Mitochondria Positioning Controls Local
Calcium Influx in T Cells
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J Immunol 2010; 184:184-190; Prepublished online 30
November 2009;
doi: 10.4049/jimmunol.0902872
http://www.jimmunol.org/content/184/1/184
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References
Christian Schwindling, Ariel Quintana, Elmar Krause and
Markus Hoth
The Journal of Immunology
Mitochondria Positioning Controls Local Calcium Influx in
T Cells
Christian Schwindling,* Ariel Quintana,*,1 Elmar Krause,† and Markus Hoth*
T
he stepwise activation of Th cells is required for an effective adaptive immune response to fight against foreign
Ags. This activation is initiated following the transport of
foreign Ags to lymph nodes by APCs. These Ags are coupled to
class II MHC molecules at the surface of APCs and corresponding
naive Ag-specific Th cells can bind to these complexes. After this
initial step, many molecular rearrangements occur in Th cells, and
the highly organized immunological synapse (IS) develops between Th cells and APC (1–4).
Formation of the IS is the trigger for several signaling pathways in
Th cells, which finally results in secretion of cytokines (e.g., IL-2),
clonal expansion, and differentiation of the Th cells (2, 5). An indispensable step during Th cell activation is the Ca2+ entry across
the plasma membrane through the opening of Ca2+ release-activated
Ca2+ (CRAC)/ORAI1 channels (6–9). These channels are opened
by STIM1 after depletion of intracellular Ca2+ stores (10, 11). To
prevent Ca2+-dependent inactivation of CRAC/ORAI1 channels,
mitochondria are able to take up the inflowing Ca2+ very efficiently
and act as Ca2+ buffers (12–19). It has previously been shown that
mitochondria translocate toward the IS during Th cell or NK cell
activation (18, 20). This mechanism results in higher intracellular
Ca2+ concentrations and more effective Th cell activation and
*Department of Biophysics and †Department of Physiology, University of the Saarland, Homburg, Germany
1
Current address: Department of Pathology, Immune Disease Institute, Harvard University, Boston, MA 02115.
Received for publication August 31, 2009. Accepted for publication October 27,
2009.
This work was supported by the Deutsche Forschungsgemeinschaft, Graduate College, “Molecular, physiological and pharmacological analysis of cellular membrane
transport,” and the Collaborative Research Center SFB 530, Project A3 (to M.H.).
Address correspondence and reprint requests to Dr. Markus Hoth, Institut für Biophysik, Gebäude 58, Universität des Saarlandes, D-66421 Homburg/Saar, Germany.
E-mail address: [email protected]
Abbreviations used in this paper: CCCP, m-carbonyl cyanidem-chlorophenylhydrazone; CRAC, Ca2+ release-activated Ca2+; ER, endoplasmic reticulum; IS, immunological synapse; TG, thapsigargin.
Copyright Ó 2009 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902872
proliferation (18, 21, 22). Interestingly, mitochondria can also accumulate at the uropod of T cells during migration and facilitate
lymphocyte chemotaxis (23).
The role of global Ca2+ signals for the regulation of gene expression in T cells (24) and subsequent T cell activation and
proliferation (21) is well established. Whether there is a physiological function for local Ca2+ entry in T cells is presently unclear.
In neurones, localized Ca2+ entry at the synapse regulates exocytosis and synaptic transmission. Th cells use two different
mechanisms to secrete different types of vesicles at different locations: one fraction including IL-2 and IFN-g is secreted at the
IS, whereas the other one including TNF and the chemokine
CCL3 is secreted everywhere at the plasma membrane (25). Huse
et al. (25) have shown that different transport proteins are associated with each pathway and have therefore proposed that different molecular mechanisms are responsible for the two modes of
secretion. Like for most types of exocytosis, it can be safely assumed that Ca2+ signals modulate the rates of vesicle secretion.
Local Ca2+ influx at the IS may therefore regulate the IS dependent secretory pathway, whereas the global secretion pathway
may depend on global Ca2+ signals.
