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PHYSIOLOGY 22: 261–268, 2007; 10.1152/physiol.00017.2007
ER-Mitochondria Communication.
How Privileged?
Clara Franzini-Armstrong
Department of Cell and Developmental Biology,
School of Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania
[email protected]
Mitochondria have a low affinity for Ca2+, but they take up these ions during normal cell activity because they are in close proximity to the sites of calcium entry
into the cell and of internal Ca2+ release. This gives mitochondria privileged
access to cytoplasmic Ca2+ without requiring a direct communication with the
endoplasmic reticulum.
Ca2+ Uptake by Mitochondria:
To Take or Not to Take (a
Historical Note)
The affinity of mitochondria for Ca2+ uptake is low:
in the 10–6–10–4M range (11, 16, 57, 71). A suggestion
that mitochondrial Ca2+ uptake might dominate Ca2
homeostasis was put forward for the cycle of contraction and relaxation of “red” muscle, which is rich
in mitochondria (8, 42, 57). This assertion, however,
was rightly met with considerable skepticism.
Mitochondria could not be expected to reduce cytoplasmic Ca2+ concentration to 10–7 M or less, as
needed for relaxation (76, 77). In addition, the sarcoplasmic reticulum (SR) alone is responsible for
the rapid Ca2+ release that initiates contraction in
muscle. To be ready for releasing calcium, the SR
must be loaded with it. If most of the Ca2+ was taken
up by mitochondria during relaxation, then the SR
would be depleted after a few contractions and e-c
coupling would fail.
Low-affinity mitochondrial Ca2+ uptake clearly does
come into play under conditions of cell damage when
cytoplasmic Ca2+ concentration can rise to dangerous
levels (6). However mitochondria do not normally
maintain a high internal calcium concentration
(61–63). Thus, until fairly recently, intracellular cycling
of Ca2+ under physiological conditions was thought to
be exclusively the task of the endoplasmic reticulum
(ER), calciosomes (44), and the SR. These organelles
bear a high-affinity Ca2+ pump in their membrane and,
acting in concert with plasmalemmal pumps and
exchangers, are ultimately responsible for returning
cytoplasmic Ca2+ to basal levels. These same
organelles release Ca2+ on command via two release
channels, the inositol (1,4,5)-trisphosphate receptors
(or IP3Rs) and ryanodine receptors (or RyRs) (3, 75).
We now know that large Ca2+ loading of the mitochondria occurs only under pathological conditions.
However, mitochondrial Ca2+ uptake can also occur
under physiological conditions, and it can be of sufficient magnitude to affect cytoplasmic Ca2+ homeostasis. Recent advances in this arena are attributed to the
availability of probes for recording Ca2+ concentrations
in various subcellular compartments and of blockers
specific for the different uptake and release pathways.
Several “mitotracker” dyes and cell-permeant Ca2+sensitive fluorophores that are taken up by mitochondria in a membrane potential-dependent manner, as
well as dyes that rapidly track changes in mitochondrial membrane potential, have become available. An
excellent review (48) covers these recent advances.
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Starting with the early demonstrations that mitochondria take up Ca2+ (5, 7, 9, 14, 55), the concept that mitochondria contribute to cellular Ca2+ homeostasis
under physiological conditions has undergone a complete cycle, from an initial proposal, through total
denial, to reconsideration based on new concepts.
Ca2+ cross the outer mitochondrial membrane, then
enter the mitochondrial matrix via a not yet fully identified “uniporter” channel located in the inner
mitochondrial membrane, driven by a large electrochemical gradient. Ca2+ ions activate respiratory
enzymes and are thus important for mitochondrial
function, but their efflux via H+ and Na+ antiporters
introduces an energy debt (25), and a large influx can
be damaging to the organelle. Overall, since the rate of
Ca2+ cycling by mitochondria is very slow in the resting
cell and since only a small fraction of Ca2+ is taken up
by mitochondria during activity, these organelles
account for only a small portion of the Ca2+ cyclingassociated energy expenditure by the cell.
