T H E J O U R N A L O F C E L L B I O L O G Y
The Journal of Cell Biology, Vol. 174, No. 7, September 25, 2006 915–921
Mitochondria have been recently established as both physiolog-
ical targets and relay points in intracellular Ca2+ signaling, con-
tributing to a spectrum of cellular events ranging from oxidative
ATP generation (Hajnoczky et al., 1995; Robb-Gaspers et al.,
1998; Jouaville et al., 1999) to apoptotic cell death (Ferri and
Kroemer, 2001; Demaurex and Distelhorst, 2003). This versa-
tile function of mitochondria depends on the generation of mi-
tochondrial matrix [Ca2+] ([Ca2+]m) signals. The [Ca2+]m signal
results from activation of the uniporter-mediated Ca2+ uptake
that shows a relatively low Ca2+ affi nity (Kirichok et al., 2004;
Nicholls, 2005). The mitochondrial Ca2+ uptake of the IP3
receptor (IP3R)–mediated Ca2+ release is facilitated locally by
the high cytoplasmic [Ca2+] ([Ca2+]c) microdomains around the
IP3Rs at focal contact areas between the ER and mitochondria
(Rizzuto et al., 1998). Notably, mitochondria exhibit structural
and functional diversity (Collins et al., 2002), and subsets of
mitochondria may interact locally with other organelles (Hoth
et al., 2000; Dolman et al., 2005). The local [Ca2+] control
between IP3Rs and mitochondria seems to occur at stable sites
between the ER and mitochondria (Filippin et al., 2003) and
displays a “quasisynaptic” organization (Csordas et al., 1999).
Mitochondria-associated ER membranes are also involved
in multiple mechanisms of joint operation between the two
organelles, in the synthesis of the mitochondrial cytochrome c
oxidase (Parimoo et al., 1982) and phospho- and glycosphingo-
lipids (Voelker, 2005).
The existence of physical links between ER and mito-
chondria have been suggested based on cosedimentation of ER
particles with mitochondria and electron microscopic observa-
tions of close associations between mitochondria and ER vesi-
cles (Shore and Tata, 1977; Meier et al., 1981; Mannella et al.,
1998). Recently, several mitochondria or ER bound proteins
have been shown to be important for maintaining the spatial re-
lationship between ER and mitochondria and, hence, have also
been implicated as possible linking elements: DLP-1/DRP1-1
(Pitts et al., 1999; Varadi et al., 2004), tumor autocrine motility
factor receptor (Wang et al., 2000), and PACS-2 and BAP31
(Simmen et al., 2005). IP3Rs have also been postulated to
interact with the Voltage-dependent anion-selective channel to
form an ER–mitochondria Ca2+ tunnel (Rapizzi et al., 2002).
Heterogeneity in the distance between the interfacing ER and outer
mitochondrial membranes (OMMs; Pacher et al., 2000) also in-
dicates that the contact formation may depend on several factors
and raises the intriguing possibility that the ER–mitochondria
Structural and functional features and signifi cance
of the physical linkage between ER and mitochondria
György Csordás,1 Christian Renken,2 Péter Várnai,3 Ludivine Walter,1 David Weaver,1 Karolyn F. Buttle,2 Tamás Balla,3
Carmen A. Mannella,2 and György Hajnóczky1
1Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107
2Resource for Visualization of Biological Complexity, Wadsworth Center, Albany, NY 12201
3Endocrinology and Reproduction Research Branch, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892
mitochondria and endoplasmic reticulum (ER). However,
the physical linkage of the ER–mitochondria interface and
its relevance for cell function remains elusive. We show
by electron tomography that ER and mitochondria are
adjoined by tethers that are ?10 nm at the smooth ER and
?25 nm at the rough ER. Limited proteolysis separates
ER from mitochondria, whereas expression of a short
he role of mitochondria in cell metabolism and sur-
vival is controlled by calcium signals that are com-
monly transmitted at the close associations between
“synthetic linker” (<5 nm) leads to tightening of the
associations. Although normal connections are necessary
and suffi cient for proper propagation of ER-derived
calcium signals to the mitochondria, tightened connections,
synthetic or naturally observed under apoptosis-inducing
conditions, make mitochondria prone to Ca2+ overloading
and ensuing permeability transition. These results reveal
an unexpected dependence of cell function and survival
on the maintenance of proper spacing between the ER
Correspondence to György Hajnóczky: email@example.com
Abbreviations used in this paper: [Ca2+]c, cytoplasmic [Ca2+]; [Ca2+]m,
mitochondrial matrix [Ca2+]; ET, electron tomography; IP3R, IP3 receptor; mRFP,
monomeric red fl uorescent protein; OMM, outer mitochondrial membrane; SBI,
soybean trypsin inhibitor; TEM, transmission EM; Tg, thapsigargin.