Data about local Ca2+ influx in T cells are sparse. Lioudyno
et al. (26) reported local Ca2+ entry at the IS due to ORAI1 and
STIM1 enrichment at the IS (26), whereas Barr et al. (27) reported
ORAI1 and STIM1 enrichment in different areas, including the IS
but also in distal capping areas. In the current study, we have
analyzed the influence of mitochondrial positioning and local Ca2+
entry on each other in Th cells.
Materials and Methods
Cells
Human Jurkat T cell lines were grown in RPMI 1640 medium supplemented
with 10% FCS and penicillin-streptomycin as described previously (28).
Cells were continuously maintained in log-phase growth at 37˚C with 5%
CO2. For the experiments, the diphtheria toxin-resistant version of the
parental cell line generated by Fanger et al. (29) was used. CD4+ T cells
were prepared and maintained as previously described (21). All experiments have been approved by the local ethics committee.
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Formation of an immunological synapse (IS) between APC and T cells activates calcium entry through ORAI channels, which is
indispensable for T cell activation. Successful proliferation and maturation of naive T cells is possible only if premature inactivation
of ORAI channels is prevented. Although it is undisputed that calcium entry through ORAI channels is required for T cell function,
it is not known if calcium influx is uniformly distributed over the plasma membrane or if preferential local calcium entry sites (for
instance, at the IS) exist. In this study, we show that mitochondrial positioning determines the magnitude of local calcium entry
anywhere in the plasma membrane by reducing local calcium-dependent channel inactivation: if mitochondria are close to any given
local calcium entry site, calcium influx is large; if they are not close, calcium influx is small. Following formation of the IS, mitochondria are preferentially translocated to the IS in a calcium influx-dependent manner but independent of the exact calcium
influx site. Mitochondrial enrichment at the IS favors local calcium entry at the IS without the necessity to enrich ORAI channels at
the IS. We conclude that local calcium entry rather than global calcium entry is the preferential mechanism of calcium entry at
stable ISs in Th cells. The Journal of Immunology, 2010, 184: 184–190.
The Journal of Immunology
Reagents
The reagents used in our experiments include: thapsigargin (TG; stock 1 mM
in DMSO, Invitrogen, Karlsruhe, Germany) anti-human CD3 (obtained
from Abd Serotec, Düsseldorf, Germany), and anti-human CD8 mAbs (BD
Bioscience, Heidelberg, Germany). All chemicals not specifically mentioned were from Sigma-Aldrich, München, Germany (highest grade).
Bead stimulation and colabeling with fura 2-AM and
MitoTracker Green FM
All procedures were carried out exactly as described previously (18).
Fluorescence microscopy, Ca2+ imaging, and rhod-2
measurements
Ca2+ and mitochondrial imaging were carried out as described (18) with
a 603 (UPlanFL, Olympus, Melville, NY; numerical aperture = 1.25, oil)
objective. The exitation wavelengths for fura 2 were 340 and 380 nm;
MitoTracker (Invitrogen) was excited at 490 nm. TG (1 mM) or anti-human
CD3/CD28 mAb coated beads were used to stimulate Jurkat T cells.
m-carbonyl cyanidem-chlorophenylhydrazone (CCCP) (1 mM) was used for
185
disrupting mitochondrial Ca2+ uptake. Rhod-2 measurements were carried
out exactly as described previously (18).
