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Mitofusin 2 regulates STIM1 migration from the Ca2+ store to the

plasma membrane in cells with depolarised mitochondria. Karthika Singaravelu, Charmaine Nelson, Daniel Bakowski. Olga Martins de Brito1, Siaw-Wei Ng, Joseph

Di Capite, Trevor Powell, Luca Scorrano1 and Anant B. Parekh*

Running title: Mitofusin2 regulates trafficking of the protein STIM1

Key words: Ca2+ channels/Mitochondria/Protein Trafficking/STIM1

Department of Physiology, Anatomy and Genetics, University of Oxford. Sherrington Building, Parks

Road, Oxford. OX1 3PT. U.K. 1 Dulbecco-Telethon Institute, Venetian Institute of Molecular Medicine, Via Orus 2, 35129 Padova, Italy.

* author for correspondence

Tel: ++44-1865-272439

Fax: ++44-1865-272488

e-mail: anant.parekh@dpag.ox.ac.uk

http://www.jbc.org/cgi/doi/10.1074/jbc.M110.174029The latest version is at JBC Papers in Press. Published on January 10, 2011 as Manuscript M110.174029

Copyright 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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Abstract

Store-operated Ca2+ channels in the plasma membrane (PM) are

activated by the depletion of Ca2+ from the endoplasmic reticulum (ER) and

constitute a widespread and highly

conserved Ca2+ influx pathway. After store emptying, the ER Ca2+ sensor

STIM1 forms multimers, which then migrate to ER-PM junctions where

they activate the CRAC channel

Orai1. Movement of an intracellular protein to such specialised sites where

it gates an ion channel is without

precedence but the fundamental question of how STIM1 migrates

remains unresolved. Here, we show that trafficking of STIM1 to ER-PM

junctions and subsequent CRAC

channel activity is impaired following mitochondrial depolarisation. We

identify the dynamin-related mitochondrial protein mitofusin 2,

mutations of which causes the

inherited neurodegenerative disease Charcot-Marie-Tooth IIa in humans,

as an important component of this mechanism. Our results reveal a

molecular mechanism whereby a

mitochondrial fusion protein regulates protein trafficking across the

endoplasmic reticulum and reveals a

homeostatic mechanism whereby mitochondrial depolarisation can

inhibit store-operated Ca2+ entry, thereby reducing cellular Ca2+

overload.

INTRODUCTION In eukaryotic cells, a variety of

different agonists including hormones,

neurotransmitters and growth factors

elicit cellular responses through a rise in

cytoplasmic Ca2+ concentration (6).

Cytoplasmic Ca2+ can be increased

following Ca2+ release form intracellular

stores, by Ca2+ entry across the plasma

membrane via Ca2+-permeable ion

channels or by both processes. In many

cell types, the emptying of intracellular

Ca2+ stores opens store-operated Ca2+

channels in the plasma membrane (28,

44, 45). The best characterised store-

operated Ca2+ channel is the CRAC

channel, which constitutes the major

Ca2+ entry pathway in immune cells (23,

67). Ca2+ entry through CRAC channels

activates a range of temporally distinct

responses including exocytosis, enzyme

activation and gene transcription (41,

45).

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Recently, the molecular basis of

store-operated Ca2+ entry has been teased

apart. Targeted RNAi screens first

identified STIM1, a single

transmembrane-spanning domain protein

found predominantly in the endoplasmic

reticulum (ER), as being essential for

CRAC channel activation (30, 52).

STIM1 has a Ca2+-binding EF-hand that

faces the lumen of the ER and site-

directed mutagenesis has revealed that

this likely senses the Ca2+ content of the

store (30, 65, 66). Upon store depletion,

Ca2+ dissociates from STIM1 and this is

thought to promote multimerization

through the N-terminal sterile α motif

(29), a step that is central to CRAC

channel activation (31). STIM1

multimers then migrate to punctate

structures < 25 nm from the plasma

membrane that correspond to ER-PM

junctions (60). At such sites, STIM1

activates Orai1, a four-transmembrane

domain plasma membrane protein that is

also required for CRAC channel activity

(11, 59, 65). Mutagenesis studies have

established that Orai1 is at least part of

the CRAC channel pore (48, 58, 62). A

cytoplasmic domain of STIM1 binds to

both the N- and C-termini of Orai1 (26,

38, 46, 64), leading to CRAC channel

activation.

Although STIM1 trafficking

towards the plasma membrane is a

critical early step in CRAC channel

activation, an important but unresolved

question is: how is the migration process

controlled? One idea is that STIM1

diffuses randomly in the ER but

becomes trapped at the ER-PM junctions

upon store depletion (61). Alternatively,

it has been suggested that STIM1

reaches the ER-PM junctions by active

transport along microtubules (56) and

STIM1 binds directly to the

microtubule-plus-end-tracking protein

EB1 (17). Interestingly, STIM1 has been

found to co-localise with alpha-tubulin

and microtubule depolymerization

reduces STIM1 puncta formation,

supporting a role for the microtubule

cytoskeleton (56). On the other hand,

STIM1 can form puncta even when

intracellular ATP levels have been

depleted, suggesting its movement is a

passive process (9).

A solid body of evidence has

demonstrated that mitochondria control

CRAC channel activity (15, 16, 20, 21,

42, 43). Although some of the effects of

mitochondria arise from their ability to

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buffer cytoplasmic Ca2+ and thus reduce

Ca2+-dependent inactivation of CRAC

channels, growing evidence suggests

that they might have an additional role in

regulating CRAC channels that is

unrelated to their ability to take up Ca2+,

produce ATP and generate reactive

oxygen species (13, 16). Mitochondria

can be positioned adjacent to ER through

interactions between proteins on the two

organelles, with a major role for the

mitochondria-shaping protein mitofusin

2 (Mfn2) (8, 10, 40). Mouse embryonic

fibroblasts lacking Mfn2 display

loosened ER-mitochondria tethering and

reduced rate of mitochondrial Ca2+

uptake following InsP3-mediated Ca2+

release from the ER (10). Whether this

physical uncoupling impacts upon

spatially more distal events is unclear.

We show here that mitochondrial

depolarization suppresses STIM1 puncta

formation and subsequent Orai1-

dependent CRAC currents and these

inhibitory effects can be partially

overcome by overexpression of either

STIM1 or a STIM1 mutant that occupies

ER-PM junctions in non-stimulated cells

with intact stores. In cells lacking Mfn2,

STIM1 puncta formation and CRAC

channel activity was independent of

mitochondrial status and analysis of

Mfn2 mutants revealed a major role for

mitochondrially targeted Mfn2. Our

results identify a new role for

mitochondria in cell biology, that is,

these organelles help regulate the

movement of an ER-resident multimeric

protein complex to the plasma

membrane. Furthermore, our findings

reveal Mfn2 as an important component

in the mechanism whereby

mitochondrial depolarisation inhibits

CRAC channel activity.

MATERIALS AND METHODS Cell Culture and Transfection- Rat basophilic leukemia (RBL-1) cells and HEK293 were bought from ATCC. RBL-1 cells were cultured (37 °C, 5% CO2) in Dulbecco’s modified Eagle medium with 10% fetal bovine serum, 2 mM L-glutamine and penicillin-streptomycin, as previously described (2). HEK293 cells were cultured in RPMI with 10% fetal bovine serum, 2 mM L-glutamine and penicillin-streptomycin. HEK293 cells were cotransfected with cDNA encoding Orai1 (Origene) and eYFP-STIM1 (gift from Dr T. Meyer) using two independent methods: lipofectamine and Amaxa systems, as described (37). eYFP-mutant STIM1 was a gift from Dr J. Putney. RBL-1 cells were transfected with RNAi against Orai1 ((39), purchased from Invitrogen) together with enhanced eYFP using the nucleofection method (Amaxa). Cells were passaged onto glass coverslips and used 36-48 h after plating. Wild type and

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mitofusin 2-deficient mouse embryonic fibroblasts (MEFs) were cultured as described in (8). Cells were grown in MEF media (DMEM, 10%FCS, 1X nonessential amino acids, 1 mM L-glutamine, penicillin/streptomycin [Life technologies/GIBCO BRL]) and transfected using LipofectamineTM 2000 (Invitrogen). ICRAC recordings-Patch-clamp experiments were conducted in the tight-seal whole-cell configuration at room temperature (20-25 0C) as previously described (2, 16). Sylgard-coated, fire-polished pipettes had d.c. resistances of 4.2-5.5MΩ when filled with standard internal solution that contained (in mM): Cs+ glutamate 145, NaCl 8, MgCl2 1, Mg-ATP 2, Ethylene glycol-bis(b-aminoethyl ether)-N,N,N’,N’,-tetraacetic acid (EGTA) 10, HEPES 10, pH 7.2 with CsOH. In some experiments, 30 µM InsP3 was added to this solution. A correction of +10 mV was applied for the subsequent liquid junction potential that arose from this glutamate-based internal solution. The composition of the extracellular solution was (in mM): NaCl 145, KCl 2.8, CaCl2 10, MgCl2 2, CsCl 10, D-glucose 10, HEPES 10, pH 7.4 with NaOH. ICRAC was measured by applying voltage ramps (-100 to +100 mV in 50 msec) at 0.5 Hz from a holding potential of 0 mV. Currents were filtered using an 8-pole Bessel filter at 2.5 kHz and digitised at 100 ms. Currents were normalised by dividing the amplitudes (measured from the voltage ramps at - 80 mV) by the cell capacitance. Capacitative currents were compensated before each ramp by using the automatic compensation of the EPC 9 -2 amplifier. For ICRAC, leak currents were subtracted by averaging 2-3 ramp currents obtained just before ICRAC had started to develop, and then subtracting