The online version of this article contains supplemental material.
JCB • VOLUME 174 • NUMBER 7 • 2006 916
distance may be controlled to affect ER and mitochondria
function. However, despite the attention paid to the structural
basis of the ER–mitochondria communication, the fundamental
question of whether direct physical linkage between ER and mi-
tochondria is required for the local [Ca2+] coupling remains to
be elucidated. Here, we visualize the ER–mitochondria tethers
and show that the local Ca2+ coupling can be weakened and
strengthened by demolition and enforcement of the interorgan-
ellar protein linkage, respectively. Furthermore, our data reveal
a novel regulatory role of the ER–mitochondria gap width in
Ca2+ signaling and in cell survival.
Results and discussion
To directly visualize the structures responsible for the physical
association of the ER with mitochondria, we used electron
tomography (ET), which can reveal fi ne structural details missed
in conventional micrographs because of overlapping densities
(Mannella et al., 1994). Tomographic analysis of isolated rat-
liver mitochondria (conventionally fi xed, plastic-embedded or
unfi xed, frozen-hydrated) show narrow particles connecting the
OMM to putative ER vesicles (Fig. 1 A and Fig. S1 A, available
These “tethers” tend to occur in clusters of six or more, spaced
13–22 nm apart, spanning intermembrane distances of 6–15 nm
with indications of increments occurring in 5-nm steps (Table S1).
Electron micrographs and tomograms of plastic-embedded liver
(not depicted) and DT40 cells (Fig. 1) indicate numerous
regions of close association between mitochondria and both
rough and smooth ER, but the noisy background (due to particle
crowding) made tethers more diffi cult to detect than in isolated
fractions. Tethers connecting OMM and smooth ER in situ
(unpublished data) have lengths (9–16 nm) similar to those
between attached vesicles and OMM of isolated mitochondria
(Table S1). Spacings between OMM and rough ER in situ begin
at 20 nm (the minimum distance to accommodate ribosomes),
and the measured tether lengths are 19–30 nm (Table S1). Of
the six tethers detected in the DT40 tomogram of Fig. 1 (B–F),
three appear to terminate at ribosomes on the ER. The DT40
cells used were IP3R triple knockout cells (IP3R-TKO), chosen
to assess the role of IP3R in the interorganellar coupling.
In electron micrographs of DT40 cells, the IP3R-TKO have
ER–mitochondria associations similar to wild-type cells (Fig. S1,
B–E), suggesting that an IP3R-independent linkage exists be-
tween ER and mitochondria.
In summary, ET has revealed direct physical links of vary-
ing length between smooth and rough ER and mitochondria,
both in normal tissue and IP3R knockout cells. Because IP3Rs
are present to mediate Ca2+ release at both smooth and rough
ER, multiple coupling elements may be relevant for Ca2+ signal
propagation from ER to mitochondria. The heterogeneity in
tether lengths points to an enticing new possibility, that ER–
mitochondria communication may be controlled by varying the
interorganellar distance. To test the functional signifi cance of
the tethers, we designed strategies for weakening and enhancing
the physical coupling.
To disrupt the ER–mitochondria physical coupling, limited
proteolysis was used. Confocal images of isolated liver mito-
chondria preparations showed abundant overlapping immuno-
reactivity for both IP3R (type 1 and 2) and cytochrome c oxidase,
an enzyme of the inner mitochondrial membrane (Fig. 2 A),
indicating that the IP3Rs reside in mitochondria-associated
ER. When this preparation was trypsinized (40 μg/ml for 150 s
followed by addition of soybean trypsin inhibitor [SBI] at
250 μg/ml) and recentrifuged, the IP3R immunoreactivity dis-
appeared (Fig. 2 B) and was recovered in the light membranes
(not depicted). The IP3- sensitive Ca2+ store was also quantifi ed
by measurement of the IP3 + thapsigargin (Tg)-induced Ca2+
release in the 10,000-g pellet (ER– mitochondria complex) and
supernatant (ER only) of both the control and trypsin-pretreated
Figure 1. Tethering structures between ER and mitochondria visualized by ET. (A, top) Slice (2 nm thick) from tomogram of a frozen-hydrated rat-liver
mitochondrion (700-nm diameter), showing several attached putative ER vesicles. Bar, 100 nm. (bottom) Higher magnifi cation (2×) slices through vesicles
1–3 showing tethers (arrowheads). Bar, 50 nm. (B) Micrograph of a DT40 TKO cell (200-nm-thick plastic section) showing ER fl anking a mitochondrion.
