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Pulsing of Membrane Potential in Individual Mitochondria:
A Stress-Induced Mechanism to Regulate Respiratory
Bioenergetics in Arabidopsis
W
Markus Schwarzländer,a,b David C. Logan,c Iain G. Johnston,d,e Nick S. Jones,d,e,f Andreas J. Meyer,b
Mark D. Fricker,a and Lee J. Sweetlovea,1
a Department
of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom
of Crop Science and Resource Conservation, University of Bonn, 53113 Bonn, Germany
c Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E2, Canada
d Department of Physics, Clarendon Laboratory, Oxford OX1 3PU, United Kingdom
e Oxford Centre for Integrative Systems Biology, Department of Biochemistry, Oxford OX1 3QU, United Kingdom
f Department of Mathematics, Imperial College London, London SW7 2AZ, United Kingdom
b Institute
Mitochondrial ATP synthesis is driven by a membrane potential across the inner mitochondrial membrane; this potential is
generated by the proton-pumping electron transport chain. A balance between proton pumping and dissipation of the
proton gradient by ATP-synthase is critical to avoid formation of excessive reactive oxygen species due to overreduction of
the electron transport chain. Here, we report a mechanism that regulates bioenergetic balance in individual mitochondria: a
transient partial depolarization of the inner membrane. Single mitochondria in living Arabidopsis thaliana root cells undergo
sporadic rapid cycles of partial dissipation and restoration of membrane potential, as observed by real-time monitoring of
the fluorescence of the lipophilic cationic dye tetramethyl rhodamine methyl ester. Pulsing is induced in tissues challenged
by high temperature, H2O2, or cadmium. Pulses were coincident with a pronounced transient alkalinization of the matrix and
are therefore not caused by uncoupling protein or by the opening of a nonspecific channel, which would lead to matrix
acidification. Instead, a pulse is the result of Ca2+ influx, which was observed coincident with pulsing; moreover, inhibitors
of calcium transport reduced pulsing. We propose a role for pulsing as a transient uncoupling mechanism to counteract
mitochondrial dysfunction and reactive oxygen species production.
INTRODUCTION
Mitochondria are an essential feature of nearly all eukaryotic
cells, providing the energy transformation capacity that is necessary to maintain and express a large genome (Lane and Martin,
2010). In addition, mitochondria perform many other essential
roles, including provision of carbon skeletons for biosynthesis
(Fernie et al., 2004), synthesis of a number of important cofactors
(e.g., cytochromes, heme, iron sulfur clusters, tetrahydrofolate;
Rebeillé et al., 1997; Meyer et al., 2005; Balk and Pilon, 2011), as
well as occupying a central position in programmed cell death
signaling (Kim et al., 2006; Scott and Logan, 2008). In green plant
tissues, mitochondria are also critically important for efficient
photosynthesis, catalyzing an essential step of the photorespiratory pathway (Maurino and Peterhansel, 2010) and providing a
sink for excess reductant generated by the chloroplast (Yoshida
et al., 2011).
1 Address
correspondence to [email protected].
The authors responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) are: Markus Schwarzländer
([email protected]) and Lee J. Sweetlove (lee.sweetlove
@plants.ox.ac.uk).
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.112.096438
The advent of live-cell fluorescent imaging has shown the
mitochondrial population in a single plant cell to be highly heterogeneous in terms of size, shape, and motility of each mitochondrion (Logan, 2010). The morphological diversity within the
mitochondrial population is also mirrored in the organization of
the mitochondrial genome, which is heteroplasmic and unevenly
distributed among the physically discrete mitochondria (Lonsdale
et al., 1988; Arrieta-Montiel et al., 2009; Woloszynska, 2010).
