceptor.
In sensitized emission FRET, the fluorescence of the acceptor is
monitored during excitation of the donor. Unlike acceptor pho-
tobleaching FRET, the sensitized emission FRET method allows
for measurement of energy transfer over a time course. Sensitized
emission FRET is also called the three-cube method, as the neces-
sary measurements for data acquisition and signal correction are
performed with three different filter sets on a fluorescence micro-
scope. In the protocol below, we outline a method for monitoring
the activation state of the integrin VLA-4 (a
4
b
1
) over time in an
adherent cell. We have coupled this FRET method with TIRF
3.1.2. K562 Cell Treatment
and FRET Measurement
3.1.3. Data Analysis
[AU1]
3.2. Sensitized
Emission FRET
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microscopy to analyze specifically integrins in close contact with
an adhesive substrate. Similar to the acceptor photobleaching
method above, we describe an experimental setup in which the
donor (CFP) and acceptor (YFP) are genetically fused to the
cytoplasmic tail of the integrin a- and b-subunits. Using this
method, we have shown that VLA-4 activation is sustained at the
leading edge of GD25 cells and migrating T lymphocytes (8).
About 5 × 10
5
GD25 cells are transfected with 2 mg a
4
-mCFP and
b
1
-mYFP using Amaxa nucleofector kit V (protocol T-24), and
the cells are then cultured overnight in DMEM containing FBS
and penicillin/streptomycin.
1. Detach adhered GD25 cells from the plate with 0.25% trypsin-
EDTA and resuspend the cells in L-15 medium supplemented
with 2 mg/ml
d-glucose.
2. Transfer the cells to a Delta T dish coated with 100 mg/ml
VCAM-1 and allow to settle for 10 min at 37°C.
3. Acquire CFP and YFP dual-emission images with CFP and
YFP excitations using a 0.5-s exposure time. Acquire images
every 10 s for 30 min through a 100× oil immersion, 1.49
NA objective lens.
Subtract the background for each fluorescence image and per-
form FRET efficiency calculations with the AutoQuant imaging
algorithm of AutoDeblur (see Note 3).
The ratiometric FRET method for measuring energy transfer from
donor to acceptor fluorophore utilizes the dependence of FRET
on the donor–acceptor ratio. As the concentration of the acceptor
fluorophore increases relative to the donor concentration, so does
the probability that an acceptor is available for energy transfer. In
the protocol below, we describe a method for measuring the spatial
proximity of the Mac-1 integrin extracellular domain headpiece to
the plasma membrane. The donor fluorophore is a FITC-conjugated
mAb directed against the integrin, and the acceptor fluorophore is
a rhodamine dye (ORB) that incorporates into the plasma
membrane. FRET is measured by holding the donor concentration
constant and varying the concentration of ORB. The fluorescence
intensity of the donor and acceptor is measured using flow cytom-
etry. By plotting the FITC mean fluorescence versus rhodamine
mean fluorescence, we obtain a curve whose slope indicates the
extent of FRET and, thus, the relative distance between donor and
acceptor for a large population of integrin molecules. Using this
method, we have shown that certain agonists, such as formyl
peptides, and activating mAbs induce the extension of the Mac-1
headpiece away from the plasma membrane.
3.2.1. GD25 Cell Culture
and Transfection
3.2.2. GD25 Cell Treatment
and FRET Measurement
3.2.3. Data Analysis
3.3. Ratiometric FRET
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14 Monitoring Integrin Activation by Fluorescence Resonance Energy Transfer
1. After allowing the reagents to reach room temperature, gently
layer 3 ml of heparinized whole blood obtained from healthy
human donors on top of 3 ml 1-Step Polymorphs in an 8-ml
round-bottom polystyrene tube.
2. Centrifuge the tubes for 45 min at 500 × g.
3. After centrifugation, the blood components will be separated
into distinct layers. The top cell layer contains mononu-
cleated cells, including monocytes and lymphocytes. The
second cell layer, separated from the top cell layer by a small
amount of clear solution, contains the neutrophils, or poly-
morphonuclear leukocytes. Erythrocytes are centrifuged to
the bottom of the tube, though there is typically a small
amount of erythrocyte contamination in the neutrophil layer.
Discard the lymphocyte layer and collect the neutrophil layer,
adding 1 ml of cells to 1 ml of NIB in each new 8-ml round-
bottom polystyrene tube. Each tube of layered blood should
provide 0.5–1 ml volume for the neutrophil layer.
