Download Regulation of mitochondrial calcium in plants versus animals

Journal of Experimental Botany Advance Access published March 21, 2016
Journal of Experimental Botany
doi:10.1093/jxb/erw100
REVIEW PAPER
Regulation of mitochondrial calcium in plants versus animals
Stephan Wagner1, Sara De Bortoli2, Markus Schwarzländer1,† and Ildikò Szabò2,†
1 Plant Energy Biology Lab, Institute of Crop Science and Resource Conservation (INRES), University of Bonn, Friedrich-Ebert-Allee 144,
D-53113 Bonn, Germany
2 Department of Biology and CNR Institute of Neurosciences, University of Padova, Viale G. Colombo 3, 35121 Padova, Italy
† Correspondence: [email protected] or [email protected]
Received 5 January 2016; Accepted 10 February 2016
Abstract
Ca2+ acts as an important cellular second messenger in eukaryotes. In both plants and animals, a wide variety of environmental and developmental stimuli trigger Ca2+ transients of a specific signature that can modulate gene expression
and metabolism. In animals, mitochondrial energy metabolism has long been considered a hotspot of Ca2+ regulation, with a range of pathophysiology linked to altered Ca2+ control. Recently, several molecular players involved in
mitochondrial Ca2+ signalling have been identified, including those of the mitochondrial Ca2+ uniporter. Despite strong
evidence for sophisticated Ca2+ regulation in plant mitochondria, the picture has remained much less clear. This is
currently changing aided by live imaging and genetic approaches which allow dissection of subcellular Ca2+ dynamics and identification of the proteins involved. We provide an update on our current understanding in the regulation
of mitochondrial Ca2+ and signalling by comparing work in plants and animals. The significance of mitochondrial Ca2+
control is discussed in the light of the specific metabolic and energetic needs of plant and animal cells.
Key words: Bioenergetics, Ca2+, ion channels, ion transporters, mitochondria.
Introduction
Plant and animal cells share a need to adjust their physiology
rapidly. This is particularly important to deal with changes
in their environment, but also to support developmental programmes. Intracellular Ca2+ transients are an essential and
universal signalling mechanism for mediating physiological
flexibility in both the short and the long term, for instance in
muscle contraction or plant pathogen defence (Escobar et al.,
1994; Cannell et al., 1995; Thor and Peiter, 2014; Keinath
et al., 2015). Animal and plant cells maintain free cytosolic
Ca2+ at much lower concentrations than most other intracellular inorganic ions, such as K+, Cl−, and Mg2+. Active extrusion of Ca2+ from the cytosol to the extracellular space, the
endoplasmic reticulum (ER) lumen, and the vacuole is necessary due to the chemical property of Ca2+ to bind anionic cellular compounds, such as organic carboxylates, phosphates,
DNA, and RNA, and to interfere with their function. This
results in steep Ca2+ gradients across the plasma membrane,
the ER membrane, and the tonoplast membrane. Since those
membranes are equipped with selective transport systems for
Ca2+, rapid and well-defined changes in intracellular concentration can be evoked through activation and inhibition of
the transporters. Changes in Ca2+ concentration can then regulate cellular processes through hundreds of cellular proteins
that change their function in response to Ca2+, for example
by binding Ca2+.
Mitochondria act as intracellular conductors of intracellular Ca2+ regulation, shaping, remodelling, relaying, and
decoding Ca2+ signals, due their ability to accumulate Ca2+
rapidly and transiently (Thayer and Miller, 1990; Friel and
Tsien, 1994; Drago et al., 2012). In the cellular response to
© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved.
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Editor: Markus Teige, University of Vienna
Page 2 of 21 | Wagner et al.
Ca2+ import into mitochondria
Outer mitochondrial membrane
Similar to other small molecules, Ca2+ is thought to pass
the outer mitochondrial membrane (OMM) freely through
VDACs (voltage-dependent anion channels, also called porins;
Fig. 1). VDACs allow flux of metabolites and ions including
Ca2+, for which mammalian VDAC1 also possesses binding
sites, as demonstrated both in vitro and in vivo (Gincel et al.,
2001; Rapizzi et al., 2002; Báthori et al., 2006; Israelson et al.,
2007; Rizzuto et al., 2009; Shoshan-Barmatz et al., 2010; De
Stefani et al., 2012). In mammals three and in Arabidopsis
four functionally distinct protein isoforms have been found
in the OMM (for recent reviews, see Shoshan-Barmatz et al.,
2010; Szabo and Zoratti, 2014; Takahashi and Tateda, 2013).
Direct electrophysiological or genetic evidence for Ca2+
uptake into mitochondria through plant VDACs is still missing, however, even though several studies described the electrophysiological properties of plant VDACs. Interestingly,
yeast two-hybrid assays have suggested that VDAC1 from
Arabidopsis (AtVDAC1) interacts with the EF-hand Ca2+sensor protein CBL1 (Li et al., 2013).
Inner mitochondrial membrane
History of Ca2+ uptake
The inner mitochondrial membrane (IMM) is tightly sealed
for Ca2+, and passage strictly requires channels/transporters.
Uptake into plant mitochondria has been studied for >50 years
after Hodges and Hanson (1965) observed Ca2+ accumulation by corn mitochondria (Fig. 2A). Since then, studies using
isolated mitochondria from different plant species and tissues
have generated a complex and, in parts, contradictory picture.
While most mitochondrial preparations take up Ca2+ (Dieter
and Marmé, 1980; Akerman and Moore, 1983), others do not
(Moore and Bonner, 1977; Martins and Vercesi, 1985). Uptake
strictly requires energization and does not take place in the presence of respiratory chain inhibitors such as antimycin A, KCN,
and NaN3 (Dieter and Marmé, 1980). Ca2+ import in most
(Hodges and Hanson, 1965; Chen and Lehninger, 1973) but not
all (Zottini and Zannoni, 1993) cases requires inorganic phosphate (Pi), and this has been interpreted as symport of Ca2+
with Pi (Day et al., 1978; Silva et al., 1992) or as a consequence
of Ca–phosphate precipitates in the mitochondrial matrix that
decrease free matrix Ca2+ and lead to a continuous drain from
the extramitochondrial space (Akerman and Moore, 1983).
Ruthenium red, an inhibitor of a general inhibitor of calciumpermeable ion channels, appears to block transport in some
(Dieter and Marmé, 1980) but not all (Akerman and Moore,
1983) instances. Conflicting findings may result from studying
Ca2+ uptake outside living cells with limited means to quantify
free Ca2+ inside mitochondria (see ‘Measuring and sensing of
mitochondrial Ca2+’ below) but could also suggest that different
uptake systems are present depending on cell and tissue type.
Although suffering from similar technical constraints, early
Ca2+ uptake studies with mammalian mitochondria provided
a clearer picture. First studied in the 1960s (Fig. 2A), transport
required respiration (DeLuca and Engstrom, 1961; Vasington
and Murphy, 1962) and was accompanied by Pi transport
(Greenawalt et al., 1964). In light of Peter Mitchell’s chemiosmotic hypothesis (Mitchell, 1961), the underlying transporter
was proposed to be an electrophoretic Ca2+ uniporter that does
not require ATP hydrolysis but makes use of the steep electrochemical gradient across the IMM (Rottenberg and Scarpa,
1974). Chemiosmotic coupling also necessitates high selectivity
of any mitochondrial Ca2+ transporter preventing energy dissipation by uncontrolled H+ influx. Once mitochondrial Ca2+
uptake could be monitored directly in intact mammalian cells
(see ‘Measuring and sensing of mitochondrial Ca2+’ below), it
became evident that free matrix Ca2+ could transiently reach
high micromolar concentrations in specific cell types (Montero
et al., 2000) and that the speed and amplitude of Ca2+ uptake
exceeded the values that had been predicted from classical bioenergetic experiments in isolated mitochondria. Subsequent
work in mammalian cells suggested an interaction of mitochondria with microdomains of high Ca2+ concentrations (Fig. 2A)
generated by localized release from the ER and the extracellular
space, allowing highly efficient uptake (Rizzuto et al., 2012).
Key properties of the Ca2+ uniporter and its identification
The functional characteristics of the uniporter have since been
investigated in fine detail. A membrane potential of –180 mV
(negative inside) generated by the respiratory chain would theoretically lead to a 1 000 000-fold accumulation of matrix Ca2+
if electrophoretic Ca2+ passage was unrestricted. Accordingly,
protonophores such as CCCP (carbonyl cyanide m-chlorophenyl hydrazone) not only trigger collapse of the membrane
potential, but also inhibit Ca2+ transport (Selwyn et al., 1970).
Ca2+ uptake into mammalian mitochondria is additionally
blocked by low concentrations of ruthenium red and Ru360,
which lead to a direct inhibition of the uniporter (Moore, 1971;
Vasington et al., 1972; Reed and Bygrave, 1974). The finding
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environmental and endogenous stimuli, mitochondria play
an integral part that goes beyond acting as passive supporters by providing the ATP required for cellular readjustment.
Instead they take an active role in Ca2+ regulation and signalling, controlling central life processes within the organelles themselves as well as the entire cell (Chalmers et al.,
2007; Colombatti et al., 2014). Despite the great interest in
identifying the molecular players of the mitochondrial Ca2+handling machinery, significant advances have been achieved
only during the last decade. This is currently opening new
doors towards a mechanistic understanding of organellar
Ca2+ signalling. Although the plant community has been at
the forefront of the study of the regulation of mitochondrial
Ca2+, the most recent burst of interest, which was sparked by
the identification of the molecular components of the mitochondrial Ca2+ uniporter, has predominantly involved animal
systems. Here we review and contrast the current insights into
the regulation of mitochondrial Ca2+ in plants and animals
side by side, to distil general principles and specific differences, and to sketch out a conceptual picture of the physiological relationship between mitochondria and Ca2+ in plants
and animals.
