Mitochondrial calcium signalling in cell death

THE THEODOR BUCHER LECTURE

Mitochondrial calcium signalling in cell death

Delivered on 1 July 2004 at the 29th FEBS Congress in Warsaw

Sara Leo1, Katiuscia Bianchi1, Marisa Brini2 and Rosario Rizzuto1

1 Department of Experimental and Diagnostic Medicine, Section of General Pathology, and Interdisciplinary Center for the Study of

Inflammation (ICSI), University of Ferrara, Italy

2 Department of Biochemistry, University of Padova, Italy

In the last few decades, much information has been

obtained on the role of calcium ions as ubiquitous sec-

ond messengers that translate the binding of signalling

molecules to plasmamembrane receptors into defined

cell activities [1]. Thanks to the development of highly

efficient probes (the intracellularly trappable fluores-

cent indicators developed by Tsien and coworkers) [2],

it was possible to investigate the calcium signals elici-

ted in a wide variety of cell types, either in culture (pri-

mary cultures and immortalized cell lines) or in situ

(organotypic slices or even the intact tissue within a

living organism) by the opening of plasmamembrane

Ca2+ channels or Ca2+ channels of intracellular reser-

voirs, cytologically identifiable with the endoplasmic

reticulum (ER) and, more recently, with the Golgi

apparatus [3,4].

The use of Ca2+ as a second messenger rests on the

maintenance of a low cytosolic Ca2+ concentration,

through the energy-consuming pumping activity of

Ca2+ ATPases located in ER ⁄SR (SERCA) or

plasmamembrane (PMCA). As to the triggering mech-

anism of the [Ca2+] rise, a route involves either the

stimulation of G-protein coupled receptors (specifically

those coupled to a G(aq) protein, that activate phos-

pholipase Cb and thus produce inositol 1,4,5-trisphos-

phate (IP3) from the hydrolysis of the lipid

phosphatidyl-inositol 4,5-diphosphate) or growth fac-

tors receptors (also causing the production of IP3

through the activation of phospholipase Cc, containingan SH2 domain that recruits it to the activated GF-R)

[1]. An alternative route for raising cytosolic Ca2+

concentration ([Ca2+]c) depends on the opening of

Keywords

apoptosis; calcium; mitochondria;

organelles; photoproteins; signal

transduction

Correspondence

R. Rizzuto, Department of Experimental and

Diagnostic Medicine, General Pathology

Section, University of Ferrara,

Via L. Borsari 46, 44100 Ferrara, Italy

Fax: +39 0532247278

Tel: +39 0532291361

E-mail: [email protected]

(Received 1 June 2005, accepted 11 July 2005)

doi:10.1111/j.1742-4658.2005.04855.x

The development of targeted probes (based on the molecular engineering

of luminescent or fluorescent proteins) has allowed the specific measure-

ment of [Ca2+] in intracellular organelles or cytoplasmic subdomains. This

approach gave novel information on different aspects of cellular Ca2+

homeostasis. Regarding mitochondria, it was possible to demonstrate that,

upon physiological stimulation of cells, Ca2+ is rapidly accumulated in the

matrix. We will discuss the basic characteristics of this process, its role in

modulating physiological and pathological events, such as the regulation of

aerobic metabolism and the induction of cell death, and new insight into

the regulatory mechanisms operating in vivo.

Abbreviations

AGC, aspartate ⁄ glutamate metabolite carrier; COX8, cytochrome c oxidase; CRAC, Ca2+ release-activated current; ER, endoplasmic

reticulum; HBx, x protein of the hepatitis B virus; IP3, inositol 1,4,5-trisphosphate; PKC, protein kinase C; PMCA, plasmamembrane Ca2+

ATPase; SERCA, sarcoplamic reticulum ⁄ ER Ca2+ ATPase.

FEBS Journal 272 (2005) 4013–4022 ª 2005 FEBS 4013

various classes of plasma membrane Ca2+ channels,

such as those directly opened by ligand binding (e.g.

the ionotropic glutamate receptors of neurons), those

opened by the depolarization of the plasmamembrane

(the wide number of voltage-dependent Ca2+ channels)

or those opened by other intracellular signals (e.g. sec-

ond messengers or the depletion of intracellular Ca2+

stores) [5,6].

The concerted action of channels with distinct spa-

tial distribution and kinetics of opening determines a

high spatio-temporal specificity of the signals elicited

by different agonists, which in turn are decoded into

radically different intracellular effects. This adds fur-

ther interest and complexity to the signalling properties

of Ca2+: not only tissue-specific functions (e.g. endo-

crine and neuro-secretion, muscle contraction and fer-

tilization), but also decisions on cell fate (proliferation,

cell death by necrosis or apoptosis) are controlled by

Ca2+ [7]. Thus, not surprisingly deregulations in intra-

cellular Ca2+ homeostasis have been implicated in the

pathogenesis of genetic (e.g. familial migraine and skin

disorders, such as Darier’s and Hailey-Hailey diseases)

and multifactorial (e.g. hypertension and diabetes) dis-

eases [8,9].

The recognition of the spatio-temporal complexity

of calcium signals and of their multiple signalling roles

has ignited interest in clarifying the molecular mecha-

nisms that allow to specifically decode different signal-

ling patterns. Extensive work in the past decades has

revealed the broad repertoire of Ca2+ effectors, i.e.

enzymes, channels or structural proteins that modify

their activity upon binding of Ca2+. At first, these

included cytosolic proteins, such as the Ca2+-depend-

ent kinases [protein kinase C (PKC), CamK] or phos-

phatases (calcineurin) and their targets [1]. In recent

years, however, it became clear that also processes

occurring within intracellular organelles (gene tran-

scription, post-translational modification of proteins

and aerobic metabolism) are modulated by [Ca2+]

changes [10,11]. Thus, Ca2+-dependent effects within

organelles are now considered a significant component

of the ‘Ca2+ symphony’, initiated by physiological or

pathological stimuli, which may influence its final out-

come.

