© 2005 Nature Publishing Group http://www.nature.

com/natureimmunology ARTICLES

The initiation of antigen-induced B cell antigen

receptor signaling viewed in living cells by fluorescence
resonance energy transfer
Pavel Tolar, Hae Won Sohn & Susan K Pierce

Binding of antigen to the B cell antigen receptor (BCR) triggers signaling that ultimately leads to B cell activation. Using
quantitative fluorescence resonance energy transfer imaging, we provide evidence here that the BCR is a monomer on the
surface of resting cells. Binding of multivalent antigen clustered the BCR, resulting in the simultaneous phosphorylation of
and a conformational change in the BCR cytoplasmic domains from a closed to an open form. Notably, the open conformation
required immunoreceptor tyrosine-activation motif and continuous Src family kinase activity but not binding of the kinase Syk.
Thus, the initiation of BCR signaling is a very dynamic process accompanied by reversible conformational changes induced by
Src family kinase activity.

B cell responses are initiated by the binding of multivalent antigens to
B cell receptors (BCRs), which triggers signaling cascades that ulti-
mately lead to B cell activation. Much has been learned about the
biochemical details of the signaling cascades1–3. However, at present
little is known concerning the molecular nature of the response of the
BCR to antigen binding that results in signaling or the time frame over
which this response occurs. The BCR is a complex that contains a
membrane immunoglobulin (mIg) with a short cytoplasmic domain
that is noncovalently associated with a disulfide-linked heterodimer of
Ig-a and Ig-b; these contain in their cytoplasmic domains immunor-
eceptor tyrosine-activation motifs (ITAMs) that couple the BCR to the
signaling apparatus4. The earliest biochemical events after multivalent
antigen binding to the BCR include a change in the detergent solubility
of the receptor5, indicating a change in the local lipid environment of
the receptor, phosphorylation of the receptor on the Ig-a and Ig-b
ITAMs by the Src family kinases, mainly Lyn, and recruitment of the
Src homology 2 domain–containing kinase Syk to the phosphorylated
ITAMs1–3. It might be assumed that antigen binding leads to con-
formational changes in the BCR that are ‘translated’ into recognizable
changes in the BCR cytoplasmic domains that promote phosphoryla-
tion of the ITAMs by Src family kinases and facilitate the assembly of a
signaling complex, as has been suggested to occur for the T cell antigen
receptor after ligand binding6,7. However, little is known about the
structure of the cytoplasmic domains of the BCR in unstimulated or
activated cells. No secondary structure is evident in the polypeptides
representing the Ig-a and Ig-b cytoplasmic domains in vitro8, although
it is not apparent how these chains function in the context of the fully
assembled BCR in the plasma membrane. In part, understanding of
the earliest events in BCR signaling is limited by reliance on conven-
tional biochemical and imaging approaches that cannot provide a
continuous view of the initiation of signaling over the time and length
scales that may be necessary for capturing the first response of the BCR
to antigen binding.
High-resolution fluorescence imaging techniques that take advan-
tage of fluorescence resonance energy transfer (FRET) between fluor-
escent proteins have been developed that offer a quantitative approach
for studying signaling from receptors in living cells9. Because of the
extreme sensitivity of the efficiency of FRET to the distance between
FRET donor and acceptor fluorescent proteins, FRET imaging has
proven to be a valuable tool for studying protein-protein interac-
tions10,11 as well as conformational changes in a protein complex12–14.
Here we report the results of studies using FRET coupled with
quantitative microscopy to describe the earliest events in BCR signal-
ing in living cells in the first several seconds after antigen binding. The
results show an unexpectedly dynamic response of the BCR intra-
cellular domains to antigen binding as signaling is initiated.
RESULTS
Expression and stoichiometry of fluorescence-labeled BCRs
To investigate the arrangement of the intracellular domains of the
BCR, we attached monomeric versions of cyan fluorescent protein
(CFP) and yellow fluorescent protein (YFP)15 to the C termini of
nitrophenyl (NP)–binding membrane immunoglobulin heavy chains
(mIgH), m and g, as well as to Ig-a and Ig-b, using short flexible
linkers. We expressed the fluorescence-labeled BCR components in
various combinations with wild-type m, g and Ig-a in the mIgH–Ig-a–

Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland 20852, USA. Correspondence
should be addressed to S.K.P. (spierce@nih.gov).
Received 10 May; accepted 12 September; published online 2 October 2005; doi:10.1038/ni1262

1168 VOLUME 6 NUMBER 11 NOVEMBER 2005 NATURE IMMUNOLOGY

 ARTICLES

Figure 1 The function and stoichiometry of the

cell surface BCR composed of CFP- and YFP- a 40
Resting
µ–YFP µ–YFP Ig-α b
containing chains. (a) Flow cytometry of wild-type Stimulated
J558L cells (0) or J558L cells stably expressing 30
Ig-a, m or g cotransfected with constructs

MFI
encoding wild-type or CFP (C)– or YFP (Y)– 20 YFP YFP Anti-IgM–Cy3
labeled BCR components; cells were stimulated
Ig-α–YFP µ Ig-α–YFP
with NP-BSA for 1 min, fixed and permeabilized, 10
and stained with monoclonal antibody specific
for phosphotyrosine. Results are presented 0

α
γ α

α
as the mean fluorescence intensity (MFI) of

α
© 2005 Nature Publishing Group http://www.nature.com/natureimmunology

γ
µ 0

+ Y
+ Y

Y
αY

βY
αY

+ Y
γC µY
+ Y

γY

αC β
γC + α

γC β
+

αC + α
µC +

µC + β

+
+
phosphotyrosine staining of the BCR+ transfected

αC
µC

αC
YFP YFP Anti-IgM–Cy3
cells. All cells had similar expression of cell J558L J558L J558L J558L
surface BCR, as measured by flow cytometry Ig-α µ γ
staining with Cy5-conjugated antibody to c d
immunoglobulin. Data represent mean ± s.d. of 20

YFP fluorescence intensity (×102)

YFP fluorescence intensity (×102)