Regulation of mitochondrial calcium | Page 3 of 21
Multiple proteins constitute and regulate the Ca2+ uniporter
MCU and MCUb MCU is a 40 kDa protein that is
inserted into the IMM via two transmembrane domains,
and oligomerizes into tetramers to form a pore that allows
Ca2+ entry into the mitochondrial matrix driven by the
electrical membrane gradient (Baughman et al., 2011;
De Stefani et al., 2011; Raffaello et al., 2013; Fig. 2C).
Recombinant MCU protein, when incorporated into an
artificial membrane, mediates Ca2+-permeable activity,
resembling the electrophysiological characteristics of the
mitochondrial uniporter (Kirichok et al., 2004; De Stefani
et al., 2011). Mammalian MCU activity can be regulated
through its paralogue MCUb. MCU and MCUb share 50%
sequence similarity, and both proteins physically interact.
MCUb carries two conserved amino acid exchanges in the
intermembrane space (IMS)-exposed loop of MCU which
is necessary to permit Ca2+ transport through MCU in lipid
bilayer experiments (Raffaello et al., 2013). In cultured
cells, MCUb forms hetero-oligomers with MCU (Fig. 2C)
and constitutes a dominant-negative regulator of MCU
transport activity.
Homologues of MCU were identified in genomes of several plant species, including maize and Arabidopsis where six
homologues are present in each (Stael et al., 2012; Meng et al.,
2015). The first proteomic evidence from Arabidopsis and
potato suggests the presence of specific MCU homologues
in mitochondrial fractions at low relative abundance, which
may be expected for an organellar ion channel (Wagner et al.,
2015a). Prediction algorithms such as TargetP (Emanuelsson
et al., 2000) assign MCU proteins a high likelihood of mitochondrial targeting across plant species. Nevertheless, chloroplast localization reaches high prediction scores in several
instances. This may be due to general similarities between
Fig. 1. Inventories of mitochondrial Ca2+ transport in animals and plants. (A) Mammalian proteins and protein complexes at both mitochondrial
membranes and their proposed impact on import or export of Ca2+. Dotted grey arrows represent effects on Ca2+ transport which may be either direct or
indirect. (B) Plant candidate proteins for the modulation of mitochondrial Ca2+ as hypothesized from their presence in plants and the proposed function of
their mammalian homologues. Experimental evidence for an involvement of the plant proteins in handling mitochondrial Ca2+ is currently lacking, except
for MCUC and GLR3.5. See main text for a detailed discussion. (This figure is available in colour at JXB online.)
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that a highly Ca2+-selective ion channel, displaying a very
small conductance of only 5 pS in 100 mM Ca2+ in vitro recapitulated the key characteristics observed for the mammalian
mitochondrial uniporter in classical bioenergetic experiments,
represented a milestone toward the molecular identification of
the uniporter (Kirichok et al., 2004; Figs 1, 2). In a next step, a
regulatory protein, mitochondrial calcium uptake 1 (MICU1),
was identified by a combination of comparative physiology,
evolutionary genomics, and organelle proteomics (Perocchi
et al., 2010). Instrumental for this approach was the MitoCarta
database, containing >1000 mitochondrial proteins as identified by subtractive proteomics and green fluorescent protein
(GFP) fusion localization studies (Pagliarini et al., 2008). This
delivered the basis for the identification of several mitochondrial calcium uniporter complex (MCUC) components in
mammals, including the central pore-forming protein MCU
(mitochondrial calcium uniporter; Baughman et al., 2011;
De Stefani et al., 2011). At the current stage, the mammalian
MCUC appears to consist of at least the pore-forming protein MCU, an MCU paralogue (MCUb), the essential MCU
regulator (EMRE), the regulatory MICU proteins, and, possibly, the mitochondrial calcium uniport regulator 1 (MCUR1;
Fig. 2B, C).
Page 4 of 21 | Wagner et al.
evidence for plant MCU proteins to act as functional channels is still missing.
mitochondrial and chloroplast targeting peptides, but could
also have biological meaning, emphasizing the need for further experimental validation (Emanuelsson et al., 2007;
Briesemeister et al., 2010). The diversification of MCU genes
in plants may thus provide regulatory flexibility on the different levels of gene expression, including transcription, translation, and post-translational organization. This is supported
by differential expression of MCU genes in Arabidopsis and
maize tissues (Stael et al., 2012; Meng et al., 2015). It appears
tempting to speculate about hetero-oligomerization of different plant MCUs to form pores of different Ca2+ transport
efficiency by analogy with the mammalian situation, where
the MCU current varies between different tissues possibly
due to the differential expression of MCU and MCUb (Fieni
et al., 2012) and/or of regulatory components. Yet empirical
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Fig. 2. The composition of the mitochondrial calcium uniporter complex
in animals and plants. (A) Number of publications per year as listed in the
PubMed literature database (http://www.ncbi.nlm.nih.gov/pubmed) as
queried for ‘mitochondrial calcium uptake’. (B) Presence of MCUC core
components (MCU, MCUb, MICU, and EMRE) and CCX family proteins
with homology to mammalian NCLX in key eukaryotic model organisms.
The taxonomic relationship of organisms is indicated by a schematic
phylogenetic tree. (C) MCUC core components as identified in human/
mouse as compared with their plant homologues based on their presence
or absence in the Arabidopsis genome. The presence of Ca2+-binding
EF-hand motifs is schematically indicated in the MICU proteins. (This figure
is available in colour at JXB online.)
MICU The mammalian MICU protein family consists of three
members that share >40% sequence identity (Fig. 2B). MICU1,
the first uniporter component identified (Perocchi et al.,
2010), is a 50 kDa protein with two functional and two pseudo
EF-hands and resides in the mitochondrial IMS (Csordás
et al., 2013; Hung et al., 2014; Patron et al., 2014; Wang et al.,
2014; Petrungaro et al., 2015). It was soon referred to as the
uniporter ‘gatekeeper’ that sets a threshold for mitochondrial
Ca2+ uptake through MCU at low extramitochondrial Ca2+
concentrations but activates the channel when surrounding
Ca2+ concentrations are high (Mallilankaraman et al.,
2012b; Csordás et al., 2013). Recent evidence that elevations
in cytosolic Ca2+ are sufficient (EC50 of 4.4 μM) to induce
rearrangement of MICU1 multimers and to trigger activation
of mitochondrial Ca2+ uptake are in agreement with this
concept (Waldeck-Weiermair et al., 2015). The initial model
for MICU function was further refined after the identification
of two additional MICU isoforms, MICU2 and MICU3
(Plovanich et al., 2013). As MICU3 was found to be almost
exclusively expressed in neural tissues (Plovanich et al., 2013),
functional characterization focused on ubiquitously expressed
MICU2. MICU2 forms a heterodimer with MICU1 through an
intermolecular disulphide bond and closes the channel at low
extramitochondrial Ca2+ concentrations (Patron et al., 2014;
Petrungaro et al., 2015). The stability of MICU2 depends on
MICU1 (Plovanich et al., 2013; Patron et al., 2014), and loss
of MICU2 in MICU1-silenced cells complicates assignment
of individual MICU1 and MICU2 functions. However, in
electrophysiological experiments, MICU2 inhibits the channel
activity, while MICU1 does the opposite in the presence of
Ca2+, in accordance with the proposed model of MICU1
and MICU2 being activator and gatekeeper, respectively
(Patron et al., 2014). Currently, two models co-exist that find
MICU1 (i) to act as a uniporter activator at high cytosolic
Ca2+ concentrations (Patron et al., 2014) or (ii) to disinhibit
the uniporter gradually with increasing Ca2+ concentrations
in the cytosol (Csordás et al., 2013). MICU is conserved in
plants, where typically one or two homologues can be found
depending on the species (Wagner et al., 2015a). Arabidopsis
possesses only a single MICU gene (Fig. 2B, C), and knockout
strongly affects mitochondrial Ca2+ dynamics, providing
molecular evidence for a functional uniporter system in plants
(Wagner et al., 2015a; Fig. 1). Arabidopsis MICU contains an
additional, third canonical EF-hand motif, which is conserved
amongst plants and protists but absent in mammalian MICU,
and may open up additional degrees of freedom for Ca2+
regulation of MCUC activity. Interestingly, one of those three
EF-hands is absent in a second splicing variant of Arabidopsis
MICU, which is expressed at much lower abundance, and
may thereby add to MICU-based fine regulation in plants.
Mitochondrial Ca2+ sensing in living roots of micu knockout
plants has suggested an inhibitory, rather than an activation,
effect of Arabidopsis MICU on plant mitochondrial Ca2+
uptake, which implies that it represents a functional homologue
of mammalian MICU2.
Regulation of mitochondrial calcium | Page 5 of 21
MCUR Not considered core components of the MCUC,
other IMM proteins have been proposed to regulate
uniport activity. MCUR1 (mitochondrial calcium
uniporter regulator 1)/CCDC90A is a 39 kDa protein with
two predicted transmembrane domains that is thought
to interact with MCU (Mallilankaraman et al., 2012a),
although later studies were unable to find support for this
interaction (Sancak et al., 2013; Paupe et al., 2015). Paupe
et al. (2015) provided evidence that MCUR1 is in fact an
assembly factor of cytochrome c oxidase and argued that
genetic manipulation modulates mitochondrial membrane
potential, imposing only a secondary effect on Ca2+
transport. In support of that, MCUR1 has an orthologue
in budding yeast which lacks core MCUC components.