Bar, 250 nm. (C) Slice (3 nm thick) from tomogram of this fi eld. (D) Surface model of ER (yellow) and OMM (red). (E) Subfi elds (130-nm diameter) from
ER–mitochondria interface regions (1–3 are from boxed region in D) with tethers indicated by arrowheads (black arrowheads indicate tethers that terminate
on ribosomes). (F) 3D models of three subregions in E, showing isodensity surfaces that best visualize the tethers (gray), membrane surfaces (OMM, red;
ER, yellow), and ribosomes (blue ellipsoids). The resolution is ?8 nm in the z (vertical or section thickness) direction.
ER–MITOCHONDRIA STRUCTURAL AND CA2+ COUPLING • CSORDÁS ET AL. 917
liver mitochondria (Fig. 2 C). Trypsin pretreatment caused
a twofold increase in Ca2+ release in the supernatants and a
signi fi cant decrease in the pellets (Fig. 2 C). Similar fi ndings
were obtained in RBL-2H3 cells (Fig. S2, A and B, available
at http://www.jcb.org/cgi/content/ full/jcb.200604016/DC1).
Colle ctively, these data suggest that limited proteolysis disrupted
the physical coupling between ER and mitochondria.
We next evaluated the effect of proteolytic treatment on
mitochondrial Ca2+ signaling. IP3R-mediated Ca2+ release ef-
fectively supports mitochondrial Ca2+ uptake in permeabilized
RBL-2H3 cells (Csordas et al., 1999). Suspensions of digitonin-
permeabilized cells were treated with proteinase K, another
serine protease (20 μg/ml for 150 s), and [Ca2+]c and [Ca2+]m
were simultaneously monitored fl uorometrically. In control
cells, 8 μM IP3 evoked an abrupt increase in [Ca2+]c that was
paralleled by a rapid and substantial increase in [Ca2+]m (Fig. 2 D,
left). In the proteinase K–pretreated cells, the IP3-induced
[Ca2+]c increase was preserved, but the [Ca2+]m increase was
practically eliminated (Fig. 2 D and Fig. S2 C). Because
proteinase K treatment did not affect IP3-induced Ca2+ release
(either in the presence or absence of mitochondrial uncouplers)
and failed to inhibit the mitochondrial uptake of directly added
Ca2+ (Fig. 2 D and Fig. S2 C), it is likely that proteinase K inhibited
the transfer of released Ca2+ from IP3Rs to the mitochondria.
Similar data were obtained when trypsin was used
instead of proteinase K (Fig. 2 E). Consistent with earlier
reports, trypsin-digested preparations retained IP3-induced
Ca2+ release (Yoshikawa et al., 1999) but almost completely
lost the IP3- induced [Ca2+]m signal, an effect that was pre-
vented by SBI (Fig. 2 E, middle). Trypsin also failed to
inhibit the [Ca2+]m increase induced by elevation of the bulk
[Ca2+]c by addition of 10 μM CaCl2 (Fig. 2 E; initial rates were
0.96 ± 0.08 for control and 1.25 ± 0.17 μM/s for trypsin, re-
spectively; n = 3). The trypsin dose dependence and time
course data (Fig. S2 C) further illustrate that the Ca2+ trans-
fer from IP3Rs to the mitochondrial matrix is very sensitive
to tryp sinolysis, whereas the Ca2+ release or mitochondrial
Ca2+ uptake by itself is hardly inhibited. Thus, limited prote-
olysis disrupts the link between ER and mitochondria and
suppresses the propagation of the IP3R-mediated Ca2+ release
to the mitochondria.
