short description of your department

							Focus Labo : Anti-Glycophorin Specificity In Human Rbc
Agglutination Characterized By Flim-Fret Microscopy.


Dr Dominique DUMAS1, Pr Bibiana D. RIQUELME 2, Natalia de Isla1.
1 Département d’Imagerie et de Biophysique Cellulaire. Laboratoire Lemta UMR CNRS 7563 et IFR CNRS

111 « Mécanique et d’Ingénierie Cellulaire et Tissulaire ». 54505 Vandoeuvre-les-Nancy, France. Tél :
(33)03 83 68 34 64 – dumas@hemato.u-nancy.
2. Facultad Cs. Bioquímicas y Farmacéuticas. Universidad Nacional de Rosario. ARGENTINA.




Short description of our department

The Laboratory of Cell and Tissue Engineering          The “Imaging and Biophysic” department of the
created by Pr Stoltz in 1996 is focused on the         group uses some advanced cytometric and
mechanobiology of cells and tissues. The primary       spectroscopic techniques to detect biological
concern of our research group (UMR CNRS 7563           events involved in these interactions including
and IFR CNRS 111) is to observe and understand         flow     cytometry,     optical      scanning
the physiological consequences of applied              microscopy/deconvolution,          multiphoton
mechanical stresses in vascular, blood and             microscopy and fluorescence lifetime imaging
cartilage cells.                                       microscopy (FLIM).

Objective

In this short study, we considered the FRET signal     Cells (RBC) by specific monoclonal antibodies
to characterize the agglutination of Red Blood         (anti-glycophorin A or B).

Introduction
         Glycophorins A, B and C are abundant in       RBC. Different approaches of FRET have been
red blood cell (RBC) transmembrane integral            developed to characterize the hemoagglutination
proteins. Their highly glycosylated nature and high    (Figure 1) based on the interaction of fluorophores
sialic acid content account for the net negative       (Alexa488TM, DiO and DiI) located in the RBC
charge of mature RBC membrane, which is                membrane or combined to secondary antibody
physiologically important because it impedes any       directed against the primary agglutining Mab.
tendency to stick together in the circulation.         Combined intensity (spectral) and Lifetime (FLIM)
Glycophorin A (GPA) is specific to RBC and is          Imaging was used to discriminate the FRET signal
found at high density on the extra-cellular surface,   of molecules on their different lifetimes whereas
possibly linked also to Band3. In this study, we       their emission spectra overlap (Alexa488TM or DIO
have tested anti-(GPA plus GPB) specific mouse         / DiI) as independent phenomena of the
monoclonal antibodies (agglutining Mab) as             fluorophore concentration and photobleaching.
primary antibody (Table I) to directly agglutinate
Methods
        For the image series and spectra                      Imaging module (Becker&Hickl, Berlin) was
measurement, a Leica TCS SP2-AOBS equipped                    interfaced (signals, Pixel Clock, Frame Sync) to
with an acousto-optical beamsplitter and Argon                the scan controller of the Leica TCS-AOBS
(457nm, 476nm, 488nm, 514nm) and HeNe                         Multiphoton laser scanning microscope. The
(543nm and 633nm) lasers were used with a                     decay analysis measured by time-correlated
x63/1,32 oil immersion. Fluorescence lifetime                 single photon counting was performed using the
measurements were performed with a time-                      SPCImage software (Becker&Hickl GmbH). The
correlated single photon counting technique using             actual time resolution depends on the detector
a Ti-sapphire laser pumped by a continuous wave               with the PMT typically built-in the microscope
argon laser (Mira 900F-Verdi 8W, Coherent,                    attached to the non-descanned port (high
pinhole fully opened, beam expander 3) with a                 sensitivity photomultiplier R6357 Hamamatsu with
pulse width from 120 fs and a repetition rate of 76           a typical rise time of 1.4 ns).
MHz. For lifetime imaging, SPC-730 TCSPC




   GR                          GR              G                   G                   G                        G
                                               R                   R                   R                        R
                          TM
              Alexa-488                                                                                    TM
                                                                                               Alexa-488