It is unclear, however, whether this structural heterogeneity
reflects heterogeneity of function, since most studies of mitochondrial bioenergetics and metabolism are based on population measurements. One fundamental functional characteristic
of mitochondria, the electrical potential across the inner mitochondrial membrane, can be assessed in vivo at the level of a
single mitochondrion by quantifying the accumulation of fluorescent lipophilic cations using fluorescence microscopy. Using
such an approach with living neurites, it was first noticed that a
small proportion of mitochondria in a cell undergo spontaneous
fluctuations of membrane potential during which membrane
potential drops by ;20 mV and then returns to the starting value
within 30 s (Loew et al., 1993). This bioenergetic phenomenon
can also be seen in isolated animal mitochondria (Hüser et al.,
1998) and is sometimes referred to as mitochondrial flickering
(Duchen et al., 1998). The underlying cause of mitochondrial
flickering remains a matter of debate and has been suggested to
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involve changes in ATP-synthase activity (Buckman and Reynolds,
2001), mitochondrial anion transport (O’Rourke, 2000), calcium
flux (Duchen et al., 1998; Vergun and Reynolds, 2004, 2005), or
the permeability transition pore (Hüser et al., 1998). Furthermore,
the observation that an initially localized induction of flickering in
a single mitochondrion can propagate in a coordinated fashion
throughout the mitochondrial population (Aon et al., 2003; Kurz
et al., 2010) is suggestive of a mobile cytoplasmic signaling
component that can diffuse between mitochondria. Recently, a
similar dynamic phenomenon, termed superoxide “flashes,” has
been reported at the level of a single mitochondrion using circularly permutated yellow fluorescent protein (cpYFP; Wang
et al., 2008), which appeared to coincide with membrane potential flickers (Wang et al., 2008; Fang et al., 2011). However, the
specificity of the cpYFP probe for superoxide under these circumstances has been challenged (Muller, 2009; Meyer and Dick,
2010; Schwarzländer et al., 2011), raising questions as to the
underlying cause of these flashes.
To date, there is no information on the bioenergetic dynamics
of individual mitochondria in plant cells. Given the extensive heterogeneity of plant mitochondria in terms of genome content,
morphology, and movement and the highly variable environment
experienced by plant cells, we propose that heterogeneity of
mitochondrial function is not only likely, but that it constitutes a
fundamental and unexplored aspect of mitochondrial physiology
and signaling in plants. To address this gap in our knowledge,
we determined the in vivo dynamics of individual mitochondrial
membrane potentials in Arabidopsis thaliana roots. Here, we report
the occurrence of spontaneous transient fluctuations of mitochondrial membrane potential, termed “pulses,” in living plant cells. In
addition, we demonstrate that these pulses are induced by environmental stress. Using isolated mitochondria, we investigate the
mechanism for the measured decrease in membrane potential
during a pulse. Based on this information, we put forward a model
that suggests there is a similar underlying cause for experimental
observations of pulsing phenomena in animal and plant cells and
discuss how fluctuations in membrane potential might play a vital
role in mitochondrial bioenergetics and signaling.
between cells, but within a cell the fluorescence intensities from
individual mitochondria were very similar. Thus, the coefficient of
variation for the ratio of TMRM to GFP signal from individual
mitochondria within a cell ranged from 7.6 to 11.7% (491 mitochondria from n = 9 cells). All organelles that showed a GFP
signal also showed a TMRM signal and vice versa, suggesting
that all mitochondria had established a membrane potential.
Analysis of the TMRM fluorescence intensity of individual
RESULTS
Transient Decrease in Membrane Potential of Single
Mitochondria in Living Root Cells
The membrane potential of mitochondria was assessed in living
plant cells by confocal laser scanning microscopy (CLSM) using
the cationic lipophilic fluorescent dye tetramethyl rhodamine
methyl ester (TMRM). TMRM accumulates reversibly in the
mitochondrial matrix in a Nernstian manner in response to the
inner mitochondrial membrane potential and therefore acts as a
dynamic reporter of mitochondrial membrane potential (Brand
and Nicholls, 2011). Arabidopsis roots expressing a mitochondrially targeted green fluorescent protein (mito-GFP; Logan and
Leaver, 2000) were used to provide a reference image of each
mitochondrion and equilibrated with 20 nM TMRM (Brand and
Nicholls, 2011). TMRM accumulated in mitochondria as indicated by colocalization of TMRM fluorescence with the mitoGFP signal (Figure 1A). The overall TMRM signal varied slightly
Figure 1. Membrane Potential of Individual Mitochondria in Arabidopsis
Root Epidermal Cells.