To tighten the physical coupling between ER and mi-
tochondria, we created a construct that encodes monomeric
red fl uorescent protein (mRFP) fused to the OMM targeting
sequence of mAKAP1 at the N terminus and fused to the ER
targeting sequence of yUBC6 at the C terminus (mAKAP1
[34–63]-mRFP-yUBC6, OMM–ER linker). Based on the size
of the fl uorescent protein (4.2 × 2.4 nm), the maximal length of
this construct is <5 nm. As a control, the above construct was
also prepared without the ER targeting sequence (mAKAP1
[34–63]-mRFP). Cells expressing the constructs showed red
fl uorescence localized to the mitochondria and displayed mi-
tochondrial aggregation in some cells (unpublished data). To
analyze the ER–mitochondria interface, the transfected cells
were sorted and prepared for transmission EM (TEM). The
mAKAP1(34–63)-mRFP transfected cells displayed numerous
associations between ER and mitochondria, but the interface
area only involved a small fraction of the mitochondrial perim-
eter (Fig. 3 A, left) similar to the situation in nontransfected
cells (Fig. 4 B). The ER–OMM distance showed bimodal
distribution, having the most frequent values at 10–15 and
25–30 nm, similar to the results of the ET analysis. In contrast,
in the OMM–ER linker transfected cells, the ER formed a
cap over large mitochondrial areas and the cleft between the
Figure 2. Limited proteolysis loosens the
structural and functional association of the
IP3R with mitochondria. (A and B) Confocal
images showing the distribution of IP3Rs (red)
and the cytochrome c oxidase (green) and
their colocalization (overlay, yellow) in a rat-
liver mitochondrial fraction attached to cover-
slips. (B) Trypsin (40 μg/ml; 150 s) was added
before attachment to the coverslips (n = 3).
(C) ER Ca2+ storage in naive and trypsin-
pretreated rat-liver mitochondrial fraction. In sus-
pensions of the particles, the capacity of the ER
Ca2+ store was determined as the sum of
the extravesicular [Ca2+] ([Ca2+]o) increases
caused by sequentially added IP3 and Tg in the
10,000-g supernatants (ER-only fraction) and
pellets (ER–mitochondria complex) after tryp-
sinization (40 μg/ml; 150 s) in the presence
(control) and absence of SBI (mean ± SEM;
n = 10). (D and E) Effect of proteinase K and
trypsin on the IP3-induced [Ca2+]c and [Ca2+]m
increase in suspensions of permeabilized RBL-
2H3 cells. (D) Control (black) and proteinase
K–pretreated cells (red) in the absence (left) or
presence (middle) of uncoupler (2 μM car-
bonyl cyanide p-trifl uoromethoxyphenylhydra-
zone + 5 μg/ml oligomycin). (right) [Ca2+]m
rise evoked by a 10 μM CaCl2 pulse (Ca; bulk [Ca2+]c increase, ?3 μM). To prevent the uptake of added Ca2+ by the ER, 2 μM Tg was added 5 s before
stimulus. (E) 100 μg/ml trypsin for 150 s (left, red) or 60 s (middle, pink). 40 μg/ml trypsin for 150 s (middle) in the absence (purple) or presence (gray)
of 250 μg/ml SBI. (right) Effect of trypsin in the presence of uncoupler.
JCB • VOLUME 174 • NUMBER 7 • 2006 918
ER membrane and the OMM was extremely narrow (Fig. 3 A,
right). On average, the ER–mitochondria distance at these sites
decreased from 24 ± 3 to 6 ± 1 nm and the interface area increased
fourfold in the presence of the OMM–ER linker (Fig. 3 B). Thus,
expression of the OMM–ER linker caused the associations to
become tighter and the interface area to increase.
To evaluate the effect of the enhanced physical coupling be-
tween the organelles on the Ca2+ transport, we conducted imaging
of [Ca2+]m and [Ca2+]c in permeabilized cells expressing either
mAKAP1(34–63)-mRFP-yUBC6 or mAKAP1(34–63)-mRFP
and the Ca2+ probe, ratiometric pericam targeted to the mitochon-
drial or nuclear matrix. Synchronous Ca2+ release evoked by
maximal IP3 induced comparable [Ca2+]c and [Ca2+]m responses
in both OMM–ER linker and control cells (unpublished data).
However, a different picture emerged when gradual Ca2+
liberation through IP3Rs was stimulated by adenophostin A
(Bird et al., 1999; Csordas and Hajnoczky, 2001). The nuclear
matrix [Ca2+] rise that closely follows the [Ca2+]c signal was un-
affected in the OMM–ER linker–expressing cells, but the [Ca2+]m
elevation was signifi cantly enhanced (Fig. 3 C, top). In particular,
the delay between the [Ca2+]nuclear and [Ca2+]m elevation was
shortened (Fig. 3 C, left). Thus, overexpression of the linker
enabled mitochondrial Ca2+ uptake during Ca2+ mobilization
conditions that normally are recognized by the mitochondria with
low effi ciency. Based on the effects of the synthetic linker, the
quantity and the length of the tethers may exert a control on the
ER–mitochondria Ca2+ coupling.