In this context, measuring Ca2+ concentrations

within organelles with accuracy and specificity has

become an important experimental task. This task has

largely been accomplished, thanks to the development

of a new class of probes that are based on Ca2+-sensi-

tive reporter proteins and take advantage of the highly

selective mechanisms that target cellular proteins to

the correct location. The first successful example has

been that of aequorin. Aequorin is a Ca2+-sensitive

photoprotein of the jellyfish Aequorea victoria, which

emits light upon binding of Ca2+ to three high-affinity

sites present in the protein sequence. The protein can

be purified from jellyfish extracts and microinjected in

cells. Using this classical indicator, seminal observa-

tions were made, such as the repetitive [Ca2+]c spiking

induced by agonist stimulation [12]. More recently, we

have taken advantage of molecular biology techniques

for developing a series of specifically targeted Ca2+

probes. The rationale was that of fusing the aequorin

cDNA with DNA sequences encoding specific target-

ing signals, i.e. the protein sequences that are necessary

and sufficient for localizing a mammalian protein to

the correct subcellular location. Figure 1A shows an

example, mtAEQ, which is the recombinant protein

developed for measuring [Ca2+] within the mitochond-

rial matrix and was instrumental in gaining new insight

into mitochondrial Ca2+ handling, i.e. the topic of this

review [13]. In the chimeric cDNA, an aequorin moiety

including an HA1 tag was fused in frame with the

N-terminal portion (including the 25 amino acids

cleavable presequence and the first eight amino acids

of the mature polypeptide) of subunit VIII of cyto-

chrome c oxidase (COX8). The fusion protein, when

expressed in mammalian cells, is entirely distributed

to the mitochondria (Fig. 1B). The localization of

mtAEQ is revealed by immunofluorescence using an

antibody that recognizes the HA1 domain.

In general, targeted aequorins (also developed for

other intracellular compartments using similar strat-

egies) have proved to be extremely valuable, and

allowed many new data and novel concepts in Ca2+

signalling to be obtained. The most important ones

A

B

Fig. 1. Schematic map of the mtAEQ construct (aequorin targeted

to the mitochondrial matrix) and its localization by immunofluores-

cence.

Mitochondrial calcium signalling in cell death S. Leo et al.

4014 FEBS Journal 272 (2005) 4013–4022 ª 2005 FEBS

not covered in this review are the estimates of ER

[Ca2+] in the near-millimolar range (� 0.5 mm)

[14,15], the role of the agonist-sensitive Ca2+ store

played by the Golgi apparatus (endowed with a resting

[Ca2+] of � 0.3 mm and rapidly emptying after agonist

stimulation) [16], the rapid equilibration of cytosolic

and nuclear [Ca2+] [17], the estimates of resting and

stimulated [Ca2+]c under the plasmamembrane, well

above those of the bulk cytosol [18]. This paper

focuses on mitochondria, as Ca2+ handling by these

organelles was not only significantly different from

that expected, but also identified them as critical

checkpoints, in which radically different effects can be

triggered by a rise in [Ca2+].

Mechanism and role of mitochondrialCa2+ homeostasis

The participation of mitochondria in Ca2+ homeostasis

is a concept that alternated periods of glory and com-

plete dismissal. Indeed, as soon as the chemiosmotic

theory was accepted as the basis of energy conservation

in mitochondria, it became obvious that these organ-

elles could, at least potentially, efficiently accumulate

Ca2+ down the electrochemical gradient established

across the inner membrane by the activity of respirat-

ory complexes. This possibility was actually directly

demonstrated by an extensive body of work carried out

with isolated mitochondria. Respiring mitochondria

can rapidly accumulate Ca2+ through an electrogenic

pathway, termed the ‘mitochondrial Ca2+ uniporter’

(MCU) [19]. This route was (and still is) undefined at

the molecular level, although very recent work by Clap-

ham and coworkers demonstrated that it is a bona fide

Ca2+ channel [20]. Ca2+ is then re-extruded by electro-

neutral exchangers (Na2+ ⁄Ca2+ and H+ ⁄Ca2+

exchangers, mostly expressed in nonexcitable and excit-

able cells, respectively) [21]. Based on this evidence,

mitochondria were thought to dynamically change the

matrix Ca2+ concentration ([Ca2+]m) in living cells

challenged with Ca2+-mobilizing agonists.

This possibility was severely questioned in the 1980s,

when the signalling pathways downstream of receptor

stimulation were clarified. It was then demonstrated

that G-protein coupled and growth factor receptors

mobilize Ca2+ from an intracellular store that proved

be the endoplasmic reticulum, not mitochondria [22].

Moreover, the accurate measurement of [Ca2+]c levels

with fluorescent indicators suggested that mitochondria

did not receive the Ca2+ released either. Indeed, both

at rest and after stimulation the [Ca2+]c values were

well below those necessary for rapid Ca2+ uptake

through the MCU. Thus, the general consensus

became that mitochondria can accumulate a significant

amount of Ca2+ only when large and sustained

[Ca2+]c increases occurred, such as those postulated to

occur in various pathological derangements (e.g. the

Ca2+ overload during neuronal excitotoxicity).

This situation was completely reversed when the tar-

geted recombinant indicators (first aequorin, then the

more recent GFP-based fluorescent probes) clearly

demonstrated that a [Ca2+]c rise elicited by a physiolo-

gical stimulation is almost invariably paralleled by

a robust [Ca2+]m increase [23], that usually largely

exceeds the values observed in the bulk cytosol and

reached values as high as 500 lm [24]. The apparent

discrepancy with the sluggish rate of Ca2+ uptake

observed in isolated mitochondria upon exposure to

Ca2+ concentrations similar to those measured in the

bulk cytosol was reconciled by postulating that mito-

chondria upon opening of the IP3-sensitive channels

are not exposed to those low [Ca2+], but rather to the

much higher values generated in the proximity of the

channel (the microdomain hypothesis) [25]. In support

of this notion, organelle labelling of the ER and mito-

chondria showed closed appositions and an aequorin

chimera located on the mitochondrial membrane detec-

ted [Ca2+] values well above those of the bulk cytosol

[26].