(A) Colocalization of TMRM (20 nM; red) with mito-GFP (green) and
merged overlay of both channels in two neighboring root cells (cell wall
shows some unspecific signal in the TMRM channel).
(B) Images at three time points of several mitochondria (same color coding
as in [A]); one of which underwent a pulse (indicated by arrow). Bars = 2 mm.
(C) Corresponding fluorescence intensity traces of TMRM (red) and mitoGFP (green) for the mitochondrion highlighted in (B). More than 300 cells
were assessed and similar events were found consistently. A typical
event is shown.
Bioenergetic Pulses in Mitochondria
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mitochondria over time showed that single mitochondria occasionally underwent a spontaneous transient decrease in membrane potential (Figures 1B and 1C; see Supplemental Video
1 online). Occurrences of decrease and recovery of membrane
potential, which we refer to as pulses, took place within a 5- to
50-s timeframe (typically ;20 s) and involved a variable degree
of TMRM loss ranging from ;20% down to background levels
of TMRM fluorescence. In an unstimulated cell, there was an
average rate of 0.15 pulses per 100 mitochondria per minute
(1980 mitochondria from n = 21 cells/roots), although rates were
quite variable between cells, leading to a spread in values (Figure
2, control). During a pulse the GFP fluorescence of the mitochondrion remained constant (Figures 1B and 1C), showing that
the mitochondrion had not simply moved out of the focal plane
of the microscope. Pulses were not an artifact caused by the
presence of TMRM because the same phenomenon was also
observed with the chemically distinct mitochondrial membrane
potential dye 3,39-dihexyloxacarbocyanine [DiOC6(3)] (Matzke
and Matzke, 1986; Liu et al., 1987) (see Supplemental Figure
1 online). Although pulses were rare events under control conditions, a first pulse for any given mitochondrion was often
followed by another pulse within seconds or minutes. In vivo
pulses frequently coincided with a spontaneous and transient
change in shape of the individual mitochondrion involving a slight
shortening of the longitudinal axis and concomitant rounding-up
of the organelle (see Supplemental Video 5 online).
Mitochondrial Pulsing Is Increased by Environmental Stress
To establish whether the extent of mitochondrial pulsing depended on the conditions experienced by the cell, Arabidopsis roots
were exposed to a variety of short- and medium-term abiotic
stresses (Figure 2). The pulsing rate was strongly induced by heat
treatments: nearly 30-fold in response to 428C for 5 min and >10fold in response to 408C for 30 min. Repetitive pulses in individual
mitochondria were common as a result of either regime. Treatments with H2O2 (10 mM) for 5 min also increased pulsing by 18fold compared with the control. Although 10 mM H2O2 is a
significant oxidative challenge, plant cells can detoxify such
concentrations within minutes (Marty et al., 2009; Schwarzländer
et al., 2009). By contrast, the pulsing rate was not significantly
changed in response to short- and medium-term salt treatments
(500 mM NaCl for 10 min or 150 mM NaCl for 60 min) nor to
osmotic treatments (1 M mannitol for 10 min or 300 mM mannitol
for 60 min). Heavy metal exposure caused a 10-fold increase in
pulsing when 3 mM CdSO4 was applied for 60 min, but not in
response to exposure to 5 mM CdSO4 for 10 min. Although
mitochondrial movement was partly inhibited in response to
these stress treatments (data not shown), this was observed to
be reversible; in addition, seedlings grew normally following
transfer to standard culture conditions (data not shown). Together, these data show that pulsing can be strongly stimulated
by certain stresses, while others had no obvious impact.