µ–YFP 1:2 1:1 µ–YFP 4:1 3:1 2:1
three experiments. (b) Confocal microcopy of Ig-α–YFP
Ig-α–YFP 15
J558L cells expressing only m–YFP and Ig-b or 15
only Ig-a–YFP and Ig-b, showing intracellular
YFP fluorescence (far left); J558L cells 10 1:1
expressing m–YFP, Ig-a and Ig-b complexes or m, 10
2:1
Ig-a–YFP and Ig-b complexes, showing cell
surface YFP fluorescence (middle, top or bottom, 3:1 5
5 1:2
4:1
respectively); or J558L cells expressing m–YFP,
Ig-a and Ig-b complexes or m, Ig-a–YFP and 0
0
Ig-b complexes stained with IgM-specific Cy3- 0 5 10 15 20 25 0 2 4 6 8 10 12
labeled antibodies and showing Cy3 fluorescence Anti-IgM–Cy3 fluorescence intensity (×102) Anti-Ig-β–Cy3 fluorescence intensity (×102)
(right, top or bottom, respectively). Original
magnification,
63. (c,d) The YFP and Cy3 surface fluorescence intensities of individual cells expressing either m–YFP, Ig-a and Ig-b complexes
(filled circles) or m, Ig-a–YFP, Ig-b complexes (open circles), stained with Cy3-labeled IgM-specific antibodies (c) or Cy3-labeled Ig-b-specific antibodies
(d). Dashed lines indicate the predicted YFP/Cy3 ratios for various stoichiometries of m:Ig-a (at ends of lines). The prediction is derived from a linear
fit to data from cells expressing m–YFP, Ig-a and Ig-b (c) and m, Ig-a–YFP and Ig-b (d); solid line is set as 1:1. Data in b–d are representative of three
independent experiments.

Ig-b+ J558L mouse cell line, which also expresses the immunoglobulin the same kinetics as the wild-type BCR after NP-BSA binding (data
l1 chain16. The emission spectra of all fluorescence-labeled BCR not shown).
components on the cell surface were identical to those of the When expressed individually in J558L cells, the m–YFP and Ig-a–
fluorescent proteins themselves. Stimulation of cells expressing fluor- YFP chains were retained in the endoplasmic reticulum and were
escent BCRs with the multivalent antigen NP–bovine serum albumin detected on the cell surfaces only when expressed together with Ig-a or
(NP-BSA) induced tyrosine phosphorylation of intracellular proteins m, respectively (Fig. 1b). This observation suggested that the cell
similar to that of cells expressing the wild-type BCR (Fig. 1a), surface contained only fully assembled BCRs and no pools of
indicating that the fluorescence-labeled mIg, Ig-a and Ig-b assembled individual chains, as predicted by studies showing that individual
into a functional BCR. As further evidence of their function, the BCR chains are not transported to the cell surface17. To determine the
fluorescence-labeled BCRs internalized to the same extent and with stoichiometry of the BCRs expressed on the cell surface, we analyzed

a First scan Second scan

b c
150 µ Ig-α–YFP 0.4
3:1 2:1
FRET channel fluorescence

µ–CFP Ig-α–YFP
458 nm 1:1
0.3
100
514 nm
EA

0.2
50 1:2

0.1

0
CFP (475–525 nm) FRET (>530 nm) YFP (>530 nm)
0.0
0 2 4 6 8 10 0.00 0.05 0.10 0.15 0.20 0.25 0.30
2 ED
YFP channel fluorescence (×10 )

Figure 2 FRET between BCR cytoplasmic domains measured by sensitized acceptor emission. (a) Excitation and emission wavelengths used to detect CFP,
FRET and YFP emissions of a cell expressing m–CFP, Ig-a–YFP and Ig-b complexes. Original magnification,
63. (b) Corrected cell surface FRET and YFP
channel fluorescence from individual cells expressing either m–CFP, Ig-a–YFP and Ig-b (open circles) or m, Ig-a–YFP and Ig-b (filled circles) as a control. Data
are representative of three independent experiments. (c) Mean EA and ED (± s.d.) calculated from cells (n ¼ 18) expressing m–CFP, Ig-a–YFP and Ig-b from
three experiments. Lines indicate the predicted EA/ED ratios for BCR complexes containing various m/Ig-a ratios (at ends of lines).

NATURE IMMUNOLOGY VOLUME 6 NUMBER 11 NOVEMBER 2005 1169

 ARTICLES

Figure 3 FRET between the cytoplasmic domains of the BCR chains

in resting B cells. (a) Average FRET efficiency (EA) for combinations of a mIgM mIgG

CFP (C)– and YFP (Y)–containing BCR chains in fully assembled mIgM 0.4
BCRs (left) and mIgG BCRs (right). For cells expressing m-CFP and m-YFP
(mCmY) or g–CFP and g–YFP (gCgY), results obtained with the equation

FRET efficiency
0.3
EMAX ¼ EA(n + 1)/n are presented, where n is the CFP:YFP stoichiometry.
Data represent mean ± s.d. of 11–28 cells from at least two experiments.
0.2
(b) Estimated distances between the centers of the fluorescent proteins
attached to BCR components. These estimates assume random orientation
of the fluorescent proteins to each other. The actual position of the 0.1
fluorescent proteins or the BCR chains cannot be determined and was set
© 2005 Nature Publishing Group http://www.nature.com/natureimmunology

arbitrarily; therefore the distances between m or g chains and Ig-a or Ig-b 0.0
in a BCR are given as a range, with the contribution of only one or equal

αY

αY
αY

γY
βY

βY
µY

βY

βY
αY
γC
µC

γC
αC

αC
µC

γC
µC
αC

αC
contribution of both mIg attached fluorescent proteins being considered.
b
the fluorescence intensities of m–YFP and Ig-a–YFP by quantitative
microscopy (Fig. 1b–d). We stained cells expressing m–YFP, Ig-a and
Ig-b and cells expressing m, Ig-a–YFP and Ig-b with indocarbocyanine µ µ γ
γ
(Cy3)–labeled IgM-specific antibodies and recorded the mean fluor- 54 Å 60 Å
escence intensities of YFP and Cy3 by confocal microscopy from 58–65 Å 55–62 Å
62–69 Å
equatorial sections of a region covering the entire plasma membrane 59–66 Å

