T cell activation requires mitochondrial translocation
to the immunological synapse
Ariel Quintana*, Christian Schwindling, Anna S. Wenning, Ute Becherer, Jens Rettig, Eva C. Schwarz, and Markus Hoth
Department of Physiology, Saarland University, Gebäude 58/59, D-66421 Homburg, Germany
Edited by Arthur Weiss, University of California School of Medicine, San Francisco, CA, and approved July 30, 2007 (received for review April 7, 2007)
T helper (Th) cell activation is required for the adaptive immune
response. Formation of the immunological synapse (IS) between Th
cells and antigen-presenting cells is essential for Th cell activation.
IS formation induces the polarization and redistribution of many
signaling molecules; however, very little is known about organelle
redistribution during IS formation in Th cells. We show that for-
mation of the IS induced cytoskeleton-dependent mitochondrial
redistribution to the immediate vicinity of the IS. Using total
internal reflection microscopy, we found that upon stimulation,
the distance between the IS and mitochondria was decreased to
values <200 nm. Consequently, mitochondria close to the IS took
up more Ca2ⴙ than the ones farther away from the IS. The
redistribution of mitochondria to the IS was necessary to maintain
Ca2ⴙ influx across the plasma membrane and Ca2ⴙ-dependent Th
cell activation. Our results suggest that mitochondria are part of
the signaling complex at the IS and that their localization close to
the IS is required for Th cell activation.
calcium 兩 lymphocyte 兩 mitochondria
T helper (Th) cell activation is central to the adaptive immune
response. The initiation of a productive Th cell-based immune
response can be divided into several steps. Foreign antigens are
transported to Th cell zones of draining lymph nodes by antigen-
presenting cells. Antigens are then presented by antigen-presenting
cells through class II MHC complexes until they encounter the
corresponding naı̈ve antigen-specific Th cells (1, 2). Molecular
rearrangements after antigen recognition lead to the formation of
an organized immunological synapse (IS), which consists of a
central cluster of Th cell receptors (TCR) [central supramolecular
activation clusters (c-SMAC)] that is surrounded by a ring of
adhesion molecules (peripheral supramolecular activation clusters)
(3–5). Whereas spatial molecular reorganization after IS formation
has been analyzed in detail, almost nothing is known about mito-
chondrial redistribution and its function during and after formation
of the IS.
After formation of the IS, several signaling cascades are initiated
in Th cells that finally lead to the secretion of IL-2 and other
cytokines and an extensive clonal expansion and differentiation
(6–8). A necessary step for the activation of Th cells after TCR
engagement is the stimulation of Ca2⫹ entry across the plasma
membrane through the opening of calcium release-activated cal-
cium (CRAC)/Orai1 channels (9–12). CRAC/Orai1 channels are
gated by Stim1 after depletion of intracellular Ca2⫹ stores (13, 14).
It is widely agreed that maximal store depletion will cause maximal
CRAC/Orai1 activation, maximal Ca2⫹ signals, maximal IL-2 pro-
duction, and maximal Th cell activation (15, 16).
Results
Efficient Ca2ⴙ-Dependent Th Cell Activation After Formation of the IS
Requires Mitochondrial Ca2ⴙ Uptake. Analyzing Ca2⫹ signals in the
Th cell line Jurkat after formation of an IS with anti-CD3 beads
[supporting information (SI) Fig. 6 A and B], we found that
increases in intracellular Ca2⫹ concentration ([Ca2⫹]i) were
more sustained after IS formation than after stimulation with the
same anti-CD3 antibody or the commonly used anti-CD3 anti-
body OKT-3 in solution (Fig. 1A), both of which do not induce
14418 –14423 兩 PNAS 兩 September 4, 2007 兩 vol. 104 兩 no. 36
IS formation. This was unexpected because anti-CD3 antibodies
in solution and anti-CD3 beads were similarly efficient to induce
the TCR-dependent signaling cascade that leads to Ca2⫹ store
depletion (Fig. 1B), which should therefore activate CRAC/
Orai1 channels to the same extent. We found similar differences
of sustained [Ca2⫹]i as shown in Fig. 1 A with anti-CD3/anti-
CD28 beads compared with the same antibodies in solution in
primary human Th cells isolated from peripheral blood (SI Fig.