Although Vais et al. (2015) recently showed that MCUR1
affects MCU activity in patch-clamp experiments, direct
regulation of Ca2+ uniport through MCUR1 is still debated.
Arabidopsis possesses two MCUR1 homologues that lack
functional characterization. Interestingly one of them has
been identified as a plant-specific subunit of complex IV
by proteome analysis (Millar et al., 2004; Klodmann et al.,
2011).
Additional components contribute to Ca2+ import
APCs Small Ca2+-binding mitochondrial carrier protein
3 (SCaMC3 or SLC25A23) is an EF-hand-containing
protein that belongs to the family of mitochondrial carriers.
Ca2+-binding mitochondrial carriers (CaMCs) are further
subdivided into two classes: aspartate/glutamate carriers
(AGCs) and ATP/Pi carriers (APCs/SCaMCs/SLCs; Del Arco
et al., 2000; Del Arco and Satrustegui, 2004; Satrustegui et al.,
2007). SCaMC3 has been shown to reduce mitochondrial
Ca2+ uptake upon knockdown in cultured mammalian cells
(Hoffman et al., 2014; Fig. 1). The same was not observed for
its paralogues SCaMC1 and 2 (SLC25A24 and SLC25A25,
respectively). As Pi in the mitochondrial matrix is critical for
free Ca2+ buffering, it is not fully resolved whether this is a
direct or indirect effect on uniporter activity (Seifert et al.,
2015). Similar to MCUR1, independent studies for SCaMC3
and MCU interaction in different cell lines have delivered
contradictory results (Sancak et al., 2013; Hoffman et al.,
2014). The Arabidopsis genome codes for three SCaMC
homologues, ATP/phosphate carriers (APC) 1–3 that all
reside in mitochondria and bind Ca2+ (Stael et al., 2011).
Reconstituted in liposomes, they transport phosphate and
adenosine nucleotides, and are regulated by Ca2+ (Monné
et al., 2015). Intriguingly, Arabidopsis APC2 has recently
been shown to transport ATP-Ca instead of ATP-Mg in vitro
(Lorenz et al., 2015). Considering the physiological baseline
concentrations of free Ca2+ and Mg2+ in the plant cytosol
(100 nM free Ca2+ versus 200–250 µM free Mg2+; Igamberdiev
and Kleczkowski, 2001; Logan and Knight, 2003; Gout
et al., 2014), it remains questionable whether this ATP-Ca2+
Fig. 3. Yellow Cameleon (YC) 3.6 as an in vivo sensor for mitochondrial Ca2+ dynamics. (A) Hypothetical model of YC3.6 based on the Protein Data Bank
(PDB) entries 1huy (for cpVenus), 1cv7 (for CFP), 2bbm (for CaM–M13), and 1cfd (for CaM in its Ca2+-unbound state). Linker segments between proteins
and the Ca2+-free M13 peptide were added manually. YC3 proteins lack one of four Ca2+-binding sites in wild-type CaM (Nagai et al., 2004). Upon
Ca2+ binding, the CaM–M13 fusion undergoes a pronounced conformational change that re-orientates the fluorescent proteins and amplifies Förster
resonance energy transfer (FRET) from CFP to cpVenus. (B) Ratiometric behaviour of YC3.6. Dynamically changing Ca2+ concentrations determine the
degree of FRET, which is low in the Ca2+-unbound state and high in the Ca2+-bound state. This manifests in changes of the relative emission intensities
of cpVenus and CFP, and their ratio in turn. (C) Expression of YC3.6 targeted to the mitochondrial matrix of Arabidopsis leaf epidermal cells. Scale
bar=10 µm. (This figure is available in colour at JXB online.)
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EMRE Another core component of the mammalian MCUC
is EMRE, a 10 kDa protein that spans the IMM with a single
transmembrane motif. EMRE has been proposed to bridge
MCU and its regulators MICU1/2 and to be indispensable for
the activity of the mammalian uniporter in vivo (Sancak et al.,
2013), although MCU alone is sufficient to form a functional
channel in vitro (De Stefani et al., 2011). EMRE is metazoan
specific and its essential role is supported by reconstitution
experiments in budding yeast that lacks an endogenous
mitochondrial Ca2+ uniporter: while expression of MCU
from the slime mould Dictyostelium alone was sufficient to
import Ca2+ into yeast mitochondria, human EMRE needed
to be expressed alongside mammalian MCU to form an
active Ca2+ uniporter system (Kovács-Bogdán et al., 2014).
Recent evidence suggests that the C-terminus of EMRE can
sense Ca2+ on the matrix side of the IMM to regulate Ca2+
uniport negatively (Vais et al., 2016). Acting in concert with
MICU, this may give rise to a sophisticated sensing module
that integrates information on Ca2+ concentration from
both sides of the IMM to avoid both Ca2+ depletion and
overload. Similar to Dictyostelium, plants possess a minimal
genetic uniporter configuration that lacks MCUb and EMRE
(Wagner et al., 2015a; Fig. 2B, C).
Page 6 of 21 | Wagner et al.
(N-methyl-d-aspartate) inotropic glutamate receptor
increased the matrix Ca2+ level in mammalian neurons
(Korde and Maragos, 2012).
transport can also take place in the living plant.
GLR3.5 Another recent study found a member of the
glutamate receptor family, AtGLR3.5, in the mitochondria
of Arabidopsis (Fig. 1). Although there is currently no
direct evidence indicating that the subfamily 3 member
AtGLR3.5 functions as a Ca2+-permeable ion channel,
a close homologue, AtGLR3.4, as well as AtGLR1.4 and
AtGLR1.1 behave as Ca2+-permeable cation channels when
expressed in heterologous systems (Tapken and Hollmann,
2008; Vincill et al., 2012; Tapken et al., 2013). AtGLR1.4
was found to be permeable to Ca2+ in a physiological
concentration range even in the presence of a physiological
concentration of K+. Whether these channels preferentially
permit the flux of Ca2+ over Na+ and K+ in vivo is still
under investigation, but studies employing knockout plants
lacking some members of the subfamily 3 indicate that
glutamate-induced Ca2+ uptake correlates with the presence
of the channel (Qi et al., 2006). In vivo measurements of
mitochondrial Ca2+ dynamics in plants lacking AtGLR3.5
indicated a contribution of the protein to Ca2+ uptake
upon wounding, which may be direct or indirect (Teardo
et al., 2015). Absence of more pronounced alterations
of mitochondrial Ca2+ dynamics may be attributed to
redundancy in mitochondrial Ca2+ uptake processes. Plants
lacking AtGLR3.5 harbour mitochondria with a strongly
altered ultrastructure. Increased AtGLR3.5 transcript
abundance in older leaves together with an early senescence
phenotype in mutant plants makes it tempting to speculate
about a developmental stage-specific role for the putative
Ca2+ transport activity of the protein (Teardo et al., 2015).
Such a hypothesis is consistent with the observation that
activation of a related mitochondrially localized NMDA
Identified uniporter components shed light on Ca2+ uptake
modes
Other mitochondrial Ca2+ uptake modes [e.g. Ca2+-selective
conductance (mCa) 2 and rapid mode of uptake (RaM)]
that have been observed in animals have currently no matching molecular identities. These uptake modes were proposed
to differ from MCUC-mediated Ca2+ uptake in terms of
Ca2+ affinity, uptake kinetics, and pharmacology (Sparagna
et al., 1995; Michels et al., 2009). Although the latter report
remains controversial, it is tempting to interpret those
observations in the light of the molecular complexity of
the MCUC that has been emerging since. Different Ca2+
uptake modes may be accommodated by MCUC existing
and operating in different functional states set by MCU–
MCUb stoichiometry, MICU regulation, and other interacting proteins. MCU knockdown efficiently abolishes Ca2+
transients in mammalian cell culture (Bondarenko et al.,
2014; Baughman et al., 2011; De Stefani et al., 2011), indicating that the MCUC can have a dominating role amongst
uptake mechanisms. In accordance with this, Ca2+ uptake
into mitochondria was almost completely abolished in the
liver of mcu animals (Pan et al., 2013). On the other hand,
this does not rule out the possibility that other mechanisms
make major contributions to Ca2+ uptake, particularly in
specialized tissues. Potential candidates include the TRPC3
channel (Feng et al., 2013; L. Wang et al., 2015) and the
mitochondrial ryanodine receptor (mRyR1). A low level
of RyR1 is detectable in heart mitochondria and provides
rapid transport of Ca2+ that is insensitive to ruthenium red
(Beutner et al., 2001, 2005).
Both TRPC3 and RyR1 have no obvious homologues in
plants (Fig. 1). The availability of several animal model systems in which MCU is genetically knocked out should help
to test the hypothesis of MCUC being responsible for different uptake modes and clarify the presence and kinetics of
co-existing uptake mechanisms. In plants, a similar rationale
is currently hampered by multiple MCU homologues with
unclear and possibly redundant function.
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Fig. 4. Concepts of shaping mitochondrial Ca2+ dynamics. (A) Cytosolic
Ca2+ transients are shaped by import and export from and to various
Ca2+ stores as well as buffering. Cytosolic transients are typically reflected
in the mitochondrial matrix but are re-modelled through the import and
export systems at the IMM aided by the electrochemical potential, and
Ca2+ buffering in the matrix. (B) In principle, matrix Ca2+ signatures may
be evoked independently of the cytosol through (1) channelled import of
Ca2+ from Ca2+ stores or (2) organelle autonomous Ca2+ uptake from the
cytosol/IMS at baseline Ca2+ driven by the steep electrochemical gradient.