Increased Pulsing Correlates with Mitochondrial Oxidation
To investigate the relationship between stress treatments and
mitochondrial pulsing further, we assessed the change in mito-
Figure 2. Effect of Abiotic Stress on the Occurrence of Pulsing and
Mitochondrial Redox Status in Arabidopsis Root Epidermal Cells.
(A) The rate of mitochondrial pulsing in epidermal root cells expressing mitoGFP and stained with TMRM (20 nM) in response to heat, oxidative, salt,
osmotic, and heavy metal stress treatments (in 0.53 MS medium). n $ 4 cells/
roots; 85 mitochondria were scored on average per biological replicate. Mean
and 95% confidence band are shown as error bars after back-transforming
from a square root transformation. *P <0.05 and **P < 0.01 (t test).
(B) Mitochondrial redox status in epidermal root cells. Percentage of
oxidized roGFP1 in mitochondria indicating the degree of oxidative
stress in response to the respective abiotic stress treatments in (A). n = 5
roots; error bars = SE, *P # 0.05 and **P # 0.01 (t test).
chondrial redox status in response to the different treatments.
Increased mitochondrial reactive oxygen species (ROS) production, leading to oxidative stress, including oxidation of cellular
redox buffers, such as the GSH pool, is a common outcome of a
wide range of unfavorable conditions. Redox-sensitive GFP
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biosensors provide a route to measure such changes with
subcellular resolution (Meyer et al., 2007; Schwarzländer et al.,
2008). Hence, mitochondrially localized roGFP1 (mt-roGFP1)
was used to assess the impact of the stressors above on
mitochondrial GSH redox status in roots (Morgan et al., 2008;
Schwarzländer et al., 2009). Strikingly, significant changes in the
oxidation of mt-roGFP1 were observed only under those conditions that also caused induction of pulsing (Figure 2B). Both heat
treatments and H2O2 exposure led to strong oxidation of mtroGFP1 in root epidermal cells, while salt and osmotic stress
caused no significant change. This was despite obvious plasmolysis confirming the effectiveness of the treatments, causing
slight detachment of the protoplast from the cell wall for 150 mM
NaCl and 300 mM mannitol, and strong protoplast shrinkage for
500 mM NaCl and 1 M mannitol. Exposure of roots to 5 mM
CdSO4 for 10 min did not cause any oxidation of mitochondrial
roGFP1, but 3 mM CdSO4 for 60 min resulted in significant
oxidation, which suggests that longer incubation times are
required to allow effective Cd2+ uptake even at relatively high
external concentrations. The observed differential oxidation of
mt-roGFP1 in response to different stressors demonstrates that
change in mitochondrial redox status is not a universal feature of
environmental stress. Moreover, the qualitative correlation between induction of pulsing and mitochondrial oxidative stress
under different abiotic stress conditions suggests a close mechanistic link between mitochondrial ROS levels, or redox status,
and pulsing.
5 to 50 s) and in terms of extent of TMRM loss (Figure 3B). These
results suggest that the pulsing phenomena observed in vivo and in
isolated mitochondria share a common underlying mechanism. It
further demonstrates that mitochondria per se can sustain pulsing
in the absence of the cellular context. The high rate of pulsing in
isolated mitochondria could be attributed to either a loss of cellular
factors that might control pulsing or to the high membrane potential
and ROS generation associated with state 2 respiration.