this from all subsequent currents. Transfected cells were identified by expression of either eYFP or eGFP. Ca2+ imaging-Ca2+ imaging experiments were carried out at room temperature using the IMAGO CCD camera-based system from TILL Photonics, as described previously (37). Cells were alternately excited at 356 and 380 nm (20 msec exposures) and images were acquired every 2 seconds. Images were analysed offline using IGOR Pro for Windows. Cells were loaded with Fura 2-AM (1 µM) for 40 minutes at room temperature in the dark and then washed three times in standard external solution of composition (in mM) NaCl 145, KCl 2.8, CaCl2 2, MgCl2 2, D-glucose 10, HEPES 10, pH 7.4 with NaOH. Cells were left for 15 minutes to allow further deesterification. Ca2+-free solution had the following composition (in mM) NaCl 145, KCl 2.8, MgCl2 2, D-glucose 10, HEPES 10, EGTA 0.1, pH 7.4 with NaOH). The rate of Ba2+ influx (on addition of 2 mM Ba2+) was obtained by measuring the initial slope of the fluorescence rise following readmission of Ba2+ to cells with depleted stores. Ca2+ signals are plotted as R, which denotes the 356/380 nm ratio. Mg2+ imaging-Cytoplasmic Mg2+ was used to measure Mg-ATP (see text). Cells were loaded with mag-fura 2-AM (1 µM) for 40 minutes at room temperature and experiments were carried out as for fura 2. TIRF microscopy-RBL-1 cells were transfected with eYFP-STIM1 and plated onto Willco thin glass bottom dishes (Intracel, UK) 48 hours in advance of experiments. Cells were bathed with the standard external solution that contained (in mM) NaCl 145, KCl 2.8, CaCl2 2, MgCl2 2, D-glucose 10, HEPES 10, pH 7.4 with

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NaOH. Images from cells using total internal reflection fluorescence (TIRF) microscopy were collected by a Hamamatsu ORCA-AG, Deep Cooled Digital Camera (Model C4742-80-12AG), connected to an inverted TE2000 microscope with a through-the-lens (prismless) TIRF imaging attachment (Nikon). Samples were viewed through a CFI Apo TIRF 60x oil-immersion high-resolution objective (1.49 N.A.) and excited by the 488 nm line of an argon laser (Spectra Physics 163-A120) via a FITC Filter Block (Excitation 465-495 nm, Dichroic 505 nm, Barrier 515-555 nm). Image collection was controlled by IPLab software (BD Biosciences) at 2s intervals for 8 minutes. Post-collection analysis consisted of each cell being selected by a Region of Interest and the mean pixel intensity calculated for each frame of the collection sequence. Electron microscopy-After specific treatments (as described in the text), eYFP-STIM1-transfected HEK cells were collected and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 30 min at room temperature. The cells were blocked with 2% BSA and 1% goat serum in PBS for 1 hr. Staining was performed by incubation with primary antibody (rabbit anti-GFP; 1:50 working dilution; Invitrogen) in PBS containing 0.2% BSA and 0.1% goat serum overnight at 4C. Cells were then incubated with biotinylated secondary antibody (goat anti-rabbit IgG; 1:200 working dilution; Vector Laboratories) for 1 hr at room temperature. The STIM1 protein was detected by using the Elite ABC peroxidase kit according to manufacturer instructions (Vector Laboratories). Cell cultures of all treatments were processed simultaneously with the same solutions

and incubation times. After post fixation with 1% osmium tetroxide for 45 min, cells were stained with 2% aqueous uranyl acetate for 1 hr. Cells were further processed as described previously (36). HEK293 cells were used for electron microscopy for two reasons. First, expression of YFP-STIM1 was much higher than in RBL-1 cells. Second, the HEK cells attached to the coverslips much better and hence less detached during the extensive washing/fixing procedures. Distribution of mitochondria (Supplementary Figure 3) was measured by computing the distance of each mitochondrion from the plasma membrane in the X-Y direction, in sequences of 100 serial sections (each of 50 nm thickness) taken across each cell. Sections were scanned into a G5 Mac computer and superimposed, in order to avoid analysing the same mitochondrion twice. Confocal microscopy-Cells were fixed in 4% paraformaldehyde in phosphate buffer for 30 minutes at room temperature, after stimulation with thapsigargin. All the washes used 0.01% Phosphate buffered saline (PBS; in mM: NaCl 137, KCl 2.7, Na2HPO4 8, KH2PO4 1). The cells were blocked with 2% BSA (bovine serum albumin) and 10% goat serum for 1 hour. Mitofusin 2 was visualized using a monoclonal antibody (kindly provided by Dr Richard Youle, NIH; used at a dilution of 1:250). The secondary anti-rabbit IgG was a HandL chain specific (goat) fluorescein conjugate (excitation at 495nm, emission at 515nm). This was used at 1:2000 in PBS for 2 hours at room temperature. The cells were mounted in Vectashield mounting medium containing a propidium iodide counterstain for DNA, (excitation 535nm, emission 615nm).

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Images were obtained using a Leica confocal microscope. ER distribution-HEK293 cells were transfected with cell light ER-RFP, Bacman 2.0 (Invitrogen) and ER distribution was viewed using confocal microscopy of fixed cells. Statistics-Data are presented as the mean±sem. Statistical significance was determined using a student t test. * denotes p<0.01. RESULTS

STIM1 migration and Orai1 activity is

regulated by mitochondria We first confirmed, using patch

clamp recordings, findings originally

made in the HEK293 expression system

(33, 47) and drosophila S2 cells (65) that

co-expression of Orai1 and STIM1 in

RBL cells increased the size of ICRAC. As

shown in supplementary Figures 1A-C,

dialysis of RBL-1 cells transfected with

cDNA for both Orai1 and eYFP-STIM1

resulted in a larger ICRAC (~ 3 to 5-fold)

than was the case with corresponding

controls transfected with eYFP or eGFP

plasmids alone. The current was

identified as ICRAC on the basis of several

characteristics including its steep inward

rectification and positive reversal

potential (supplementary Figure 1B), its

absence when external Ca2+ was

removed (data not shown), inhibition by

the CRAC channel blocker 2-APB (50

µM, data not shown) and ability to

activate when stores were depleted

passively (10 mM EGTA,

supplementary Figure 1C). Transfection

with eYFP-STIM1 alone increased Ca2+

influx < 1.5-fold compared with the

control response (Supplementary Figure

2A). On the other hand, as observed

previously (33, 59), overexpression of

Orai1 alone reduced Ca2+ influx by ~

35% (Supplementary Figure 2A). Hence

recombinant co-expression of STIM1

and Orai1 increases the size of ICRAC. It is

important to note that the increase in

RBL cells is very modest, when

compared with common expression

systems like HEK293 cells where a >

500-fold increase in current can

routinely be obtained (33, 47, 57, 65).

We designed experiments to see if Orai1

also contributed to native ICRAC and

agonist-evoked Ca2+ entry. Knockdown

of Orai1 using an RNAi approach that

we have described recently (39) reduced

the amplitude of ICRAC by ~ 70%

(supplementary Figure 1D). We altered

the expression levels of Orai1 before

challenging intact cells, loaded with the

Ca2+-sensitive fluorescent dye fura-2,

with the endogenous P2Y receptor

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agonist ATP. Cells were stimulated with

agonist in Ca2+-free solution and then

Ba2+ was applied. Ba2+ permeates CRAC

channels but, unlike Ca2+, is not

transported out of the cytoplasm by

Ca2+ATPase pumps and therefore

provides a good indication of CRAC

channel activity (5, 45, 53). Exposure to

Ba2+ after challenge with ATP in Ca2+-

free solution resulted in prominent Ba2+

influx (supplementary Figure 1E,

labelled control). The rate and extent of

Ba2+ influx was significantly larger in

cells co-expressing eYFP-STIM1 and

Orai1 (supplementary Figure 1E).

Although STIM1 has been reported to

control non-store-operated Ca2+ entry

pathways, this requires the protein to be

in the plasma membrane (34). eYFP-

STIM1 is not inserted into the plasma

membrane and remains within the ER

where it specifically regulates CRAC

channels (33). Knockdown of

endogenous Orai1 reduced agonist-

evoked Ba2+ influx compared with

control cells (supplementary Figure 1E).

Similar findings were observed when

cells were stimulated with different

concentrations of ATP (supplementary

Figure 1F), demonstrating that ICRAC is

the dominant source of Ca2+ entry over a

range of stimulus intensities.

Mitochondria regulate CRAC

channel activity in several cell types (42,

43, 45). In RBL cells co-expressing

Orai1 and eYFP-STIM1, both store-

operated Ca2+ influx in intact cells

(Figures 1A and B) and ICRAC (Figures

1C and D) were substantially reduced

following mitochondrial depolarisation

by inhibition of complex III of the

respiratory chain with antimycin A

(together with oligomycin to prevent the

F1-F0ATPsynthase from running in

reverse). Similar findings were seen

when mitochondria were depolarised

with the protonophore FCCP (ICRAC was

reduced by 79±4%, data not shown).