of individual cells. The ratio of YFP to Cy3 fluorescence intensities was β

constant over the entire range of expression (Fig. 1c). Similarly, α 70 Å α 70 Å

staining the cells with Cy3-labeled Ig-b-specific antibodies showed a

constant ratio of YFP to Cy3 fluorescence intensities (Fig. 1d). These
data agreed best with a 2:1 stoichiometry of m:Ig-a, directly supporting
the reported 1:1 stoichiometry of mIgM:Ig-a–Ig-b of the complex
determined by biochemical means18. Thus, the transfected cells
expressed on their surfaces functional, fluorescence-labeled BCRs
that were assembled with consistent stoichiometry.
Measurement of FRET in resting cells
To determine the molecular organization of the cytoplasmic domains
of mIgH, we measured Ig-a and Ig-b FRET between the FRET donor
CFP and FRET acceptor YFP attached to each of the mIgH, Ig-a and
Ig-b chains of the BCR complex and expressed in pairs in J558L cells.
We also measured FRET in cells that expressed Ig-a–CFP and Ig-a–YFP
together to detect higher-order assemblies of the BCR. We quantified
FRET efficiencies by calibrated sensitized acceptor emission19,20. The-
oretically, measured FRET efficiencies between a pair of fluorescent
probes depend on the distance between the probes, their orientation
and the stoichiometry of the complex. Experience with attachment of
CFP and YFP to proteins of interest has suggested that the fluorescent
centers assume an almost random orientation to each other, probably
because of their attachment to protein through flexible linkers12.
Because the stoichiometry of the chains in the BCR is constant, the
changes in FRET efficiency in the BCR complex should consequently
depend mainly on the distance between the fluorescent proteins.
We measured FRET between m–CFP and Ig-a–YFP from the plasma
membrane in individual cells (Fig. 2). We collected fluorescence from
three channels: CFP (CFP excitation and CFP emission), FRET (CFP
excitation and YFP emission), and YFP (YFP excitation and YFP
emission; Fig. 2a). Control experiments showed that after correction
for crosstalk between channels, no signal was detected in the FRET
channel in cells expressing only the individual CFP or YFP or in cells
expressing CFP and YFP together (data not shown). Similarly, no
FRET was detected in cells expressing a BCR containing m, Ig-a–YFP
and Ig-b (Fig. 2b). In contrast, a strong FRET signal was detected in
cells expressing a BCR containing m–CFP, Ig-a–YFP and Ig-b
(Fig. 2b). The linearity of the data (Fig. 2b) indicated a constant
FRET efficiency over the range of BCR expression. To determine the
actual FRET efficiency from the data, we made calculations based on
calibrated FRET ratios19,20, yielding FRET efficiencies for both the
donor (ED), and the acceptor (EA). As the ratio of EA to ED is equal to
the stoichiometry of CFP to YFP for EA greater than 0 and ED greater
than 0, this allowed us to monitor the BCR assembly in each cell21. In
addition, FRET efficiency is sensitive to the relative amounts of the
interacting partners, and direct estimation of the stoichiometry
is important for interpretation of the data. In cells expressing
m–CFP, Ig-a–YFP and Ig-b, EA/ED was 1.97 ± 0.19 (mean ± s.d. of
n ¼ 18; Fig. 2c), confirming the 1:1 ratio of mIgM to Ig-a determined
above by direct comparisons of fluorescence intensities (Fig. 1c,d). For
the experiments described below, EA is presented as the FRET
efficiency, because EA provided more reliable FRET measurements as
it depended less on the detection of the noticeably dimmer CFP.
In resting cells, we did not detect much FRET in cells expressing m
and both Ig-a–CFP and Ig-a–YFP plus Ig-b (Fig. 3a), indicating that
on the surface of resting cells, the BCR was mainly monomeric and
was not present in higher-ordered clusters or oligomers. In contrast,
we obtained specific FRET values for all other combinations of CFP-
and YFP-containing BCR components, indicating that the BCR chains
were assembled into a complex with defined intracellular organization.
The surface BCR in cells expressing m–CFP, m–YFP, Ig-a and Ig-b had
a broad range of calculated CFP:YFP stoichiometries, as expected from
random pairing of the m–CFP and m–YFP chains into mIgM. Plotting
of the calculated EA values versus the stoichiometry showed that the
data fit the description of a homodimer (data not shown); therefore,
we have presented maximum efficiency (EMAX)22 values (Fig. 3a).
With that correction, we detected strong FRET between the m
chains, consistent with close proximity of the short intracellular tails
in the dimeric structure. As demonstrated above, expression of m–CFP,
Ig-a–YFP and Ig-b together led to assembly of BCRs with uniform
stoichiometry. In contrast, cells expressing m–CFP, Ig-a, Ig-b–YFP and
Ig-b and cells expressing m, Ig-a–CFP, Ig-b–YFP and Ig-b had variable
CFP/YFP ratios of greater than 2 and greater than 1, respectively,
indicating that both exogenous Ig-b–YFP and endogenous Ig-b
participated in assembly of the BCR complex, resulting in variable

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 ARTICLES

Figure 4 Antigen-induced clustering of BCR mIgM mIgG

extracellular domains. (a) FRET efficiency a 0.7
1.0 1.0
between Cy3- and Cy5-labeled NP-CGG bound 0.6

Relative binding
FRET efficiency

FRET efficiency
to the surface of cells expressing mIgM or mIgG

Relative binding
0.8 0.2 0.8
0.5
(filled circles) and total antigen bound to the
0.6 0.6
cell relative to the maximum bound during the 0.4
time course of the experiment (open circles). 0.3 0.4 0.1 0.4
Antigens were added after the first scan.
FRET 0.2 FRET 0.2
(b) FRET efficiency measured between Cy3- and 0.2
Binding Binding
Cy5-labeled Fab of immunoglobulin-specific 0.1 0.0 0.0 0.0
antibodies on the surfaces of mIgM- and mIgG- 0 100 200 300 400 500 600 0 100 200 300 400 500 600
© 2005 Nature Publishing Group http://www.nature.com/natureimmunology

expressing cells after the addition of NP-BSA Time (s) Time (s)
after the first scan. Data represent mean ± s.d. of b 0.5
six to eight cells from at least two experiments. 0.5