Bringing the ER closer to mitochondria by the physiologi-
cal tethers effectively supports local Ca2+ signaling. However,
sustaining a gap between the organelles by the tethers may also
bear signifi cance for other cell functions. A too-close associa-
tion between ER and mitochondrial membranes might cause
continuous mitochondrial Ca2+ uptake during background Ca2+
release and, in turn, could facilitate mitochondrial Ca2+ over-
loading and membrane permeabilization (Bernardi, 1999; Green
and Kroemer, 2004). Also, the extent of the anchorage of ER to
the mitochondria is relevant to the motility of the mitochondria,
which allows dynamic redistribution of the mitochondrial ATP
production and Ca2+ buffering throughout the cell (Yi et al.,
2004; Brough et al., 2005). To test the idea that tightening of the
ER–mitochondria coupling may affect the cells’ ability to re-
spond to challenges, RBL-2H3 cells expressing the ER–OMM
linker were exposed to Tg, which gradually mobilizes the ER
Ca2+ store and, in turn, stimulates the store-operated Ca2+ entry
(as a control, either the mitochondria- or the ER-targeted part
of the linker was overexpressed). In the cells expressing the
OMM–ER linker, the [Ca2+]c signal showed an initial elevation
followed by a partial decay to a plateau. After a longer period of
time, a gradual elevation appeared turning to a steep and robust
[Ca2+]c rise (Fig. 4 A, red). This second [Ca2+]c rise began at
different time points in the individual cells, causing a more
gradual rise in the mean response (Fig. 4 A, bottom). In control
cells, the fi rst [Ca2+]c rise was similar to those in the OMM–ER
linker cells; however, the second [Ca2+]c rise developed much
more slowly (Fig. 4 A, black). Analysis of [Ca2+]c signals in
single cells showed an early onset of both the gradual [Ca2+]c
elevation and the steep and robust [Ca2+]c increase (Fig. 4 A,
right) in the cells expressing the OMM–ER linker. The second
[Ca2+]c elevation was prevented by the addition of either 5 μM
carbonyl cyanide p-trifl uoromethoxyphenylhydrazone and 2.5
μg/ml oligomycin or by 5 μM cyclosporin A, a drug interfering
with the Ca2+-dependent activation of the permeability transi-
tion pore, suggesting that it depended on mitochondrial Ca2+
uptake and was a result of Ca2+ release from Ca2+-overloaded
mitochondria (unpublished data). Hence, mitochondria were
susceptible to Ca2+ overloading and permeabilization in cells
where the ER–mitochondria coupling was tightened by the
The mitochondrial Ca2+ dysregulation was regularly fol-
lowed by detachment of the cells, indicating the loss of viability.
Because Ca2+ transfer to the mitochondria is a key step in induc-
tion of many forms of cell death, we reasoned that tightening of
the ER–mitochondria coupling may contribute to the execution
Figure 3. Enhancement of the ER–mitochondria association and Ca2+
coupling by a synthetic linker protein. (A) Electron micrographs of RBL-2H3
cells expressing the mAKAP1(34–63)-mRFP-yUBC6 or mAKAP1(34–63)-
mRFP with red arrows showing the ER–mitochondria contacts. (B) Dimen-
sions of the ER–mitochondria interface in each condition. (C) [Ca2+]m and
nuclear [Ca2+] ([Ca2+]nu) responses to submaximal doses of adenophostin
(AP) recorded using pericam in cells transfected with OMM-mRFP (black)
or OMM–ER linker-mRFP (red). Adenophostin evokes gradual Ca2+ libera-
tion through IP3Rs and a [Ca2+]m increase characterized with a gradual
slow phase when the sarcoplasmic/endoplasmic reticulum calcium ATPase
pumps are blocked (Csordas and Hajnoczky, 2001). As a reference, a
20-μM CaCl2 pulse (Ca) was applied. (right) [Ca2+]c and [Ca2+]m increases
60 s after adenophostin stimulation. Data are normalized to the response
evoked by Ca (P > 0.01; n = 15–16). Error bars indicate SEM.