Numerous studies then followed that demonstrated,

both in cell lines and in intact tissues, the occurrence

of rapid [Ca2+]m transients in cells as diverse as HeLa,

hepatocytes, cardiac and skeletal muscle and neurons

[27]. A striking example is that of cardiac muscle, in

which a [Ca2+]m transient was detected at every con-

tractile cycle [28]. This implies that both the uptake

and the release mechanism are highly efficient in situ,

and allow the completion of a Ca2+ cycle within the

short time frame of a single contraction.

What is the role of mitochondrial Ca2+ homeosta-

sis? A first obvious function stems from long-standing

biochemical evidence, i.e. the demonstration by Den-

ton, McCormack and Hansford in the 1960s that three

key metabolic enzymes (the pyruvate, a-ketoglutarateand isocitrate dehydrogenases) are activated by Ca2+,

by different mechanisms. In the case of pyruvate dehy-

drogenase, this is through a Ca2+-dependent dephos-

phorylation step, whereas in the latter two cases this is

through the direct binding of Ca2+ to the enzyme

complex [29,30]. Thus, a [Ca2+] rise in the matrix may

allow the up-regulation of aerobic metabolism and

tuning of ATP production to the increased needs of

stimulated cells. This could be demonstrated by the

direct measurement of mitochondrial ATP levels with

a targeted chimera of the ATP-sensitive photoprotein

luciferase. Parallel measurements of Ca2+ and ATP

S. Leo et al. Mitochondrial calcium signalling in cell death


levels showed that the Ca2+ signal within the mito-

chondria is responsible for the enhanced ATP produc-

tion, an effect that lasts longer than the Ca2+ signal

itself, highlighting a novel form of cellular ‘metabolic

memory’ [31].

Interestingly, recent work indicates that other Ca2+-

dependent metabolic checkpoints are operative.

Namely, the aspartate ⁄ glutamate metabolite carriers

(AGCs) were shown to include EF-hand domains, and

Ca2+ binding to these sites was shown to increase their

activity [32]. In turn, recombinant expression of wild

type AGCs enhanced ATP production upon cell stimu-

lation, an effect that was not observed with truncated

mutants lacking the Ca2+-binding domain [33].

Substantial evidence has built up in recent years

indicating that metabolic regulation is only one of the

roles of the mitochondrial Ca2+ signal. It now appears

evident that massive Ca2+ loading (as in the case of

glutamate excitotoxicity of neurons) and ⁄or the com-

bined action of apoptotic agents or pathophysiological

conditions (e.g. oxidative stress) can induce a profound

alteration of organelle structure and function [34–36].

As a consequence, bioenergetic dysfunction and ⁄orrelease into the cytosol of proteins acting as caspase

cofactors, such as cytochrome c [37,38], AIF [39] and

Smac ⁄Diablo [40], may lead the cell to necrotic or

apoptotic cell death. In relation to this effect, the anti-

oncogene Bcl-2 was shown to reduce the steady state

Ca2+ levels in the ER (and thus dampen the pro-apop-

totic Ca2+ signal) [41,42].

Some of these data, which refer to our work on Bcl-

2 overexpression in HeLa cells [41], are presented in

Fig. 2A–C. HeLa cells were transiently transfected

with a cytomegalovirus-driven expression plasmid for

Bcl-2 and Ca2+ homeostasis at the subcellular level

was investigated 36 h after transfection using organ-

elle-targeted aequorin chimeras. In Bcl-2 overexpress-

ing cells a steady-state [Ca2+]er level of � 350 lm was

measured, compared to � 450 lm of control cells

(Fig. 2A). Accordingly, when the cells were stimulated

with an IP3-generating agonist (ATP 100 lm), the

[Ca2+] increases evoked in the cytoplasm and in mito-

chondria were significantly smaller (Fig. 2B,C). This

‘reduction’ of cellular Ca2+ signals has a protective

effects toward a variety of inducers of cell death, such

as the lipid mediators ceramide or oxidative stress.

When ER was partially depleted of Ca2+ independ-

ently of Bcl-2, e.g. by inhibiting the SERCA, over-

expressing the PMCA or reducing the [Ca2+] of the

extracellular medium, survival upon ceramide treat-

ment was markedly enhanced. As to the downstream

targets of the Ca2+ effect, mitochondria seem to play

an important role. Ceramide treatment causes large

scale morphological rearrangements (fragmentation,

swelling), consistent with both apoptotic (release of

cytochrome c) and necrotic (bioenergetic dysfunction)

hallmarks and probably linked to the opening of the

permeability transition pore. Conversely, organelle

morphology is preserved if ceramide is applied to cells

in which [Ca2+]er is reduced by one of the experimen-

tal procedures described above. This is apparent from

the experiment shown in Fig. 2D, in which mitochon-

dria are visualized by transfected mtGFP in cells trea-

ted with ceramide while being maintained in KRB

A

B

C

D

Fig. 2. Upper panel: analysis of Ca2+

homeostasis (using different targeted

aequorins. (A) erAEQ; (B) cytAEQ; (C)

mtAEQ) in control vs. Bcl-2 overexpressing

HeLa cells (modified from [41]). Lower

panel: effects of ceramide application on

mitochondrial structure at different

extracellular [Ca2+] (modified from [41]).



supplemented with physiological (1 mm; left) or a

lower (0.05 mm; right) Ca2+ concentration, which cau-

ses a partial Ca2+ depletion of the ER. Overall, the

data indicate that the anti-apoptotic protein Bcl-2, by

reducing Ca2+ signals evoked by physiological and

pathological stimuli, counteracts the efficacy of death

pathways acting through the mitochondrial check-

point.