(This figure is available in colour at JXB online.)
UCPs Preceding the molecular identification of MCUC
components, additional Ca2+ uptake mechanisms in
mammalian mitochondria were proposed. Uncoupling
proteins 2 and 3 (UCP2/3) have been deemed essential
components of mitochondrial Ca2+ uniport (Trenker
et al., 2007; Fig. 1). This view was challenged (Brookes
et al., 2008), and indirect effects on Ca2+ uptake into
mitochondria have been proposed (De Marchi et al., 2011).
Despite a convincing case against a direct role for UCPs
as Ca2+ transporters, the discussion of UCP2/3 function
is still ongoing (Bondarenko et al., 2015). Although an
MCU homologue of tomato is up-regulated when the
plants overexpress an Arabidopsis UCP protein (Barreto
et al., 2014), potential functional interplay has not yet been
investigated in plants.
Regulation of mitochondrial calcium | Page 7 of 21
Matrix Ca2+ buffering
Mitochondrial Ca2+ export
Described as the mitochondrial ‘Ca2+ cycle’ (Carafoli, 1979),
Ca2+ can be extruded from mitochondria by an antiport
mechanism, to regulate matrix Ca2+ concentrations and
to avoid overload, which can be deleterious for mitochondrial function (see below when discussing PTP). In the late
1970s, two Ca2+ export systems were discussed: a Na+/Ca2+
exchanger (Crompton et al., 1977, 1978) and a H+/Ca2+
exchanger (Akerman, 1978; Fiskum and Lehninger, 1979).
NCLX exports Ca2+ in exchange for Na+
While the molecular identity of the latter remains unclear, the
mammalian protein NCLX (Na+/Ca2+/Li+ exchanger; Palty
et al., 2010) has been proposed as the underlying molecular
entity of electrogenic transport of one Ca2+ against three Na+
(Fig. 1). De Marchi et al. (2014) have recently made a strong
case for NCLX to represent the long-sought mediator of Ca2+
export from the mitochondrial matrix. Arabidopsis possesses
five homologues that belong to the cation/Ca2+ exchanger
(CCX) family (Fig. 2B; Emery et al., 2012). However, these
proteins reach lower prediction scores for mitochondrial targeting than for localization in the secretory pathway [based
on The SubCellular Proteomic Database SUBA3 (Tanz et al.,
2013) and Aramemnon (Schwacke et al., 2003)]. In agreement
with this, a GFP fusion of CCX3 was found in the endomembrane system, where it was suggested to mediate H+/K+
exchange (Morris et al., 2008). On a physiological level, the
involvement of Na+ raises further questions about a corresponding antiport situation in plants, for which, in contrast
to animals, Na+ is not essential (Blumwald, 2000).
LETM proteins as exporters for matrix Ca2+?
Mammalian LETM1 was thought to act as an a K+/H+
exchanger (Nowikovsky et al., 2004; Dimmer et al., 2008)
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Once inside the mitochondrial matrix, Ca2+ predominantly
exists as insoluble Ca–phosphate precipitate but is also
bound to proteins and inorganic acids. This sequestration
allows isolated mitochondria to accumulate large amounts of
total Ca2+ up to a concentration of 1 M, with bound Ca2+
exceeding free Ca2+ by 150 000-fold (Chalmers and Nicholls,
2003). In neurons in the resting state, the ratio between the
bound and free form reaches values of ~4000 in the mitochondrial matrix compared with values of ~100 in the cytosol
(Neher, 1995; Babcock et al., 1997). Yet, concentrations of
free Ca2+ in the resting state are similar between the cytosol and the mitochondrial matrix {animals: [Ca2+]m and
[Ca2+]c=100–200 nM (Rizzuto et al., 1992; Babcock et al.,
1997); Arabidopsis: [Ca2+]m=200 nM, [Ca2+]c=100 nM (Logan
and Knight, 2003)}. The exact chemical states of bound Ca2+
inside the matrix of the living cell and the relative contributions of proteins, metabolites, and Pi are largely unclear in
both plants and animals.
before a genome-wide RNAi screen for proteins mediating
mitochondrial Ca2+ dynamics identified LETM1 as a Ca2+/H+
antiporter with Ca2+ affinity in the physiologically meaningful range (~200 nM; Fig. 1; Jiang et al., 2009). Follow-up work
pointed to a function in mitochondrial Ca2+ uptake or export
dependent on the balance between intra- and extramitochondrial Ca2+ concentrations (Waldeck-Weiermair et al., 2011;
Doonan et al., 2014; Tsai et al., 2014), in agreement with electroneutral antiporter activity found in vitro (Tsai et al., 2014).
This model has been challenged by Nowikovsky and Bernardi
(2014) who put forward strong arguments in favour of K+/
H+ exchange through LETM1: budding yeast mitochondria
lacking a uniporter complex for rapid Ca2+ uptake possess
a LETM1 homologue (Mdm38; Nowikovsky et al., 2004).
Inactivation of Mdm38 leads to mitochondrial swelling, which
points to abnormal accumulation of K+ in the mitochondrial
matrix (Lodish et al., 2000; Rodriguez-Navarro, 2000). This
phenomenon is also associated with LETM1-like proteins from
other species (Hasegawa and van der Bliek, 2007; McQuibban
et al., 2010; Hashimi et al., 2013) and can be reverted by the
ionophore nigericin that specifically mediates K+/H+ exchange
(Nowikovsky et al., 2004). Intriguingly, the yeast Mdm38 lacks
Ca2+-binding EF-hands, and a dual function for LETM1-like
proteins in antiport of H+ against both K+ and Ca2+ cannot be
fully excluded. Recent results suggest that both mitochondrial
Ca2+ influx and efflux rates are impaired in LETM1 knockdown that did not affect the expression level of MCUC proteins. Expression of the ΔEF-hand LETM1 mutant largely
prevented Ca2+ uptake (Doonan et al., 2014). Yet, such observations generally need to be interpreted with a systems view
on mitochondrial physiology, considering indirect effects of
LETM1 removal on Ca2+ homeostasis.
Ca2+ uptake by electroneutral exchange for protons, as
observed in vitro (Ca2+ in; 2 H+ out; Tsai et al., 2014), is thermodynamically implausible in an actively respiring mitochondrion considering a proton gradient of up to 10-fold
(i.e. 1 pH unit) as part of the inner membrane electrochemical gradient. Only at very high cytosolic/IMS free Ca2+ concentrations could the exchanger mechanism allow uptake.
A conclusive physiological case is currently missing and the
thermodynamic argument appears striking enough for all
genetic or biochemical evidence to be interpreted in its light.
In contrast, a H+-driven Ca2+ export function for LETM1 is
thermodynamically plausible, which is a necessary but not
a sufficient argument for export to be mediated by LETM1
under physiological conditions. Indeed a recent study has
provided genetic evidence against such a role by showing that
overexpression of LETM1 did not increase Ca2+ export rates,
while overexpression of NCLX did (De Marchi et al., 2014).
Although some of the proposed activities appear unlikely in
vivo, the mechanism accounting for the impact that LETM1
has on mitochondrial Ca2+ homeostasis remains unknown.
The Arabidopsis genome contains two genes with homology to LETM1 (Fig. 1), and a double knockout is not viable
(Zhang et al., 2012). Both Arabidopsis proteins, LETM1 and
LETM2, reside in the IMM, contain EF-hands, but lack, like
yeast Mdm38, the leucine zipper domain of animal LETM1like proteins (Zhang et al., 2012). Partial depletion of LETM in
Page 8 of 21 | Wagner et al.
a letm1-1(−/−) LETM2-1(+/−) line does not compromise mitochondrial morphology but rather mitochondrial protein translation (Zhang et al., 2012). Such an effect is also associated with
absence of LETM1 in yeast (Frazier et al., 2006; Bauerschmitt
et al., 2010) where dysfunctional mitochondrial translation was
proposed to be a secondary effect of disrupted K+ homeostasis
(Hashimi et al., 2013), based on the observation that nigericin
rescued the translation phenotype in cultured cells.
Opening of the mitochondrial permeability transition
pore for extrusion of matrix Ca2+?
Measuring and sensing of mitochondrial
Ca2+
Ca2+ dyes
Our understanding of mitochondrial Ca2+ dynamics relies
on the development and optimization of Ca2+-sensing tools.
Genetically encoded Ca2+ sensors
Compartment-specific studies across species (Davies and
Terhzaz, 2009; Laude and Simpson, 2009; Stael et al.,
2012) largely benefited from the development of genetically
encoded, protein-based Ca2+ probes. The luminescent protein
aequorin binds Ca2+ via EF-hands and undergoes an irreversible conformation change upon Ca2+ binding triggering
the emission of a photon (Johnson and Shimomura, 1972).
While the protein initially had to be isolated from the jellyfish
Aequorea in the absence of Ca2+ and injected into the cells to
be imaged, cloning of its cDNA (Inouye et al., 1985; Prasher
et al., 1985) allowed recombinant expression and targeting
to subcellular compartments, including the mitochondria
(Rizzuto et al., 1992). Native and modified aequorins cover a
wide range of free Ca2+ concentrations, from ~100 nM to the
millimolar range and, compared with Ca2+ dyes, introduce a
very low Ca2+ buffering themselves (Bonora et al., 2013). As
luminescence yield can be calibrated to deduce absolute Ca2+
concentration (Bonora et al., 2013), aequorin has allowed
measurement of mitochondrial Ca2+ concentrations in animals (Rizzuto et al., 1992) and plants (Logan and Knight,
2003; Mehlmer et al., 2012).