6C). Accordingly, formation of the IS was also more efficient to
induce proliferation of primary Th cells than the same antibodies
in solution (Fig. 1C). The sarcoendoplasmic Ca2⫹ ATPase
inhibitor thapsigargin (TG) has been shown to slowly but max-
imally and irreversibly deplete Ca2⫹ stores (17), which leads to
maximal activation of CRAC/Orai1 channels and maximal
[Ca2⫹]i increases (18, 19). Although CRAC channels were
maximally activated under these conditions, TG-induced in-
creases of [Ca2⫹]i were also lower than increases of [Ca2⫹]i after
formation of the IS (Fig. 1 A).
The sustainability of the [Ca2⫹]i rise induced by the IS was
completely abolished by inhibition of mitochondrial Ca2⫹ uptake
with carbonyl cyanide m-chlorophenylhydrazone (Fig. 1D), a com-
bination of antimycin/oligomycin (SI Fig. 6D), ruthenium red (SI
Fig. 6E), or ruthenium 360 (SI Fig. 6F). Under these conditions,
formation of the IS reduced steady state [Ca2⫹]i to the same degree
as did TG or anti-CD3 antibodies in solution. This indicates that
mitochondrial Ca2⫹ uptake plays an essential role to maintain an
efficient Ca2⫹-dependent Th cell stimulation after formation of the
IS. Mitochondrial Ca2⫹ uptake has previously been shown to
sustain CRAC/Orai1 channel activity by reducing its Ca2⫹-
dependent Ca2⫹ inactivation after nonphysiological activation of
channels (20, 21). Mitochondria likely dissipate the microdomain of
high [Ca2⫹]i beneath the plasma membrane and thereby prevent
slow CRAC/Orai1 channel inactivation (20, 22). For this reason,
mitochondria should take up a larger amount of Ca2⫹ after IS
formation than after TG stimulation, which in turn would result in
a more efficient reduction of Ca2⫹-dependent CRAC/Orai1 chan-
nel inactivation and thereby sustain Ca2⫹ influx through channels
for an extended period. Using the mitochondrial Ca2⫹ indicator
rhod-2/AM, we tested this hypothesis. To facilitate rhod-2 mea-
surements, cells were electroporated after rhod-2/AM loading,
which removes most of the remaining cytosolic rhod-2, allowing a
better resolution of mitochondrial Ca2⫹ measurements. As ex-
pected, mitochondria took up significantly more Ca2⫹ after IS
formation than after TG stimulation (Fig. 1 E and F). Mitochon-
Author contributions: A.Q., E.C.S., and M.H. designed research; A.Q., C.S., A.S.W., and U.B.
performed research; A.Q., C.S., A.S.W., U.B., J.R., E.C.S., and M.H. analyzed data; and A.Q.,
J.R., E.C.S., and M.H. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Abbreviations: IS, immunological synapse; Th, T helper; CRAC, calcium release-activated
calcium; TG, thapsigargin; TCR, Th cell receptor; c-SMAC, central supramolecular activation
cluster; TIRF, total internal reflection microscopy.
*To whom correspondence should be addressed. E-mail: ptaqgo@uks.eu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0703126104/DC1.
© 2007 by The National Academy of Sciences of the USA
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0703126104

Fig. 1. Efficient Ca2⫹-dependent Th cell activation after formation of the IS
requires mitochondrial Ca2⫹ uptake. (A) Average [Ca2⫹]i of Jurkat T cells. The
[Ca2⫹]i plateau (sustained [Ca2⫹]i) at 800 –900 s was analyzed for anti-CD3 beads
(1,361 ⫾ 7 nM, 183 cells), 1 ␮M TG (735 ⫾ 39 nM, 1,525 cells), 5 ␮g/ml OKT-3 (470 ⫾
15 nM, 321 cells), or 5 ␮g/ml anti-CD3 mAbs (464 ⫾ 8 nM, 170 cells). Bead-induced
[Ca2⫹]i was significantly higher than TG-, OKT-3-, or anti-CD3-induced [Ca2⫹]i
(unpaired Student’s t test, P ⬍ 2.5 ⫻ 10⫺16). (B) Average [Ca2⫹]i of Jurkat T cells in
0 mM Ca2⫹ Ringer’s solution to assess the efficiency of Ca2⫹ store depletion. (C)
Average of cell proliferation of Th cells from four blood donors after a 5-day
incubation with 2 ␮g/ml phytohemagglutinin plus 10 units/ml IL-2 (positive
control), anti-intercellular adhesion molecule beads (negative control), 5 ␮g/ml
anti-CD3 and anti-CD28 mAbs (costimulation) in solution, or anti-CD3/CD28
beads. Data are shown as percentage of Th cell proliferation regarding the cell
proliferation measured at day 0. Bead stimulation was significantly better than
antibody stimulation (Mann–Whitney test, P ⫽ 0.000032). (D) Average [Ca2⫹]i of
Jurkat T cells in the presence of 1 ␮M carbonyl cyanide m-chlorophenylhydrazone
(CCCP). No significant difference was found between the sustained Ca2⫹ signals
(P ⬎ 0.1). (E) Infrared images and rhod-2 fluorescence pictures of cells stimulated
by either TG or anti-CD3 beads. Warmer colors indicate higher rhod-2 fluores-
cence. (F) Statistical analysis of the normalized rhod-2 fluorescence from T cells
stimulated by either TG (37 mitochondrial spots) or anti-CD3 beads (90 mitochon-
drial spots).