Interestingly, the link between Ca2+ signalling and

cell death has been reinforced by the study of an unre-

lated pro-apoptotic protein, the x-protein of the hepa-

titis B virus (HBx), which also conceptually extended

the molecular mechanisms through which the Ca2+

effect can be tuned [43]. In this case, when HeLa and

liver-derived HepG2 cells were transfected with HBx, a

marked enhancement of the cytosolic Ca2+ responses

evoked by cell stimulation was detected (Fig. 3A). This

alteration (the opposite effect of Bcl-2) was not due to

an alteration in ER Ca2+ handling (both the steady

state levels and the release kinetics were the same in

HBx-transfected and control cells), but rather to the

caspase-dependent cleavage of PMCA, the most effect-

ive molecular route for rapidly returning [Ca2+]c to

basal values (Fig. 3B). The Ca2+ signalling alteration

leads to major morphological alterations of mitochon-

dria (Fig. 3C) and spontaneous apoptosis. Indeed cell

death was blocked by treatment with caspase-3 inhibi-

tors (100 lm ZVAD-fmk), loading of a Ca2+ buffer

(2 lm BAPTA-AM) or overexpression of Bcl-2

(Fig. 3D).

On the cytosolic side, mitochondrial Ca2+ uptake

exerts two different effects. In the first, the spatial

clustering of mitochondria in a defined portion of the

cell represents a physiological ‘fixed spatial buffer’ that

prevents (or delays) the spread of cytoplasmic Ca2+

waves. In this case, mitochondria act as a ‘firewall’

that shields some cell domains from the Ca2+ signal

elicited by submaximal agonist stimulation. This was

clearly shown in pancreatic acinar cells, in which only

high agonist doses (that overwhelm the mitochondrial

firewall) induce a [Ca2+]c rise in the basal portion of

the cell, containing the nucleus [44].

The second mechanism through which mitochondria

may modulate cytosolic Ca2+ transients refers to

events occurring in signalling microdomains, in which

mitochondria are placed in close contact with Ca2+

channels that are under feedback control by Ca2+

itself. In this case, mitochondrial Ca2+ accumulation

participates in clearing Ca2+ from the microenviron-

ment of the channel, thus reducing the (positive or

negative) feedback on channel activity. The first

demonstration of such an effect was obtained by

Lechleiter and coworkers in Xenopus oocytes, in which

the energization state (and thus the capacity to accu-

mulate Ca2+) was shown to influence the spatio-tem-

poral pattern of the typical propagating Ca2+ waves

induced by IP3 [45]. In mammals, several similar exam-

ples have been reported. In permeabilized hepatocytes

the decrease in [Ca2+]er evoked by submaximal IP3

was enhanced when mitochondrial Ca2+ uptake was

blocked [46]. Ca2+ uptake by mitochondria thus sup-

presses the local positive feedback effects of Ca2+ on

the IP3R, giving rise to subcellular heterogeneity in IP3

sensitivity and IP3R excitability. Similarly, in astro-

A B

C D

Fig. 3. Effects of HBx overexpression on

Ca2+ homeostasis (A,B), mitochondrial mor-

phology (C) and cell viability (D). See text for

details (modified from [43]).



cytes inhibition of mitochondrial Ca2+ uptake almost

doubled the rate of propagation of the calcium wave

across the cell [47]. In contrast, in BHK cells inhibition

of mitochondrial Ca2+ uptake resulted in reduction of

ER Ca2+ release [48], indicating that in this case

mitochondria play a major role in preventing the

Ca2+-depended inhibition of the InsP3 channel. This

effect is not limited to ER Ca2+ channels. Indeed, sev-

eral papers from the groups of Lewis and Parekh have

demonstrated that Ca2+ uptake by energized mito-

chondria relieves the Ca2+-dependent inhibition of

Ca2+ release-activated current (CRAC) channels, i.e.

those activated by the emptying of intracellular Ca2+

stores [49,50]. This notion explains the high buffering

(mimicking mitochondrial activity) required to observe

ICRAC in many experimental conditions. A complex

role, somewhat similar to that proposed for the modu-

lation of capacitative Ca2+ influx, has been proposed

by Nicholls and coworkers for the modulation of

Ca2+ activation-inhibition of glutamate and voltage

operated Ca2+ channels in cerebellar granule cells [51].

Specific regulatory pathways formitochondrial Ca2+ homeostasis

Mitochondria can thus be regarded as critical check-

points in Ca2+ signalling, acting as membrane-bound

Ca2+ buffers, in which Ca2+ itself plays a regulatory

role. In this situation, the possibility that their uptake

capacity (kinetics, amplitude) is tuned by converging

signalling pathways may add further complexity (and

option for regulation) to Ca2+-mediated signal trans-

duction.

We investigated this possibility, focusing on two

aspects: the role of the broad family of PKC kinases and

of the three-dimensional structure of mitochondria, in

turn controlled by the activity of large GTPases indu-

cing organelle fusion or fission (mitofusins, dynamin-

related protein-1).

PKC comprises a family of serine-threonine kinases

that are involved in the transduction of a wide number

of extracellular signals. Based on their biochemical

properties, they are divided into classical (e.g. a, bI,bII, c), activated by Ca2+ and diacylglycerol, novel

(e.g. d, e, g, h), activated by diacylglycerol, and atyp-

ical (e.g. f, k), insensitive to both Ca2+ and diacylglyc-

erol. Different isozymes are coexpressed in the various

cell types giving rise to a highly flexible molecular rep-

ertoire, which can mediate radically different intra-

cellular effects, e.g. isoforms belonging to the same

group, such as d and e, have been reported to play

opposite effects on apoptosis. To evaluate specific

effects of PKC isoforms on cellular Ca2+ homeostasis,

we overexpressed PKC–GFP chimeras in HeLa cells

and investigated agonist-dependent Ca2+ signals in

the cytosol and mitochondria, using organelle-targeted

aequorin chimeras [52]. An interesting scenario

emerged, with distinct roles for the various isoforms.