FRET efficiency

FRET efficiency
0.4
0.4
ED (data not shown). EA, however, was inde-
0.3
pendent of the stoichiometry, indicating that
0.3
the presence of the endogenous Ig-b did not
0.2
affect the accuracy of FRET measurements.
0 100 200 300 400 500 600 0 100 200 300 400 500 600
Higher FRET between m–CFP and Ig-b–YFP
Time (s) Time (s)
than between m–CFP and Ig-a–YFP (0.27
and 0.20; P ¼ 0.0001) indicated that m was
closer to the C terminus of Ig-b than to the
C terminus of Ig-a. Finally, low FRET detected between Ig-a–CFP compared the intracellular structure with that of the mIgM BCRs.
and Ig-b–YFP indicated that the Ig-a and Ig-b intracellular As for mIgM BCRs, there was no FRET detected between g, Ig-a–YFP,
domains were separated by a considerable distance. Although the Ig-b complexes and g, Ig-a–CFP, Ig-b complexes, indicating that the
exact position of the fluorescent proteins could not be calculated mIgG BCR was mainly in a monomeric form in resting cells (Fig. 3a).
from these measurements, we estimated the individual distances Comparison of the FRET values obtained for cells expressing all other
that would correspond to each FRET value to provide a relation- combinations of CFP- and YFP-containing chains of mIgG BCRs
ship to the dimensions of the BCR (Fig. 3b). Assuming random showed that the intracellular organization of mIgM BCRs and mIgG
orientation of the fluorescent centers, these estimates suggest that BCRs were very similar with some small but significant differences.
the data are consistent with the size and possible orientation of the These suggested that in the mIgG BCR, the g intracellular domains
BCR complex. were further apart (FRET efficiency, 0.24 versus 0.36; P ¼ 0.0001) and
During differentiation, B cells express different mIg isotypes, some protruded deeper toward Ig-a (0.25 versus 0.20; P ¼ 0.004) and Ig-b
of which have unique signaling properties23,24. We measured FRET (0.33 and 0.27; P ¼ 0.006) than did m intracellular domains in mIgM
between the intracellular domains of mIgG-containing BCRs and BCR (Fig. 3).

a 0 20 s 60 s 100 s 360 s 580 s c mIgM mIgG

0.5

CFP

0.4

YFP
FRET efficiency

0.3
FRET efficiency

0.4
0.3
0.2
0.1 0.2
0

0.40
b
FRET efficiency

0.1
0.35

0.30
0.0
0 100 200 300 400 500 600 0 100 200 300 400 500 600
0.25 Time (s) Time (s)
Ig-α–CFP Ig-α–YFP mIgH–CFP Ig-β–YFP
0 100 200 300 400 500 600 Ig-α–CFP Ig-β–YFP mIgH–CFP mIgH–YFP
Time (s) mIgH–CFP Ig-α–YFP

Figure 5 Dynamic antigen-induced changes in FRET between the BCR intracellular domains. (a) CFP, YFP and calculated FRET efficiency images of a cell
expressing g–CFP, Ig-a–YFP and Ig-b at various time points after antigen binding. Original magnification,
63. (b) FRET efficiency calculated from the
images in a, plotted against time. (c) FRET efficiency of cells expressing CFP and YFP attached to various chains (key; mIgH indicates m or g) of fully
assembled surface mIgM (left) or mIgG (right) BCRs; cells were stimulated with antigen after the first scan. EA is plotted as the FRET efficiency at the cell
surface over time. Data represent mean ± s.d. from six to eleven cells from at least three experiments.

NATURE IMMUNOLOGY VOLUME 6 NUMBER 11 NOVEMBER 2005 1171

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mIgM mIgG
b 0.25 WT WT
YY/FF 0.20 YY/FF

FRET efficiency
0.20

a 0.15
0.45 Control 0.15
PP2
0.40 0.10
0.10
FRET efficiency

0.35
© 2005 Nature Publishing Group http://www.nature.com/natureimmunology

0 100 200 300 400 500 600 0 100 200 300 400 500 600
0.30
WT 0.55 WT
0.45
YY/FF YY/FF
0.25
0.50

FRET efficiency
PP2 from 100 s 0.40
0.20 0.45
0 100 200 300 400 500 600
0.35
Time (s) 0.40
0.30
0.35

0.25 0.30

0 100 200 300 400 500 600 0 100 200 300 400 500 600
Time (s) Time (s)

Figure 6 The open conformation of the BCR cytoplasmic chains requires phosphorylation of the Ig-a and Ig-b ITAMs by Src kinases. (a) FRET efficiencies
of cells expressing g–CFP, Ig-a–YFP and Ig-b stimulated with antigen after the first scan in the absence (Control; filled circles) or presence (open circles)
of PP2. Gray circles represent the addition of PP2 100 s after antigen stimulation. Data represent mean ± s.d. of FRET efficiencies from four to six cells
from at least two independent experiments. (b) FRET efficiencies of cells expressing BCRs with Ig-a and Ig-b ITAM residues altered from tyrosine to
phenylalanine (YY/FF): top, m, Ig-aYY/FF–CFP and Ig-bYY/FF–YFP or g, Ig-aYY/FF–CFP and Ig-bYY/FF–YFP; bottom, m–CFP, Ig-aYY/FF and Ig-bYY/FF–YFP or g–CFP,
Ig-aYY/FF and Ig-bYY/FF–YFP. Cells were stimulated with antigen after the first scan. WT, wild-type counterparts. Data represent mean ± s.d. of FRET
efficiencies from five to nine cells from two experiments.