Combined fluorescence microscopy and patch clamp
Cells were loaded with 2 mM fura 2-AM and 200 nM MitoTracker Green
FM simultaneously for 30 min at room temperature. After two washing
steps, cells were kept in Ca2+-free solution containing 1 mM TG to deplete
Ca2+ stores irreversibly and activate CRAC channels. To induce local Ca2+
influx in T cells, a cell-attached patch was then formed with the patch
pipette containing 1 mM Ca2+ Ringer solution. The membrane potential
was changed from +150 mV to 2100 mV for Jurkat T cells and +60 mV to
260 mV for CD4+ T cells to induce Ca2+ influx via the patch pipette. Fura
2 ratios and mitochondrial localization were detected simultaneously using
an Olympus IX70 microscope with a 603 UPlanApo objective (Olympus;
numerical aperture = 1.25, oil immersion). A TILL monochromator (TILL
Photonics, Gräfelfing, Germany) was used for excitation; the dichroic
mirror was a 500 LP and the emission filter a 515 LP.
Actin staining
T cells were washed two times with PBS (pH 7.4), fixed in 3.7% formaldehyde solution for 10 min at room temperature, washed two or more
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FIGURE 1. Mitochondrial translocation toward the IS requires calcium influx across the plasma membrane. A, Infrared and MitoTracker (Invitrogen)
fluorescence images from a single MitoTracker Green AM–loaded Jurkat T cell in the presence of 1 mM external Ca2+. The cell is stimulated by an antiCD3/anti-CD28–coated bead (indicated by the green circle). B, Same conditions as in A but in Ca2+-free solution. C, Statistical analysis of the subplasma
membrane localization of mitochondria (,0.99 mm beneath the plasma membrane) in Jurkat T cells (n = 13) near the IS (taken from the red area in A) and
under the rest of plasma membrane (black trace) after anti-CD3/anti-CD28 bead stimulation in the presence of 1 mM external Ca2+ (t test). D, Same as in C
but in Ca2+-free conditions (n = 9 cells) (t test). E, Infrared and fluorescence images from single Jurkat T cells in which the actin cytoskeleton was stained
by Texas Red-X phalloidin after 30-min stimulation with anti-CD3/anti-CD28 in the absence or presence of external Ca2+.
186
MITOCHONDRIA CONTROL LOCAL CALCIUM INFLUX IN T CELLS
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. The
staining procedure was performed on the stage of the microscope after the
combined patch clamp/imaging experiments were finished.
Data analysis
Data were analyzed using TILL Vision (TILL Photonics), Igor Pro
(WaveMetrics, Lake Oswego, OR) and Microsoft Excel (Microsoft, Redmond, WA). All values are given as mean 6 SEM (n, number of cells). If
data points were normally distributed, a paired or unpaired two-sided
Student t test was used. If normal distribution could not be confirmed,
a nonparameterized test (Mann-Whitney) was carried out.
Results
Ca2+ influx is necessary for the translocation of mitochondria
to the IS
FIGURE 2. Control of local Ca2+ entry with
a cell-attached patch pipette. A, Experimental
setup: a T cell is patched in a cell-attached patch
configuration and stimulated by TG. Ca2+ can
enter the cell only through the pipette over the
patched membrane because Ca2+ is absent from
the bath solution. B, Setup as in A. Fura 2 ratio
values of a Jurkat T cell treated with TG at different membrane potentials (+150 mV and 2100
mV). Ca2+ influx can be triggered by changes of
the membrane potential via the cell-attached
patch. C, Infrared, fluorescence and fura 2 ratio
images of a Jurkat T cell patched in a cellattached patch configuration. After changing of
the membrane potential from +150 mV to 2100
mV, Ca2+ enters the cell at the site of the patch
area and diffuses/is transported into the rest of
the cytosol.
Local Ca2+ entry in T cells
To test whether local Ca2+ entry at the IS was required to translocate
mitochondria to the IS, we applied a technology previously used in
pancreatic acinar cells to induce local Ca2+ entry (30). A cellattached patch was formed on a cell bathed in 0 mM Ca2+ solution,
which contained 1 mM TG to deplete intracellular Ca2+ stores and
activate store-operated CRAC channels. The patch pipette contained 1 mM Ca2+ and was thus the only extracellular Ca2+ source
(Fig. 2A). The patch was clamped at +150 mV immediately after
establishing the on-cell configuration to avoid any Ca2+ influx over
the patch. A patch potential change from +150 mV to 2100 mV
induced Ca2+ entry over the patch, resulting in a global Ca2+ signal
in the T cell (Fig. 2B). The intracellular Ca2+ concentration increase
showed a Ca2+ overshoot typical for T cells and was terminated
completely by depolarizing the patch membrane back to +150 mV.