Overexpression of PKCe did not modify the amplitude

and the kinetics of either the cytosolic and mitochond-

rial Ca2+ transients evoked by histamine stimulation,

indicating that this PKC isoform does not influence

either Ca2+ signalling globally in the cell or mito-

chondrial Ca2+ accumulation. Conversely, PKCaoverexpression greatly reduces the agonist-evoked

[Ca2+] increase both in the cytosol and in the mito-

chondria. The monitoring of ER [Ca2+] demonstrated

that this is due to the sharp reduction of Ca2+ release

from the organelle. These data are in keeping with pre-

vious demonstration that phorbol esters inhibited ER

Ca2+ release [53], suggesting that PKCa is the isoform

responsible for this effect. As to the target, the demon-

stration of the phosphorylation of the IP3 receptor by

PKC [54] indicates that the ER Ca2+ release channel

itself is a plausible site of action for the PKCa effect.

Interestingly, some isoforms appear to have an

effect on mitochondrial, but not on cytosolic, Ca2+

handling. Indeed, in cells overexpressing PKCb (and

to a smaller extent PKCd), the mitochondrial but not

the cytosolic [Ca2+] rise is reduced. Conversely, over-

expression of PKCf enhances the mitochondrial Ca2+

responses to agonist stimulation, while still leaving

cytosolic [Ca2+] increases unaffected. These effects do

not depend on alterations of mitochondrial structure

(monitored by mtRFP labelling), an important deter-

minant of organelle responsiveness, nor to significant

changes in the driving force for Ca2+ accumulation

(the mitochondrial membrane potential, DYm). A pos-

sibility is that the Ca2+ uptake machinery of

the organelle is directly modulated, but the demonstra-

tion of this possibility awaits the molecular characteri-

zation of the uptake process. As to the functional

significance of this regulatory mechanism, ‘mitochond-

rial Ca2+ desensitization’, i.e. the sharp reduction of

[Ca2+]m peaks upon repetitive cell stimulation, was

proposed to be responsible for phenomena, such as

the down-regulation of insulin secretion in pancreatic

b-cells [55]. Interestingly, inhibition of PKCb greatly

reduces the [Ca2+]m reduction upon repetitive agonist

stimulation, indicating that it could represent the

molecular route for Ca2+-dependent inhibition of cel-

lular responses.

The other physiological regulation of mitochondria

that has been studied in relation to Ca2+ signalling is

the state of fusion and fission. The study of mito-

chondrial structure, and of the mechanisms that



dynamically control it in living cells, is a field of study

that literally boomed in the past decade. Indeed,

although the idea that mitochondria can form an inter-

connected reticulum was shown by reconstructing elec-

tron microscopy images in the late 1970s [56] and

more recently by using fluorescent GFP-based probes

and high-resolution digital imaging systems [26], it was

only after the identification of the molecules involved

that it became clear that the morphology of mitochon-

dria is tightly controlled by a dedicated cellular

machinery. Large GTPases, such as dynamin-related

protein-1 (Drp-1; a mechanoenzyme involved in mem-

brane constriction and fission), OPA1 (a dynamin rela-

ted GTPase, involved in fusion and mostly located in

the inner mitochondrial membrane) and mitofusins

(also involved in fusion and homologous to the first

element described, the ‘fuzzy onion’, fzo, protein of

Drosophila melanogaster [57]), and docking proteins,

such as Fis-1 (a transmembrane protein of the outer

mitochondrial membrane that participates in recruiting

Drp-1 to the organelle during organelle fragmenta-

tion). It is beyond the scope of this review to describe

in detail this fascinating field, and we refer to excellent

reviews on this topic [58–60]. Recent work gives some

insight into the dynamic regulation of the process, and

suggests that Ca2+ could be involved: (a) Ca2+ release

from the ER promotes the translocation of Drp-1 from

the cytoplasm to the outer mitochondrial membrane

[61]; (b) treatment with the Ca2+ ionophore, A23187,

triggers mitochondrial fission [62]; and (c) a novel

group of rho-GTPases have been described (mitoch-

ondrial rho 1 and 2; miro-1 and miro-2), that promote

mitochondrial fusion and include EF-hand Ca2+ bind-

ing sites in their sequence [63].

In our work, we took advantage of the molecular

knowledge of the fusion ⁄fission machinery for actively

modifying the three-dimensional structure of mitochon-

dria, and verified the impact on organelle Ca2+ hand-

ling. Specifically, given that in HeLa cells mitochondria

mostly form an interconnected network, we over-

expressed the fission protein Drp-1. As expected, this

caused massive fragmentation of the mitochondrial net-

work, while leaving the ER morphology unaffected

(Fig. 4A). Studies carried out with targeted aequorins

showed that Ca2+ release from the ER and the ensuing

[Ca2+]c increases are not modified by Drp-1-dependent

mitochondrial fragmentation; conversely, the [Ca2+]mpeak was drastically reduced (Fig. 4B–D). Single-cell

analysis of [Ca2+]m dynamics, using the GFP-based

periCaM probe [64,65] and a high-speed imaging system

showed that in control cells intramitochondrial Ca2+

waves originate from focal points and gradually diffuse

through the mitochondrial network. These waves were

blocked in the Drp-1-fragmented network, excluding

some individual mitochondria from the [Ca2+] rise and

thus reducing the average [Ca2+]m response. Conse-

quently, in Drp-1 overexpressing cells the apoptotic

efficacy of ceramide, which causes a Ca2+-dependent

perturbation of mitochondrial structure and function,

was drastically reduced [66].