Antigen-induced clustering of the BCR ectodomains
To investigate the response of the BCR to binding of multivalent
antigen, we exposed cells expressing m, Iga–YFP and Igb complexes or
g, Iga–YFP and Igb complexes to a mixture of Cy3-labeled (FRET
donor) and indodicarbocyanine (Cy5)–labeled (FRET acceptor) NP–
chicken g-globulin (NP-CGG) and measured FRET efficiency. We
determined the time course of the binding of Cy3 and Cy5 to the cells
simultaneously. Antigen binding to the cells was essentially saturated
within the first minute after addition to the cells (Fig. 4a). There was
also an immediate increase in FRET between the Cy3- and Cy5-labeled
antigens (Fig. 4a). Similarly, we detected a rapid increase of FRET after
antigen stimulation in cells in which surface BCR was stained with a
mixture of Cy3- and Cy5-labeled Fab fragments of antibodies specific
for immunoglobulin (Fig. 4b). In both cases, FRET increased quickly
in the first 2 min of stimulation, with subsequent slower or no
increases. These results collectively demonstrate in living cells that
the binding of a multivalent antigen induces stable clustering of the
ectodomains of the BCR.
Antigen-induced changes in the BCR cytoplasmic domains
To detect possible rearrangements in the intracellular portion of
the BCR induced by antigen binding, we stimulated cells expressing
combinations of CFP- and YFP-tagged BCR components with
NP-BSA and measured FRET over time. An example of the images
of an individual cell expressing g–CFP, Ig-a–YFP and Ig-b complexes
showed that antigen binding resulted in the appearance of
microscopic clusters of the BCR on the surface of the cells beginning
at 20 s that became larger with time, through 580 s (Fig. 5a). The
FRET efficiency, calculated between CFP and YFP, seemed to reach
a maximum 20 s after the addition of antigen and decreased
thereafter. These data plotted as FRET efficiency versus time showed
a notable pattern (Fig. 5b). The FRET efficiency peaked immediately
(about 20 s) after the addition of the antigen and rapidly
decreased afterward but did not drop below the FRET between
BCR chains in the unstimulated BCR monomer (Fig. 5b). Calculation
of many traces from such time-lapse images of individual cells
confirmed that antigen induced a more complex pattern of
FRET changes between the cytoplasmic domains than was expected
from simple clustering of the ectodomains (Fig. 5c). Most mIgM-
and mIgG-expressing cells produced a characteristic pattern of
FRET changes over time, consisting of an immediate increase (within
20 s) followed by a drop on a time scale of less than 2 min, then
either a second phase of increase in FRET (in the case of mIgM) or a
plateau in FRET (in the case of mIgG). The magnitude of the
immediate peak of FRET was also slightly higher for mIgG BCR
than for mIgM BCR. This pattern was independent of the position
of the fluorescent proteins in the BCR for both mIgM and
mIgG (Fig. 5c). Notably, we found the same pattern of FRET in
cells expressing m or g and both Ig-a–CFP and Ig-a–YFP plus Ig-b that
detects the intermolecular interactions between the cytoplasmic
domains of two different BCRs. These observations indicate that the
immediate gain and subsequent loss in FRET were most likely due to
the initial clustering of the cytoplasmic domains of individual BCR
monomers and the subsequent opening up of this cluster. Consistent
with that conclusion, we noted that after antigen binding, the FRET
rose above but did not fall below that measured between BCR
components in the monomer. The BCR cytoplasmic domains showed
the dynamic gain and loss of FRET despite the fact that the BCR
ectodomains remained stably clustered (Fig. 4). Finally, after antigen-
induced clustering, the FRET measured between the Ig-a and Ig-b
components of the BCR and either m or g did not decrease below that
of the monomeric BCR, indicating that over the time course of the
experiment there was no measurable dissociation of the mIg molecules
from the Ig-a–Ig-b heterodimer.

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a Control b Syk recruitment e 0.40

Control
Piceatannol 0.45 IgG-Ig-α FRET 1.0 Latrunculin

Relative Syk recruitment

0.40
0.9
0.40

FRET efficiency
FRET efficiency
0.35
FRET efficiency

0.35 0.8
0.35
0.30 0.7 0.30
0.30
0.6
0.25 0.25 0.25
0.5
0.20 0.20 0.4 0.20
© 2005 Nature Publishing Group http://www.nature.com/natureimmunology

0 100 200 300 400 500 600 0 100 200 300 400 500 600 0 100 200 300 400 500 600
Time (s) Time (s) Time (s)

c WT FRET WT Syk recruitment d WT Control

∆ FRET ∆ Syk recruitment ∆ 0.40 MDC

0.35 1.4 0.50

Relative Syk recruitment

0.30 0.35
1.2 FRET efficiency

FRET efficiency
FRET efficiency

0.25 0.45

0.20 1.0
0.30
0.40
0.15
0.8
0.10
0.35 0.25
0.05 0.6

0.00 0.4
0.30 0.20
0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 500 600
Time (s) Time (s) Time (s)

0.50 Control
Figure 7 Syk activity and binding is not required for the drop in FRET, but recruitment of Syk 0.45
MβCD
correlates with the change in FRET. (a) FRET efficiencies of cells expressing g–CFP, Ig-a–YFP and

FRET efficiency
Ig-b complexes. Cells were stimulated with antigen after the first scan in the absence (Control) or 0.40

presence of the Syk inhibitor piceatannol. Data represent the mean ± s.d. of FRET efficiencies from 0.35
four to six cells from at least two experiments. (b) Syk recruitment in cells expressing g, Ig-a–YFP 0.30
and CFP–Syk. Cells were stimulated with NP-BSA after the first scan; relative Syk membrane
0.25
recruitment is expressed as the membrane/cytoplasmic CFP ratio. Syk recruitment over time is
superimposed with FRET data from cells expressing g–CFP, Ig-a–YFP and Ig-b. Data represent 0.20
mean ± s.d. of six cells from three experiments. (c) Syk recruitment and interaction with Ig-b 0 100 200 300 400 500 600
assessed in cells expressing Ig-a and Ig-b with deletion of two amino acids between ITAM tyrosine Time (s)
residues (D). Cells expressing g, Ig-aD and Ig-bD–YFP (D) or the wild-type counterparts (WT)
together with CFP–Syk were stimulated with antigen. Syk recruitment and interaction with Ig-b are presented as membrane/cytoplasmic CFP ratio or FRET
efficiency, respectively. Data represent mean ± s.d. from four to six cells from two experiments. (d) FRET efficiencies of cells expressing g–CFP, Ig-aD
and Ig-bD–YFP (D) or the wild-type counterparts (WT), stimulated with antigen. Data are expressed as mean ± s.d. from six to eight cells from three
experiments. (e) FRET efficiency of cells expressing g–CFP, Ig-a–YFP and Ig-b complexes. Cells were stimulated with antigen after the first scan in the
absence (Control) or presence of methyl-b-cyclodextrin (MbCD), latrunculin or monodansylcadaverine (MDC). Data represent mean ± s.d. of FRET efficiencies
from four to six cells from at least two experiments.