Ca2+ imaging of cells under these conditions revealed that the
global Ca2+ signal cannot be explained by simple Ca2+ diffusion
away from the Ca2+ entry source. Complicated diffusion patterns
and Ca2+ gradients imply that diffusion barriers and/or Ca2+
transport through organelles must exist (Fig. 2C).
The technology of focal Ca2+ entry in T cells was now used to
test whether Ca2+ influx at the IS was required to induce translocation of mitochondria to the IS. Fig. 3A shows a model T cell
that is patched on the upper side of the cell. In addition, an antiCD3/anti-CD28 Ab-coated bead is brought into contact with the
cell at the opposite side to stimulate the cell and induce IS formation. IS formation was confirmed by actin cytoskeleton reorganization toward the cell-bead contact point (Fig. 3B). Using
this setup, we could now separate the location of the IS formation
and Ca2+ entry from each other. Analyzing mitochondrial movement under these conditions, we found that mitochondria were
clearly moved toward the IS and not toward the Ca2+ entry site
(Fig. 3C). In the example shown, small Ca2+ entry was activated
by the potential change after 20 s. Translocation of mitochondria
to the IS was observed immediately after activation of Ca2+ entry
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We have previously shown that Ca2+ influx through CRAC
channels regulates the translocation of mitochondria to the plasma
membrane (17) and to the IS (18) during and after its formation
with Ab-coated beads. In this study, we confirm these findings and
show in addition that Ca2+ influx is necessary for the translocation
of mitochondria during the early and late phase of IS formation.
Fig. 1A shows an example of anti-CD3/anti-CD28 Ab-coated
bead–induced mitochondrial translocation to the IS in the presence of Ca2+ entry from the extracellular space. In the absence of
external Ca2+, the release of Ca2+ from stores was not sufficient to
promote translocation of mitochondria to the IS (Fig. 1B). The
statistical analysis in the presence and absence of extracellular Ca2+
confirms that Ca2+ influx is needed for mitochondrial translocation
to the IS (Fig. 1C, 1D). However, Ca2+ influx is necessary but not
sufficient because in the absence of actin cytoskeleton rearrangement during IS formation, Ca2+ influx alone was not able
to induce mitochondrial transport to the IS (18). The same is true
for the rearrangement of the actin cytoskeleton, because actin fibers moved to the IS in the absence of extracellular Ca2+ (Fig. 1E),
and mitochondrial translocation toward the IS was completely
absent (Fig. 1B, 1D). In conclusion, both the actin rearrangement
and Ca2+ entry are necessary for mitochondrial accumulation at
the IS; none of the two can therefore be sufficient.
The Journal of Immunology
187
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FIGURE 3. Mitochondria translocate to the IS and not to the calcium influx source. A, Experimental setup: same as in Fig. 2A, only in addition, an IS is
induced distal to the patch by an anti-CD3/anti-CD28 Ab-coated bead. B, Infrared and fluorescence images from a Jurkat T cell. The actin cytoskeleton was
stained by Texas Red-X phalloidin after 30 min of stimulation with an anti-CD3/anti-CD28 Ab-coated bead. The membrane potential across the patch was
changed after 20 s from +150 mV to 2100 mV. C, Infrared, fluorescence, and fura 2 ratio images from a Jurkat T cell loaded with MitoTracker Green FM
and fura 2-AM. The membrane potential across the patch was changed after 20 s from +150 mV to 2100 mV. D, Statistical analysis of cells as shown in C.