The use of aequorin is constrained, however, by low light
emission limiting microscopic applications, such as in a specific cell or mitochondrion, in particular. A variety of fluorescent protein-based sensors has been developed to overcome
this limitation. The cameleon family of FRET-based Ca2+
sensors, for instance, was introduced by Miyawaki et al.
(1997) and has been optimized since then (Miyawaki et al.,
1999; Griesbeck et al., 2001; Nagai et al., 2004; Horikawa
et al., 2010). The Yellow Cameleon (YC) 3.6, a particulary
popular variant, consists of calmodulin (CaM) and a CaMbinding M13 peptide, both of which are inserted between the
sequences on an enhanced cyan fluorescent protein (ECFP)
and a cpVenus FRET pair (Fig. 3A). The CaM can bind
Ca2+ through three competent EF-hands which induces binding to the M13 peptide, triggering a conformational change
that increases FRET, which is measureable as a change in
the relative intensity of both fluorescent proteins (Fig. 3B).
Pericams (Nagai et al., 2001), GCaMPs (Nakai et al., 2001),
and GECOs (Zhao et al., 2011) use a circularly permuted
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Transient opening of the mitochondrial permeability transition pore (PTP) has been proposed to cause release of Ca2+
from mammalian mitochondria (Bernardi and von Stockum,
2012; Fig. 1). Such a mechanism appears attractive to counteract matrix Ca2+ overload. Under specific conditions, plant
mitochondria have also been observed to undergo permeability
transition (Arpagaus et al., 2002; Petrussa et al., 2004; Vianello
et al., 2012; for a recent review, see Zancani et al., 2015). Yet,
the physiological consequences that a Ca2+ release function of
the PTP implicate appear drastic. Extrusion of Ca2+ through a
PTP-like pore would need to rely on Ca2+ outflow that is thermodynamically plausible; that is, the gradient between free Ca2+
levels in the matrix and the cytosol/IMS would need to exceed
the electrical potential. The electrical potential can be expected
to be, at least partially, dissipated in the first place by the transient
PTP opening, leaving the gradient of free Ca2+ as a main driver.
On partial loss of membrane potential only a large Ca2+ gradient, as expected at Ca2+ overload, would allow Ca2+ extrusion.
Yet, active export coupled to the electrochemical gradient may
be more effective and offer better control. More importantly, a
partially or fully dissipated electrochemical gradient would not
only allow Ca2+ extrusion, but would also severely interfere with
matrix physiology, including ATP/ADP exchange, Pi uptake,
metabolite shuttling, and also Ca2+ extrusion via the NCLX,
which strictly depend on the proton motive force. Transient
variations in membrane potential that have been observed in
both plants and animal cells and occasionally been interpreted
as transient PTP opening have been shown to coincide with
an increase in the pH gradient, implying that the underlying
mechanism does not involve an unselective pore and that the
proton motive force overall remains intact during the transients
(Schwarzländer et al., 2012a, b; Santo-Domingo et al., 2013).
The question of whether the drastic situation of opening a large
unspecific pore, that impacts severely, albeit transiently, on the
characteristic physiological makeup of the mitochondrion, can
fulfil a physiological housekeeping function like Ca2+ export, or
rather is reserved for extreme pathological situations, remains
to be thoroughly tested and validated.
Early uptake studies relied on radiolabelled 45Ca2+ (DeLuca
and Engstrom, 1961), but deduction of kinetic parameters
came with technical pitfalls (Borle, 1981). The first Ca2+
dyes used in the 1960s and 1970s, such as murexide (Mela
and Chance, 1968), partially overcame this issue, but many
of them lacked the properties to quantify Ca2+ specifically.
A more sophisticated generation of Ca2+ dyes was introduced
in the late 1970s (Tsien, 1980). These 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-related
dyes were developed to be membrane permeable and trappable in cells (Tsien, 1981). However, only few chemical dyes are
cell compartment specific, limiting their applicability for in
vivo Ca2+ measurements in mitochondria. Rhod-2 that accumulates in the mitochondrial matrix constitutes an important
exception and has been extensively used.
Regulation of mitochondrial calcium | Page 9 of 21
Mitochondrial re-modelling of Ca2+
transients
Mitochondria shaping cytosolic Ca2+ transients
In animal cells, mitochondria were the first intracellular
organelle to be associated with Ca2+ handling. Their ability to sense Ca2+ signals rapidly and to act as localized
Ca2+ capacitors has long been recognized. By changing the
Ca2+ concentration in its direct vicinity, mitochondrial Ca2+
uptake can influence the frequency and amplitude of cytosolic Ca2+ transients, which depend on release channels that
are regulated by a Ca2+-mediated feedback mechanism. For
example, Ca2+ flux across both the calcium release activated
channel CRAC (Orai1/Stim1) on the plasma membrane/ER
and the inositol-1,4,5-trisphosphate receptor on the ER are
influenced by the physical proximity of mitochondria. This
proximity, sustained by specific mitochondria-associated
membrane (MAM) contacts via chaperones, such as sigma
receptor 1, has been reported to set the extent and duration
of mitochondrial Ca2+ increase. In addition, recruitment of
mitochondria to specific regions has been suggested to constrain Ca2+ signals to defined cell domains, which may be particularly relevant in large cells. In support of those concepts,
mitochondrial Ca2+ uptake has been shown to be associated
with numerous pathophysiological processes including insulin secretion, neuronal excitotoxicity, cardiomyocyte function, and tumorigenesis (see recent reviews by Rizzuto et al.,
2012; Foskett and Philipson, 2015).
In plant cells, most research on how Ca2+ transients are
generated and shaped has been performed with a focus on
cytosolic signatures (Knight et al., 1991; Johnson et al.,
1995; Knight et al., 1997; Wymer et al., 1997; Kiegle et al.,
2000; Allen et al., 2001; Y. Wang et al., 2015). The interplay
between influx, buffering, and export shapes the spatiotemporal properties of the transients which are thought to contribute specificity to intracellular Ca2+ signalling (Fig. 4A).
This explains the large diversity of cytosolic Ca2+ signatures
that have been observed. Yet, the Ca2+ transients inside the
mitochondrial matrix take regulatory complexity of Ca2+
dynamics to another level.
Controlling matrix Ca2+ dynamics at the inner
mitochondrial membrane level
The available in vivo data suggest that, similarly to the situation in animal cells, matrix Ca2+ transients in plants generally
follow transients in the cytosol. This may be seen as evidence
that Ca2+ is first released into the cytosol to then be taken up
from there in a secondary step (Fig. 4A). Alternatively, direct
Ca2+ influx from extracellular, vacuolar, or ER stores may
occur via contact sites, while Ca2+ may coincidently also be
released into the cytosol (Fig. 4B). There is evidence for both
scenarios in mammalian cells (Lawrie et al., 1996; Rizzuto
et al., 1998; Csordás et al., 1999), which can be extended to
plant cells in principle, where the ER can also form physical
contacts with mitochondria (Stefano et al., 2014). Both scenarios have in common that Ca2+ needs to pass the mitochondrial membranes, via specific uptake machineries discussed
above. It is not clear if propagation of transients across the
OMM may modify the signature, but its impact is often
assumed to be minor at most. Much control, regulation, and
integration occur at the level of the IMM, which specifically
choreographs the resulting matrix transient through its influx
and efflux machineries. As such, the IMM acts as an intracellular integration platform that processes and re-shapes
cytosolic/IMS Ca2+ signatures while passing them on into the
matrix, which also impacts on the signature through its specific Ca2+-buffering environment that differs from that of the
cytosol.
Experimental data from living plant and animal cells confirm this additional level of complexity and regulation. The
data consistently show obvious differences between the spatiotemporal properties of cytosolic and matrix Ca2+ transients at a given stimulus, such as extracellular application
of ATP (eATP), glutamate, or histamine (Loro et al., 2012,
2013; Logan et al., 2014; Waldeck-Weiermair et al., 2015).
The first aequorin-based measurements in the cytosol and
the mitochondrial matrix of Arabidopsis seedlings revealed
slightly higher baseline Ca2+, slower onset, lower amplitude,
and longer recovery times for matrix transients triggered by
environmental stimuli as compared with their cytosolic counterparts (Logan and Knight, 2003). This pattern could be
confirmed using cameleon sensors (Loro et al., 2012; Wagner
et al., 2015a). Similar steady-state concentrations of free Ca2+
in the cytosol and the matrix in the presence of a steep electrochemical gradient are evidence of the remarkable degree
of control through the interplay of a tightly sealed IMM with
the necessity for Ca2+ activation of Ca2+ uptake (via MICU)
by an otherwise low affinity channel (MCU), a high buffering
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fluorescent protein inserted in between Ca2+-binding CaM
and the M13 peptide. Here the Ca2+-induced conformational
change alters the chemical environment of the chromophore,
its protonation state, and its fluorescent properties in turn.