Ca2+ Microdomains: Prime
Mitochondrial Real Estate
The observations by Miyata et al. (36) and the ingenious experiments of Rizzuto et al. (49) provided initial
breakthroughs in our understanding of mitochondrial
Ca2+ uptake under physiological conditions. In the latter study, a modified aequorin was targeted to the
mitochondrial matrix in cultured epithelial cells to
directly monitor changes in mitochondrial Ca2+ in a
living cell. Surprisingly, these experiments show that
mitochondrial Ca2+ signals are relatively rapid and
transient (on a second time scale, 49) and of a larger
magnitude than previously thought (36). Block of
1548-9213/05 8.00 ©2007 Int. Union Physiol. Sci./Am. Physiol. Soc.
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mitochondrial Ca2+ uptake by an uncoupler confirms
the signal as originating from intact mitochondria
(49).
In solving the apparent discrepancy between mitochondria’s low affinity for Ca2+ uptake and their ability
to accumulate Ca2+ ions under physiological condi-
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FIGURE 1. Close relationship between SR calcium release sites and mitochondria in adult mammalian
striated muscles
In mouse, fast mitochondria-rich (“red”) skeletal muscle fibers (A) and cardiac muscle cells (B), mitochondria (M) are in
close proximity to calcium release units (triads dyads, arrows), the sites of interaction between sarcoplasmic reticulum
(SR) and T-tubules, where Ca2+ is released during muscle activation. Note extensive cristae in cardiac muscle mitochondria. C: frequency of close appositions between triads and mitochondria. Shown is a merged confocal image of a
single adult mouse FDB fiber co-loaded with Hoechst 34580 stain (blue, nuclei), mitotracker green (green, mitochondria), and Di-8-ANNEPS (red, T-tubules, the central elements of the triads). Courtesy of Ann E. Rossi and Robert T.
Dirksen. D: diagram of the close apposition between Ca2+ release sites and mitochondria in muscle. SR Ca2+ release
channels or RyRs (red) are located in the SR membrane facing the T-tubule. Although the membranes delimiting SR
and mitochondrion are at a short distance from each other (~10 nm), calcium ions (yellow dots) must travel a relatively
large distance (100 nm or more) from the sites of release (through RyRs) to the mitochondrion. Ca2+ crosses the outer
mitochondria membrane (through VDAC channels, green) and then enters the mitochondrial matrix through a uniporter (blue). Access to the uniporter is hindered by the narrow openings of the cristae.
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A Structural Basis for ERMitochondria Interaction
The proximity of mitochondria to Ca2+ sources is the
most important parameter in determining Ca2+ uptake
by these organelles. In the quest for a structure-function
correlation underlying this spatially close interaction,
many observations and speculations have come into
play. Domains of the ER in many cells, including the
specialized SR of muscle, are closely associated with
mitochondria (FIGURE 1; Refs. 12, 33, 58). This proximity is not random but instead seems to be a coordinated
feature of cell organization. In some mammalian muscle cells, for example, the mitochondria are arranged in
a highly organized fashion (FIGURE 1; Ref. 73). This
arrangement is not simply due to limited space availability but rather is developmentally regulated (54). In
early (<1 wk) postnatal mammalian muscle, mitochondria are randomly clustered and exhibit no specific spatial relationship with the SR. In the postnatal weeks,
however, the SR and mitochondria are coordinately
rearranged and acquire a specific position relative to
the myofibrils and to each other. In this process, the
mitochondria become closely associated to the Ca2+
release sites of the SR required for mitochondrial Ca2+
uptake during rapid and brief Ca2+ release events.
Further evidence for a coordinated ER-mitochondrial association is the discovery, in liver, of numerous
sites of close proximity between the two organelles
that can survive cell fractionation (33, 35, 60) and the
observation, in liver and muscle, that physical links
tether the outer mitochondrial membrane to the adjacent ER/SR (4, 12). The links vary considerably in
length (~10 nm for smooth ER and 25 nm for rough ER
in liver, and ~10 nm for SR in mammalian skeletal
muscle) and thus may be comprised of different components in different cells and/or cell domains.
Although mitochondria are known to have a good
degree of motility in many cell types, tethers indicate
that some mitochondria may be trapped in specific
positions, although perhaps not permanently. The
extent of contact is quite variable. For example,
FIGURE 1 shows extensive contacts between mitochondria and Ca2+ release units in a “red” skeletal
muscle fiber and a very short connection in a cardiac
muscle, where short physical links are also visible.