ER–MITOCHONDRIA STRUCTURAL AND CA2+ COUPLING • CSORDÁS ET AL. 919
of the cells induced by certain proapoptotic stimuli. To this end,
RBL-2H3 cells were exposed to apoptotic conditions (serum
starvation and tunicamycin treatment) and were fi xed for EM at
24 h, before the onset of cell detachment. Analysis of the dimen-
sions of the ER–mitochondria interface showed shortening of
the mean distance between ER and mitochondria in both the
serum-starved and tunicamycin-treated cells and an increase in
the frequency of tight associations (<6 nm distance; Fig. 4 B).
The high incidence of the tight associations could not be attributed
to the condensation of the apoptotic cells because the perimeter
or area of the cell cross sections has not been altered yet. These
results suggest that narrowing of the ER–mitochondria gap
occurs in intact cells and may be an important step in the execution
of some apoptotic mechanisms.
The scheme in Fig. 4 C illustrates the novel aspects of
the ER–mitochondria signaling uncovered in the present work.
The association between ER and mitochondria is due to the
presence of tethers that link both smooth and rough ER to the
mitochondria. The length of the tethers displays some diversity,
giving rise to varying distances between ER and mitochondria.
In response to apoptotic agents the ER–mitochondria gap narrows,
indicating dynamic regulation of the interorganellar junction.
In healthy cells, the ER–mitochondria tethering ensures the
propagation of IP3R-linked Ca2+ signals to the mitochondria to
coordinate ATP production with the stimulated state of the cell
and to enable the mitochondrial Ca2+ buffering. However, the
gap between the organelles is suffi ciently wide to isolate mito-
chondria from the slow Ca2+ leakage from the ER. Relaxing the
ER–mitochondria coupling suppresses the Ca2+ signal propaga-
tion to the mitochondria, putting at risk the Ca2+-dependent
control of mitochondrial metabolism. In contrast, tightening
of the coupling invokes mitochondria in the handling of Ca2+
Figure 4. Relevance of the tight ER–
mitochondria association for cell survival.
(A) Sensitization to Tg-induced Ca2+ overload-
ing and mitochondrial membrane permeabili-
zation in RBL-2H3 cells expressing either the
OMM–ER linker or OMM- or ER-only targeted
control. (left) Overlaid fura2FF fl uorescence
images recorded at 340 nm (red)/380 nm
(green) excitation (56 × 60 μm area) and the
fura2FF ratio graphs for individual cells
(middle) and for the mean (bottom) show two
sequential [Ca2+]c elevations evoked by 2 μM
Tg in OMM–ER linker-mRFP (red) and OMM-
mRFP (black) overexpressing cells (n = 74 and
100, respectively). (right) Lag time distribu-
tions for the steep second [Ca2+]c rise in the
experiment shown in the left (bottom) and the
fraction of cells exhibiting a [Ca2+]c rise above
the fi rst peak at 30 min of stimulation for
the series of experiments (n = 3; top). (B) EM
images (red arrowheads depicting the
close contacts) and measurements of the ER–
mitochondria interface in cells exposed to pro-
apoptotic conditions (0% serum starvation and
10 μg/ml tunicamycin). (C) Dependence of
mitochondrial function on the gap width be-
tween ER and mitochondria. In the scheme, the
effects on [Ca2+]m, ATP production, and mem-
brane permeabilization are shown for the
normal (middle), loose (left), and tight (right)
variations in ER–mitochondria physical coupling.
Error bars indicate SEM.
JCB • VOLUME 174 • NUMBER 7 • 2006 920
under resting conditions, sensitizing mitochondria to Ca2+ over-
loading and leading to permeabilization and committing the
cells to a cell death pathway. Tightening of the connections
seems to be relevant for several mechanisms of cell death. Thus,
these results reveal an unexpected dependence of cell function
and survival on the maintenance of a proper spacing between
the ER and mitochondria.
Materials and methods
To construct the OMM–ER linker, mRFP was targeted to the ER by using the
C-terminal ER localization sequence of the yeast UBC6 protein (X73234,
residues 233–250: M V Y I G I A I F L F V G L F M K ), through the linker (S G L R S R A Q-
A S N S R V ; Varnai et al., 2005). This construct was complemented with the
N-terminal mitochondrial localization sequence of the mouse AKAP1 pro-
tein (V84389, residues 34–63: M A I Q L R S L F P L A L P G L L A L L G W W W F F S R K K )
with the linker (D L E L K L R I L Q S T V P R A R D P P V A T ). Ratiometric pericam targeted
to the mitochondrial or nuclear matrix was provided by A. Miyawaki
(Institute of Physical and Chemical Research, Wako-city, Japan).
