An alternative route for raising cytosolic Ca2+ concentration [Ca2+]c depends on the opening of Keywords apoptosis; calcium; mitochondria; organelles; photoproteins; signal transduction
Trang 1Mitochondrial calcium signalling in cell death
Delivered on 1 July 2004 at the 29th FEBS Congress in Warsaw
Sara Leo1, Katiuscia Bianchi1, Marisa Brini2and 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 recepfac-tors (also causing the production of IP3 through the activation of phospholipase Cc, containing
an 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: r.rizzuto@unife.it
(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, Ca 2+
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 Ca 2+
ATPase; SERCA, sarcoplamic reticulum ⁄ ER Ca 2+ ATPase.
Trang 2various 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+]cspiking 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.
Trang 3not 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 mitochondrial
Ca2+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+]cincreases 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+]crise 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+]mtransients 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-ketoglutarate and 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
Trang 4levels 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⁄ or
release 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 Ca 2+
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 [Ca 2+ ] (modified from [41]).
Trang 5supplemented 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
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]).
Trang 6cytes 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 for
mitochondrial 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, PKCa overexpression 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
Trang 7dynamically 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 locarela-ted 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+]cincreases are not modified by Drp-1-dependent mitochondrial fragmentation; conversely, the [Ca2+]m peak 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]
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]).
Trang 8A 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+]crise 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)
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