These links have not yet been described in other cells,
but specific locations of mitochondria have been
noted: e.g., immediately below the plasmalemma in
smooth muscle (23) and in a semicircle in pancreatic
acinar cells (FIGURE 2; Ref. 72)
Given the proximity between sites of Ca2+ release and
mitochondria, an obvious question is whether the interaction between the two may be quite specific. For example, is there a direct intermolecular link allowing
molecule-to-molecule cross talk of the type proposed
for the reciprocal interaction between T-tubule and SR
proteins in skeletal muscle e-c coupling (38)? Such a
privileged membrane-to-membrane communication
pathway has been proposed at the sites of close ER and
mitochondrial apposition (30, 31). The intriguing recent
suggestion (68) of a direct connection between IP3Rs of
ER and the channel that facilitates Ca2+ entry across the
outer mitochondrial membrane (VDLAC; voltage-
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tions, the old cell biology concept of restricted cellular
microdomains (or nanondomains for the supercool
scientist) has come to the forefront in a new form (48).
Ca2+ entry sites at the plasma membrane and internal
release sites are not uniformly distributed within cells.
Thus the Ca2+ signal can vary greatly depending on the
distance from the Ca2+ source. If the mitochondria
happen to be very close to a Ca2+ source, then they will
be exposed to a relatively high [Ca2+] for a short period
of time. In addition to being spatially heterogeneous,
changes in cytoplasmic Ca2+ can also be temporally
variable. Factors that can govern the Ca2+ environment
experienced by the mitochondria include diffusion,
binding to cytoplasmic buffers, and Ca2+ clearance by
other Ca2+ removal processes.
Numerous experiments have confirmed and refined
the idea that the Ca2+ microenvironment determines
mitochondrial uptake. For example, neither internal
perfusion of a cell with Ca2+ nor induction of a slow Ca2+
leak from the ER by blocking the Ca2+ pump is sufficient
to trigger rapid mitochondrial Ca2+ uptake, whereas a
fast and more localized Ca2+ release through activation
of IP3Rs in the ER does (50, 51). In some cells, a fairly
prolonged Ca2+ release via IP3Rs is needed to effectively activate mitochondrial uptake (66), but in others (see
below) the uptake may start within milliseconds. A possible microdomain-independent uptake has also been
suggested (71), but this may be an exception.
Muscle is a tissue of choice for the study of the mitochondrial Ca2+ microenvironment because it exhibits
large cytoplasmic Ca2+ transients and often a stereotypical co-location of SR Ca2+ release sites and mitochondria (FIGURE 1; Ref. 73). In the presence of a slow Ca2+
buffering system, release of Ca2+ from the SR by caffeine induces synchronous cytoplasmic and mitochondrial Ca2+ signals (59). Even with introduction of a fast
Ca2+ buffering system sufficient to greatly decrease the
cytoplasmic Ca2+ signal, ~70% of mitochondria still
exhibit Ca2+ uptake. Due to their proximity to the Ca2+
source, these mitochondria, it is argued, can “see” the
Ca2+ release even in the presence of a fast Ca2+ buffer
(59). Thus mitochondrial positioning accounts for the
speed and amplitude of the mitochondrial Ca2+ concentration increase. These experiments (51, 59) also
show that not all mitochondria are equivalent within
the cell, since only a fraction have privileged position
near the Ca2+ source. Mitochondria in cardiac muscle
are also abundant and in close proximity to sites of calcium release (58). In isolated rat cardiac myocytes, Ca2+
release from the SR results in synchronous cytoplasmic
and mitochondrial Ca2+ signals (58).
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distance quoted above, because Ca2+ needs to diffuse
out of the junctional space and around the SR cisternae before reaching the mitochondrion (FIGURE 1;
Ref. 58). The Ca2+ release apparatus of muscle is
designed to provide rapid diffusion of Ca2+ into the
cytoplasm to activate myofibrils within milliseconds.
The cloud of Ca2+ released even by a single RyR quickly expands and provides a sufficiently high [Ca2+] for
mitochondrial uptake.