Oligomycin alone (15 minutes pre-

treatment) had no inhibitory effect (data

not shown; see also [26]). Importantly,

the extent of Ca2+ release from the stores

was not compromised by mitochondrial

depolarisation (Figure 1A, see also (16)).

Mitochondria therefore target a step

distal to store depletion. Since STIM1

movement occurs after store depletion,

we looked to see whether its migration

towards the plasma membrane was

affected by impairing mitochondrial

activity. Analysis of eYFP-STIM1

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movement using confocal microscopy on

fixed cells showed that very little STIM1

was located at the cell periphery in

resting cells (Figure 1E, first panel,

labelled Control). Store depletion with

thapsigargin (applied in Ca2+-free

solution) resulted in strong staining of

the cell periphery together with the

appearance of punctate-like structures in

the cytoplasm (Figure 1E, second panel).

Strikingly, mitochondrial depolarisation

dramatically reduced staining around the

cell surface and increased the number of

punctate-like structures in the cytoplasm

(Figure 1E, third panel), presumably

reflecting formation of intracellular

STIM1 multimers that were unable to

traffic to the plasma membrane.

Inhibition of ATP synthesis with

oligomycin did not affect eYFP-STIM1

movement induced by store depletion

(Figure 1E, fourth panel), consistent

with a recent report that oligomycin does

not impair puncta formation in a breast

cancer cell line (9). Analysis of eYFP-

STIM1 location together with

mitochondria (identified with

mitotracker red) in the same cells

revealed very little co-localisation,

ruling out significant location of STIM1

on mitochondria (Figure 1F).

Mitochondria do not migrate to the

plasma membrane In T cells, mitochondria migrate to the

cell periphery following Ca2+ entry

through CRAC channels (50). To

quantify the distribution of mitochondria

in more detail and to examine whether

they also migrated to the cell periphery

like STIM1 after store depletion, we

compared mitochondrial location in

resting and store-depleted RBL-1 cells

using electron microscopy

(supplementary Figure 3). We measured

the number of mitochondria as a

function of distance from the plasma

membrane, obtained from quantitative

analysis of 100 serial sections, taken

every 50 nm and thus spanning the entire

cell thickness. Within 100 nm of the

plasma membrane, there was a paucity

of mitochondria. To see whether Ca2+

influx through CRAC channels changed

the pattern of mitochondrial distribution,

we stimulated cells with thapsigargin for

10 minutes (in the presence of 2 mM

external Ca2+) and then cut serial

sections. The pattern was similar to that

seen in control cells, indicating that store

depletion and subsequent CRAC channel

activation did not change the

mitochondrial profile near the plasma

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membrane, at least with our stimulation

protocol. Most mitochondria were found

around 500 nm to 1 micrometre from the

cell surface. Consistent with these

structural findings, functional evidence

against close apposition of mitochondria

with the plasma membrane was provided

by measuring the rate and extent of rapid

inactivation of CRAC channels. Ca2+

permeation through CRAC channels

results in the build-up of a microdomain

of elevated Ca2+ which can feedback to

inactivate the channels partially (12, 22,

68). The Ca2+ binding site is thought to

reside within 10 nm of the channel pore

because it is reduced by the fast chelator

BAPTA but not the slower EGTA.

Although ICRAC was reduced by

mitochondrial depolarisation, the rate

and extent of fast inactivation was

unaffected (supplementary Figure 3D;

measured using Ca2+ as the charge carrier

and with matched peak amplitudes

between control and

antimycin/oligomycin-treated cells),

suggesting that the organelle was not

sufficiently close to the plasma

membrane to impact upon the buildup of

local microdomains emanating from

open CRAC channels. Collectively,

these findings demonstrate that

mitochondria do not migrate with

STIM1 to the cell periphery following

store depletion. In addition, our results

show that mitochondria can regulate

CRAC channels without needing to be

located very close to the plasma

membrane. This is in good agreement

with a recent report that found

mitochondria were absent from plasma

membrane regions containing active

store-operated Ca2+ channels (14).

STIM1 puncta formation is regulated by mitochondria

To characterize the role for

mitochondria on STIM1 trafficking in a

more quantitative way, we monitored

eYFP-STIM1 movement with total

internal reflection fluorescence (TIRF)

microscopy, which detects events

restricted to within ~100 nm of the

plasma membrane. Stimulation of RBL-

1 cells with thapsigargin resulted in

eYFP-STIM1 movement to the cell

periphery (Figure 2A) and this was

substantially reduced by mitochondrial

depolarisation with antimycin A and

oligomycin (Figure 2A) or FCCP and

oligomycin (supplementary Figure 2B)

but not oligomycin alone (supplementary

Figure 2B). Aggregate data comparing

the total fluorescence rise in TIRF

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microscopy mode for cells stimulated

with thapsigargin in the absence and

presence of antimycin A and oligomycin

are compared in Figure 2B, and the half-

times to peak of the eYFP-STIM1

movement is compared in Figure 2C.

Mitochondrial depolarisation

significantly reduced both the rate and

extent of STIM1 trafficking to the

plasma membrane in RBL cells.

No role for mitochondrial Ca2+

buffering in STIM1 migration We designed experiments to

address the mechanism whereby

depolaised mitochondria inhibits STIM1

migration. One important role for

mitochondria is to buffer a rise in

cytoplasmic Ca2+. However, several

pieces of evidence militate against such

a role here. First, with our protocol to

deplete stores (thapsigargin in Ca2+-free

solution for ~5 minutes), very little Ca2+

is taken up by mitochondria (7).

Consistent with this, the amplitude and

time course of Ca2+ release was

unaltered following mitochondrial

depolarisation (Figure 1A; see also (7)).

Second, increasing cytoplasmic Ca2+

buffering by loading cells with the fast

Ca2+ chelator BAPTA virtually

suppressed the cytoplasmic Ca2+ rise

evoked by thapsigargin in Ca2+-free

solution compared with control non-

BAPTA-loaded cells (Figure 3A) but

this did not affect migration of STIM1 to

the cell periphery (compare middle

panels of Figures 3B and 3C).

Movement of STIM1 to the plasma

membrane does not therefore require a

cytoplasmic Ca2+ rise. Importantly, the

robust redistribution of STIM1 to the

periphery in BAPTA-loaded cells was

much less pronounced following

mitochondrial depolarisation (Figure

3C). Therefore mitochondrial

depolarisation impairs STIM1

trafficking even in the absence of a

cytoplasmic Ca2+ rise and therefore in

the absence of the organelle’s ability to

take up Ca2+. Finally, we overexpressed

Orai1 and STIM1 and then measured the

Na+ flux through the CRAC channels

that occurs when cells are exposed to

divalent-free external solution (3, 4, 22,

49). Following dialysis with InsP3 and

10 mM BAPTA, a large Na+ current

developed (Figure 3D), which showed

the characteristics of Na+ flux through

CRAC channels (Figure 3E; inwardly

rectifying current-voltage relationship,

reversal potential of ~ + 60 mV, low

current noise). Mitochondrial

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depolarisation substantially reduced the

size of this Na+ current (Figures 3D-F).

Similar results were seen when cells

were dialysed with BAPTA alone. Since

cells were dialysed with 10 mM BAPTA

and no Ca2+ was present outside, the

involvement of mitochondria can clearly

be separated from a Ca2+ buffering role.

No role for mitochondrially-derived ATP

Another important function of

mitochondria is ATP production.

However, several arguments can be

raised against a role for mitochondrially-

derived ATP in STIM1 trafficking. First,

mast and RBL-1 cells are glycolytically

competent and depolarisation of

mitochondria with antimycin A and

oligomycin does not reduce cellular ATP

levels provided glucose is available (35).

In our experiments, we always had 10

mM glucose present. We nevertheless

measured cytoplasmic ATP levels in

single cells using cytoplasmic Mg2+ as

an indicator of Mg-ATP (27, 54). As

Mg-ATP is consumed, free Mg2+ rises

because the hydrolytic product ADP has

significantly lower affinity for Mg2+. In

cells loaded with mag-fura, treatment

with antimycin A and oligomycin failed

to generate a clear rise in Mg2+ levels in

the presence of glucose (Figure 3G). On

the other hand, replacement of glucose

with the non-metabolisable analogue 2-

deoxyglucose together with iodoacetate

(an inhibitor of glycolysis; used at 1

mM) resulted in a substantial rise in

cytoplasmic Mg2+, consistent with

depletion of Mg-ATP. Although mag-

fura can also bind Ca2+, it does so with

an affinity of ~ 50 µM. Experiments

with fura 2 (affinity of ~ 200 nM)

revealed a small and inconsistent rise in

cytoplasmic Ca2+ following exposure to

antimycin A and oligomycin, which was

~ 200 nM in amplitude. This is therefore

too small to impact on the mag-fura

signals of Figure 3G. Although these

global measurements fail to demonstrate

a fall in ATP upon mitochondrial

depolarisation provided glycolysis is

intact, we were concerned that ATP

below the plasma membrane might have

been reduced, especially as this is the

site for STIM1 translocation. To test this

possibility, we stimulated cells with

thapsigargin in Ca2+-free solution and

measured the time course of decay of the

Ca2+ signal (Figure 3H), since the latter

reflects extrusion of Ca2+ by the plasma

membrane Ca2+ATPase. The time

constant of decay was unaffected by

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antimycin A and oligomycin (Figure 3I),

arguing against significant depletion of

subplasmalemmal ATP. Finally, block of

the mitochondrial F1F0ATP synthase

with oligomycin failed to affect eYFP-

STIM1 trafficking to the cell periphery

(Figure 1E). Collectively, these results

show that the inhibition of STIM1

trafficking to the cell periphery upon

mitochondrial depolarisation is not due

to cytoplasmic ATP depletion.