Criteria to select the most suitable sensor depend on the specific question and have been extensively reviewed (Palmer
et al., 2011; Perez Koldenkova and Nagai, 2013). The pH stability of the sensor is of particular concern for mitochondria
where matrix pH can naturally fluctuate, particularly during Ca2+ transients (Santo-Domingo and Demaurex, 2012;
Marland et al., 2015). pH stability together with a Ca2+ dissociation constant close to the resting matrix concentration
(Kd=250 nM; Nagai et al., 2004) has made YC3.6 a particularly powerful sensor for matrix Ca2+ dynamics in animals (Yi
et al., 2011) and the current standard in plants (Fig. 3C; Loro
et al., 2012; Behera et al., 2013; Teardo et al., 2015; Wagner
et al., 2015a, b). R-GECO1, the most recent genetic Ca2+
sensor to be introduced into Arabidopsis for cytosolic measurements, allows for higher sensitivity and multiplexing with
other blue, green, and yellow fluorescent sensors, due to its
large spectroscopic response range and its red colour (Keinath
et al., 2015; Y. Wang et al., 2015). The intensiometric, rather
than ratiometric, readout of the sensor and its pronounced
pH sensitivity will require careful optimization, however,
before exploiting it also for mitochondrial measurements.
Page 10 of 21 | Wagner et al.
potential for Ca2+ in the matrix, and efficient export against
the electrical gradient with high affinity.
An increase in Ca2+-selective IMM permeability by activation of a transporter is required to generate a matrix transient. This level offers many options for integration and
tuning, which is reflected in the makeup and composition of
the uptake systems that appear to include multiple potential
channel classes of variable relative abundances, affinities,
conductivities, and regulators integrating different stimuli.
At the level of the MCUC this plasticity is apparent, and far
from fully understood.
Ca2+ regulation of mitochondrial Ca2+ uptake
Understanding of mitochondrial Ca2+ control in vivo
To understand how a matrix Ca2+ transient is generated and
tuned in a realistic physiological context, in vivo monitoring
of Ca2+ dynamics is currently indispensable. Direct deductions are not without problems, however. Combination
with genetic approaches, such as heterologous expression
or removal of involved proteins, has already been intensely
exploited for the functional analysis of MCUC components on matrix Ca2+ physiology in intact animal cells and
tissues (Perocchi et al., 2010; Baughman et al., 2011; De
Stefani et al., 2011; Plovanich et al., 2013; Raffaello et al.,
2013; Sancak et al., 2013). Similarly, the assessment of Ca2+
dynamics in Arabidopsis mutants of MICU was the basis for
deducing an inhibitory function of MICU in plants, based
on increased steady-state concentrations and more rapid
transients reaching higher peaks (Wagner et al., 2015a).
In the current model, inhibition of uptake can be lifted by
cytosolic/IMS Ca2+ binding to the regulatory EF-hands of
MICU. This allows MICU to shape matrix Ca2+ dynamics
by throttling influx, dependent on the properties of cytosolic/IMS Ca2+. Although in vivo sensing of subcellular Ca2+
dynamics in mutants can deliver new mechanistic insights,
conclusions need to be drawn with caution. It is in fact likely
that manipulation of expression of any component from the
Ca2+ regulation machinery of the mitochondrion will have
system effects and might also alter expression of the other
regulatory components, as has been shown for MICU1 and
MICU2 in mammals (Patron et al., 2014). Functional redundancy can provide a back up for the absence or inhibition
of even those players that may be centrally important in the
wild-type scenario. Even when Ca2+ dynamics are modified, as in the case of the Arabidopsis micu lines, it remains
unclear to what extent the status of the Ca2+ handling system, being particularly dynamic and delicate, is comparable
with the wild-type situation.
A combination with pharmacological approaches can circumvent acclimation, but may introduce off-target effects.
Established inhibitors of mitochondrial Ca2+ uptake, such
as ruthenium red and lanthanum, act with low specificity.
Investigation of the structure–activity relationship of the
known players and their comparison across systems, such
as plants and animals, offers a handle for rational improvement and the development of novel, more specific regulators
with promise for clinical use. Reports on the structures of a
truncated MCU variant (Lee et al., 2015) and MICU1 (Wang
et al., 2014) have provided first insights, but further improvements towards a high quality MCUC structure are urgently
needed.
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The empirical observation of a slightly delayed Ca2+ increase
in the matrix as compared with the cytosol supports the concept of elicitation of channel activity by Ca2+ itself, while
lowered matrix amplitudes and delayed recovery to baseline
are in general agreement with high matrix buffering and
Ca2+ export driven by and against the electrochemical gradient. Interestingly, however, lower amplitudes of matrix
Ca2+ appear not to hold true for all stimuli in Arabidopsis.
Auxin application can stimulate much higher amplitudes in
the matrix than in the cytosol (Wagner et al., 2015a). This
could be interpreted as channelled Ca2+ flux from the store
directly into the matrix with only minor involvement of the
cytosol (see above; Fig. 4B). The assumption of channelling
is not critically required, however, since it is thermodynamically plausible that free Ca2+ in the matrix accumulates to
relatively high levels while cytosolic free Ca2+ remains low.
Minor Ca2+ elevation in the cytosol may trigger transport,
and the steep electrochemical gradient across the IMM can
in turn drive uptake to much higher levels in the matrix than
in the cytosol/IMS. This means that minor Ca2+ transients
can be ‘magnified’ in the matrix, and it is intriguing to speculate how this may be harnessed by the cell to generate Ca2+
signatures that specifically act in mitochondria, but not in
other cell compartments. For MICU as gatekeeper for the
uniporter, those observations imply that even low cytosolic
Ca2+ elevations can be sufficient to activate MCUC activity,
and it is interesting to note that particularly high Ca2+ binding affinity has been estimated for Arabidopsis MICU in vitro
(Kd ~1 µM; Wagner et al., 2015a) as compared with mammalian MICU1 (16–21 µM and 4.4 µM; Wang et al., 2014;
Waldeck-Weiermair et al., 2015), which may be linked to the
presence of an additional EF-hand (see section on ‘MICU’
above). Even more, the electrochemical gradient across the
IMM may drive the generation of matrix Ca2+ transients
without the need for a cytosolic transient to occur in the first
place (Fig. 4B). Activation of transport with adequately high
affinity would then be sufficient to trigger uptake of Ca2+
from the cytosol/IMS as a ‘low-concentration Ca2+ store’.
This would, however, require overcoming the MICU-based
inhibition of uptake by increasing its Ca2+ binding affinity or
by a non-Ca2+-binding mechanism. Alternatively, the activity
of a hypothetical channel other than MCUC would be necessary. Without the need for a primary cytosolic Ca2+ transient, such a scenario predicts autonomous Ca2+ transients
in individual mitochondria. Interestingly spontaneous fluctuations in the chemiosmotic gradient of single mitochondria have indeed been observed in plant and animal cells and
linked to influx of Ca2+ (Duchen et al., 1998; Schwarzländer
et al., 2012a; Hou et al., 2013), although the chemiosmotic
fluctuations did not coincide with matrix Ca2+ transients in
other cases (Santo-Domingo et al., 2013; Breckwoldt et al.,
2014).
Regulation of mitochondrial calcium | Page 11 of 21
Despite more and more structural, pharmacological, biochemical, physiological, and genetic data from the mitochondrial Ca2+ machineries of animals and plants, it is just
emerging how matrix Ca2+ transients are shaped in vivo.
Modelling approaches may offer an elegant way to make use
of the existing information to start dissecting the choreography that occurs at the IMM. Such a strategy could generate testable hypotheses about the properties of the players
involved and inform synthetic approaches to generate and
manipulate subcellular Ca2+ signatures rationally, with the
potential to re-wire intracellular Ca2+ signalling in a targeted
manner.
Physiological relevance of mitochondrial
Ca2+
Matrix Ca2+ tunes mitochondrial metabolism in
mammals
EF-hands make mitochondrial proteins candidates for
Ca2+ regulation
Intracellular Ca2+ can be sensed by either Ca2+ sensor relays or
sensor responders (Sanders et al., 2002). While sensor relays
undergo a conformational rearrangement on Ca2+ binding
that is passed on to a target protein, Ca2+ binding changes the
function of a sensor responder directly. The EF-hand helix–
loop–helix motif, which arranges four amino acid residues (X,
Y, Z, and –Z in Fig. 5C, D) to co-ordinate Ca2+, is a typical
feature of Ca2+ sensors in animals and plants. Yet, not every
Ca2+-binding protein carries an EF-hand (e.g. annexins and
proteins carrying a C2 domain) and not every EF-hand binds
Ca2+ (e.g. Gelhaye et al., 2004). The human genome encodes
at least 83 EF-hand proteins, and the Arabidopsis genome
250 (Day et al., 2002). For an appraisal of the role that Ca2+
plays in regulating mitochondrial function in mammals and
plants, we queried the recently updated ‘MitoCarta’ list of
human mitochondrial proteins (Pagliarini et al., 2008; Calvo
et al., 2015) and an Arabidopsis mitochondrial proteome
data set (Wagner et al., 2015a) for EF-hand motifs (ProSite
pattern PS00018 and PS50222) using the ProSite algorithm
(De Castro et al., 2006). Both data sets contain 10 EF-hand
proteins each, associated with similar protein classes including proteins associated with Ca2+ transport, such as APC/
AGC carrier proteins, LETM-like proteins, and MICU proteins (Hajnóczky et al., 2014) (Table 1; Fig. 5A, B).
EF-hands appear in functionally related dehydrogenase
systems in animals and plants
Not common to both sets are several dehydrogenases
(Table 1; Fig. 5A, B): human mitochondrial glycerol-3-phosphate (G-3-P) dehydrogenase (mtGPDH/GDP2) resides on
the outer surface of the IMM where it acts as part of the
‘G-3-P shuttle’, which consumes cytosolic NADH to generate G-3-P from dihydroxyacetone phosphate, subsequently
re-oxidized by mtGDPH at the IMM, transferring electrons
to the mitochondrial ubiquinone pool. The G-3-P shuttle has
been thoroughly characterized in animals and yeast (Larsson
et al., 1998; Rigoulet et al., 2004; Mráček et al., 2013).