A B

C

D

Fig. 4. Effects of Drp-1 overexpression on

organelle morphology (A), and Ca2+ homeo-

stasis (B–D). See text for details (modified

from [66]).



Conclusions

A large body of experimental work shows that in vir-

tually every cell type the [Ca2+]c increases elicited in

the cytosol, by the opening of plasma membrane or

ER ⁄SR Ca2+ channels, is paralleled by a large

increase in the [Ca2+] of the mitochondrial matrix.

This process has two important functional conse-

quences. The first is that part of the Ca2+ entering the

cytoplasm is rapidly removed. Mitochondria thus act

as Ca2+ buffers, and this activity influences both the

local microdomain (hence affecting local inhibitory or

activatory effects of Ca2+ itself on the channel) or the

kinetics of the diffusion across the cytosol (thus acting

as a barrier limiting the [Ca2+]c rise to one portion of

the cell). Mitochondrial role is not limited, however, to

Ca2+ buffering. Indeed, within the organelle Ca2+ can

regulate functions as diverse as aerobic metabolism

(through Ca2+ sensitive enzymes and metabolite trans-

porters) and cell death (probably by activating the per-

meability transition pore). Given this high functional

plasticity, it is not surprising that we start to obtain

evidence that different mechanisms can finely tune

amplitude and kinetics of the mitochondrial Ca2+

responses. We have described a few intriguing exam-

ples (PKC, the fusion ⁄fission machinery), but it is rea-

sonable to predict that in the near future we will learn

much about these signalling routes and their cross-talk,

and get to know (finally!) the mitochondrial targets,

namely the channels, the regulatory elements and the

scaffolding proteins.

Acknowledgements

Experimental work in the authors’ laboratory was sup-

ported by grants from the Italian Ministry of Education

(FIRB, PRIN, local interest grants), the PRRIITT pro-

gram of the Emilia-Romagna region (ER-GenTech),

Telethon-Italy, the Italian Association for Cancer

Research (AIRC) and the Italian Space Agency (ASI).

References

1 Berridge MJ, Bootman MD & Roderick HL (2003) Cal-

cium signalling: dynamics, homeostasis and remodelling.

Nat Rev Mol Cell Biol 4, 517–529.

2 Tsien RY, Pozzan T & Rink TJ (1982) Calcium homeo-

stasis in intact lymphocytes: cytoplasmic free calcium

monitored with a new, intracellularly trapped fluores-

cent indicator. J Cell Biol 94, 325–334.

3 Pozzan T, Rizzuto R, Volpe P & Meldolesi J (1994)

Molecular and cellular physiology of intracellular cal-

cium stores. Physiol Rev 74, 595–636.

4 Petersen OH, Tepikin A & Park MK (2001) The endo-

plasmic reticulum: one continuous or several separate

Ca(2+) stores? Trends Neurosci 24, 271–276.

5 Montell C (2001) Physiology, phylogeny, and functions

of the TRP superfamily of cation channels. Sci. STKE

2001, re1.

6 Parekh AB & Putney JW Jr (2005) Store-operated cal-

cium channels. Physiol Rev 85, 757–810.

7 Orrenius S, Zhivotovsky B & Nicotera P (2003) Regula-

tion of cell death: the calcium-apoptosis link. Nat Rev

Mol Cell Biol 4, 552–565.

8 Carafoli E (2004) Calcium-mediated cellular signals: a

story of failures. Trends Biochem Sci 29, 371–379.

9 Rizzuto R & Pozzan T (2003) When calcium goes

wrong: genetic alterations of a ubiquitous signaling

route. Nat Genet 34, 135–141.

10 Duchen MR (2000) Mitochondria and calcium: from

cell signalling to cell death. J Physiol 529 Part 1, 57–68.

11 Dolmetsch R (2003) Excitation-transcription coupling:

signaling by ion channels to the nucleus. Sci STKE

2003, pe4.

12 Woods NM, Cuthbertson KS & Cobbold PH (1986)

Repetitive transient rises in cytoplasmic free calcium in

hormone-stimulated hepatocytes. Nature 319, 600–602.

13 Rizzuto R, Simpson AW, Brini M & Pozzan T (1992)

Rapid changes of mitochondrial Ca2+ revealed by spe-

cifically targeted recombinant aequorin. Nature 358,

325–327.

14 Montero M, Brini M, Marsault R, Alvarez J, Sitia R,

Pozzan T & Rizzuto R (1995) Monitoring dynamic

changes in free Ca2+ concentration in the endoplasmic

reticulum of intact cells. EMBO J 14, 5467–5475.

15 Barrero MJ, Montero M & Alvarez J (1997) Dynamics

of [Ca2+] in the endoplasmic reticulum and cytoplasm

of intact HeLa cells: a comparative study. J Biol Chem

272, 27694–27699.

16 Pinton P, Pozzan T & Rizzuto R (1998) The Golgi

apparatus is an inositol 1,4,5-trisphosphate-sensitive

Ca2+ store, with functional properties distinct from

those of the endoplasmic reticulum. EMBO J 17, 5298–

5308.

17 Brini M, Marsault R, Bastianutto C, Pozzan T &

Rizzuto R (1994) Nuclear targeting of aequorin: a new

approach for measuring nuclear Ca2+ concentration in

intact cells. Cell Calcium 16, 259–268.

18 Marsault R, Murgia M, Pozzan T & Rizzuto R (1997)

Domains of high Ca2+ beneath the plasma membrane

of living A7r5 cells. EMBO J 16, 1575–1581.

19 Carafoli E (2003) Historical review: mitochondria and

calcium: ups and downs of an unusual relationship.

Trends Biochem Sci 28, 175–181.