Percentage of mitochondria at the IS (C, red area) compared with the percentage of mitochondria at the Ca2+ influx site (C, blue area) at 0 min and 30 min.
by the membrane potential jump during IS formation (data not
shown). The qualitative analysis revealed that translocation of
mitochondria to the IS was observed in .90% of the cells. Mitochondrial fluorescence was on average increased by a factor of
two, whereas it was constant at the Ca2+ entry site (Fig. 3D). We
conclude that mitochondria are moved to the IS independent of the
position of the Ca2+ entry source.
Mitochondria control local Ca2+ entry in T cells
We have previously shown that global Ca2+ entry (over the whole
plasma membrane) in T cells is controlled by mitochondria Ca2+
uptake (13, 18). To test whether the same is true for local Ca2+ entry,
a cell-attached patch was formed on a cell bathed in 0 mM Ca2+
solution, which contained 1 mM TG to deplete intracellular Ca2+
stores and activate store-operated CRAC channels. Changing the
patch potential from +150 to 2100 mV, we found that intracellular
Ca2+ concentration increased much more in the case in which mitochondria were by chance close to the patch and the local Ca2+
influx source compared with the case in which mitochondria were
by chance far away from the patch (Fig. 4A). In the case in which
mitochondria were close to the Ca2+ entry source, the Ca2+ influx
during steady state was much bigger compared with the case in
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MITOCHONDRIA CONTROL LOCAL CALCIUM INFLUX IN T CELLS
which mitochondria were far away from the Ca2+ entry source (Fig.
4B). The statistical analysis reveals a clear difference between the
Ca2+ signals in cells with mitochondria close to or away from the
patch (Fig. 4C). A similar result was found in primary human Th
cells (Fig. 4D). The effect of mitochondria on CRAC/ORAI channel
activity can be explained by capability of mitochondria to take up
large amounts of Ca2+ (31), which enable them to reduce the local
accumulation of Ca2+ near the sites that govern Ca2+-dependent
channel inactivation efficiently (13, 18). Indeed, the larger Ca2+
signals were reduced if mitochondrial Ca2+ uptake was inhibited by
1 mM CCCP in cells with mitochondria close to the patch (Fig. 4C).
These experiments clearly indicate that mitochondrial positioning
controls local Ca2+ entry in T cells.
To analyze how much Ca2+ was taken up by mitochondria depending on their position, we used rhod-2 loading by an electroporation technique established previously (18, 19). As expected,
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FIGURE 4. Local Ca2+ entry is influenced by the localization of mitochondria relative to the Ca2+ entry site. A, Examples of two Jurkat T cells treated
with TG and loaded with MitoTracker Green AM and fura 2-AM. Ca2+ influx was activated by changing the patch potential across the patch from +150 mV
to 2100 mV. The upper panel shows a Jurkat T cell where the mitochondria were by chance localized close to the patch (as seen by the MitoTracker picture
excited at 470 nm); the lower panel shows a Jurkat T cell where the mitochondria were by chance localized far away from the patch (as seen by the
MitoTracker picture excited at 470 nm). B, Fura 2 ratios of two Jurkat T cells under experimental conditions as in A. The patch potential was changed after
20 s from +150 mV to 2100 mV. C, Average fura 2 ratios during the Ca2+ plateau (450–500 s) of cells with mitochondria localized close to the patch pipette
(n = 19) or cells with mitochondria localized away from the patch pipette (n = 12). Values were significantly different (two-sided unpaired t test, p , 0.05).
The third bar shows cells with mitochondria localized close to the patch pipette (n = 15) but in addition treated with 1 mM CCCP to disrupt the mitochondrial Ca2+ uptake. D, Average fura 2 ratios from 450 to 500 s of primary CD4+ T cells using the same experimental conditions as in A and B, only the
patch potential was changed after 20 s from +60 mV to 260 mV (the patches of the primary cells were not stable enough at more extreme potentials). Ten
cells with mitochondria close to the patch and three cells with mitochondria away from the patch were analyzed. E, Infrared and fluorescence images of
a Jurkat T cell treated with TG and patched in a cell-attached patch configuration. Mitochondrial Ca2+ concentrations were visualized with rhod-2. Ca2+
influx was activated by changing the patch potential across the patch after 20 s from +150 mV to 2100 mV. The arrows highlight mitochondrial clusters
located near or away from the Ca2+ influx site. F, Statistical analysis of cells as the one shown in E. Mitochondria located near (red trace; n = 9 cells) or
away (black trace; n = 7 cells) from the Ca2+ influx site were analyzed.