Relationship of FRET changes to BCR signaling residues in Ig-a and Ig-b ITAMs that were phosphorylated by Lyn
We sought to determine if the loss in FRET was dependent on the were altered from tyrosine to phenylalanine. These alterations resulted
activity of the first kinase in the BCR signaling pathway, Lyn, and if in a complete loss of inducible tyrosine phosphorylation of intracel-
the tyrosine residues in the ITAMs of the cytoplasmic domains of the lular substrates (data not shown). We measured FRET after antigen
Ig-a–Ig-b complex that are phosphorylated by Lyn were necessary for binding and compared it in each case to the FRET of cells expressing
the FRET loss. We treated cells expressing g–CFP, Ig-a–YFP and Ig-b the corresponding wild-type Ig-a and Ig-b chains. The alterations
complexes with the Src family kinase inhibitor PP2 (4-amino-5-(4- of ITAM tyrosine residues in the transfected Ig-a and Ig-b were
chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; ref. 25) before sufficient to eliminate the drop in FRET, resulting in a simple
adding NP-BSA. Inhibiting Lyn had a considerable effect on the clustering pattern (Fig. 6b).
FRET changes, converting the dynamic clustering and subsequent To determine if activity of Syk (the Src homology 2 domain–
opening pattern of the cytoplasmic domains to one of simple cluster- containing kinase recruited to the phosphorylated ITAMs of Ig-a and
ing (Fig. 6a). To determine if the observed opening of the cyto- Ig-b) was required for the drop in FRET, we treated cells with the Syk
plasmic domains required the continued presence of Lyn activity, inhibitor piceatannol before adding antigen26. Piceatannol had no
we added PP2 to the cells 100 s after adding NP-BSA. The addition of detectable effect on the FRET pattern (Fig. 7a), indicating that activity
PP2 at 100 s, just after the decrease in FRET occurred, resulted in a of Syk was not required for maintenance of the open conformation of
rapid increase in FRET, indicating that the oligomerized BCR cyto- the clustered BCR cytoplasmic domains. To determine the relationship
plasmic domains returned to a closed conformation (Fig. 6a). Thus, between the recruitment of Syk and the opening of the clustered BCR,
the open conformation of the cytoplasmic domains was reversible and we transfected a fluorescent construct that had CFP attached to the
required the continuous activity of Lyn, indicating the association of a N terminus of Syk (CFP–Syk) into cells expressing m, Ig-a–YFP and
phosphatase with the BCR. We noted a similar pattern for cells Ig-b or cells expressing g, Ig-a–YFP and Ig-b and monitored the
expressing Ig-a and Ig-b chains in which critical tyrosine ITAM recruitment of CFP–Syk to the BCR after antigen stimulation

NATURE IMMUNOLOGY VOLUME 6 NUMBER 11 NOVEMBER 2005 1173

 ARTICLES
(Fig. 7b). Most CFP–Syk translocated to BCR clusters 40 s after the
addition of antigen, as shown for the mIgG BCR, correlating with the
onset of the drop in FRET between intracellular BCR domains. To
abolish Syk binding without affecting BCR phosphorylation, we
deleted two amino acids in between the ITAM tyrosine residues of
Ig-a and Ig-b27. This deletion led to a considerable reduction in Syk
membrane recruitment and FRET between CFP–Syk and Ig-b–YFP in
both resting and stimulated cells expressing g and the Ig-a and Ig-b–
YFP with deletion of amino acids between ITAM tyrosine residues
© 2005 Nature Publishing Group http://www.nature.com/natureimmunology
(Fig. 7c). Correspondingly, we noted 65% reduction in the phosphor-
ylation of intracellular substrates (data not shown). However, this
deletion had no detectable effect on the pattern of FRET changes
(Fig. 7d), indicating that the open conformation of the BCR was
independent of Syk binding. We obtained similar results for mIgM
(data not shown).
To further probe the requirements for the open conformation of the
clustered BCR, we treated cells with methyl-b-cyclodextrin to remove
cholesterol from the membrane and to disrupt the function of the
cholesterol-rich membrane microdomains called ‘lipid rafts’28.
Methyl-b-cyclodextrin blocked the drop in FRET, indicating that
lipid rafts may be required for opening of the clustered BCR
cytoplasmic domains (Fig. 7e). Treatment with latrunculin, which
blocks actin polymerization, thereby inhibiting the function of the
actin cytoskeleton29, did not affect the FRET pattern, indicating that
the actin cytoskeleton was not required for the clustering of the BCR
or for the opening of the cytoplasmic domains. Treatment of the cells
with monodansylcadaverine to block internalization of the BCR30 had
no effect on the drop in FRET, indicating that the observed changes in
FRET occurred in the absence of internalization of the receptor at the
plasma membrane.
DISCUSSION
The studies described here provide a new view of the earliest response
of the BCR to antigen binding in living cells and also allow compar-
isons with data obtained in studies of these early events by conven-
tional imaging and biochemical approaches. First, using quantitative
fluorescence microscopy, we determined that the BCR is a monomer
on the surface of cells in the absence of antigen. Biochemical data
acquired from native PAGE have suggested that the BCR is an
oligomer on the surface of resting cells18, and it has been proposed
that antigen, rather than clustering of the BCR to initiate signal,
disrupts pre-existing oligomers to trigger a response31. It is possible
that the oligomer detected in native PAGE forms as a result of the
treatment of the cells with detergent. It is also possible that the
oligomer detected in native PAGE reflects a higher-order structure
that may form after multivalent antigen binding. If so, the structure of
this oligomer would be of considerable interest.
Second, the results of our studies reported here measuring
FRET between individual BCR chains have shown that the BCR
mIg, Ig-a and Ig-b chains assembled into a complex with defined
intramolecular distances between the chains and that those intramo-
lecular distances were not altered detectably by antigen binding.
Biochemical studies have provided evidence that the BCR mIg and
Ig-a–Ig-b chains disassociate after binding of antigen at the cell
surface, rendering the mIg signaling incompetent, suggesting that
destabilization of the BCR complex may be a means of regulating
BCR signaling32,33. Here, analyses of FRET between BCRs that con-
tained mIg–CFP and Ig-a–YFP showed that during antigen-induced
clustering of the BCRs, the FRET between mIg–CFP and Ig-a–YFP was
never less than the FRET measured before addition of antigen. Thus,
over the time course of this experiment there was no evidence of
1174
dissociation of mIg and the Ig-a–Ig-b complex. It may be that the
observed dissociation of mIg and the Ig-a–Ig-b complex in the
biochemical studies32,33 reflected a conformational change in
the BCR that rendered the complex more detergent soluble. Indeed,
the BCR does demonstrate a decrease in FRET after the initial FRET
gain due to oligomerization, and it is possible that the BCR in the
open form is more easily disrupted by detergent.
The results from quantitative FRET imaging demonstrated a very
dynamic response of the BCR to antigen binding. Based on our results,
we propose that in resting cells the BCR appears as a monomer with a
defined orientation of the ectodomains relative to the cytoplasmic
domains. Antigen binding clusters the receptors that simultaneously
are phosphorylated by Lyn and undergo a conformational change in
the cytoplasmic domain from a closed to an open form. We imagine
this conformational change resembles an umbrella opening. Finally,
Syk is recruited to the phosphorylated ITAMs in the open conforma-
tion. Phosphorylation of the closed conformation by Lyn could cause
the opening of the cytoplasmic domains by, for example, recruiting
some component to the phosphorylated cytoplasmic domains. Alter-
natively, the clustered BCR may be rapidly ‘breathing’ (opening and
closing), and the phosphorylation of the cytoplasmic domains by Lyn
may lock the oligomer in an open conformation. Notably, the open
conformation of the BCR was maintained only with continued activity
of Lyn, indicating that the BCR must be associated with a phosphatase
whose activity opposes Lyn’s activity. It may be that early in the
activation of the BCR signaling cascades, there is a competition
between the activity of the activating kinases such as Lyn and the
deactivating phosphatases that requires continued phosphorylation of
the receptor until ‘downstream’ events are fully triggered34. Finally, the
conformational change does not require the activity or binding of Syk,
the actin cytoskeleton or the internalization of the BCR.
We noted that the antigen-clustered BCR did not undergo a
conformational change in cells depleted of cholesterol that would
disrupt detergent-insoluble lipid rafts. Other studies have shown by
biochemical means that in resting cells, the BCR is present in
detergent-soluble membranes that do not contain Lyn and that
after antigen binding the BCR is associated with cholesterol-
dependent, detergent-insoluble membranes along with Lyn and is
phosphorylated by Lyn in those membranes35. It has been proposed
that the clustering of the BCR by antigen results in a coalescing of the
raft-like microdomains that contain Lyn around the BCR oligomer
that, in contrast to the monomer, ‘prefers’ the ordered environment of
the saturated raft lipids. This coalescing of the Lyn-containing raft
domains would allow time for the receptor to be phosphorylated by
Lyn. We now propose that the BCR phosphorylation and conforma-
tion change occur in lipid raft domains. The relationship between
lipid rafts and the proposed phosphatase activity is not known but
should be able to be explored using the same FRET imaging
technology described here.
Here we studied the BCR constructs expressed in the J558L cell line
that, like many cell lines, may not have all the components known to
regulate BCR signaling in normal B cells. Indeed, J558L cells do not
express certain regulators of BCR signaling such as CD22, CD45 and
CD19 (refs. 36–38). It would be useful to express the constructs
described here in normal B cells and B cell precursors to study the
BCR in these contexts. In summary, the results presented here provide
evidence that FRET technology can be applied to explore signaling
processes in living cells in real time. The use of this technology has
provided a new view of the earliest response of the BCR to antigen
binding over a time and length scale that could not be obtained before
by conventional imaging and biochemical approaches.
VOLUME 6 NUMBER 11 NOVEMBER 2005 NATURE IMMUNOLOGY