20 Kirichok Y, Krapivinsky G & Clapham DE (2004) The

mitochondrial calcium uniporter is a highly selective ion

channel. Nature 427, 360–364.



21 Cox DA & Matlib MA (1993) Modulation of intramito-

chondrial free Ca2+ concentration by antagonists of

Na+–Ca2+ exchange. Trends Pharmacol Sci 14, 408–

413.

22 Streb H, Irvine RF, Berridge MJ & Schulz I (1983)

Release of Ca2+ from a nonmitochondrial intracellular

store in pancreatic acinar cells by inositol-1,4,5-trisphos-

phate. Nature 306, 67–69.

23 Rizzuto R, Bastianutto C, Brini M, Murgia M &

Pozzan T (1994) Mitochondrial Ca2+ homeostasis in

intact cells. J Cell Biol 126, 1183–1194.

24 Montero M, Alonso MT, Carnicero E, Cuchillo-Ibanez

I, Albillos A, Garcia AG, Garcia-Sancho J & Alvarez J

(2000) Chromaffin-cell stimulation triggers fast millimo-

lar mitochondrial Ca2+ transients that modulate secre-

tion. Nat Cell Biol 2, 57–61.

25 Rizzuto R, Brini M, Murgia M & Pozzan T (1993)

Microdomains with high Ca2+ close to IP3-sensitive

channels that are sensed by neighboring mitochondria.

Science 262, 744–747.

26 Rizzuto R, Pinton P, Carrington W, Fay FS, Fogarty

KE, Lifshitz LM, Tuft RA & Pozzan T (1998) Close

contacts with the endoplasmic reticulum as determinants

of mitochondrial Ca2+ responses. Science 280, 1763–

1766.

27 Brini M, De Giorgi F, Murgia M, Marsault R,

Massimino ML, Cantini M, Rizzuto R & Pozzan T

(1997) Subcellular analysis of Ca2+ homeostasis in

primary cultures of skeletal muscle myotubes. Mol Biol

Cell 8, 129–143.

28 Rudolf R, Mongillo M, Magalhaes PJ & Pozzan T

(2004) In vivo monitoring of Ca2+ uptake into mito-

chondria of mouse skeletal muscle during contraction.

J Cell Biol 166, 527–536.

29 McCormack JG, Halestrap AP & Denton RM (1990)

Role of calcium ions in regulation of mammalian

intramitochondrial metabolism. Physiol Rev 70, 391–

425.

30 Hansford RG (1994) Physiological role of mitochondrial

Ca2+ transport. J Bioenerg Biomembr 26, 495–508.

31 Jouaville LS, Pinton P, Bastianutto C, Rutter GA &

Rizzuto R (1999) Regulation of mitochondrial ATP

synthesis by calcium: evidence for a long-term metabolic

priming. Proc Natl Acad Sci USA 96, 13807–13812.

32 Palmieri L, Pardo B, Lasorsa FM, del Arco A,

Kobayashi K, Iijima M, Runswick MJ, Walker JE,

Saheki T, Satrustegui J et al. (2001) Citrin and aralar1

are Ca2+-stimulated aspartate ⁄ glutamate transporters in

mitochondria. EMBO J 20, 5060–5069.

33 Lasorsa FM, Pinton P, Palmieri L, Fiermonte G,

Rizzuto R & Palmieri F (2003) Recombinant expression

of the Ca2+-sensitive aspartate ⁄ glutamate carrier increa-

ses mitochondrial ATP production in agonist-stimulated

Chinese hamster ovary cells. J Biol Chem 278, 38686–

38692.

34 Szalai G, Krishnamurthy R & Hajnoczky G (1999)

Apoptosis driven by IP(3)-linked mitochondrial calcium

signals. EMBO J 18, 6349–6361.

35 Pinton P, Ferrari D, Di Virgilio F, Pozzan T & Rizzuto

R (2001) Molecular machinery and signalling events in

apoptosis. Drug Dev Res 52, 558–570.

36 Pinton P, Ferrari D, Rapizzi E, Di Virgilio FD, Pozzan

T & Rizzuto R (2001) The Ca2+ concentration of the

endoplasmic reticulum is a key determinant of cera-

mide-induced apoptosis: significance for the molecular

mechanism of Bcl-2 action. EMBO J 20, 2690–2701.

37 Kluck RM, Bossy-Wetzel E, Green DR & Newmeyer

DD (1997) The release of cytochrome c from mitochon-

dria: a primary site for Bcl-2 regulation of apoptosis.

Science 275, 1132–1136.

38 Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J,

Peng TI, Jones DP & Wang X (1997) Prevention of

apoptosis by Bcl-2: release of cytochrome c from mito-

chondria blocked. Science 275, 1129–1132.

39 Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow

BE, Brothers GM, Mangion J, Jacotot E, Costantini P,

Loeffler M et al. (1999) Molecular characterization of

mitochondrial apoptosis-inducing factor. Nature 397,

441–446.

40 Du C, Fang M, Li Y, Li L & Wang X (2000) Smac,

a mitochondrial protein that promotes cytochrome

c-dependent caspase activation by eliminating IAP inhi-

bition. Cell 102, 33–42.

41 Pinton P, Ferrari D, Magalhaes P, Schulze-Osthoff K,

Di Virgilio F, Pozzan T & Rizzuto R (2000) Reduced

loading of intracellular Ca2+ stores and downregulation

of capacitative Ca(2+) influx in Bcl-2-overexpressing

cells. J Cell Biol 148, 857–862.

42 Foyouzi-Youssefi R, Arnaudeau S, Borner C, Kelley

WL, Tschopp J, Lew DP, Demaurex N & Krause KH

(2000) Bcl-2 decreases the free Ca2+ concentration

within the endoplasmic reticulum. Proc Natl Acad Sci

USA 97, 5723–5728.