The Journal of Immunology
Discussion
We have shown that Ca2+ influx anywhere in the plasma membrane is
required to induce the accumulation of mitochondria at the IS. Mitochondrial positioning allows local Ca2+ entry only at sites where
mitochondria are localized. Therefore, we conclude that sustained
Ca2+ entry in T cells, which is essential to induce and maintain NFAT
translocation in the nucleus, occurs preferentially at the IS.
In mast cells, local Ca2+ entry is coupled to specific cellular
functions like 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14cis-eicosatetraenoic acid secretion (32) or gene expression (33, 34).
This further highlights the potential importance of localized Ca2+
entry in immune cells to control secretion and gene expression. One
potential mechanism to generate local Ca2+ entry at a given site of
a cell is the enrichment of STIM and ORAI at a given location.
Lioudyno et al. (26) found that STIM1 and ORAI1 were accumulated at the IS in T cells following their formation. This accumulation favored Ca2+ influx at the IS and higher Ca2+ signals in the
subcellular areas close to the IS. However, using high-resolution
microscopy, Barr et al. (27) reported a diverse localization of
STIM1 and ORAI1 throughout the plasma membrane after IS
FIGURE 5. Local Ca2+ entry at the IS is influenced
by the localization of mitochondria with respect to the
Ca2+ entry site at the IS. A, Experimental setup: same
as in Fig. 2A, only that in addition, an IS is induced at
the site of the patch by including anti-CD3/anti-CD28
Abs in the patch pipette. B, Infrared and fluorescence
images from a Jurkat T cell. The actin cytoskeleton was
stained by Texas Red-X phalloidin after 30 min of
stimulation with the patch pipette. The membrane potential across the patch was changed after 20 s from
+150 mV to 2100 mV. C, Fura 2 ratios of two Jurkat
T cells under experimental conditions as in A with
mitochondria localized close or away from the patch.
The latter case was extremely rare because in .90% of
the cells, mitochondria were translocated to the IS
(compare to Fig. 3C). D, Average fura 2 ratios during
the Ca2+ plateau (450–500 s) of cells as in C with
mitochondria close to the patch (n = 15) or with mitochondria away from the patch (n = 3).
formation. They found that STIM1 and ORAI1 often clustered in
caps, which were often localized at the distal pole and not at the IS.
In addition, they also found that caplike structures were able to
move to sites close to the IS. This dynamic led Barr et al. (27) to
speculate that the caps could be used to assemble pre-existing
ORAI1–STIM1 complexes for use at newly established synapses.
Alternatively, the caps could also prevent STIM1–ORAI1 complexes to participate actively in Ca2+ entry outside the IS. Unfortunately, it cannot be finally resolved at the moment if and where
ORAI and STIM are localized during and after IS formation. Because we have shown that mitochondrial positioning determines
local Ca2+ entry at the IS, only ORAI1 channels at the IS would stay
open for extended times. Thus, the mitochondrial localization at the
IS induces localized and sustained Ca2+ influx.
In many cell types, it has been demonstrated that mitochondria
can sense microdomains of Ca2+ released from the endoplasmic
reticulum (ER) (31). The close interaction between ER and mitochondria influences Ca2+ release from the ER and Ca2+-dependent
cell functions. In T cells, mitochondria appear not to have such
a drastic role in controlling Ca2+ release from the ER (12, 28).