drial modulation of CRAC/Orai1 channel activity has already been
reported after a directed translocation of mitochondria toward the
plasma membrane, which allows mitochondria to take up a larger
amount of incoming Ca2⫹ directly beneath the mouth of channels
and thereby significantly reduces the Ca2⫹-dependent CRAC/
Orai1 channel inactivation (23). Therefore, we analyzed mitochon-
drial localization within Th cells after IS formation.
The IS and Mitochondria Form a Signaling Complex. Usually, mito-
chondria appear to be evenly distributed throughout the cytosol in
most cell types. This is, for instance, the case in HeLa cells, in which
many thin tubular mitochondrial structures have been observed by
using confocal microscopy (24–26) (Fig. 2A). Mitochondrial local-
ization in T cells is different, because they are often localized
preferentially in one area of the cell as seen in the confocal and EM
pictures (Fig. 2 B and C). This polarized mitochondrial localization
may be caused by the rather large nucleus in T cells. It is difficult
Quintana et al.
to resolve the dense interconnected mitochondrial network in T
cells even with confocal microscopy. To get an estimate of the
localization of all mitochondrial structures within T cells over longer
times without significant bleaching, we used epifluorescence mea-
surements such as the one depicted in Fig. 2D.
Fig. 3A shows that mitochondrial localization was changed after
formation of the IS. In almost all Jurkat or primary human Th cells
we observed a directed movement of mitochondria to the plasma
membrane as exemplified in Fig. 3A. Quantitative analysis revealed
that many mitochondrial structures moved into an area not farther
than 1 ␮m away from the plasma membrane as indicated by the
yellow lines in Fig. 3A and the bar graphs in Fig. 3B. After IS
formation, mitochondria were, however, not only translocated to
the plasma membrane but preferentially to the IS itself, as is evident
from the epifluorescence and two-photon pictures in Fig. 3C.
Approximately 50% (in case of Jurkat Th cells) or even 70% (in case
of primary human Th cells) of the mitochondria were found in the
small area around the IS marked in red (Fig. 3D). To analyze how
intimate this potential interaction between the IS and mitochondria
is, we used total internal reflection microscopy (TIRF), which limits
detection of fluorescence to the immediate vicinity (⬍200 nm) of
the plasma membrane. Th cells were seeded onto coverslips coated
with either anti-CD3 (to induce IS formation on the coverslip) or
IgG (as control) antibodies. Mitochondrial translocation to the IS
was observed with anti-CD3- but not with anti-IgG-coated cover-
slips (Fig. 3E). Quantification of data from all cells revealed a clear
difference between anti-CD3 antibody-coated coverslips compared
with anti-IgG antibody-coated coverslips (Fig. 3F). To analyze
whether mitochondrial translocation depends on Ca2⫹ influx
through CRAC/Orai1 channels, we separated Ca2⫹ release and
influx during TIRF experiments. Ca2⫹ stores were depleted for 15
min by allowing contact with the anti-CD3-coated coverslip in the
IMMUNOLOGY
absence of extracellular Ca2⫹. At time 0, Ca2⫹ influx was initiated
by the readdition of 20 mM Ca2⫹. We observed a large translocation
of mitochondria to the IS, which was significantly reduced in the
presence of the CRAC/Orai1 channel blockers 2-APB or BTP2
(Fig. 3G). Therefore, we conclude that mitochondrial translocation
into the immediate vicinity (distance ⬍200 nm) of the IS depends
on Ca2⫹ influx.
The intimate contact between mitochondria and IS should lead
to an enhanced uptake of Ca2⫹ by mitochondria in the vicinity of
the IS. We tested this in rhod-2-loaded cells. Whereas in most T
cells mitochondria were only found close to the IS, we found cells
in which some mitochondrial structures were close to the IS and
others farther away. Fig. 3H depicts such a cell, and it is obvious that
mitochondria close to the IS had higher rhod-2 fluorescence than
the mitochondria farther away from the IS. The analysis of rhod-2
fluorescence over time of all cells reveals a clear difference between
mitochondria close to and farther away from the IS. The higher
intramitochondrial Ca2⫹ is probably caused by the intimate contact
between mitochondria and the IS, which exposes mitochondria to
higher microdomains of [Ca2⫹]i.