43 Chami M, Ferrari D, Nicotera P, Paterlini-Brechot P

& Rizzuto R (2003) Caspase-dependent alterations of

Ca2+ signaling in the induction of apoptosis by hepa-

titis B virus X protein. J Biol Chem 278, 31745–

31755.

44 Tinel H, Cancela JM, Mogami H, Gerasimenko JV,

Gerasimenko OV, Tepikin AV & Petersen OH (1999)

Active mitochondria surrounding the pancreatic acinar

granule region prevent spreading of inositol trispho-

sphate-evoked local cytosolic Ca(2+) signals. EMBO J

18, 4999–5008.

45 Jouaville LS, Ichas F, Holmuhamedov EL, Camacho P

& Lechleiter JD (1995) Synchronization of calcium

waves by mitochondrial substrates in Xenopus laevis

oocytes. Nature 377, 438–441.

46 Hajnoczky G, Hager R & Thomas AP (1999) Mito-

chondria suppress local feedback activation of inositol



1,4, 5-trisphosphate receptors by Ca2+. J Biol Chem

274, 14157–14162.

47 Boitier E, Rea R & Duchen MR (1999) Mitochondria

exert a negative feedback on the propagation of intra-

cellular Ca2+ waves in rat cortical astrocytes. J Cell

Biol 145, 795–808.

48 Landolfi B, Curci S, Debellis L, Pozzan T & Hofer AM

(1998) Ca2+ homeostasis in the agonist-sensitive inter-

nal store: functional interactions between mitochondria

and the ER measured in situ in intact cells. J Cell Biol

142, 1235–1243.

49 Hoth M, Fanger CM & Lewis RS (1997) Mitochondrial

regulation of store-operated calcium signaling in T lym-

phocytes. J Cell Biol 137, 633–648.

50 Gilabert JA & Parekh AB (2000) Respiring mitochon-

dria determine the pattern of activation and inactivation

of the store-operated Ca2+ current I (CRAC). EMBO J

19, 6401–6407.

51 Budd SL & Nicholls DG (1996) Mitochondria, calcium

regulation, and acute glutamate excitotoxicity in cultured

cerebellar granule cells. J Neurochem 67, 2282–2291.

52 Pinton P, Leo S, Wieckowski MR, Di Benedetto G &

Rizzuto R (2004) Long-term modulation of mitochon-

drial Ca2+ signals by protein kinase C isozymes. J Cell

Biol 165, 223–232.

53 Montero M, Lobaton CD, Gutierrez-Fernandez S,

Moreno A & Alvarez J (2003) Modulation of histamine-

induced Ca2+ release by protein kinase C. Effects on

cytosolic and mitochondrial [Ca2+] peaks. J Biol Chem

278, 49972–49979.

54 Vermassen E, Fissore RA, Nadif KN, Vanderheyden V,

Callewaert G, Missiaen L, Parys JB & De Smedt H

(2004) Regulation of the phosphorylation of the inositol

1,4,5-trisphosphate receptor by protein kinase C. Bio-

chem Biophys Res Commun 319, 888–893.

55 Maechler P, Kennedy ED, Wang H & Wollheim CB

(1998) Desensitization of mitochondrial Ca2+ and insu-

lin secretion responses in the beta cell. J Biol Chem 273,

20770–20778.

56 Bakeeva LE, Chentsov Y & Skulachev VP (1978)

Mitochondrial framework (reticulum mitochondriale) in

rat diaphragm muscle. Biochim Biophys Acta 501, 349–

369.

57 Hales KG & Fuller MT (1997) Developmentally regu-

lated mitochondrial fusion mediated by a conserved,

novel, predicted GTPase. Cell 90, 121–129.

58 Bossy-Wetzel E, Barsoum MJ, Godzik A,

Schwarzenbacher R & Lipton SA (2003) Mitochondrial

fission in apoptosis, neurodegeneration and aging. Curr

Opin Cell Biol 15, 706–716.

59 Scorrano L (2003) Divide et impera: Ca2+ signals, mit-

ochondrial fission and sensitization to apoptosis. Cell

Death Differ 10, 1287–1289.

60 Perfettini JL, Roumier T & Kroemer G (2005) Mito-

chondrial fusion and fission in the control of apoptosis.

Trends Cell Biol 15, 179–183.

61 Breckenridge DG, Stojanovic M, Marcellus RC & Shore

GC (2003) Caspase cleavage product of BAP31 induces

mitochondrial fission through endoplasmic reticulum

calcium signals, enhancing cytochrome c release to the

cytosol. J Cell Biol 160, 1115–1127.

62 Duncan CJ, Greenaway HC, Publicover SJ, Rudge MF

& Smith JL (1980) Experimental production of

‘septa’ and apparent subdivision of muscle

mitochondria. J Bioenerg Biomembr 12, 13–33.

63 Fransson A, Ruusala A & Aspenstrom P (2003) Atypi-

cal Rho GTPases have roles in mitochondrial homeosta-

sis and apoptosis. J Biol Chem 278, 6495–6502.

64 Nagai T, Sawano A, Park ES & Miyawaki A (2001)

Circularly permuted green fluorescent proteins engi-

neered to sense Ca2+. Proc Natl Acad Sci USA 98,

3197–3202.

65 Filippin L, Magalhaes PJ, Di Benedetto G, Colella M

& Pozzan T (2003) Stable interactions between mito-

chondria and endoplasmic reticulum allow rapid accu-

mulation of calcium in a subpopulation of

mitochondria. J Biol Chem 278, 39224–39234.

66 Szabadkai G, Simoni AM, Chami M, Wieckowski MR,

Youle RJ & Rizzuto R (2004) Drp-1-dependent division

of the mitochondrial network blocks intraorganellar

Ca2+ waves and protects against Ca2+-mediated apop-

tosis. Mol Cell 16, 59–68.



Mitochondrial calcium signalling in cell death

Documents

Transcript of Mitochondrial calcium signalling in cell death