   A                                       B                                       C




Figure 1. Confocal images of Red Blood Cells. 1A. For control, RBC agglutination has been verified in the presence
of agglutining antibody and revealed with a secondary antibody combined to Alexa488 TM. 1B. Two RBC populations
have been separately labeled with 1-1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiO, 5.7 mM,
Molecular Probes) or 3,3’ – dioctadecyloxacarbocyanine perchlorate (DiI, 2.15 mM, Molecular Probes) and
agglutination of a mixture of both population has been made in the presence of agglutining Mab. 1C. The DiI-RBC
were agglutinated in the presence of Mab and revealed by secondary antibody combined to Alexa488 TM. This is a
donor (Alexa-488TM)- acceptor (DiI) couple in a Forster resonance energy transfer process (FRET), whereby the
fluorescence of the donor is quenched by the acceptor whose fluorescence is enhanced provided these fluorophores
are in close proximity and the fluorescence of the donor overlaps the absorption spectrum of the acceptor. Then 33 µL
of the stock solution were added to RBC. In agreement with the low absorbance of DiI at 488 nm (  8000 M-1 cm-1)
as compared to that of Alexa-488TM (  65000 M-1 cm-1), their similar quantum yield of fluorescence and
concentration ratio ([Alexa488TM]= 20[DiI]), the contribution of the DiI fluorescence at 565 nm upon excitation at
488 nm is only due to the FRET process.
Results
         All the antibody used was found to directly   fluorescence alone from donor (without acceptor
agglutinate the human RBC. In view of the              excitation) or acceptor (without detection of
fluorescence properties depicted in RBC for the        fluorescence from unpaired donor). To avoid
pair of fluorophore Alexa-488TM (donor) and DiI        these limitations in the case of multi-labelling
(acceptor), it may be expected that upon               experiments, FLIM in dynamic-state provides a
agglutination of RBC, effective FRET should be         discrimination of molecules in their fluorescence
observed. FRET is a distance-dependent                 lifetime, which allows to evaluate the underlying
interaction between electronic states and depends      mechanism of energy transfer process. Figure 3
on the inverse sixth power of the intermolecular       demonstrates upon excitation at 780 nm the
interaction distance. For 3-17 Mab (Anti-GPA), the     FLIM-FRET from Alexa488TM to DiI for the 3-16
proximity requirement for the occurrence of FRET       and 3-46 Mabs only with a lifetime distribution in
cannot be met upon excitation at 488 nm as             the picosecond range. Similarly, effective FRET
shown in Figure 2 which displays the fluorescence      was not observed for the 3-17 and 3-47 Mabs.
spectrum (without acceptor emission at 565 nm).        The contrast in measured lifetime image (Figure
The FRET from Alexa-488TM to DiI was found for         4) is a reliable indicator for spatial variations in
3-16 Mab with detection of fluorescence from           donor-acceptor association. All together, these
acceptor. For 3-46 and 3-47 Mab, the spectra           results only revealed by FRET-FLIM strongly
analysis cannot solve the problems of spectral         support the importance of the specific reactivity
contaminations that arise in steady-state              with Glycophorin A or B of agglutining Mab.
(intensity) measurements, as specific detection of
Figure 2 : Fluorescence spectra of RBC stained with DiI, then agglutinated with different anti-glycophorin A (3-17
and 3-47) or anti-glycophorin B (3-16 and 3-46) antibodies and revealed by secondary IgG antibody combined to
Alexa 488TM. Excitation wavelength was respectively 488 nm for FRET in all the cases and adjusts to minimize the
excitation of the acceptor. Fluorescence spectra were recorded in selected region on the RBC agglutinate to show
different profiles (in and out the closed contact on the membrane of two RBC face to face).
Figure 3: FRET image of RBC agglutinated with 3-16 Mab. The color is determined by the amplitude of the
fluorescence lifetime values from 0 to 3 ns.

Acknowledgement

The authors would like to thank Dr. Blanchard (EFS Nantes - France) for the anti-GPA and anti-GPB specific
mouse monoclonal antibodies supply.



                         700

                         600

                         500
                                                                                              3-16
                         400
                                                                                              3-46
                    UA




                         300                                                                  3-47
                                                                                              3-17
                         200

                         100

                          0
                               0     500       1000        1500     2000       2500
                                                      ps




Figure 4 : Temporal histogram depicting the Alexa-488TM fluorescence lifetime distribution obtained by a
biexponential fit of the fluorescence decay at each position in an image of RBC aggregate. We assume that the
average lifetime measured is the sum of the true lifetimes of the donor in bound and unbound state, weighted by their
respective population. The average lifetime of the alone fluorophore (Alexa488 TM) is around of 4,13 ns. When the
Alexa488TM fluorophore is combined to secondary Mab (IgG), its average fluorescence lifetime is in the nanosecond
range (1,7-2,2 ns) but upon transferring energy, it may drop in picoseconds (350-750 ps). The reduced lifetime of
Alexa-488TM (indirectly combined to 3-16 and 3-46 Mab) indicates a molecular interaction with DiI incorporated in
the RBC membrane. The proximity requirement for the occurrence of FRET was not met for the 3-47 and 3-17 Mab.
References

Wouters, F.S et al. (2001) Imaging biochemistry inside cells. TRENDS in Cell Biology.11, 203-211.

Elangovan, M et al. (2003) Characterization of one-and two-photon excitation fluorescence resonance
energy transfer microscopy. Methods. 29, 58-73.

Jakobs, S et al. (2000) EGFP and DsRed expressing cultures of Escherichia coli imaged by confocal, two
photon and fluorescence lifetime microscopy. FEBS Letters. 479, 131-135.