In cells other than muscle, the situation is less fully
defined at the moment. A close association between
IP3R-containing ER membranes and mitochondria
has been clearly shown (12). However, the exact position of IP3Rs relative to the mitochondrial outer membrane is not known. Indirect evidence indicates that a
similarity to muscle may exist. The beautiful EM tomographs of Mannella (12) show detailed images of
tethers connecting ER to the mitochondria’s outer
membrane but find no evidence for the presence of
IP3R in the ER membrane directly facing the mitochondria. The cytoplasmic domains of the IP3 receptor project for some distance into the cytoplasm and
have a clear tetrameric structure (29); thus the presence of even a few IP3 receptor channels should not be
easily missed in a tomograph. Direct “tunneling” of
Ca2+ from ER to mitochondria, essentially bypassing
the cytoplasm, has been proposed (13). However, for
the moment, there is no direct evidence for a location
of IP3R in close apposition to and/or in direct contact
with the outer mitochondrial membrane, and diffusion distances for Ca2+ may be similar to those in cardiac muscle, which is at least tens of nanometers. If
that is the case, it is not clear how changing the length
of the ER-mitochondrion tethers by a few nanometers
may strongly affect Ca2+ uptake by mitochondria (12).
Is there more than meets the eye?
Mitochondrial Contribution to
Cellular Ca2+ Homeostasis
FIGURE 2. Active mitochondria surround the secretory granule area of
pancreatic exocrine (acinar) cells and constitute a barrier to Ca2+ diffusion
Confocal transmitted light (A and C) and fluorescence images (B and D) of freshly isolated acinar cells illustrating the intracellular distribution of mitochondria. A and C: the
cell polarity: zymogen granules concentrated at the secretory poles are segregated
from the basolateral parts of the cells. In B and D, mitochondria are labeled with a
mitochondrial-specific dye (MitoTracker Green). Most mitochondria are located within a
belt surrounding the secretory pole, but some distinct spots in the vicinity of the plasma membrane are also seen. Figure is reproduced from Ref. 72 with permission.
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That mitochondria can take up Ca2+ is quite clear, but
in terms of cell function an important question is how
significant is this removal mechanism? The answer,
obviously, will vary from cell to cell. An interesting
example comes from gonadotropes (28), where hormones induce oscillations in cytoplasmic Ca2+.
Abolishing the mitochondrial membrane potential
with a protophore, thus annulling the driving force for
Ca2+ uptake, only changes the basal cytoplasmic Ca2+
concentration by a small amount but significantly
alters the shape and frequency of the oscillations
(FIGURE 3A). The mitochondrial inner membrane
potential oscillates in synchrony with the cytoplasmic
[Ca2+] oscillations due to the two competing factors of
Ca2+ entry and the work of the proton pump
(FIGURE 3B). Although mitochondria occupy only
~2% of the cell volume of the gonadotrope, they affect
the rate of exocytosis without uncontrollably loading
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dependent large anion channel) (46) would indeed support this hypothesis. However, the structural considerations described below suggest that, to the contrary, Ca2+handling interactions between the SR (and possibly also
ER) and mitochondria is quite indirect.
Although the mitochondria are closely tethered to
the SR domains that release Ca2+ in muscle, the release
channels or RyRs occupy the SR surface that faces
toward the T tubules, away from the mitochondria,
and are therefore at some distance from the mitochondria’s surface (Ref. 3; FIGURE 1). The nearest distance “as the crow flies” between RyRs and the outer
mitochondrial membrane is shorter in cardiac muscles, where the SR cisternae are flat (~37 nm; Ref. 58)
and more variable and mostly larger in skeletal muscle
(up to ~130 nm in mouse leg muscles). Because the SR
lumen separates RyRs from the mitochondrial outer
surface, there is no chance for a direct molecular link
between the RyRs and the mitochondrion. It must
therefore be assumed that mitochondrial Ca2+ uptake
in muscle depends on diffusion of Ca2+ released from
the SR to the mitochondrial surface. The actual distance for diffusion is much larger than the minimum
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gonadotropes and slower muscles fibers (56), on the
other hand, mitochondria have a positive influence,
since they operate on a relevant time scale.
A fascinating example of mitochondria’s role in controlling cytoplasmic Ca2+ is revealed in the exocrine (acinar) cells of the pancreas (72, 64). In these cells, the Ca2+
containing ER is present throughout the cell and sends
long projections between the zymogen-containing
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with Ca2+ (1). As in gonadotropes, the contribution of
mitochondria to Ca2+ handling is also well detectable
and even of large amplitude in chromaffin cells (26,
37) and hippocampal neurons (74). In contrast, mitochondrial Ca2+ uptake is almost negligible in mouse
pancreatic beta cells in vivo (10). A comparative look
at frequency and size of ER-mitochondria appositions
in these cells would be of great interest.