Homologues of GPDH have also been described in plants
(Shen et al., 2003, 2006), but the EF-hand for direct Ca2+
binding and activation of mammalian mtGPDH (Hansford
and Chappell, 1967; Klingenberg, 1970; MacDonald and
Brown, 1996) is absent in related proteins from plants, yeast,
and fungi (Brown et al., 1994; Satrustegui et al., 2007). Plants
possess a particularly large diversity of mitochondrial dehydrogenases (Schertl and Braun, 2014), including additional
dehydrogenases to mediate oxidation of cytosolic NAD(P)H
through mitochondrial electron transport (Rasmusson et al.,
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In mammals, Ca2+ elevations in the mitochondrial matrix
stimulate respiration and ATP synthesis to cover temporarily high energy needs of cells (Denton, 2009). Ca2+ overload,
in contrast, can trigger cell death (Duchen, 2000). Increased
biosynthesis rates of ATP rely on the activation of three mitochondrial dehydrogenases by Ca2+ (McCormack et al., 1990).
Pyruvate dehydrogenase (PDH; Denton et al., 1972), NADisocitrate dehydrogenase (NAD-ICDH; Denton et al., 1978),
and oxoglutarate dehydrogenase (OGDH; McCormack and
Denton, 1979) are activated by physiologically relevant Ca2+
concentrations (100 nM and 1 µM) in mitochondria isolated
from mammalian tissues (Denton and McCormack, 1980;
Denton et al., 1980). Ca2+ elevations in intact cells result in
NAD(P) reduction (Duchen, 1992; Pralong et al., 1992), supporting a central role for Ca2+-dependent regulation of mitochondrial metabolism. In animals and plants, PDH activity
is regulated through reversible phosphorylation (Holness
and Sugden, 2003; Tovar-Méndez et al., 2003). The involved
phosphatase of mammals, PDP1, is Ca2+-dependent, and an
increase in free matrix Ca2+ switches PDH from an inactive to
an active state, boosting the rate of oxidative phosphorylation.
Knockout of the MCUC regulator MICU1 that results in an
increased basal Ca2+ concentration in the matrix of cultured
mammalian cells accordingly reduced PDH phosphorylation
(Mallilankaraman et al., 2012b). Vice versa, lower levels of
basal matrix Ca2+ in MCU−/− mice increased PDH phosphorylation (Pan et al., 2013). In contrast, PDH phosphatase
in plants is not activated by Ca2+ in vitro or in intact mitochondria (Miernyk and Randall, 1987; Budde et al., 1988).
Comparative studies further found that while the activity of
the tricarboxylic acid (TCA) cycle enzymes NAD-ICDH and
OGDH from various vertebrate sources (human heart, frog,
and pigeon) is increased in the presence of Ca2+, the same
does not hold true for the respective homologues from insect
flight muscle, yeast, Escherichia coli, potato, and the spadix of
Arum (McCormack and Denton, 1981; Nichols et al., 1994).
Prediction of alternative physiological targets of Ca2+ in
plant mitochondria is complicated by the fact that Ca2+ often
exerts an indirect regulatory effect or the mechanism of Ca2+
regulation remains unknown, due to lack of obvious Ca2+binding motifs and Ca2+-binding interactors. For instance,
mammalian PDH is activated through Ca2+-controlled PDH
phosphatase, while NAD-ICDH and OGDH do not contain
any typical Ca2+-binding motifs and it remains unclear how
their regulation by Ca2+ works mechanistically.
Page 12 of 21 | Wagner et al.
2008). NDB-type NAD(P)H dehydrogenases in plants are
also located at the outer surface of the IMM (Douce et al.,
1973; Luethy et al., 1995; Rasmusson et al., 1999; Elhafez
et al., 2006) and also contain a conserved EF-hand (Table 1;
Fig. 5B; Michalecka et al., 2003). Arabidopsis NDB1 and
NDB2, which were both identified as EF-hand proteins in
the Arabidopsis proteome (Table 1), are specific for NADPH
(NDB1) and NADH (NDB2), respectively, and the activities of both are controlled by Ca2+ (Geisler et al., 2007). The
activating effect of Ca2+ on NDB2 additionally depends on
cytosolic pH (Hao et al., 2015), putting this protein at the
interface between cytosolic and mitochondrial metabolism,
Ca2+ signalling, and redox regulation (Wallström et al.,
2014).
EF-hands are acquired and lost during the course of
evolution
Glutamate dehydrogenase 2 (GDH2) was identified as a
plant mitochondrial EF-hand protein (Table 1; Fig. 5B).
Arabidopsis possesses three NAD(H)-dependent GDHs
(Fontaine et al., 2012), with GDH2 being the only one to
carry an EF-hand (Fig. 5C, D). GDHs from multiple plant
species have been shown to be activated by Ca2+ (Garland
and Dennis, 1977; Kindt et al., 1980; Yamaya et al., 1984;
Das et al., 1989; Itagaki et al., 1990; Turano et al., 1997).
In plants and animals, GDHs reversibly convert glutamate
to the TCA cycle intermediate 2-oxoglutarate and connect
nitrogen and carbon metabolism. In plants, Ca2+ seems
mostly to activate the amination reaction (Garland and
Dennis, 1977; Turano et al., 1997; Yamaya et al., 1984), but
it is an open question whether Ca2+ activates GDH in planta,
since high micromolar Ca2+ concentrations were required for
maximal activation of GDH in vitro (Turano et al., 1997),
while matrix Ca2+ transients peak in the low micromolar
range (Zottini and Zannoni, 1993; Logan and Knight, 2003;
Wagner et al., 2015a). Two human homologues of plant
GDH, GLUD1 and GLUD2, localize predominantly to the
mitochondrial matrix (Mastorodemos et al., 2009) but lack
EF-hands (Fig. 5B–D).
Miro GTPases, detected in both data sets (Table 1; Fig. 5),
are EF-hand proteins that decorate the OMM and mediate mitochondrial motility and morphology in animals and
plants (Boldogh and Pon, 2007; Yamaoka and Leaver, 2008;
Yamaoka and Hara-Nishimura, 2014).
A CaM protein was among the mitochondrial proteome from
Arabidopsis (Table 1; Fig. 5B). CaM proteins are exceptionally
highly conserved Ca2+ sensor relays, of which seven genes in
Arabidopsis encode four protein isoforms that are considered
to be genuine CaMs due to their high similarity to vertebrate
CaMs (McCormack and Braam, 2003). These isoforms differ
by a maximum of four amino acids and are thus indistinguishable in our proteomic data set. In contrast to animals, plants also
possess CaM-like proteins (CMLs) that harbour 2–6 EF-hands
Downloaded from http://jxb.oxfordjournals.org/ at uni padova on June 16, 2016
Fig. 5. Mitochondrial EF-hand proteins in humans and Arabidopsis as targets of Ca2+ regulation. (A) Submitochondrial localization of selected
EF-hand-containing proteins detected in human and Arabidopsis data sets of mitochondrial proteins (Table 1). Topology of EF-hands in Arabidopsis
transmembrane proteins is inferred from their mammalian homologues. (C) Structure model of the canonical EF-hand motif. Amino acid positions X, Y, Z,
and –Z are responsible for Ca2+ co-ordination. (D) Conservation of positions X, Y, Z, and –Z in human mtGPDH, Arabidopsis GDH2, and their EF-handlacking homologues. Purple background indicates compatibility of the amino acid with EF-hand function according to ProSite. Numbers in grey indicate
the total sequence similarity between related proteins. (This figure is available in colour at JXB online.)
Regulation of mitochondrial calcium | Page 13 of 21
Table 1. Mitochondrial EF-hand proteins in animals and plants
Proteins in the human MitoCarta and an Arabidopsis mitochondrial proteome data set (Wagner et al., 2015a) that possess Ca2+-binding
EF-hands according to ProSite. Protein IDs refer to UniProt entries (human) and AGI codes (Arabidopsis). References refer to protein and/or
EF-hand localization studies. Due to a lack of data, localization of EF hands is not further specified for Arabidopsis proteins. In the MitoCarta,
additional proteins (NEFA (NUCB2), RCN2 (ERC55), and FKBP10) were found, but are not shown since they probably represent false positives
and localize to the Golgi apparatus or ER instead (Patterson et al., 2000; Weis et al., 1994; Nesselhut et al., 2001).