STIM1 translocation to ER-

plasma membrane junctions has been

reported to require the C-terminal

polybasic motif which, in other proteins,

binds polyphosphoinositides in the

plasma membrane (29). We do not think

mitochondrial depolarisation depletes

the plasma membrane phosphoinositide

pool because agonist-evoked PIP2

hydrolysis and subsequent InsP3-

dependent Ca2+ release is unaffected by

antimycin A and oligomycin (16).

Furthermore, the amplitude of the

inward rectifier, a K+ current regulated

by phosphoinositides, was unaffected by

mitochondrial depolarisation

(supplementary Figure 4)

We also considered that

mitochondrial depolarisation might alter

the distribution of the endoplasmic

reticulum (ER), the organelle in which

STIM1 is embedded. We used an RFP

construct targeted to the ER (ER-RFP) to

monitor ER distribution. ER-RFP

labelling revealed a reticular network

(supplementary Figure 5) and this was

unaffected by store depletion with

thapsigargin. Mitochondrial

depolarisation with antimycin A and

oligomycin for 15 minutes also had no

discernible effect on ER distribution

(supplementary Figure 5).

Strong overexpression of STIM1

rescues CRAC channel activity

In T cells, two thirds of the

specialised ER-PM junctions where

STIM1 accumulates upon store depletion

are thought to be already pre-formed in

resting cells (60). We reasoned that

extensive overexpression of STIM1

might therefore result in an increased

probability of STIM1 occupying some of

these pre-existing ER-PM junctions, thus

overcoming the inhibitory effects of

mitochondria on STIM1 trafficking and

store-operated influx. HEK293 cells are

an ideal system for testing this idea

because of their high transfection

efficiency and ability to overexpress

recombinant proteins massively. Indeed,

we find absolute eYFP-STIM1

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fluorescence in HEK293 cells to be at

least 10 times greater than in RBL-1

cells, consistent with the very large size

of ICRAC seen in HEK293 cells compared

with RBL-1 cells following

overexpression of Orai1 and STIM1

(HEK current ~ -100 to -500 pA/pF, see

also (33, 47); RBL ~ -10 to -12 pA/pF).

We first confirmed that store-operated

Ca2+ entry in HEK293 cells was reduced

by mitochondrial depolarisation. As

shown in Figure 4A, readmission of

external Ca2+ to store-depleted wild type

cells (exposed to thapsigargin in Ca2+-

free external solution at t=0) resulted in

robust Ca2+ entry, which was

significantly reduced by mitochondrial

depolarisation. Ca2+ influx in cells with

depolarised mitochondria has been

normalised to the corresponding control

influx in this and subsequent panels.

Overexpression of eYFP-STIM1 alone

partially rescued Ca2+ influx in cells with

depolarised mitochondria (Figure 4B).

Co-expression of Orai1 and eYFP-

STIM1 resulted in more substantial

recovery of store-operated Ca2+ influx

(Figure 4C), and the rate and extent of

Ca2+ influx in cells expressing Orai1 and

STIM1 with depolarised mitochondria

was larger than in wild-type non-

transfected cells. One interpretation of

these results is that strong

overexpression of STIM1 in HEK cells

rescues the inhibition of Ca2+ entry seen

when mitochondria are compromised.

We were nevertheless concerned that the

Ca2+ signals, particularly after

overexpression of Orai1 and STIM1,

were so large that they saturated the dye

fura 2. Hence the apparent recovery of

the Ca2+ signal could be misleading,

because the control store-dependent

Ca2+ responses (following expression of

either STIM1 or STIM1 together with

Orai1) have been underestimated

because of dye saturation. We therefore

used the lower affinity dye fura 5F to

test this. Although store-operated Ca2+

entry recovered somewhat in cells with

depolarised mitochondria following

overexpression of STIM and Orai1

(Figure 4D), the extent of rescue was

less than that detected with fura 2 as the

Ca2+-sensitive dye (Figure 4C). These

results serve as a salutary warning in

quantifying cytoplasmic Ca2+ signals

following overexpression of

STIM1/Orai1 based on measurements

using high affinity dyes such as fura 2.

We confirmed that overexpressed

STIM1 was able to traffic to the cell

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periphery in HEK293 cells even after

mitochondrial depolarisation by

monitoring puncta formation with TIRF

microscopy. Compared with non-

stimulated cells (Figure 4E), store

depletion with thapsigargin led to

numerous puncta being formed (Figure

4F) and these were still prominent when

cells were pre-treated with antimycin A

and oligomycin prior to thapsigargin

(Figure 4G), although these STIM1

puncta appeared to coalesce into larger

structures after mitochondrial

depolarisation (Figure 4G). Fewer,

larger puncta were observed in the

presence of antimycin A and oligomycin

and thapsigargin compared with the

numerous, smaller ones seen in

thapsigargin-treated cells. We analyzed

puncta intensity by measuring eYFP-

STIM1 fluorescence in 1 µm2 sections

spanning the entire cell footprint.

Whereas intensity was largely a normal

distribution in cells exposed to

thapsigargin (Figure 4H, upper panel),

the pattern changed dramatically

following mitochondrial depolarisation

(Figure 4H, lower panel). Now,

numerous high intensity regions formed,

indicative of the merging of puncta.

Nevertheless, partial rescue of Ca2+

influx occurred (Figure 4D).

Electron microscopy provides the

highest spatial resolution for resolving

protein location. We identified the

position of eYFP-STIM1 in serial ultra

thin sections of HEK293 cells prepared

for electron microscopy using the ABC

coupled horseradish peroxidase system

(see Methods for more details).

Following expression of eYFP-STIM1,

cells were fixed and then exposed to

anti-GFP primary antibody followed by

incubation with biotinylated secondary

antibody. We then applied avidin-

coupled horseradish peroxidase, which

bound to the secondary antibody. The

complex was visualised using the DAB

chromagen, which is oxidised by

peroxidase to form an electron dense

precipitate at the site of the reaction,

thereby revealing the location of STIM1.

In micrographs from non-stimulated

HEK293 cells expressing eYFP-STIM1,

we saw very little protein near the

plasma membrane. Instead, most dark

deposits were seen around the nuclear

membrane and ER-like tubular structures

(Figure 5A, box indicated in the left

hand panel has been magnified in the

right hand panel). On the other hand,

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store depletion with thapsigargin led to

the formation of electron dense deposits

at the cell periphery (Figure 5B), with a

loss of deposit from the nuclear

membrane. Store depletion after pre-

treatment with antimycin A and

oligomycin resulted in some labelling at

the cell surface, although this was less

extensive (Figure 5C). We compared the

fraction of the cell periphery that stained

for STIM1 between non-stimulated cells,

cells stimulated with thapsigargin and

cells pre-treated with antimycin A and

oligomycin prior to thapsigargin

exposure (Figure 5D). Whereas very

little staining was detectable in control

cells, a substantial portion of the

periphery contained STIM1 after store

depletion. The extent of this was reduced

by antimycin A and oligomycin,

although staining was still prominent.

Because of lateral diffusion, the DABS

reaction product is not restricted just to

the site of HRP. Hence we were unable

to quantify the lateral extent of puncta in

electron micrographs between

thapsigargin-treated cells and those first

exposed to antimycin A and oligomycin,

to see how this related to the changes

observed in the TIRF microscopy

experiments (Figure 4G).

Collectively, these results

confirm that a fraction of the STIM1

pool migrates up to the plasma

membrane following store depletion

after mitochondrial depolarisation in

HEK cells, when STIM1 is strongly

overexpressed.

Ca2+ influx to a STIM1 mutant that

accumulates in ER-PM junctions in non-stimulated cells is insensitive to

mitochondrial depolarization

An explanation of the results so

far is that mitochondrial depolarisation

impairs STIM1 trafficking to the plasma

membrane, but not the events that arise

once STIM1 is at ER-PM junctions. To

test this more directly, we expressed the

mutant D76A STIM1, which localizes to

ER-PM junctions even when stores are

full. This construct has a point mutation

in the Ca2+-binding EF-hand domain, so

that the expressed protein forms

punctate-like structures in cells with

replete Ca2+ stores, resulting in

constitutive store-operated Ca2+ influx

(30, 55, 66). Expression of D76A

STIM1 in HEK293 cells resulted in

formation of puncta close to the cell

periphery (Figure 5E, left hand panel)

and these structures were unaffected by

mitochondrial depolarisation (Figure 5E,

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right hand panel). Constitutive Ca2+

influx was revealed by briefly exposing

cells to Ca2+-free external solution for 90

seconds and then readmitting external

Ca2+. The subsequent store-operated Ca2+

influx was unaffected by mitochondrial

depolarisation (Figure 5F; aggregate data

is summarised in Figure 5G), at least

over the duration of mitochondrial

depolarisation we have used (10-15

minutes in this study).