Coupling Between the IS and Mitochondria Is Required for Ca2ⴙ
Signaling. We have previously shown that microtubules are involved
in the translocation of mitochondria toward the plasma membrane
after artificial stimulation of Ca2⫹ influx through CRAC/Orai1
channels with TG. After nocodazole treatment to disrupt micro-
tubule-based transport, mitochondrial translocation toward the
plasma membrane after TG stimulation was completely abolished
(23). In contrast, after bead stimulation, intact microtubule-based
transport was not necessary for the translocation of mitochondria
toward the IS (Fig. 4 A Left and B) and also not required for the
Ca2⫹-dependent Th cell activation (Fig. 4C). This shows that, after
formation of the IS, other transport mechanisms for mitochondria
must exist. Microfilaments (also called actin filaments or actin
cytoskeleton) are also used for mitochondrial transport within the
cell (27, 28). Latrunculin B is known to inhibit actin polymerization,
PNAS 兩 September 4, 2007 兩 vol. 104 兩 no. 36 兩 14419
Fig. 2. Different intracellular localization profile of mitochondria in HeLa and T cells. (A) Confocal fluorescence image from a single MitoTracker
Green/AM-loaded HeLa cell. (B) Confocal fluorescence image of MitoTracker/AM/di-8-ANEPPS colabeled Jurkat T cells. (C) Two examples of EM images obtained
from Jurkat T cells. N, nucleus; M, mitochondria; PM, plasma membrane. (D) Infrared and MitoTracker fluorescence images from MitoTracker Green/AM-loaded
Jurkat T cells obtained by epifluorescence microscopy. Scale bars indicate magnifications.
thereby disrupting actin-dependent cellular functions (29). The
treatment of Th cells with latrunculin B significantly reduced
mitochondrial translocation toward the plasma membrane (Fig. 4 A
Right and B) and sustained Ca2⫹ signals after bead stimulation (Fig.
4C), demonstrating the importance of mitochondrial localization
relative to the plasma membrane for Ca2⫹-dependent Th cell
activation.
Although we did not find any side effect of latrunculin B on
TCR-dependent store-operated Ca2⫹ signals using anti-CD3 anti-
bodies in solution, CRAC channel activity, or mitochondrial Ca2⫹
uptake (SI Fig. 7 A–D), latrunculin B clearly reduced IS-induced
Ca2⫹ release (Fig. 4D), which could in part explain the low Ca2⫹
signals in Fig. 4C, because reduced Ca2⫹ release from stores may
not activate CRAC channels efficiently enough to allow sustained
[Ca2⫹]i elevations. The reduced Ca2⫹ release was likely due to a
lower number of TCR that could be focally stimulated by the
anti-CD3 bead at the IS, because latrunculin B disrupted the actin
rearrangement and subsequent TCR translocation toward the
cell–bead contact point (Fig. 4E) as reported previously (29). A
reduced TCR engagement probably decreased the InsP3 produc-
tion and thereby the subsequent InsP3 receptor-dependent Ca2⫹
release, which in turn lead to a less efficient CRAC channel
activation.
To analyze the importance of the actin cytoskeleton-dependent
translocation of mitochondria to the IS independent of the amount
of Ca2⫹ store release, we repeated the Ca2⫹ imaging experiments
after maximal store depletion with TG (Fig. 4F Inset). Stores were
depleted in the presence of TG in 0-Ca2⫹ solution, and Ca2⫹ influx
was assessed by the change to 1 mM Ca2⫹ solution. Under these
conditions, peak [Ca2⫹]i signals were generally higher (23) because
Ca2⫹ ATPases in the plasma membrane initially cannot counter-
balance the massive simultaneous Ca2⫹ influx through fully acti-
vated and opened CRAC channels, because the activity of Ca2⫹
ATPases is only up-regulated slowly (30). The important result,
however, is that the sustained Ca2⫹ signals (magnified in Fig. 4F)
have the highest amplitude if cells were stimulated by TG and beads
in the absence of latrunculin B. In this case, in addition to maximal
store depletion, a functional IS is generated and mitochondria are
translocated to the IS. With latrunculin B present, the IS cannot
form (Fig. 4E), and sustained Ca2⫹ signals are lower despite
14420 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0703126104
maximal store depletion. TG, in the presence or absence of
latrunculin B, did of course also not induce IS formation, and,
consequently, sustained Ca2⫹ signals were also lower. We therefore
conclude that an actin cytoskeleton-dependent translocation of
mitochondria to the IS is required for sustaining Ca2⫹ signals that
are necessary for efficient Ca2⫹-dependent Th cell activation and
subsequent cell proliferation.