Human MitoCarta
Process
Name
ID
Location (protein/EF hand)
Transport-related
APC1, SCAMC1, SLC25A24
APC2, SCAMC3, SLC25A23
APC3, SCAMC2, SLC25A25
AGC1, SLC25A12
AGC2, SLC25A13
LETM1
MICU1
Q6NUK1
Q9BV35
Q6KCM7
O75746
Q9UJS0
O95202
Q9BPX6
IMM/IMS (Nosek et al., 1990;
Del Arco and Satrústegui, 2004)
MICU2
Q8IYU8
mtGPD2
MIRO1, RHOT1
MIRO2, RHOT2
NDUFAB1, SDAP
P43304
Q8IXI2
Q8IXI1
O14561
EFHD1, mitocalcin
GRP75, HSPA9, PBP74, mortalin
Q9BUP0
P38646
Associated with complex I and/or matrix-localized/? (Runswick et al.,
1991; Cronan et al., 2005)
IMM/? (Tominaga et al., 2006)
Matrix and OMM/? (Dahlseid et al., 1994; Szabadkai et al., 2006)
IMM/matrix (Nowikovsky et al., 2012)
IMS/IMS (Csordás et al., 2013; Sancak et al., 2013;
Petrungaro et al., 2015)
IMS/IMS (Sancak et al., 2013; Patron et al., 2014;
Petrungaro et al., 2015)
IMM/IMS (Klingenberg, 1970; MacDonald and Brown, 1996)
OMM/cytosol (Fransson et al., 2006)
Arabidopsis mitochondrial proteome data set
Process
Name
ID
Location (protein)
Transport-related
APC1
APC3
LETM1
LETM2
MICU
GDH2
NDB1
NDB2
MIRO1
Calmodulin
AT5G61810
AT5G07320
AT3G59820
AT1G65540
AT4G32060
AT5G07440
AT4G28220
AT4G05020
AT5G27540
AT1G66410
AT2G27030
AT2G41110
AT3G43810
AT3G56800
AT5G21274
AT5G37780
IMM (Stael et al., 2011)
IMM (Stael et al., 2011)
IMM (Zhang et al., 2012)
IMM (Zhang et al., 2012)
IMS (Wagner et al., 2015a)
Matrix (Ito et al., 2006)
IMM (Elhafez et al., 2006)
IMM (Elhafez et al., 2006)
OMM (Duncan et al., 2011)
?
Dehydrogenase
Other
and share at least 15% sequence identity with CaMs without
having other identifiable functional domains (McCormack and
Braam, 2003). Applying these criteria, 50 genes were predicted
to code for Arabidopsis CMLs and they are involved in various processes covering growth and development, abiotic stress
response, and pathogen defence (Perochon et al., 2011; Bender
and Snedden, 2013). Although CaMs and CMLs are considered mostly nucleo-cytoplasmic, individual isoforms, as well as
matching CaM-binding proteins (Bussemer et al., 2009), have
been found within other cell compartments including the mitochondrion (Yamaguchi et al., 2005; Chigri et al., 2012).
Three additional EF-hand proteins, NDUFAB1/SDAP,
GRP75/HSPA9/PBP74, and EFHD1, were found in the
human data set without EF-hand-containing counterparts in
Arabidopsis (Table 1). Briefly, they act as an acyl carrier protein (NDUFAB1/SDAP), at the physical interface between
mitochondria and the ER (GRP75/HSPA9/PBP74), and in
apoptosis and differentiation of mammalian neuronal and
muscle precursor cells (EFHD1). The importance of Ca2+
binding in these processes is unclear.
The simple comparison of EF-hand proteins in protein
data sets of human and Arabidopsis mitochondria results in
a remarkably coherent picture. Although each of the the 10
proteins found is likely to represent only a subset of the full
Ca2+-related inventory, there are clear parallels between the
respective protein functions. Mitochondrial dehydrogenases
Downloaded from http://jxb.oxfordjournals.org/ at uni padova on June 16, 2016
Dehydrogenase
Other
IMM/IMS (Palmieri et al., 2001)
Page 14 of 21 | Wagner et al.
appear to be able to obtain and lose EF-hand motifs in a
modular manner in the course of evolution (Fig. 5D). This
may correlate with the particular lifestyles and environments
of plants versus mammals. On the functional level, similarities between the different dehydrogenases are striking, however, suggesting that a link between mitochondrial Ca2+ and
respiratory redox metabolism is conserved between plants
and animals. Notably, the EF-hands of most identified proteins are not exposed to the mitochondrial matrix (Fig. 5A,
B). This does not mean, however, that regulation of these proteins is restricted to cytosolic Ca2+. Local IMS Ca2+ levels are
likely to be dependent on the local import and export dynamics, giving rise to microdomains, which further emphasizes
the complexity of the mitochondrion as a cellular integration
platform of Ca2+ regulation.
Phenotypes of animals and plants with defective
mitochondrial Ca2+ regulation
Conclusions and future perspectives
While several key players of the regulation of mitochondrial Ca2+ have been identified in animal systems, the situation remains less clear for plants. Comparative approaches
with concerted research efforts in both systems side-by-side
represent a promising strategy if we are to understand the
complexity of the machineries involved and to distil the minimal set of components and regulatory mechanisms required.
The use of genetically encoded Ca2+ sensors combined with
genetic manipulation of the organism has proven fruitful to
dissect the individual contributions of the different players in
live cells and whole organisms. Yet, the generation of genetic
models to study mitochondrial Ca2+ in animals under in vivo
conditions remains technically challenging. Arabidopsis
T-DNA insertion collections offer a strong advantage, which
is partly offset by genetic diversification with multiple family
members, however, as is the case for MCU. Both constraints
may be overcome by emerging techniques, such as genome
editing by CRISPR–Cas9, which are usable in a wide range
of organisms and potentially allow multigene targeting.
Pleiotropic and compensatory effects during development
Downloaded from http://jxb.oxfordjournals.org/ at uni padova on June 16, 2016
The sophistication of the regulation of mitochondrial Ca2+
at the molecular and cell physiological level, as well as the
existence of several Ca2+-regulated mitochondrial proteins
predicts that dysfunction gives rise to severe defects at the
organismal level. It came as a surprise, therefore, when an
initial report described the generation of viable and healthy
MCU knockout mice, with only mild alterations at the
whole-organism level (Pan et al., 2013). Their mitochondria
lacked any capacity for rapid Ca2+ uptake and their cytosolic Ca2+ signatures were altered, indicating dysfunctional
Ca2+ handling at the cellular level. Those results have been
heavily debated, since viable mice could only be obtained in
a mixed genetic background (Pendin et al., 2014). Several
recent in vivo studies in mammals suggest that alterations
in mitochondrial Ca2+ dynamics by interference with MCU
function are indeed linked to various pathologies. Post-natal
manipulation of MCU levels in mice demonstrated the contribution of MCUC to the regulation of skeletal muscle
tropism. MCU overexpression or down-regulation caused
muscular hypertrophy and atrophy, respectively, probably independently of metabolic alterations, but associated
with Ca2+-dependent mitochondria-to-nucleus signalling
(Mammucari et al., 2015). Finally, in mice with myocardial
MCU inhibition by transgenic expression of a dominantnegative MCU, a strong correlation between MCU function,
oxidative phosphorylation, and correct pacemaker cell function was found (Wu et al., 2015). In zebrafish (Prudent et al.,
2013) and Trypanosome brucei (Huang et al., 2013), genetic
manipulation of MCU also resulted in major developmental and energetic defects. Homozygous human patients carrying a loss-of-function mutation of MICU1 suffer from
myopathy, cognitive impairment, and extrapyramidal movement disorder (Logan et al., 2014). At the cellular level,
they show increased agonist-induced mitochondrial Ca2+
uptake at low cytosolic Ca2+ concentrations and decreased
cytosolic Ca2+ transient amplitudes. However, at least under
resting conditions, the fibroblasts from affected individuals
do not display any severe metabolic defects. Instead, chronic
elevation of the mitochondrial matrix Ca2+ load seems to
lead to mitochondrial stress, resulting in fragmentation of
the mitochondrial network.
There are currently no corresponding phenotypic observations for plants lacking MCU. A complete loss-of-function line of Arabidopsis may require knockout of all six
MCU homologues (see section on ‘MCU’ above; Fig. 2B).
However, with a better understanding of the individual plant
MCU homologues, their subcellular localization. and their
differential expression (Stael et al., 2012; Meng et al., 2015),
less complex genetic lines may allow the first focused analyses over the next years. In Arabidopsis lines lacking MICU
(see section on ‘MICU’ above), pronounced changes in
matrix Ca2+ dynamics in root tips correlate with changes in
mitochondrial ultrastructure and adjustments in the respiratory machinery, while no gross developmental phenotype
was found (Wagner et al., 2015a). In contrast, Arabidopsis
lines lacking mitochondrial GLR3.5 show abnormal mitochondrial ultrastructure and an early senescence phenotype
without strong alterations of matrix Ca2+ dynamics, making
it difficult to draw direct mechanistic links at present (see
section on ‘GLR3.5’ above). The general incoherence of the
current picture in both animals and plants may be partly
explained by compensatory mechanisms operating through
the subcellular physiological network (Schwarzländer and
Finkemeier, 2013). Alternatively, acclimation between the
cellular and whole-organism scale may be responsible for
the observed robustness (see ‘Understanding of mitochondrial Ca2+ control in vivo’ above). Both levels are currently
insufficiently understood for complex biological systems,
including animals and plants. Contrary to common argument, there is no sound mechanistic basis to justify correlations between the importance of most cell physiological
players or processes and gross developmental phenotypes
that may be induced by their impairment on the organismal
level.
Regulation of mitochondrial calcium | Page 15 of 21
may be counteracted by inducible loss- or gain-of-function
approaches. Active interaction across the animal and the plant
disciplines appears to hold particular promise to unravel further the fundamental roles that mitochondrial Ca2+ signalling
plays in vivo.
Acknowledgements
We thank Marco Zancani (Università degli Studi di Udine) for critical reading of the manuscript. MS thanks the Deutsche Forschungsgemeinschaft
(DFG) for support through the Emmy-Noether programme (SCHW1719/11), the Research Training Group 2064, and a grant (SCHW1719/5-1) as
part of the package PAK918, and IS thanks the Ministero dell’Istruzione,
dell’Università e della Ricerca for funding through the PRIN project
(2010CSJX4F), to the Human Frontiers Science Program (HFSP 0052) and
to the Italian Association for Cancer Research (IG 11814).
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