4. Add 4 ml of NIB to each tube, bringing the volume to 6 ml,
and then centrifuge the tubes at 200 × g for 7 min.
5. Discard the supernatant. At this point, cells from multiple
tubes can be combined into a single tube. Gently resuspend
the cell pellet and wash once with 6 ml of NIB. At this point,
cells from multiple tubes can be combined into a single tube.
Centrifuge the tubes at 200 × g for 7 min.
6. Discard the supernatant. Gently resuspend the cell pellet.
Lyse contaminating erythrocytes by adding 4.5 ml of HELB
and gently inverting the tubes several times over 30 s.
Immediately add 1.35 ml of normalization buffer and gently
invert the tubes several times. Centrifuge the tubes at 200 × g
for 7 min.
7. Discard the supernatant. Gently resuspend the cell pellet and
wash once with 6 ml of NIB. Count cells using a hemocytom-
eter. You should be able to purify approximately 1–2 × 10
6
cells/ml whole blood. Centrifuge the tubes at 200 × g for
7 min.
1. Discard the supernatant. Gently resuspend the cell pellet in
NLB at a concentration of 10 × 10
6
cells/ml.
2. In 1.5-ml microfuge tubes, treat 2 × 10
6
neutrophils per
experimental group with integrin activation agonists/antago-
nists. Place the tubes on ice.
3. Label the cells with a FITC-conjugated mAb directed against
the extracellular Mac-1 integrin head domain for 1 h. Retain
a control group of cells that are unlabeled for use in determining
3.3.1. Neutrophil Isolation
3.3.2. Neutrophil Treatment
and Labeling
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base level signal and signal compensation during flow cytometry
acquisition.
4. Wash the cells three times with NLB containing the appropriate
experimental agonists/antagonists used during the treatment
step. During the washes, centrifuge the tubes at 200 × g for
5 min at 4°C.
1. For each experimental group, resuspend cells (2 × 10
6
) in 2 ml
of NLB and aliquot into four flow cytometry tubes in 0.5-ml
volume, keeping the tubes on ice.
2. For each set of four tubes in an experimental group, add 0,
0.25, 0.5, or 1.0 ml of the 200 mM ORB stock. This gives
final ORB concentrations of 0, 100, 200, and 400 nM. Mix
well and incubate the tubes for 20 min on ice before acquisi-
tion on flow cytometer (see Notes 5 and 6). Acquire data for
at least 10,000 cells.
1. The relationship between the donor FITC fluorescence and
the concentration of ORB in the membrane depends on the
distance between the donor and the membrane surface, L, to
the inverse fourth power (9):
DA D
/ (1 [ORB]),=+FF S
64
0
/2 ,SR L
where F
DA
is the donor (FITC) fluorescence in the presence
of acceptor (rhodamine), F
D
is the donor fluorescence in
the absence of acceptor, (ORB) is the concentration of
acceptor in the cell membrane (assumed to be proportional
to the ORB fluorescence), and S is the slope factor, which
depends on the Forster radius (R
0
) and distance between
donor and acceptor (L). The relative change in spatial sep-
aration of the donor and acceptor between two experimen-
tal conditions, called the distance ratio, can be calculated
as follows:
2. Using flow cytometry data analysis software, such as FlowJo
(Tree Star), gate on the neutrophil population. Obtain mean
fluorescence intensity (MFI) values for both the donor (FITC)
and the acceptor (rhodamine) for all samples.
3. For each experimental group, plot the data: acceptor MFI
(x-axis) and donor MFI (y-axis). Use the curve-fitting tool in
your graphing software to obtain a curve fit of data points in
each experimental group according to the equation: y = b/
(1 + ax). In the derived equation for the curve, b should have
3.3.3. Flow Cytometry
Data Acquisition
3.3.4. Data Analysis
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14 Monitoring Integrin Activation by Fluorescence Resonance Energy Transfer
a value similar to your initial donor MFI (F
D
), and a repre-
sents your slope factor, S, and should be on the order of
0.001–0.01.
1. Leu-221 in pECFP-N1 and pEYFP-N1 was replaced with
Lys to produce the monomeric mutant (10).
2. For all CFP/YFP fluorescence image acquisitions, a 1/8 neu-
tral density filter and 0.5-s exposure time is used.