In aggregate, these results have two

important implications. Firstly,

mitochondria are not involved in the late

stages of CRAC channel activation

because i) constitutive store-operated

Ca2+ influx after D76A mutant STIM1

expression was unaffected by impairing

mitochondria (Figure 5 E-G) and ii)

overexpression of eYFP-STIM1 and

Orai1 could partially overcome the block

by mitochondrial depolarisation (Figure

4D). Presumably, the interaction

between STIM1 and Orai1 at the ER-PM

junctions (46) can activate CRAC

channels without a requirement for

mitochondria. Secondly, the tools used

for inducing mitochondrial

depolarisation do not interfere with

store-operated Ca2+ entry non-

specifically or with the CRAC channels

themselves, otherwise neither rescue by

Orai1 and STIM1 nor constitutive Ca2+

influx to the mutant STIM1 would have

been seen in cells with depolarised

mitochondria.

Mitofusin 2 (Mfn 2) regulates STIM1

trafficking Mitochondria can be tethered to

the ER through the mitochondrial

dynamin-related protein Mfn 2 (10). Mfn

2 is found mainly in the outer

mitochondrial membrane, with a small

fraction in the ER. It is particularly

abundant within the contact sites

between the mitochondria and ER,

where it forms transorganellar

homotypic and heterotypic interactions

between mitofusin 1 or 2 on

mitochondria and Mfn 2 on the ER (10).

In mouse embryonic fibroblasts (MEFs)

lacking Mfn 2, mitochondria are

physically uncoupled from ER and the

spatial distance between them is

increased (10, 40). We considered the

possibility that mitochondrial tethering

to the ER might hinder STIM1

movement to the cell periphery. To test

this, we compared STIM1 migration and

store-operated Ca2+ influx between

control mouse embryonic fibroblasts and

those in which Mfn 2 had been knocked

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out (10). Stimulation with thapsigargin

in control cells resulted in prominent

STIM1 puncta formation (Figure 6A).

Store-operated Ca2+ entry was also

present (Figures 6C and 6E). Both

STIM1 puncta formation (Figure 6A)

and store-operated Ca2+ entry (Figure

6C) in wild type cells were suppressed

by antimycin A and oligomycin pre-

treatment (aggregate data is summarised

in Figure 6E). Wild-type mouse

embryonic fibroblasts therefore behave

in a manner similar to RBL and HEK

293 cells. In mouse embryonic

fibroblasts lacking Mfn 2, numerous

STIM1 puncta formed after exposure to

thapsigargin (Figure 6B) and this was

followed by store-operated Ca2+ entry

(Figure 6D). The rate of Ca2+ entry was

~2-fold faster than in wild-type cells

(Figures 6D and E). Importantly, in Mfn

2-deficient cells STIM1 puncta

formation (Figure 6B) and store-

operated Ca2+ entry (Figures 6D and E)

were unaffected by mitochondrial

depolarisation. Hence STIM1 puncta

formation and store-operated entry are

insensitive to mitochondrial

depolarisation when Mfn 2 is absent.

It is important to note that the

extent of Ca2+ release and the rate of

recovery of the Ca2+ signal (to

thapsigargin in Ca2+-free solution) were

similar between wild type MEF cells and

those lacking Mfn 2. Hence stores are

loaded with Ca2+ and sufficient ATP is in

the cytoplasm to support Ca2+ATPAse

activity in Mfn 2-/- cells.

Endogenous Mfn 2 has a patchy

distribution throughout the cytoplasm in

resting MEF cells ((10); Figure 7A).

Stimulation with thapsigargin did not

alter the pattern of Mfn 2 and, unlike the

case with STIM1, no clear punctate-like

structures were formed (Figure 7A). We

also transfected cells with a Mfn 2-GFP

construct. However, the distribution of

Mfn 2-GFP was drastically different

from endogenous Mfn 2 in that it formed

aggregates around the nucleus (not

shown), This is in agreement with other

studies that have found that

overexpression of tagged Mfn 2 leads to

a non-physiological distribution (1, 24,

63).

Re-expression of untagged Mfn 2

into MEF Mfn2-/- cells resulted in an

accelerated decline of the thapsigargin-

evoked Ca2+ signal, reflecting a loss of

store-operated Ca2+ entry (Figure 7B).

As with mitochondrial depolarisation,

the inhibitory effect of Mfn 2 could be

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overcome by transfecting STIM1

together with Mfn 2 in the MEF Mfn 2-/-

cells (Figure 7B).

Mfn 2 is located on both

mitochondria and ER. This prompted us

to ask whether mitochondrial Mfn 2, ER

Mfn 2 or both were involved in

regulating store-operated Ca2+ entry. To

address this, we expressed Mfn 2

constructs that are selectively expressed

in either the ER (Mfn 2-IYFFT) or

mitochondria (Mfn 2-ActA) in Mfn2-/-

cells and then looked to see whether

either construct could re-introduce

sensitivity to mitochondrial

depolarisation. Cells were stimulated

with thapsigargin in Ca2+-free solution in

the presence of antimycin A and

oligomycin and then Ca2+ was

readmitted. Whereas Mfn 2-/- cells

responded by generating robust Ca2+

influx in the presence of depolarised

mitochondria, Ca2+ entry was

substantially reduced by expression of

the Mfn 2-ActA construct (Figure 7Ci

and ii). Ca2+ release was only slightly

reduced and in several cells it was

indistinguishable from control release

yet was followed by much less Ca2+

entry. On the other hand, store-operated

Ca2+ entry was largely unaffected by

mitochondrial depolarisation following

expression of Mfn 2-IYFFT (Figure 7Di

and ii). These results reveal that Mfn 2

restricted to mitochondria renders store-

operated entry susceptible to

mitochondrial depolarisation whereas an

ER-resident Mfn 2 is less effective.

DISCUSSION

Activation of the ubiquitous

store-operated Ca2+ influx pathway by

the ER Ca2+ sensor STIM1 is a three step

process (29, 31). First, upon store

depletion STIM1 monomers come

together to form multimers in the ER

membrane. Second, the oligomers then

migrate to specialised ER-PM junctions,

resulting in punctate-like structures at

the cell periphery. Finally, STIM1

activates the plasma membrane CRAC

channels by binding to the N- and C-

termini of Orai1 (46, 64). Although

recent studies have provided insight into

how STIM1 senses store depletion and

interacts with Orai1 channels, little is

known about mechanisms that regulate

STIM1 migration to the cell periphery.

Our new results reveal that

mitochondrial depolarisation selectively

regulates trafficking of STIM1

multimers to the plasma membrane, and

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in a manner dependent on the

mitochondrial protein Mfn 2.

A substantial body of evidence

has established that mitochondrial

depolarisation inhibits store-operated

Ca2+ entry through CRAC channels and

that energised mitochondria increase the

size of the CRAC current (15,16, 20,

21). A major factor contributing to this

effect involves mitochondrial buffering

of cytoplasmic Ca2+ (42,43).

Mitochondrial Ca2+ uptake reduces the

extent of Ca2+-dependent inactivation of

CRAC channels, and thus leads to an

enhanced Ca2+ entry. Our new findings

add a further component to

mitochondrial gating of CRAC channels

that is independent of the Ca2+ buffering

action and involves an action on STIM1

trafficking.

How might mitochondrial

depolarisation be relayed to STIM1

proteins in the ER? The two organelles

are held in close proximity to one

another through interaction between

proteins spanning the respective

membranes, with a major role for the

dynamin-related protein Mfn 2 (8, 10).

Mfn 2 is abundant at contact sites

between ER and mitochondria, where it

forms transorganellar homotypic and

heterotypic interactions. Knockout of the

Mfn 2 gene in mouse embryonic

fibroblasts uncouples mitochondria from

ER and increases the distance between

them (8, 10). By using Mfn 2-deficient

cells, we found that STIM1 trafficking

and store-operated Ca2+ entry were no

longer impaired by mitochondrial

depolarisation. This suggests that Mfn2

is required to confer sensitivity of store-

operated Ca2+ entry to mitochondrial

depolarisation, at least in MEF cells.

Mfn2 is expressed both in mitochondria

and ER. By expressing Mfn 2 constructs

in either mitochondria or ER in Mfn 2-

deficient MEF cells, we found that

mitochondrial Mfn 2 rendered store-

operated Ca2+ entry sensitive to

mitochondrial depolarisation. It is

unlikely that Mfn 2 senses mitochondrial

depolarisation directly because it is

expressed in the outer mitochondrial

membrane (51). However, it can interact

with inner mitochondrial membrane

proteins including OPA1 (18, 40). Hence

changes in mitochondrial potential could

be relayed to the adjacent ER through

protein-protein interactions with

mitofusin 2 acting as a transducer. How

does mitofusin 2 control STIM1

trafficking and thereby CRAC channel

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activation following mitochondrial

depolarisation? Strong overexpression of

Mfn 2 leads to the formation of

aggregates of mitochondria that are

uncoupled from one another and clump

around the nucleus (1, 24). These

mitochondria have a decreased

membrane potential (24). De-energised

mitochondria are less able to buffer

cytosolic Ca2+, and this would result in

stronger Ca2+-dependent inactivation of

CRAC channels (15, 20). Whilst this

mechanism can help account for the

reduced Ca2+ influx seen upon over

expression of Mfn 2, it fails to explain i)

why STIM1 movement after store

depletion is impaired by mitochondrial

depolarisation in the absence of Ca2+

entry and when cytoplasmic Ca2+ is

buffered with BAPTA, conditions that

would prevent Ca2+-dependent

inactivation of CRAC channels and ii)

why STIM1 overexpression, at least

partially, rescues Ca2+ entry in Mfn 2-

overexpressing cells. An alternative

mechanism involves a direct or indirect

physical block on STIM1 movement by

Mfn 2. This could be due either to steric

hindrance whereby mitofusin 2 needs to

be displaced from a site in order for

STIM1 multimers to migrate towards

ER-PM junctions or that a component of

the mitochondrial tethering complex

(which includes Mfn 2, voltage-

dependent anion channel, grp75, sigma 1

receptor and PACS-2; (40)) binds to

STIM1, thus impeding its movement

towards the periphery. In either case,

mitochondrial depolarisation would be

predicted to stabilise this interaction.