Discussion
The main result of this study is that efficient Ca2⫹-dependent
activation and clonal expansion of Th cells require an intimate
coupling between mitochondria and the IS. Formation of a matured
IS does not only induce sustained TCR stimulation by confining the
TCR and agonist peptide–MHC complex to a small region of the
plasma membrane (4, 31), but it also activates and sustains mito-
chondrial relocalization to the IS. Both process are required to
sustain Ca2⫹ signals through CRAC/ORAI1 channels, which in
turn allows the efficient production of cytokines that occurs over a
period of hours (3, 32).
Although a quick, directed, and active mitochondrial transloca-
tion along microtubules toward the plasma membrane was already
observed after Ca2⫹ influx through CRAC channels in T cells (23),
we never detected significant increases of mitochondria into an area
within 200 nm from plasma membrane after TG or OKT3 stimu-
lation (data not shown) as described here after formation of IS.
Such an intimate contact between mitochondria and plasma mem-
brane allows mitochondria to reduce the local accumulation of
Ca2⫹ close to the sites that govern CRAC channel inactivation more
efficiently and thereby prolong Ca2⫹ entry for an extended period.
This intimate contact was mediated by the actin cytoskeleton. In
agreement with these results, disruption of actin cytoskeleton
reduced Ca2⫹ signals in T cells only after formation of the IS but
not after TG or OKT3 stimulation despite maximal store depletion
(Fig. 4D and SI Fig. 7). We propose that mitochondria move actively
along microtubules toward the membrane until they reach the
cortical actin cytoskeleton, where mitochondria are transferred
from microtubules to actin cytoskeleton and then translocated to
the IS in an actin cytoskeleton-dependent manner.
The efficiency of an antigen-coated surface to activate T cells
compared with antigens in solution has been mostly explained by a
Quintana et al.

Fig. 3. Focal stimulation of TCR activates mitochondrial translocation not only
toward the plasma membrane but also to the IS. (A) Infrared and fluorescence
images from MitoTracker Green/AM-loaded Jurkat T cells before and 15 min after
stimulation with anti-CD3 beads. (B) Statistical analysis of the subplasma mem-
brane localization of mitochondria (⬍0.99 ␮m beneath the plasma membrane) in
Jurkat (31 cells) and CD4⫹ (18 cells) T cells before (resting) and 15 min after
stimulation with anti-CD3 beads. Bead stimulation was significantly different
from resting conditions (paired Student’s t test; Jurkat, P ⫽ 7 ⫻ 10⫺5; CD4⫹, P ⫽
3 ⫻ 10⫺6). (C) Epifluorescence microscopy and infrared pictures of MitoTracker
Green during anti-CD3 bead stimulation. The yellow ring labels the plasma
membrane. Shown are two-photon microscopy pictures of a Jurkat T cell stained
with MitoTracker/AM and anti-CD45-Alexa Fluor 488 mAb (for the plasma mem-
brane). (D) Percentage of mitochondria at the IS (see Inset, area limited by red
line) compared with the net subplasma membrane MitoTracker fluorescence
(⬍0.99 ␮m beneath the plasma membrane) in Jurkat and CD4⫹ T cells after
anti-CD3 bead stimulation. (E) TIRF microscopy pictures of single MitoTracker
Green/AM-loaded Jurkat T cells that were settled on anti-CD3 or anti-IgG mAb-
coated coverslips in the absence of extracellular Ca2⫹ solution for 4 min and then
(at 0 min) exposed to 20 mM Ca2⫹. (F) Statistical analysis of the normalized
MitoTracker fluorescence from 21 and nine cells analyzed as the one in E. (G)
Statistical analysis of the normalized MitoTracker fluorescence from TIRF exper-
iments. Jurkat T cells were settled on anti-CD3 mAb-coated coverslips in the
absence of extracellular Ca2⫹ solution for 15 min and then (at 0 min) exposed to
20 mM Ca2⫹ solution. Control (47 cells), 2-APB-treated (50 ␮M, 10 cells) or
BTP2-treated (100 nM overnight incubation, 20 cells) cells are shown. (H) Infrared
images and rhod-2 pictures of T cells stimulated by anti-CD3 beads. Red arrow
indicates the position of a mitochondrial cluster beneath the IS, and green arrows
indicate the position of mitochondrial populations farther away from the IS. Also
shown is kinetic analysis of the normalized rhod-2 fluorescence from mitochon-
dria localized either close to or farther away from the IS (seven cells).
prolonged TCR stimulation. A faster down-regulation of TCR
induced by antibodies in solution has been observed, leading to a
less sustained TCR signaling (33, 34). However, it has also been
Quintana et al.