METHODS
Cell lines, antigens, antibodies and phosphotyrosine staining. J558L cell lines
were maintained as described39. NP-BSA containing 18–26 NP groups per BSA
molecule and NP-CGG containing 30 NP groups per CGG molecule were
prepared as described40. IgM- and IgG-specific antibodies and Fab fragments
labeled with Cy3 and Cy5 were from Jackson ImmunoResearch. HM-79
monoclonal antibody to Ig-b was provided by B. Vilen (University of North
Carolina, Chapel Hill, North Carolina). Cy3 or Cy5 labeling kits (Pierce) were
used for fluorescent labeling of NP-CGG and antibody to Ig-b using the
protocol suggested by the manufacturer. For measurement of intracellular
© 2005 Nature Publishing Group http://www.nature.com/natureimmunology
tyrosine phosphorylation, cells were stimulated for 1 min at 37 1C with 1 mg/ml
of NP-BSA, were fixed successively by 4% paraformaldehyde and ice-cold
methanol, were stained with the phosphotyrosine-specific monoclonal anti-
body PY-20 coupled to phycoerythrin (BD Pharmingen) and were analyzed
with a FACSCalibur (Becton Dickinson) and FlowJo software (TreeStar).
Constructs and transfection. Monomeric versions of CFP and YFP15 were
attached to the C termini of mouse NP-specific B1-8 m or g mIg heavy chains
(gifts from M. Shlomchik, Yale University, New Haven, Connecticut and
G. Kelsoe, Duke University, Durham, North Carolina, respectively), Ig-a and
Ig-b, or were attached to the N terminus of rat Syk by a six–amino acid linker
(GGGAAS) by PCR. Quickchange (Stratagene) was used for tyrosine-to-
phenylalanine alterations in the ITAMs of Ig-a (tyrosines 182 and 193) and
Ig-b (tyrosines 195 and 206) and amino acid deletions between ITAM tyrosine
residues (amino acids 187–188 of Ig-a and 200–201 of Ig-b). The resulting
constructs as well as wild-type BCR components were cloned into the pcDNA3
series of vectors (BD Clontech). Electroporation and selection of stable clones
were used to create cell lines expressing m, g and Ig-a, which, together with
wild-type J558L cells, were subsequently transiently transfected with the desired
combinations of fluorescent constructs using nucleofection (Amaxa). Images
were obtained 36–48 h after transfection. Monomeric CFP and YFP in pMSCV
(BD Clontech) and a CFP-YFP fusion protein in pECFP (provided by
A. Grammer, National Institutes of Health, Bethesda, Maryland) were used
to generate stable cell lines from the PT67 retroviral packaging cells (BD
Clontech) for control measurements.
Imaging and image processing. Cells were resuspended in a buffer of HBSS
plus 0.5% BSA and were allowed to attach to poly-L-lysine-coated coverslip
chambers (Labtek). For antibody staining, cells were fixed by 20 min of
incubation in 4% paraformaldehyde. A Zeiss 510 Meta confocal microscope
equipped with a heated stage, a heated air circulation system and an objective
heater (Carl Zeiss) was used for fluorescence microscopy. All imaging of living
cells was done at 37 1C. A Zeiss Plan-Apochromat 63
oil-immersion objective
was used exclusively for image acquisition. For inhibitor studies, 50 mM PP2,
50 mM piceatannol, 2 mg/ml of latrunculin B (Calbiochem) or 100 mM
monodansylcadaverine (Sigma) was added 10 min before stimulation or during
the experiment. Independent experiments showed that these doses lead to a
complete block of all intracellular phosphorylation (PP2), phosphorylation of
the Syk substrate BLNK (piceatannol) or internalization (latrunculin and
monodansylcadaverine). Methyl-b-cyclodextrin (10 mM; Sigma) was used
30 min before stimulation. For antigen stimulation, 50 ml of diluted NP-BSA
or NP-CGG was added between the first and second scan to a final concentra-
tion of 5 mg/ml, and the contents of the chamber were gently mixed to allow
rapid and simultaneous stimulation of the cells.
For measurements in resting cells, the Zeiss ‘emission fingerprinting’
technique was used. Emission was simultaneously collected into eight spectrally
continuous channels, followed by linear unmixing to separate the signal into
traditional channels based on spectral profiles of the individual fluorophores
using Zeiss LSM Software. For YFP-Cy3 imaging, cells were excited with
488-nm light and emission was collected in the range of 512–597 nm. For
CFP-YFP FRET imaging, two sequential scans were acquired: first with 458-nm
excitation to collect emission in the range of 469–555 nm and then with
514-nm excitation and an emission range of 523–608 nm.
For time-lapse imaging of stimulated cells, the regular channel mode was
used with low laser power to prevent bleaching of the specimen. Cy3-Cy5 FRET
imaging was done with sequential excitation at 543 nm and collection of Cy3
(554–619 nm) and FRET (661–758 nm) emission, followed by 633-nm
NATURE IMMUNOLOGY VOLUME 6 NUMBER 11 NOVEMBER 2005
ARTICLES
excitation and Cy5 (661–758 nm) detection. CFP-YFP FRET imaging was done
with sequential excitation at 458 nm and collection of CFP (475–525 nm) and
FRET (4 530 nm) emission, followed by excitation at 514 nm and collection of
YFP emission (4 530 nm). Laser switching was done after each line of the
image so that individual pixels had less than 10 ms between the two scans.
All resulting images were horizontally realigned and corrected for lateral lack
of homogeneity in illumination using Zeiss LSM Software. No vertical