Loss of Mfn 2 might therefore be

expected to increase the number of

STIM1 multimers that successfully

migrate to ER-PM junctions, resulting in

increased Ca2+ entry. Consistent with

this, the rate of Ca2+ influx was slightly

higher in Mfn 2-deficient cells (Figure

7E).

Although overexpression of

STIM1 led to partial recovery of store-

operated Ca2+ influx in HEK293 cells

with depolarised mitochondria, the

pattern of puncta formation was

strikingly different from that seen in

normal cells. After mitochondrial

depolarisation, electron micrograph

analysis revealed that ~40% less STIM1

migrated to the cell periphery after store

depletion (Figure 5D). TIRF

measurements showed that although

fewer puncta formed, they had a higher

STIM1 intensity suggesting coalescence

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of individual puncta into larger

structures. Despite such changes, store-

operated Ca2+ influx still developed,

albeit to a lesser extent. Hence it would

appear that formation of puncta per se is

sufficient to activate several Orai1

channels, in that numerous discrete

puncta are only moderately(~ 2-fold)

more effective in evoking Ca2+ entry that

larger, merged structures. This would be

consistent with the finding that

disaggregation of microfilaments results

in fewer, larger puncta being formed but

without any effect on CRAC channel

activity (32).

What might be the physiological

relevance for Mfn 2 regulation of STIM1

movement? Mfn 2 is not essential for the

store-operated pathway because store-

operated Ca2+ entry in both MEF

fibroblasts and RBL cells (data not

shown) was prominent despite its

knockdown. Therefore Mfn 2 has a

regulatory role, but not an essential one.

Rather, its effects are manifest only after

mitochondrial depolarisation. Our results

suggest Mfn 2 might serve as a brake,

inhibiting Ca2+ entry only after

mitochondria depolarize. Although

significant fluctuations in the

mitochondrial membrane potential have

been reported in some intact cells, the

sustained mitochondrial depolarization

we have evoked is more typically seen

following glutamate excitotoxicity in

neurons. From a teleological standpoint,

Mfn 2 regulation of STIM1 migration

and subsequent CRAC channel opening

might serve to oppose Ca2+ overload

under conditions where mitochondrial

Ca2+ buffering is compromised, due to

collapse of the mitochondrial membrane

potential.

Precisely how Mfn 2 controls migration

of STIM1, its impact, if any, on Ca2+

entry under physiological levels of

stimulation and whether this mechanism

contributes to mitofusin-related diseases

such as Charcot-Marie tooth neuropathy

await further study.

Acknowledgements. This work was supported by the Medical

Research Council (U.K.) and the British Heart Foundation.

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and Meyer, T. (2005) Curr Biol 15, 1235-1241. 31. Luik, R.M., Wang, B., Prakriya, M., Wu, M.M. and Lewis RS. (2008) Nature 454, 538-542 32. Luik, R.M., Wu, M.M., Buchanan, J. and Lewis, R.S. (2006) J Cell Biol 174, 815-825. 33. Mercer, J.C., DeHaven, W., Smyth, J.T., Wedel, B., Boyles, R.B., Bird, G.S. and Putney, J.W.Jr. (2006) J Biol Chem 281, 24979-24990. 34. Mignen, O., Thompson, J.L. and Shuttleworth, T.J. (2007) J Physiol 579, 703-715. 35. Mohr, F.C. and Fewtrell, C. (1990) Am J Physiol (Cell Physiol) 258, C217-226. 36. Moreau, B., Nelson, C. and Parekh, A.B. (2006) Curr Biol 16, 1672-1677. 37. Moreau, B., Straube, S., Fisher, R.J., Putney, J.W,Jr and Parekh, A.B. (2005) J Biol Chem 280, 8776-8783 38. Muik, M., Fahrner, M., Derler, I., Schindl, R., Bergsmann, J., Frischauf, I., Groschner, K. and Romanin, C. (2009) J Biol Chem 284, 8421-8426 39. Ng, S-W., DiCapite, J.L., Singaravelu, K. and Parekh, A.B. (2008) J Biol Chem 283, 31348-31355 40. Parekh, A.B. (2009) Curr Biol 19, R200-R203 41. Parekh, A.B. (2007). Cell Calcium 42, 111-121 42. Parekh, A.B. (2008) Cell Calcium 44, 6-13 43. Parekh, A.B. (2003) J Physiol (Lond) 547, 333-348 44. Parekh, A.B,and Penner, R. (1997) Physiol Revs 77, 901-930 45. Parekh, A.B. and Putney, J.W.Jr. (2005) Physiol Revs 85, 757-810 46. Park, C.Y., Hoover, P.J., Mullins, F.M., Bachhawat, P., Covington, E.D., Rausner, S., Walz, T.,

Garcia, C., Dolmetsch, R.E. and Lewis, R.S. (2009) Cell 136, 1-15 47. Peinelt, C., Vig, M., Koomoa, D.L., Beck, A., Nadler, M.J.S., Koblan-Huberson, M., Lis, A., Fleig, A., Penner, R. and Kinet, J-P. (2006) Nat Cell Biol 8, 771-773 48. Prakriya, M., Feske, S., Gwack, Y., Srikanth, S., Rao, A. and Hogan, P.G. (2006) Nature 443, 230-233 49. Prakriya, M. and Lewis, R.S. (2001) J Physiol (Lond) 536, 3-19 50. Quintana, A., Schwarz, E.C., Schwindling, C., Lipp, P., Kaestner, L. and Hoth, M. (2007) J Biol Chem 281, 40302-40309 51. Rojo, M., Legros, F., Chateau, D. and Lombes, A. (2002) J Cell Science 115, 1664-1674 52. Roos, J., DiGregorio, P.J., Yeromin, A.V., Ohlsen, K., Lioudyno, M., Zhang, S., Safrina, O., Kozak, J.A., Wagner, S.L., Cahalan, M.D., Velicelebi, G. and Stauderman, K.A. (2005) J Cell Biol 169, 435-445 53. Schilling, W.P., Rajan, L. and Strobl-Jager, E. (1989) J Biol Chem 264, 12838-12848 54. Silverman, H.S., Di Lisa, F., Hui, R.C., Miyata, H., Sollott, S.J., Hanford, R.G., Lakatta, E.G. and Stern, M.D. (1994) Am J Physiol (Cell Physiol) 266, C222-233 55. Smyth, J.T., DeHaven, W.I., Bird, G.S. and Putney, J.W. (2008) J Cell Sci 121, 762-772 56. Smyth, J.T., Dehaven, W.I., Bird, G.S. and Putney, J.W. (2007) J Cell Sci 120, 3762-3771 57. Soboloff, J., Spassova, M.A., Tang, X.D., Hewavitharana, T., Xu, W. and Gill, D.L. (2006) J Biol Chem 281, 20661-20665 58. Vig, M., Beck, A., Billingsley, J.M., Lis, A., Parvez, S., Peinelt, C., Koomoa, D.L., Soboloff, J., Gill, D.L.,

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Fleig, A., Kinet, J.P. and Penner. R. (2006) Curr Biol 16, 2073-2079 59. Vig, M., Peinelt, C., Beck, A., Koomoa, D.L., Rabah, D., Koblan-Huberson, M., Kraft, S., Turner, H., Fleig, A., Penner, R. and Kinet, J-P. (2006) Science 312, 1220-1223 60. Wu, M.M., Buchanan, J., Luik, R.M. and Lewis, R.S. (2006) J Cell Biol 174, 803-813 61. Wu, M.M., Luik, R.M. and Lewis, R.S. (2007) Cell Calcium 42, 163-172 62. Yeromin, A.V., Zhang, S.L., Jiang, W., Yu, Y., Safrina, O. and Cahalan, M.D. (2006) Nature 443, 226-229 63. Yoon, E., Beom, S., Cheong, H., Kim, S., Oak, M., Cho, D. and Kim, K.M. (2004) BBRC 325, 117-123 64. Yuan, J.P., Zeng, W., Dorwart, M.R., Choi, Y.J., Worley, P.F. and Muallem, S. (2009) Nat Cell Biol 11, 337-343 65. Zhang, S.L., Yeromin, A.V., Zhang, XH-F., Yu, Y., Safrina, O., Penna, A., Roos, J., Stauderman, K.A. and Cahalan, M.D. (2006) Proc Natl Acad Sci USA 103, 9357-9362 66. Zhang, S.L., Yu, Y., Roos, J., Kozak, J.A., Deerinck, T.J., Ellisman, M.H., Stauderman, K.A. and Cahalan, M.D. (2005) Nature 437, 902-905 67. Zweifach, A. and Lewis, R.S. (1993) Proc Natl Acad Sci USA 90, 6295-629 68. Zweifach, A. and Lewis, R.S. (1995) J Gen Physiol 105, 209-226 FIGURE LEGEND Figure 1. Mitochondrial depolarisation

inhibits ICRAC following overexpression

of STIM1 and Orai1. (A), In fura 2-

loaded RBL-1 cells coexpressing eYFP-

STIM1 and Orai1, readmission of

external Ca2+ to cells treated with

thapsigargin (2 µM) results in Ca2+

influx and this is inhibited by

depolarising mitochondria with

antimycin A (5 µg/ml) and oligomycin

(0.5 µg/ml), applied to cells 10 minutes

before thapsigargin. (B), The rate of Ca2+

entry (measured from experiments as in

panel A) is compared (each bar

represents > 60 cells). (C), The large

ICRAC in cells coexpressing eYFP-STIM1

and Orai1 is substantially reduced by

mitochondrial depolarisation and

aggregate data is plotted in panel (D).