Fig. 4. Sustained [Ca2⫹]i after IS formation requires actin cytoskeleton rear-
rangement-dependent mitochondrial translocation to the IS. (A) Infrared and
MitoTracker fluorescence images from single MitoTracker Green/AM-loaded
Jurkat T cells before and after stimulation with anti-CD3 beads in the presence of
2 ␮M nocodazole after a preincubation period for 35 min or in the presence of 10
IMMUNOLOGY
␮g/ml latrunculin B after a preincubation period for 15 min. (B) Statistical analysis
of the subplasma membrane localization of mitochondria in Jurkat T cells (⬍0.99
␮m beneath the plasma membrane): resting (86 cells), beads (31 cells), beads plus
nocodazole (36 cells), and beads plus latrunculin B (19 cells). Bead stimulation was
significantly different from either resting conditions (unpaired Student’s t test,
P ⫽ 2 ⫻ 10⫺6) or cells stimulated in the presence of latrunculin B (P ⫽ 0.009). (C)
Average [Ca2⫹]i of Jurkat T cells stimulated with anti-CD3 beads (control, 1,361
cells) or with anti-CD3 beads in the presence of nocodazole (178 cells) or latrun-
culin B (106 cells). (D) Average [Ca2⫹]i of Jurkat T cells stimulated with anti-CD3
beads (control, 104 cells) or with anti-CD3 beads in the presence of nocodazole (52
cells) or latrunculin B (38 cells). (E) Infrared, fluorescence, and merged images
from single Jurkat T cells in which the actin cytoskeleton was stained by Texas red
phalloidin after the stimulation with anti-CD3 beads (control) or with anti-CD3
beads in the presence of nocodazole or latrunculin B. (F) Average [Ca2⫹]i of Jurkat
T cells stimulated with TG or TG plus anti-CD3 beads in the presence (290 or 270
cells, respectively) or absence (260 or 300 cells, respectively) of latrunculin B.
(Inset) The complete experiments. The influx and sustained phase of the Ca2⫹
signals are magnified.
reported that the down-regulation of TCR in the plasma membrane
is not significantly altered by antibodies in solution compared with
antibodies coated on a surface (32). Liu et al. (35) demonstrated
that reduction of TCR on the cell surface is caused by the disruption
of TCR-containing vesicle recycling to the membrane rather than
enhancement of TCR internalization and subsequent degradation.
The down-regulation of ⬇20% of TCR at the plasma membrane
took ⬇30 min (35). In agreement with this result, we did not find
any differences in the efficiency of Ca2⫹ store depletion by anti-
bodies in solution compared with bead stimulation. Thus, the
decreased Ca2⫹ signals and the inefficient Th cell activation by
antibodies in solution are not explained by a faster down-regulation
of TCR or by disruption of the signalosome.
Recent evidence suggests that TCR microclusters rather than the
c-SMAC initiate and sustain TCR signaling (5, 29, 36, 37). TCR
microclusters are dynamically generated in the peripheral supramo-
lecular activation clusters and move to the c-SMAC where their
signaling is switched off after a while. These results raise the
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Fig. 5. Model for Ca2⫹-dependent T cell activation. After formation of the
IS, many mitochondria are translocated into the vicinity of the IS. This close
coupling facilitates a larger and more sustained Ca2⫹ influx and the concom-
itant activation of transcription factors like NFAT, AP1, and NF-␬B by reducing
the Ca2⫹-dependent CRAC/Orai1 channel inactivation of channels localized
close to or within the IS. CRAC/Orai1 channels localized far away from the IS
(and most likely not close to mitochondria) inactivate much more rapidly and
do not contribute to sustained Ca2⫹ signals.
question of why Th cells allow the formation of c-SMAC in the first
place, if it is needed neither to initiate nor to sustain TCR signaling.