The content of mitochondria and their participation
in Ca2+ handling vary considerably in muscle. Under
experimental conditions, application of ruthenium
red (a blocker of the mitochondrial Ca2+ uniporter)
affects the relaxation rate of skinned “red” (mitochondria rich) fibers, but not that of “white” (mitochondria
poor) fibers (24). In cardiac muscle, mitochondria also
affect the transverse propagation of Ca2+ waves by
buffering Ca2+ at the level of the Z line, where they are
closely apposed to the SR Ca2+ release sites (65).
Under physiological conditions, Ca2+ transients are
quite rapid in muscle, so the next questions are do
mitochondria take up Ca2+ in muscle during a contraction-relaxation cycle, and how much do they affect the
time course of relaxation? In cardiac muscle, mitochondrial Ca2+ signals are detected in response to brief
SR release events (40,70), and actual beat-to-beat
tracking of Ca2+ by mitochondria occurs (52). In a
mammalian skeletal muscle, with contraction-relaxation cycles of 120–150 ms, Ca2+ uptake and release by
mitochondria can keep up with the cytoplasmic Ca2+
transients, with only a few milliseconds delay (56). The
direct effect of this uptake on the time course of relaxation has not been measured under in vivo conditions,
but it may be significant. During an in vivo twitch, the
transient increment in Ca2+ concentration in the mitochondrial matrix is almost as large as the average increment in the cytoplasm (56). The mitochondrial volume
is up to 5-fold that of the SR in red skeletal muscle
fibers (19) and up to 16-fold in mouse cardiac muscle,
and the matrix constitutes a good portion of that volume. So, even though the matrix volume is less than
that of the entire mitochondrion and has less rapid calcium buffering than the SR, the uptake of even a small
fraction of the released Ca2+ by the mitochondria is
likely to shorten the time course of relaxation.
Nature offers an interesting example of how cells position mitochondria to suit their needs. In cardiac muscle
and in most mitochondria-rich skeletal muscle fibers of
mammals, most of the mitochondria are in close proximity to Ca2+ release sites (FIGURES 1 AND 4A). In contrast, in some super-fast muscle fibers, mitochondria are
excluded from the intermyofibrillar areas where the Ca2+
release sites are located (FIGURE 4B; Refs. 20, 47). These
fibers perform very rapid and repeated force oscillations
that require synchronous and very fast (<10 ms) Ca2+
release and reuptake (53). If mitochondria take up and
release Ca2+ at rates that are very different from those of
the SR, then their activity would hinder the rapid oscillations. In cells with slower Ca2+ transients, such as
FIGURE 3. Mitochondria shape Ca2+ oscillations
in secretory cells and are transiently depolarized
A: the shape and frequency of cytoplasmic calcium oscillations induced by hormonal stimulation of a
gonadotrope change when a protonophore (CCCP) is
added, reducing the mitochondria inner membrane
potential and thus inhibiting Ca2+ uptake by these
organelles. CCCP elevated the cytoplasmic Ca2+ concentration and slowed the falling phase of the oscillation. B:
changes in TMRE (a mitochondrial membrane potential
indicator) fluorescence (top trace) and in cytoplasmic
Ca2+ concentrations (bottom trace) are synchronous during hormonal stimulations. Depolarization with CCCP at
the end of the experiment provides a reference. Note,
however, that mitochondria steadily accumulate calcium
during these oscillations and release it later. Figure is
reproduced from Ref. 28 with permission.
PHYSIOLOGY • Volume 22 • August 2007 • www.physiologyonline.org
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granules (22, 41). Application of an agonist that
induces Ca2+ release from the ER via IP3Rs results in a
cytoplasmic Ca2+ signal that remains strictly localized
to the apical region of the cell and results in apical exocytosis of granules. Located in a ring surrounding the
apical region, the mitochondria act as guardians,
blocking the diffusion of Ca2+ to the basal regions of the
cells and thus limiting intracellular spread of Ca2+
(FIGURE 2; Refs. 72, 64). The dire consequences of tampering with this cellular organization are evidenced in
pathological conditions induced by alcoholism.