disparities between channels were found. Regions of interest were designed
to cover the whole plasma membrane in equatorial sections of the cells and
mean fluorescent intensities with background subtracted were exported to
Microsoft Excel spreadsheets for subsequent calculations. Background values
were determined from plasma membrane regions of nonfluorescent cells.
FRET calculation. The techniques for FRET determination from sensitized
acceptor emission have been described in detail19,20. We used simultaneously
both FRET efficiency for the donor (ED) and for the acceptor (EA), calculated
as follows:
F  bD  ð1  bdÞgA
EA ¼
ð1  bdÞgAKA
F  bD  ð1  bdÞgA
ED ¼ ;
KD ðD  dFÞ + F  bD  ð1  bdÞgA
where D, F, and A are fluorescent intensities in the donor, FRET and acceptor
channels, respectively. In these equations, b is a correction factor defined from
cells with donor only as b ¼ F/D, and g and d are correction factors defined
from cells with acceptor only as g ¼ F/A and g ¼ D/F. For imaging using
emission fingerprinting, b, d is 0; for Cy3-Cy5 imaging, d is 0. KA and KD
characterize the conversion of fluorescence ratios to FRET efficiency and are
defined as KA ¼ eD/eA and KD ¼ QAGF/QDGD, where eD and eA are the
extinction coefficients of the donor and acceptor, respectively, at the donor
excitation wavelength; QD and QA are the quantum yields of the donor and
acceptor, respectively; GF is a constant characterizing the efficiency of detection
of the fluorescence of the acceptor with the FRET filter set; and GD is a constant
characterizing the efficiency of detection of the donor fluorescence in the donor
filter set. For our purposes, KA and KD were determined from cells with equal
amounts of the donor and acceptor at distance close enough to produce
nonzero FRET. For CFP and YFP imaging, this was done with cells expressing a
CFP-YFP fusion protein; for Cy3 and Cy5 imaging, surface BCR was stained
with Cy3- and Cy5-labeled antibodies together in conditions with equimolar
concentrations of Cy3 and Cy5. Then the following equations were used:
F  bD  ð1  bdÞgA
KA ¼
ð1  bdÞgAEbleaching
F  bD  ð1  bdÞgA
KD ¼  F  bD  ð1  bdÞgA;
ðD  dFÞEbleaching
where Ebleaching is FRET efficiency determined by donor ‘dequenching’ after
acceptor photobleaching using the following equation:
Dafter  Dbefore  dF
1  bd
Ebleaching ¼
Dafter
where Dbefore and Dafter are fluorescence intensities in the donor channel before
and after complete acceptor photobleaching. As expected, b, g, d and KD
depended on the image acquisition settings and were determined indepen-
dently for each experiment, whereas KA was constant at 12.6 ± 1.1 for Cy3-Cy5
imaging or 2.6 ± 0.29 for CFP-YFP imaging. For CFP-YFP imaging, we found
that b, g and d were indistinguishable whether isolated cytoplasmic CFP and
YFP or CFP- and YFP-tagged BCR constructs were used for their determina-
tion. Because of the better signal/background ratio, b, g, d, KA and KD were
determined with the fibroblastoid cell line PT67.
For time-course FRET imaging, we found that repeated scanning of
unstimulated cells lead to exponential decay of EA and ED, as expected from
1175
 ARTICLES
photobleaching of the donor and the acceptor. Experimental data from
stimulated cells were therefore corrected by normalization to the decay
determined from resting cells for each experiment. For cells expressing Ig-a–
CFP and Ig-a–YFP with no FRET in the resting state, we used the same
correction as for cells expressing Ig-a–CFP and Ig-b–YFP. Although all
corrections for photobleaching during imaging of dynamic FRET are approxi-
mate20, we ensured that the small amount of photobleaching (typically less
than 1% of EA per scan) in our experimental conditions did not affect the
antigen-induced patterns presented here. For the generation of FRET image,
original images were ‘masked’ to retain only areas with substantially higher
© 2005 Nature Publishing Group http://www.nature.com/natureimmunology
signal over background, were ‘smoothened’ to remove noise and then were
processed on a pixel-by-pixel basis with the equations presented above. The
distance R between CFP and YFP pairs from FRET data was calculated with the
following formula:
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
6
6 R0  EA R0
6
R¼ ; 86, 2517–2529 (2004).
EA
where R0 is 49.2 Å (ref. 41) assuming random orientation of the fluorophores.
For FRET of Ig-a or Ig-b with the dimer of mIgH, a range is given from the
value above to the value calculated as follows:
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
6
6 2ðR0  EA R0 Þ
6 23. Martin, S.W. & Goodnow, C.C. Burst-enhancing role of the IgG membrane tail as a
R¼ ; molecular determinant of memory. Nat. Immunol. 3, 182–188 (2002).
EA
assuming contribution of only one or equal contribution of both, respectively,
mIgH-associated fluorescent proteins.
ACKNOWLEDGMENTS
We thank T. Jin for the initial setup of the imaging system and J. Coligan
for help with transfections. Supported by the Intramural Research program
of the National Institutes of Health, National Institute of Allergy and
Infectious Diseases.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Published online at http://www.nature.com/natureimmunology/
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/
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