Pipette solution contained InsP3 + 10

mM EGTA. Number of cells is 11 for

STIM1 + Orai1 alone and 9 in the

presence of antimycin A and

oligomycin. (E), Images from confocal

microscopy showing the distribution of

eYFP-STIM1 in cells before (labelled

Control) and then after exposure to 2 µM

thapsigargin in Ca2+-free solution for the

various conditions shown. (F), The

distribution of eYFP-STIM1 and

mitochondria (detected with mitotracker

red) are compared. Co-localisation is

indicated in yellow.

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Figure 2. STIM1 trafficking to the

plasma membrane is impaired by

mitochondrial depolarisation. (A), TIRF

microscopy images from an RBL-1 cell

expressing eYFP-STIM1. The left hand

panel shows a resting cell and the middle

panel the same cell after stimulation with

thapsigargin (2 µM) in Ca2+-free solution

for 180 seconds. The right hand panel

shows the response to thapsigargin (after

180 seconds) after exposure to antimycin

A and oligomycin. (B), The total

increase in eYFP-STIM1 fluorescence

measured with TIRF microscopy is

compared between cells stimulated with

thapsigargin in the absence (6 cells)

versus presence (5 cells) of antimycin A

and oligomycin. (C), The time at which

this fluorescence reached 50% of its

maximum value (half-time) is compared

for the two conditions.

Figure 3. STIM1 migration does not

depend on mitochondrial Ca2+ buffering

or ATP production. (A), Loading cells

with BAPTA impaired the cytoplasmic

Ca2+ rise evoked by thapsigargin. Cells

were loaded with either fura 2-AM and

0.1% DMSO (control) or fura 2-AM and

BAPTA-AM (10 µM) prior to

stimulation with thapsigargin. (B),

Confocal images from a non-treated cell

showing eYFP-STIM1 distribution at

rest, and then following stimulation with

thapsigargin in the absence and then

presence of antimycin A and

oligomycin. Images represent different

cells. (C), Loading cells with BAPTA

does not impair migration of eYFP-

STIM1 to the cell periphery but

mitochondrial depolarisation still

reduces translocation in BAPTA-loaded

cells. (D), Time course of Na+ current

through CRAC channels in whole cell

patch clamp recording from cells

overexpressing Orai1 and eYFP-STIM1.

Filled circles denote a control cell and

open circles one after exposure to

antimycin A and oligomycin. (E),

Corresponding current-voltage

relationships from panel D are shown,

taken when the currents had peaked. (F),

Aggregate data from 6 control cells and

5 antimcycin A/oligomycin-treated cells

are compared. Amplitude was measured

at -80 mV. (G), Mitochondrial

depolarisation (labelled +Anti./oligo)

does not affect intracellular Mg-ATP

levels (measured through Mg2+

concentration) provided glycolysis is

intact. 2-DOX denotes 10 mM 2-

deoxyglucose. (H), Time course of

decay of the Ca2+ signal to thapsigargin

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is unaffected by mitochondrial

depolarisation. This time course

represents Ca2+ removal by the ATP-

dependent plasma membrane Ca2+ pump.

(I), Aggregate data from several

experiments as in panel H is summarised

(control denotes 76 cells and Anti./oligo

85 cells).

Figure 4. Store-operated Ca2+ entry can

be partially rescued by overexpressing

STIM1 in HEK293 cells with

depolarised mitochondria. In panels (A-

C), HEK293 cells were stimulated with

thapsigargin in Ca2+-free solution (at

t=0) and then 2 mM external Ca2+ was

readmitted as shown. Only the Ca2+ entry

component is shown for simplicity. The

extent of Ca2+ release was similar for the

different conditions. (A), Store-operated

entry in wild-type cells is inhibited by

antimycin A and oligomycin. (B),

Overexpression of eYFP-STIM1 results

in modest recovery of store-operated

Ca2+ entry in cells with depolarised

mitochondria. (C), Overexpression of

eYFP-STIM1 and Orai1 results in

substantial rescue of store-operated Ca2+

entry in the presence of antimycin A and

oligomycin. For each graph, the

response in antimycin A and oligomycin

has been normalised to the

corresponding control response. Control

response has been re-calculated as ((R-

R0)/Rpeak)*100%, where R is the

measured ratio (356/380) at any time

point following Ca2+ readmission, Rpeak

is the peak ratio measured in the cell

(maximal response) and R0 is the basal

ratio (resting Ca2+). (D), Ca2+ influx

following overexpression of Orai1 and

STIM1 is compared before and after

mitochondrial depolarisation in cells

loaded with Fura 5F. (E-H), TIRF

images comparing puncta formation in a

resting HEK cell shown in panel (E), and

then after store depletion with

thapsigargin for 4 minutes (F) and after

store depletion following exposure to

antimycin A and oligomycin for 15

minutes (G). (H) The histograms plot the

absolute fluorescence intensity measured

in 1µm2 segments against the frequency

of occurrence. Upper panel represents

cells exposed to thapsigargin and the

lower panel analyses cells pre-exposed

to antimycin A and oligomycin prior to

thapsigargin challenge.

Figure 5. Ca2+ influx following

expression of a mutant STIM1is

unaffected by mitochondrial

depolarization. Scale bar in this and

subsequent left hand panels is 1 µm and

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is 0.2 µm in each right hand panel. (A),

Electron micrograph from a HEK293

cell expressing eYFP-STIM1 in the

absence of store depletion. The right

hand panel shows a magnified view of

the box marked in the left hand panel.

(B) and (C), Electron micrographs after

stimulation with thapsigargin in the

absence (B) and presence (C) of

antimycin A/oligomycin. STIM1

location was identified using the DAB

reaction, which is visible as dark

deposits. (D), The fraction of the cell

periphery stained for STIM1-eYFP is

compared for control cells, cells

stimulated with thapsigargin and cells

pre-treated for 15 minutes with

antimycin A and oligomycin prior to

exposure to thapsigargin. (E), Mutant

STIM1 localizes to the cell periphery in

HEK293 cells in the absence of store

depletion and this is not altered by

mitochondrial depolarisation. (F),

Constitutive store-operated Ca2+ entry,

revealed by transiently removing and

then readmitting 2 mM external Ca2+, is

unaffected by mitochondrial

depolarisation. (G), Aggregate data,

measuring the rate of Ca2+ influx from

experiments as in panel F are

summarised. Each bar denotes >50 cells.

Figure 6. STIM1 puncta formation and

subsequent store-operated Ca2+ entry are

unaffected by mitochondrial

depolarisation in mitofusin 2-deficient

cells. A, EYFP-STIM1 distribution is

compared between control (resting)

mouse embryonic fibroblasts and those

stimulated with thapsigargin, in the

absence and then presence of antimycin

A plus oligomycin. WT above the

images denotes wild type fibroblasts. B,

Experiments were conducted as in panel

A but now using mitofusin 2-deficient

(Mfn2-/-) mouse embryonic fibroblasts.

C, After depleting Ca2+ stores with

thapsigargin (2µM) in Fura-2 loaded

cells, readmission of external Ca2+ (2

mM) resulted in a rapid increase in

cytosolic Ca2+ concentration (control)

that was suppressed by depolarising

mitochondria. (D), Ca2+ influx was

prominent in mitofusin 2-deficient cells

and was unaffected by impairing

mtochondria. (E) Bar chart compares

the rate of cytosolic Ca2+ rise upon Ca2+

readmission in control and mitofusin 2-

deficient cells, in the absence and

presence of antimycin A plus

oligomycin. Mitochondrial

depolarisation reduces the rate of Ca2+

entry in WT, but not in mitofusin 2-

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deficient cells. The rate of Ca2+ influx in

wild type fibroblasts has been taken as

100%, for comparative purposes.