3. For the sensitized emission FRET measurement, cross talk
between CFP and YFP that occurs with the CFP/YFP dual-
band filter set (CFP
Ex
/YFP
Em
) needs to be calculated and
accounted for. This is done using GD25 cells transfected with
a
4
-mCFP/wt b
1
or a
4
-mYFP/wt b
1
.
4. We have used FITC-conjugated anti-CD11b clone ICRF44
mAb (Ancell) to observe Mac-1 extension on neutrophils.
This is a mAb directed against the ligand-binding I domain in
the headpiece of the a-subunit of Mac-1.
5. To complete the experiment in a reasonable amount of time
and thus avoid variance in the signal from degradation of
the FITC-conjugated mAbs, we found it is best to stagger
the addition of ORB to each tube by 3 min. This allows
3 min for data acquisition to be performed for each tube on
the flow cytometer at the 20-min time point of incubation
with ORB.
6. In our experience, we have found that a negligible amount of
compensation is needed for FL-2, the acceptor channel,
whereas a significant amount of compensation is required
for FL-1, the donor channel. The user should determine the
appropriate compensation values using single-labeled cells.
References
4. Notes
1. Hynes, R. O. (2002) Integrins: bidirectional,
allosteric signaling machines. Cell 110,
673–687.
2. Evans, R., Patzak, I., Svensson, L., De Filippo,
K., Jones, K., McDowall, A., and Hogg, N.
(2009) Integrins in immunity. J. Cell. Sci. 122,
215–225.
3. Luo, B. H., Carman, C. V., and Springer, T. A.
(2007) Structural basis of integrin regulation and
signaling. Annu. Rev. Immunol. 25:619–647.
4. Takagi, J., Petre, B. M., Walz, T., and
Springer, T. A. (2002) Global conformational
rearrangements in integrin extracellular
domains in outside-in and inside-out signal-
ing. Cell 110, 599–511.
5. Wennerberg, K., Lohikangas, L., Gullberg,
D., Pfaff, M., Johansson, S., and Fassler, R.
(1996) Beta 1 integrin-dependent and -inde-
pendent polymerization of fibronectin. J. Cell
Biol. 132, 227–238.
6. Kim, M., Carman, C. V., and Springer, T. A.
(2003) Bidirectional transmembrane signaling
by cytoplasmic domain separation in integrins.
Science 301, 1720–1725.
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7. Miyawaki, A., and Tsien, R. Y. (2000)
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8. Hyun, Y. M., Chung, H. L., McGrath, J. L.,
Waugh, R. E., and Kim, M. 2009. Activated
integrin VLA-4 localizes to the lamellipodia
and mediates t cell migration on VCAM-1. J.
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9. Holowka, D., and Baird, B. (1983) Structural
studies on the membrane-bound immuno-
globulin E-receptor complex. 1. Characterization
of large plasma membrane vesicles from rat
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amphipathic fluorescent probes. Biochemistry
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10. Zacharias, D. A., Violin, J. D., Newton, A. C., and
Tsien, R. Y. (2002) Partitioning of lipid-modified
monomeric GFPs into membrane microdomains
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Author Queries
Chapter No.: 14 0001273593
Query Details Required Author’s Response
AU1 Please provide the placement of the reference number.
    • "The resting state of β 2 integrins shows a bent structure shaped like an inverted " V " with the low affinity headpiece closely approaching the plasma membrane [154,168], experimentally verified in live leukocytes by Förster resonance energy transfer (FRET) [169,170], in which FRET from α I domain (FITC-conjugated antibodies) to plasma membrane (Octadecyl rhodamine B, ORB) was observed in resting leukocytes and disappeared when the cells were activated. The bent ectodomain of β 2 integrins is about 11 nm above the plasma membrane, whereas the extended ectodomain is about 23 nm (with α I domain) [78]. "
    [Show abstract] [Hide abstract] ABSTRACT: Integrins are a group of heterodimeric transmembrane receptors that play essential roles in cell-cell and cell-matrix interaction. Integrins are important in many physiological processes and diseases. Integrins acquire affinity to their ligand by undergoing molecular conformational changes called activation. Here we review the molecular biomechanics during conformational changes of integrins, integrin functions in leukocyte biorheology (adhesive functions during rolling and arrest) and molecules involved in integrin activation.
    Full-text · Article · Dec 2015
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