RBL-2H3 cells were cultured as described previously (Csordas et al., 1999).
Cells were transfected with cDNA by means of electroporation in suspensions
(4.5 × 106 cells + 20 μg of each cDNA in 250 μl medium). Electroporation
was performed in a BTX-830 square-pulse generator in a 4-mm gap cu-
vette using a single 250-V 13-ms pulse. For FACS sorting (MoFlo FACS
sorter [DakoyCytomation] equipped with a 488-nm laser), 8.5–12.5 × 106
cells transfected with a construct of interest and EGFP were cultured
for 24 h.
DT40 (wild type and IP3R knockouts alike were a gift from T. Kurosaki,
Kansai Medical University, Moriguchi, Japan) cells were cultured in
suspension, in RPMI 1640 with glutamine supplemented with penicillin/
streptomycin, 2 mM L-glutamine, 10% heat inactivated FCS, and 1%
chicken serum (Invitrogen) in 5% CO2 and 95% air at 40°C.
Fluorometric measurements of [Ca2+]c and [Ca2+]m in suspensions
of permeabilized RBL-2H3 cells
Experiments were performed as described earlier (Csordas and
Fractionation of RBL-2H3 cells
Cells grown overconfl uent in tissue culture fl asks (?7 × 107 cells) were
loaded with fura2FF/AM, harvested using trypsin/versene, and washed
with Na-Hepes/EGTA. All further steps were performed at 4°C. The cells
were exposed to hyposmosis for 10 min (14 ml intracellular medium [ICM;
120 mM KCl, 10 mM NaCl, 1 mM KH2PO4, 20 mM Tris-Hepes, 2 mM
MgATP, and 1 μg/ml each of antipain, leupeptin, and pepstatin, pH 7.2])
diluted fi vefold with dH2O and supplemented with 200 μM EGTA and
5 mM MgCl2). Subsequently, the cells were homogenized in a dounce
glass/glass homogenizer (30–35 strokes, tight pestle). To restore osmolarity,
3 vol of 100% ICM supplemented with 125 mM sucrose, 200 μM EGTA,
and 5 mM MgCl2 was added. To eliminate unbroken cells and nuclei, the
homogenate was centrifuged at 1,000 g for 10 min. The supernatant was
further centrifuged at 10,000 g for 15 min, and the pellet (mitochondrial
fraction) was resuspended in 400–500 μl ICM plus protease inhibitors
and 10 μM EGTA and stored on ice. Attachment to CellTak (BD Biosci-
ences) coated coverslips was performed at room temperature for 5 min in
the presence of 2 mM Mg2+ATP in 25–50 μl vol.
Fractionation of rat liver
The protocol was adapted from Meier et al. (1981). The liver of a
350–400-g normal male Sprague-Daily rat was perfused with ?200-ml
Na-Hepes/EGTA and was removed. All the further steps were done at 4°C.
The liver was cut up to small pieces with scissors and washed with ICM.
After determination of the wet weight, a 1:4 homogenate was prepared in
350 mM sucrose containing 2.5 mM magnesium acetate and 10 mM Tris
maleate, pH 7.4. Homogenization was performed in a 60-ml glass-Tefl on
homogenizer (11 strokes at 900 rev/min). The homogenate was fi ltered
through two layers of sterile gauze and once more through one layer of
Miracloth (Calbiochem). The mitochondrial fraction was obtained by centri-
fuging the supernatant of the 900-g (10 min) fraction at 8,000 g for 15 min.
Particles were attached to CellTak-coated coverslips as described above in
the previous paragraph.
Immunostaining of the mitochondrial fractions
The membrane fractions attached to coverslips were fi xed in 3% parafor-
maldehyde. A monoclonal anti-human cytochrome c oxidase complex IV
subunit 1 antibody (Invitrogen) was used to visualize mitochondria and
polyclonal anti–IP3R-1 and -2 antibodies (Affi nity BioReagents, Inc.) were
used to visualize the IP3Rs. The secondary antibodies were fl uorescently
labeled (Alexa Fluor 488 and 568). Images were acquired using a con-
focal system (Radiance 2001; Bio-Rad Laboratories), and colocalization
was evaluated using Lasersharp software (Bio-Rad Laboratories; Csordas
and Hajnoczky, 2001).