Figure 7. Mitochondrial Mfn 2 is

involved in regulating store-operated

calcium entry after mitochondrial

depolarisation. A, Confocal images show

distribution of endogenous Mfn 2 in

control, non-stimulated, MEF cells and

following stimulation with thapsigargin

(8 minutes). (B), Comparison of Ca2+

signals to thapsigargin in wildtype MEF

cells, MEF cells transfected with Mfn 2

and MEF cells transfected with Mfn 2

and STIM1. (Ci), Store-operated Ca2+

entry was measured in Mfn 2-/- cells

(black curve) and in Mfn 2-/- cells

(dotted curve) transfected with the Mfn

2-ActA construct, which is expressed in

mitochondria. (Cii), Summary of

aggregate data from several cells (41 for

Mfn 2-/- and 38 for Mfn 2-/- and ActA).

(D), As in panel C but now Mfn 2-/-

cells were transfected with the Mfn 2-

IYFFT construct that expresses

exclusively in the ER (35 cells for Mfn

2-/- and 36 for Mfn 2-/- and IYFFT). In

panels C and (D), cells were pre-treated

with antimycin A and oligomycin for 10

minutes before challenge with 2 µM

thapsigargin.

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8006004002000Time (secs)

Thap. 2 mM Ca2+

STIM1 + Orai1

Antimycin A + oligomycin

10

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0Rate

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flux

((R/

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0

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150100500Time (seconds)

STIM1 + Orai1

Antimycin A + oligomycin

Control Anti. A + oligo

Control Antimycin A + oligomycin

Thap.

EYFP-STIM1 Merge Mitotracker Red Oligomycin

Anti. A + oligo

Control

Figure 1A B C D

E F

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0.10

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A

B

B CFigure 2

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anti/oligo Thapanti/oligo anti/oligo

Thap

*

*

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150100500Time (seconds)

40

20

0Na+ c

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nt (p

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3002001000Time (seconds)

+2-DOX+iodoacetate+oligo

+Anti/oligo

Thap./0Ca2+

Figure 3

mV

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-15

-50 +50

controlcontrol

Anti/oligoAnti/oligo

control Anti/oligo

A B C

RestingThap.

D E F

R (3

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0

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1.2

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Control

BAPTA-loadedAnti./oligo. Resting

Thap.Anti./oligo.

BAPTA-loaded

*

Control

G H I

200 400 600Time (seconds)

0.4R

0 200 400Time (seconds)

1.2

1.4R

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control Anti/oligo

100

200

300

0

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R (3

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450400350300Time (sec)

Nor

mal

ised

R (3

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450400350300Time (sec)

Nor

mal

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R (3

56/3

80)

450400350300Time (sec)

100

0

A B C

Wild-type

Antimycin A + Oligomycin

STIM1

Antimycin A + Oligomycin STIM1+Orai1

Antimycin A

+ Oligomycin

50 50 50

00

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420Fr

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Figure 4

Control Thap. Anti/oligo then thap.

Thap.

Anti/oligo then thap.

Thap.Anti/Oligo

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A B C D

6004002000Time (seconds)

6x10-3

4

2

0

Rate

of C

a2+ en

try (R

/s)

E F G

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0Frac

tion

occu

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Figure 5

Control Thap. Anti/oligo then Thap.

1.0

0.9

0.8

Control

Anti/Oligo

ControlAnti/Oligo

Anti/OligoControl

Thap.

+ AntiOligoControl

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100

0Rate

of C

a2+ in

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(%)1.1

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80)

1000800600400200Time (sec)

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56/3

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1000800600400200Time (sec)

WT Mfn2-/-

A B

CThap./ 0Ca2+

D E

Antimycin A + oligomycin

Control

Thap. Thap.

2 Ca2+Thap./ 0Ca2+

2 Ca2+

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anti.+oligo

anti.+oligo

Antimycin A + oligomycin

Control

anti.+oligo

anti.+ oligo

ctrl

ctrl

WT Mfn2-/-Figure 6

*

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1.10

1.05

R(35

6/38

0)

10007505002500Time (seconds)

6x10-3

4

2

0

R/s

Mfn 2-/- Mfn 2-/-+ IYFFT

Mfn 2-/-

Mfn 2-/-+ IYFFT

1.3

1.2

1.1

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0)

10007505002500Time (seconds)

Mfn 2-/-+ ActA

Mfn 2-/-10x10-3

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A

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3002001000Time (seconds)

Wild type

Mfn 2 + STIM 1

Mfn 2

Thapsigargin + 2 Ca2+

Thap/0Ca2+

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2Ca2+

2Ca2+

B

Control Thap

Figure 7

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3002001000Time (seconds)

100 µM ATP +0 Ca2+

Control

STIM1 + Orai1

RNAi to Orai1

Control

STIM1 + Orai1

RNAi to Orai1

20

15

10

5

0Rate

of C

a2+ in

flux

((R/

s)*1

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10080604020[ATP] µM

-10

-5

0I C

RAC

(pA

/pF)

150100500Time (seconds)

Control

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15

10

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0

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**

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1.1

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80)

800700600500Time (sec)

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+FCCP (2 µ M)+ Oligo

+Oligo

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60

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-15

-100 +100 mV

-30

-45

-60 pA/pFcontrol

+ anti.+ oligo.

A B

Resting cell Anti./oligo.Thapsigargin

Supplementary Figures.

Figure 1. Co-expression of Orai1 with

eYFP-STIM1 increases the size of ICRAC

following store depletion by InsP3 or

agonist. (A), Time course of

development of ICRAC following dialysis

with InsP3 and 10 mM EGTA is

compared between a control (eYFP-

transfected) cell and one expressing

eYFP-STIM1 and Orai1. (B), The

current-voltage relationship for the two

recordings in panel a are shown (taken

once the currents had peaked). (C), The

histogram compares the amplitude of

ICRAC (measured at -80 mV) for control

cells (n=11) and those co-expressing

eYFP-STIM1 with Orai1 (14 cells)

following dialysis with either 30 µM

InsP3 and 10 mM EGTA or 10 mM

EGTA alone (5 cells). (D), RNAi to

Orai1 substantially reduces the extent of

ICRAC. Cells were dialysed with InsP3 in

10 mM EGTA. Number of cells is 8 for

each condition (E), Agonist-evoked

store-operated Ca2+ entry is increased in

cells overexpressing eYFP-STIM1 and

Orai1 and is reduced by RNAi to Orai1

(each point is > 50 cells). (F), Agonist-

evoked cation entry is dependent on

Orai1 over a wide range of agonist

concentrations (>30 cells per point).

ATP was applied in Ca2+-free solution

and then 2 mM Ba2+ was added 100

seconds after the Ca2+ release transient

had ended. R denotes the (356/380)

ratio.

Figure 2. (A), Store-operated Ca2+ entry,

measured with fura 2, is increased

following overexpression of eYFP-

STIM1 and reduced by overexpression

of Orai1.

Thapsigargin was applied in Ca2+-free

solution at t=0. Control denotes cells

transfected with eYFP alone. (B),

Fluoresence measurements of eYFP-

STIM1 with TIRF microcsopy showing

that oligomycin alone does not affect

STIM1 migration to the plasma

membrane, whereas FCCP and

oligomycin does. Thap. control and

Thap + oligo represent 3 independent

experiments; FCCP + oligomycin are 2

experiments. Data have been normalised

to the thapsigargin control response.

Thapsigargin was applied in Ca2+-free

solution for 120-150 seconds.

Figure 3. Distribution of mitochondria

before and after store depletion. (Ai),

Electron micrograph from a non-

stimulated RBL-1 cell. A(ii), The

histogram shows the distribution of

mitochondria from the plasma

membrane, measured in a stack of 100

serial sections (50 nm apart) taken from

resting RBL-1 cells. (Bi), Electron

micrograph taken through a RBL-1 cell

that had been stimulated with

thapsigargin for 5 minutes. (Bii) plots

the corresponding distribution of

mitochondria from the plasma

membrane. (C), A cumulative plot of the

number of mitochondria as a function of

distance from the plasma membrane (up

to 4000 nm) is compared between

control cells and those pre-exposed to

thaspigargin. No difference was found

between the two groups. D, Ca2+-

dependent fast inactivation was

compared between a control cell and one

exposed to antimycin A and oligomycin.

Membrane potential was stepped from 0

to -120 mV for 250 msec. The pipette

solution contained 10 mM EGTA and

thapsigargin.

Figure 4. Mitochondrial depolarisation

has no effect on the inwardly rectifying

K+ current in RBL-1 cells. A, I-V

relationship for the inward rectifier in

the absence and presence of

mitochondrial depolarisation. B,

aggregate data from several cells is

summarised (6 cells for each condition).

Bath solution contained standard

external ringer with KCl raised to 45

mM and NaCl lowered to 100 mM.

Pipette solution was K-glutamate (145

mM) based.

Figure 5. ER distribution after

mitochondrial depolarisation. The ER

marker ER-RFP was used to identify the

distribution of ER in RBL cells. Cells

were transfected with and images were

taken 36 hours post transfection. The

left hand panel depicts a confocal image

of a resting cell. The middle panel shows

the ER distribution following stimulation

with thapsigargin. The right hand panel

reveals little change in ER distribution

after depolarising mitochondria for 15

minutes with antimycin A and

oligomycin.

Siaw-Wei Ng, Joseph Di Capite, Trevor Powell, Luca Scorrano and Anant B. ParekhKarthika Singaravelu, Charmaine Nelson, Daniel Bakowski, Olga Martins de Brito,

in cells with depolarised mitochondria.Mitofusin 2 regulates STIM1 migration from the Ca2+ store to the plasma membrane

published online January 10, 2011J. Biol. Chem.

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