The Use of Inhibitors to Explore the Mechanism of Pulsing in
Isolated Mitochondria
To explore the mechanism responsible for pulsing, we exploited
the isolated mitochondrial system to test a range of mitochondrial effectors. Mitochondrial membrane potential is determined
Isolated Mitochondria Show High Rates of Membrane
Potential Pulsing
The fact that mitochondrial pulsing in vivo is sensitive to environmental stress suggests that pulsing has physiological significance. To understand the role of pulsing and its physiological
impact, it is necessary to understand what causes pulsing at the
biochemical level. For this purpose, we chose to study isolated
mitochondria, which offer practical advantages over in vivo
experiments, including the ability to control the bioenergetic
state through the provision of different substrates, straightforward application of inhibitors, particularly those that cannot be
reliably used in vivo due to insufficient uptake by the plant cell,
and improved quantitative imaging. This is particularly important
in plants where mitochondria in vivo are highly motile, hindering
quantitative imaging of single organelles over a time course,
whereas isolated mitochondria can be immobilized by gentle
centrifugation onto a cover slip. We therefore tested whether
membrane potential pulsing could be observed in isolated mitochondria. Freshly isolated, well-coupled Arabidopsis mitochondria were provided with succinate as respiratory substrate and
100 nM TMRM and imaged by CLSM. Under these conditions,
respiration operates in state 2, as ATP-synthase cannot operate
in the absence of ADP and phosphate. In state 2, membrane
potential is high (allowing TMRM imaging) but the electron
transport chain (ETC) is also reduced, leading to a high rate of
ROS production (Murphy, 2009). All mitochondria showed high
rates of pulsing (Figure 3; see Supplemental Video 2 online).
Pulses in isolated mitochondria displayed similar characteristics
to those observed in vivo, both in terms of pulse interval (typically
Figure 3. Membrane Potential Pulsing in Isolated Arabidopsis Mitochondria.
(A) Mitochondria isolated from mito-GFP seedlings and energized with
succinate (10 mM) in minimal medium containing 100 nM TMRM. Red,
TMRM; green, mito-GFP. Bar = 2 mm.
(B) and (C) Fluorescence intensity traces of six representative mitochondria shown in (A). TMRM signal (B) and corresponding mito-GFP
reference signal (C).
Bioenergetic Pulses in Mitochondria
by the balance of proton pumping by the ETC and the influx of
protons (and other cations) back into the matrix due to transport
mechanisms, such as the ATP-synthase, uncoupling protein,
and a variety of ion channels. In principle, therefore, pulsing
could either be caused by a rapid decrease in ETC activity or by a
rapid electrogenic flux of protons or other ions. Distinguishing
between these mechanisms provided the focus for an inhibitor
screen. To get a rough quantitative measure of pulsing activity,
the TMRM signal for individual mitochondria over a time course
was extracted from the CLSM imaging data. The mean of the
coefficient of variation (mean CV) of the TMRM signals over time
for individual mitochondria were used to determine the effect of
treatments on pulsing (Figure 4). Mitochondria that are inhibited
in pulsing will exhibit lower CV values than those that pulse
dramatically and frequently. A large number of single mitochondria were analyzed for each sample (typically >150 mitochondria
from $2 experimental repeats).
Pulsing is dependent on ETC activity to establish a membrane
potential in the first place. However, respiratory components can
be specifically inhibited or bypassed while maintaining active
proton pumping and a membrane potential. This allows testing
whether the activity of a specific ETC complex is responsible for
pulsing. We compared pulsing rates under five different modes
of electron transport (Figure 4A; see Supplemental Table 1 online)
that engage different combinations of the ETC complexes and
are known to drive differential ROS production. The highest
pulsing activity (mean CV) was observed during succinate-driven
respiration (complex II substrate). Pulsing was significantly less
pronounced when ferrocyanide (complex IV substrate) was used
as substrate, while a combination of the tricarboxylic acid cycle
intermediates, pyruvate and malate, which provide reductant to
complexes I and II, led to intermediate pulsing activity. Bypassing complex III and diverting electron flow to the alternative
oxidase by myxothiazol treatment resulted in reduced pulsing
under pyruvate/malate respiration to the level measured during
ferrocyanide respiration (succinate cannot be used with myxothiazol as the activity of at least one proton pumping complex is
required to maintain a membrane potential). Inhibition of complex IV with potassium cyanide further reduced pulsing to a small
but significant degree. While the mechanistic link between pulsing and mode of electron transport is not clear, pulsing activity
correlates with the ROS production that has been associated
with the different ETC modes (Murphy, 2009). While pulsing
activity was influenced by electron transport mode, pulsing
continued under all combinations of substrates and inhibitors
tested, suggesting that the pulsing mechanism does not involve
changes in activity of any single ETC complex.