Furthermore, we could not detect an influence of mitochondrial
mobility on Ca2+ release from the ER (18). In conclusion, Ca2+
release from the ER appears not to have the same functional importance in T cells as in other cells (16), highlighting the role of Ca2+
entry at the IS as the major source of Ca2+ (22).
The duration of an IS can range from seconds or minutes up to
several hours (35, 36). Naive CD4+ or CD8+ cells have to continuously scan their environment to find matching APCs. Very
short IS durations are beneficial to allow as many contacts as
possible to find a potentially rare match with an Ag-presenting
dendritic cell. Because T cells still show net migration during the
formation of such a short IS, Dustin (37) termed such a short-lived
synapse “kinapse” (from “kinetic synapse”). Kinapses have been
implicated to be important in the induction of tolerance in case
naive T cells only contact low-potency MHC-bound Ags. T cells
did not change migration pattern under these conditions and were
never arrested to form a stable long-lasting IS, but they finally
became tolerant after forming several kinapses (36). Longer IS
durations of 20–60 min have been observed when CTLs kill target
cells (38). Even longer lasting stable synapses are required for the
maturation and proliferation of naive T cells. Given the great
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mitochondria close to the Ca2+ influx source took up more Ca2+
earlier and at a higher rate than the ones further away from the influx
source after Ca2+ influx was activated by the patch potential change
from +150 mV to 2100 mV, as evident from the pictures in Fig. 4E
and the time course of the relative fluorescence signals in Fig. 4F.
Finally, we induced IS formation and local Ca2+ influx at the
same location. To do this, we included anti-CD3/anti-CD28 Abs in
the patch pipette (Fig. 5A). Again, a cell-attached patch was
formed with Ca2+ included in the patch solution but no Ca2+ in the
bath solution. IS formation at the position of the cell-attached
patch under these conditions was confirmed by actin cytoskeleton
rearrangement (Fig. 5B). Comparing the many cells that had mitochondria localized close to the IS with the few ones that had
mitochondria localized away from the IS and the focal Ca2+ entry,
we found again that Ca2+ signals were significantly higher if
mitochondria were close to the IS (Fig. 5C, 5D). In summary, we
conclude that mitochondrial positioning determines where focal
Ca2+ entry is induced. Following their accumulation at the IS,
mitochondria therefore induce local Ca2+ entry through CRAC/
ORAI channels at the IS.
189
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MITOCHONDRIA CONTROL LOCAL CALCIUM INFLUX IN T CELLS
Acknowledgments
We thank Eva C. Schwarz for help with primary cells, and we thank Bettina
Strauß and Anja Ludes for excellent technical support.
Disclosures
The authors have no financial conflicts of interest.
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importance of Ca2+ signals for all aspects of T cell activation,
mitochondrial positioning may be very important to determine
synaptic efficacy by regulating ORAI1 activity.
Mitochondria-dependent restricted Ca2+ influx at the IS would
preferentially induce Ca2+ signals close to the IS. This may initially restrict Ca2+-dependent vesicle exocytosis to the area of the
IS. However, if the IS persists for extended times, mitochondria
would also transport the Ca2+ away from the IS to distant cytosolic
sites and induce vesicle release there. In the case of effector CD4+
T cells, it has been indeed shown that different mechanisms exist
to release vesicles restricted either to the IS only or anywhere at
the plasma membrane (25). Therefore, we believe that mitochondria-dependent local Ca2+ entry could be used to determine
the place and timing of vesicle release.
In the case of naive CD4+ cells, they have to first continuously
scan their environment to find matching APCs. Very short IS
durations are beneficial to allow them as many contacts as possible
to find a potentially rare match with an APC. However, once the
right APC is found, longer IS (16–24 h) are required to sustain
translocation of NFAT into the nucleus. Therefore, it is very important to analyze IS durations under physiological conditions to
fully integrate the basic mechanisms that control Ca2+ signaling.
Local Ca2+ entry may be an important tool to modulate T cell
activation and function.