Our data suggest a role for the c-SMAC during the activation of Th
cells. We postulate that one important function of the c-SMAC is
to maintain a high polarization of Th cells to initiate and sustain the
actin cytoskeleton-dependent localization of mitochondria in the
immediate vicinity of the IS (Fig. 5). The IS–mitochondria signaling
complex sustains CRAC/Orai1 activity, which is required for the
Ca2⫹-dependent activation of transcription factors and proper Th
cell function. Hence, CRAC/Orai1 channels localized at the IS
would be longer active than those far away from the IS. In
agreement with this prediction we found that mitochondria close to
the IS take up significantly more Ca2⫹ than mitochondria farther
away from the IS. In addition, it is likely that CRAC/Orai1 channels
are enriched at the IS as is the case for other membrane proteins
after cell polarization. Such an enrichment of CRAC/Orai1 chan-
nels would make sense because their long-lasting activity requires
an efficient reduction of their Ca2⫹-dependent inactivation, which
is achieved only by the mitochondrial Ca2⫹ uptake in the immediate
vicinity of the channel.
Our results stress the importance of the actin cytoskeleton to
coordinate the relative distance between IS and mitochondria.
Rather than being part of the signal transduction machinery itself,
as has been proposed in T cells and other cells, the actin cytoskel-
eton may exert much of its ‘‘signaling’’ power through changing the
relative distances between signalosomes and/or organelles. Mito-
chondrial relocalization during IS formation can thus be used to
control the Ca2⫹-dependent activation of transcription factors (38).
Experimental Procedures
Research carried out for this study with human material was
approved by the local ethics committee.
Cells. Human Jurkat T cell lines were grown as described previously
(39). Human peripheral blood lymphocytes and CD4⫹ T cells were
isolated and cultured as previously described (40). To avoid pre-
stimulation, Th cells (CD4⫹) were negatively purified with the T
Cell Negative Isolation Kit from Invitrogen (Karlsruhe, Germany)
according to the manufacturer’s instructions.
Reagents. All chemicals not specifically mentioned were of the
highest grade from Sigma–Aldrich (Deisenhofen, Germany). Other
reagents used in our experiments include fura-2/AM, rhod-2/AM,
MitoTracker Green FM, latrunculin B, TG (Invitrogen), OKT3
14422 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0703126104
(ATCC CRL-8001), mouse anti-human CD43-FITC mAb (Dako
Cytomation, Hamburg, Germany), ruthenium 360 (Calbiochem,
Darmstadt, Germany), CGP37157 (Tocris, Ellisville, MO), and
BTP2 (Nycomed, Konstanz, Germany).
Fluorescence Microscopy and Ca2ⴙ Imaging. Ca2⫹ and mitochondrial
imaging were carried out as described (23) with a ⫻40 (Uplan/Apo,
N.A. 1.0 oil) or ⫻100 (Uplan/Apo, N.A. 1.35 oil) objective. TG (1
␮M), OKT3 (5 ␮g/ml), anti-human CD3 mAbs (5 ␮g/ml), or
anti-CD3 beads were used to stimulate Jurkat T cells. An anti-
human CD3 mAb/anti-human CD28 mAb (5 ␮g/ml) mixture or
anti-CD3/anti-CD28 beads were used to stimulate peripheral blood
lymphocytes. Carbonyl cyanide m-chlorophenylhydrazone (1 ␮M),
an antimycin (2 ␮M)/oligomycin (1 ␮M) mixture, ruthenium red
(100 ␮M), or ruthenium 360 (50 ␮M, 45-min preincubation) were
used for disrupting mitochondrial Ca2⫹ uptake. Because of the
reported incapability of ruthenium red to cross the plasma mem-
brane (41), cells were electroporated as previously reported (42) to
introduce it into cells.
For mitochondrial Ca2⫹ measurements, cells were loaded at
22–23°C for 45 min with 10 ␮M rhod-2/AM in culture medium with
10 mM Hepes added, washed with fresh medium, electroporated as
reported (42) to remove the cytosolic rhod-2, washed with fresh
medium twice, stored at room temperature for 10 min, and used
immediately. Cells were illuminated at 550 nm with the Polychrome
IV Monochromator (TILL Photonics, Gräfelfing, Germany) using
SP 546/10 as excitation filter and DCLP 555 as dichroic mirror. The
fluorescence emissions at ⬇580 nm (HQ 575/30) were captured
with a CCD camera (TILL Photonics), digitized, and analyzed using
TILL Vision software. Cells were illuminated every 5 s. To facilitate
Ca2⫹ measurements in mitochondria with rhod-2, mitochondrial
Ca2⫹ export was inhibited by 10 ␮M CGP 3715. Rhod-2 fluores-
cence was background-subtracted and normalized to the initial
fluorescence values.