Through a chain of indirect events, alcoholism impairs
pancreatic mitochondrial function and results in large
Cross sections of muscle fibers from the “red” bundle of the rat sternomastoid (A) and
from the swimbladder muscle of the toadfish (B). The latter is one of the fastest known
muscles, responsible for a sound-producing vibration at frequencies higher than 100
Hz. A: long, thin mitochondria, overlaid in yellow, form extensive necklaces around all
myofibrils at the I band level, where they establish extensive close contacts with triads
(compare with FIGURE 1). B: mitochondria (yellow overlay) are far fewer and also are
segregated away from the area occupied by myofibrils where triads are located.
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The Selfish Mitochondrion
Mitochondria do contribute to Ca2+ homeostasis, but
their contribution is more limited in magnitude and
less favorable in terms of energy used per ion cycled
than that of other Ca2+ sequestering organelles. Entry
of Ca2+ into the inner mitochondrial matrix is
metabolically expensive because it reduces the mitochondrial membrane potential and thus the energy
available for the operation of the ATP synthase,
although it also stimulates ATP production. In addition, release of Ca2+ is far slower than that of the ER/SR
(it may take several seconds to be completed in some
cells), and it also requires expenditure of energy in the
maintenance of the Na+ and H+ gradients. Thus mitochondria should be seen as reluctant participants in
Ca2+ homeostasis, since Ca2+ entry is necessary for
their function (34), but it extracts a high price (25) and
can be damaging if in large amounts (15, 17, 18, 27, 39,
67, 69). Mostly the mitochondrion protects itself from
the dangerous accumulation of Ca2+ by limiting entry
to relatively high cytoplasmic concentrations by the
relatively rapid extrusion of this ion mostly through
the electroneutral Na-Ca exchange (28, 56, 58, 70) and
by a selective position in the cell. Internal mitochondrial Ca2+ buffering systems also come into play.
Although it is clear that Ca2+ sequestration by mitochondria affects cell function, this also has some
limitations. Unlike the ER and SR, mitochondria accumulate Ca2+ in a transient manner in vitro (16) as well
as in vivo (56). Mitochondria release Ca2+ on their own
time scale, not necessarily at the cell’s service, and
Ca2+ taken up by the mitochondria is not available for
stimulated release. Thus mitochondria may participate in calcium uptake but cannot be relied on to
deliver calcium when needed by the cell. Some structural features of the mitochondria and of their interactions with the ER/SR seem designed for limiting either
Ca2+ uptake or its negative effects. Mitochondrial
cristae are very abundant and tightly packed in mitochondria of the most active cells (e.g., myocardium),
and their very large surface area results in a relatively
high total membrane capacitance and a proportional
damping effect on the potential change resulting from
Ca2+ crossing the inner mitochondrial membrane. The
mitochondria themselves are often very large, even if
small in diameter: in FIGURE 3A, each individual
mitochondrion wraps itself around many myofibrils.
The cristae are pleomorphic and have very complex
shapes, and most importantly their connections to the
inner boundary membrane are scarce and via narrow
tubules, a design appropriate for limiting diffusion of
Ca2+ from the mitochondrion periphery to the sites
where uniporters are located on the cristae (FIGURE 1D;
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FIGURE 4. Muscle fibers tailor mitochondria positioning to
their requirements
prolonged Ca2+ signals that invade the whole cell. This
leads to trypsin activation within the secretory granules
and pancreatitis, a major health problem (43).
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How Privileged?
To summarize, proximity between ER/SR and mitochondria allows the latter to take up calcium under
physiological conditions. The opportunity for communication is determined by the developmentally regulated programming of an appropriate proximity
between Ca2+ sources and mitochondria.
Communication is more likely to be by an indirect
pathway via diffusion of Ca2+ over distances that are
short in term of diffusion but long in terms of a direct
molecular contact. In answer to the question of
whether ER-mitochondrial communication is privileged in the sense of “it is a privilege to have an exclusive line of communication with you and get your
direct input” or “it is a privilege to sit near you and get
some of your crumbs,” my vote would be for the latter.
Given a robust Ca2+ release event, the crumbs will do
fine, thank you very much. „
I thank Cecilia Gold for teaching me how to write, Dr.
Robert T. Dirksen for numerous useful hints and a critical
review of the manuscript, and Dr. Simona Boncompagni
for help with the literature.
Supported by National Heart, Lung, and Blood Institute
Grant RO1 HL-48093.
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