Fluorometric monitoring of extravesicular [Ca2+] in suspension of rat-liver
A 20–25-μl aliquot of the crude mitochondrial fraction was transferred to
800 μl ICM supplemented with 1.5 μM fura2/FA, 2 mM Mg-ATP, 2 mM
succinate, and protease inhibitors in a stirred cuvette at 35°C. Ratiometric
recording of fura2 fl uorescence was performed as described for the
Fluorescence imaging of [Ca2+] in single cells and in adherent
RBL-2H3 cells or mitochondrial fractions attached to coverslips were placed
in 1 ml buffer to the heated stage (35° C) of a microscope (IX70 [Olympus];
40×; UApo340) connected to a cooled charge-coupled device camera
(PXL; Photometrics). Ratiometric imaging of fura2FF and pericam was used
to monitor [Ca2+]c and [Ca2+]m as described previously (Csordas and
Hajnoczky, 2001; Yi et al., 2004).
TEM and ET
For embedding, a standard protocol was used (Pacher et al., 2000). Ultrathin
sections for TEM were poststained with UA and sodium bismuth (Pacher
et al., 2000). The sections were examined with either a scanning transmis-
sion electron microscope (model 7000; Hitachi) or a digital transmission
electron microscope (Tecnai 12; Philips) driven by Gatan software.
For ET of DT40 cells, 150–300-nm-thick sections were cut from
epoxy blocks, and 15-nm colloidal gold particles were applied to one side
as alignment markers. Tilt series were collected on an AEI EM7 high-voltage
electron microscope operated at an accelerating voltage of 1,000 kV.
Images were serially recorded around two orthogonal tilt axes, over angular
ranges of ±60° at 2° intervals.
For cryo-EM of isolated mitochondria, 3–5-μl aliquots of mitochon-
drial suspensions (10–20 mg/ml in 0.225 M manitol and 0.075 M sucrose)
containing 10-nm colloidal gold particles were deposited on freshly glow-
discharged 300-mesh copper grids with holey carbon fi lms. Grids were
blotted with fi lter paper and immediately plunged into liquid ethane cooled
by liquid nitrogen. Tilt series were collected over an angular range of ±60°
at 2° intervals (total dose ?24 electrons/Ǻ2) using a transmission electron
microscope (JEM-4000FX; JEOL) equipped with Gatan cryo-transfer unit
and a TVIPS 1024 × 1024 cooled charge-coupled device camera.
Images were aligned and tomographic reconstructions calculated
as previously described (Penczek et al., 1995), using the weighted back-
projection method as implemented in the SPIDER image processing system
(Frank et al., 1996). 3D models were generated by density thresholding
using Iris Explorer (Numerical Algorithms Group) or surface rendering in
Iris Explorer after manual membrane tracing in Sterecon (Marko and Leith,
1996). In the case of plastic sections, the z dimension (section thickness) of
the fi nal models was increased by 20% to compensate for radiation-
induced thinning of the plastic section. Lengths of tethers connecting mito-
chondrial outer membranes and ER membranes were determined using
Online supplemental material
Table S1 shows the dimensions of the ER–mitochondria interaction areas.
Fig. S1 shows tight ER–mitochondria associations in quick frozen and
chemically fi xed isolated liver mitochondria and in wild-type and IP3R-TKO
DT40 cells. Fig. S2 demonstrates protein linkage between ER and mito-
chondria in RBL-2H3 cells. Online supplemental material is available at
We thank Drs. David Birk and Ted Taraschi for access to EM equipment.
ET at the Resource for Visualization of Biological Complexity is supported
by a grant from the National Center for Research Resources of the National
Institutes of Health (RR01219). T. Balla and P. Varnai were supported in part
ER–MITOCHONDRIA STRUCTURAL AND CA2+ COUPLING • CSORDÁS ET AL.921 Download full-text
by the Intramural Research Program of the National Institute of Child Health
and Human Development of the National Institutes of Health. P. Varnai was
supported in part by the Senior Fellowship Program administered by the Oak
Ridge Institute for Science and Education through an interagency agreement
between the U.S. Department of Energy and the National Institutes of Health.
This work was supported by a grant from the National Institutes of Health to
G. Hajnoczky (DK51526).
Submitted: 5 April 2006
Accepted: 15 August 2006
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