Bead Stimulation. We followed the standard procedure for absorb-
ing proteins on polystyrene microparticles (size ⬎0.5 ␮m) estab-
lished by Polysciences (Eppelheim, Germany). Two alterations in
the procedure were required to optimize our results. In step 1 we
used 100 ␮l of a 2.5% suspension of beads (⬇2.1 ⫻ 107 beads), and
in step 7 we added 50 ␮g of the protein to be absorbed. Chemical
composition and pH of buffers were the same as recommended in
the protocol.
Azid free anti-human CD3, CD28, or intercellular adhesion
molecule mAbs (Euroclone, Lugano, Switzerland) were passively
coupled to microparticles (diameter ⫽ 5.83 ␮m) to produce stim-
ulatory (anti-CD3 beads, 50 ␮g), costimulatory (anti-CD3/CD28
beads, either 25 ␮g/25 ␮g or 12.5 ␮g/37.5 ␮g), and nonstimulatory
(anti-intercellular adhesion molecule beads, 50 ␮g) beads, respec-
tively. They were stored at 4°C in the specified storage buffer until
use. Beads were washed twice with PBS before resuspending them
in the Ringer’s solution used for the experiments. The ratio of beads
to cells was between 2:1 and 1:1.
Cell Proliferation. Proliferation experiments were carried out in
96-well cell culture plates (black/transparent bottom, catalog no.
353948; Becton Dickinson, Heidelberg, Germany). Data points
were measured in triplicates. A total of 50,000 cells per well were
used in a total volume of 200 ␮l in each well. Plates were incubated
for 4 h (day 0) or 120 h (day 5) at 37°C, 5% CO2, and 95% humidity.
After incubation time, the number of living cells was determined by
the CellTiter-Blue assay (catalog no. G8081; Promega, Madison,
WI) with a GeniosPro universal microplate reader (Tecan, Crail-
sheim, Germany) following the manufacturer’s instructions, and
results are expressed as percentage of relative fluorescence units.
Cells were stimulated by phytohemagglutinin (2 ␮g/ml, PHA-P,
catalog no. L9132; Sigma–Aldrich) and human IL-2 (10 units/ml,
catalog no. 1204700; Roche, Mannhein, Germany) as a positive
Quintana et al.
control, anti-CD3/CD-28 beads, 5 ␮g/ml anti-CD3, and 5 ␮g/ml
anti-CD28 antibodies in solution (not coated on the plate). As
negative control anti-intercellular adhesion molecule-coated beads
or unstimulated cells were analyzed.
Actin Staining. For labeling F-actin, stimulated Th cells were pre-
cipitated and washed two times with Ringer’s solution before
allowing them to adhere to polyL-ornithine-coated (0.1 mg/ml in
distilled water; Sigma–Aldrich) glass coverslips for 10–15 min at
room temperature. Cells were washed twice with PBS (pH 7.4),
fixed in 3.7% formaldehyde solution for 10 min at room temper-
ature, washed two or more times with PBS, treated with 0.1%
Triton X-100 for 3–5 min, washed two or more times with PBS, and
stained with Texas red-X phalloidin (catalog no. T7471; Invitrogen)
for 20 min at room temperature. After the staining period, cells
were washed at least twice before taking pictures.
Two-Photon Imaging. Two-photon imaging was carried out exactly as
described previously (23).
Evanescent-Wave Imaging. The TIRF setup was based on an IX70
microscope (Olympus, Hamburg, Germany) equipped with a
⫻100/1.45 N.A. Plan Apochromat Olympus objective, a TILL-
TIRF condenser (TILL Photonics), and an argon 180 laser (Spectra
Physics, Darmstadt, Germany) emitting at 488 nm. Images were
acquired with a Micromax 512BFT camera (Princeton Instruments,
Ottobrunn, Germany) controlled by MetaMorph (Visitron, Göt-
tingen, Germany). The acquisition rate was 0.5 Hz, and the expo-
sure time was 200 ms. Pixel size was 130 nm. The penetration depth
was measured as follows. A low density of 100-␮m Ø TetraSpec
Beads (Invitrogen) was suspended in water containing 0.05% agar
to immobilize them. Other beads were attached to a glass coverslip,
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C9, and C12; Deutsche Forschungsgemeinschaft Grant HO 2190/1-2; and
Graduate College Grant 845 ‘‘Molecular, Physiological, and Pharmacolog-
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PNAS 兩 September 4, 2007 兩 vol. 104 兩 no. 36 兩 14423