There are several mechanisms downstream of electron transport and proton pumping that could lead to pulsing, including (1)
increased proton influx via ATP-synthase or uncoupling protein
(Smith et al., 2004); (2) specific influx of other cations, such as
potassium (Pastore et al., 1999) or calcium (Duchen et al., 1998);
(3) efflux of anions via the inner mitochondrial membrane anion
channel (O’Rourke, 2000); or (4) ionic equilibration due to the
opening of a nonspecific pore, such as the permeability transition
pore (mtPTP) (Hüser et al., 1998; Hüser and Blatter, 1999; Zorov
et al., 2000; Jacobson and Duchen, 2002; for a review of the
mtPTP in plants, see Logan, 2008). Proton translocation by ATP-
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Figure 4. A Screen for Inhibition of Pulsing in Isolated Arabidopsis
Mitochondria.
Pharmacological manipulation of mitochondrial electron transport (A)
and inner membrane channels (B). The mean CV of the TMRM signal of
single mitochondria is used as a measure of pulsing activity. Mitochondria in minimal medium were supplemented with a respiratory substrate,
oligomycin (25 mM), to inhibit ATP-synthase and BSA (0.1% [w/v]) to
sequester free fatty acids that can cause uncoupling.
(A) Pulsing activities during different electron transport modes driven by
either succinate (succ), ferrocyanide (fer), or pyruvate/malate (pyr/mal) as
respiratory substrates and inhibition of complex III or IV by myxothiazol
(myx) or potassium cyanide (KCN), respectively (see Supplemental Table
1 online). n > 450; error bars = SE.
(B) Change of pulsing activity, as measured by fold change in mean CV,
in response to different inhibitors of mitochondrial inner membrane
channels and redox-active compounds (see Supplemental Table 2
online). Atr, Atractosylide; CsA, cyclosporin A; DIDS, dithiocyanostilbene
disulfonate; DTE, dithioerythritol; EGTA 150, 150 mM EGTA; EGTA 300,
300 mM EGTA; Glyb, glyburide; LaCl3, lanthanum chloride; NEM,
N-ethylmaleimide; RuRed, ruthenium red; Ryan, ryanodine; succ, succinate; Temp, Tempol. Respiration was driven by 10 mM succinate. n >
150. Error bars = 95% confidence interval (logarithmic transformation) on
the ratio of mean CV (with effector treatment) to mean CV (in corresponding control experiment). **P # 0.01 (Bonferroni corrected).
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synthase is unlikely as a pulsing mechanism because ATPsynthase is inactive under the control conditions due to the
absence of ADP and the inclusion of the inhibitor oligomycin. To
test some of the other possible mechanisms, pulsing activity was
measured during treatment with 19 pharmacological effectors,
targeting different mitochondrial channels (uncoupling protein,
K+ channel, Ca2+ channels, inner mitochondrial membrane anion
channel, and mtPTP), redox active compounds, and ROS (see
Supplemental Table 2 online), the rationale being that treatments
that lead to a strong decrease, or arrest, of pulsing are likely to
affect mitochondrial components that mediate pulsing. This
screen was limited to treatments that did not strongly affect the
mitochondrial membrane potential and, thereby, TMRM uptake.
Although pulsing activity was changed by a number of treatments, there was no consistent behavior of all the agonists and
antagonists that were expected to influence a specific target
(Figure 4B). Interestingly, nine effectors caused an increase
compared with the control, but only three caused a significant
decrease in mean CV. Lanthanum chloride and mitoQ caused a
close to complete arrest of observable pulsing (see Supplemental Videos 2 to 4 online). Calcium chelation led to a significant
decrease in pulsing with 300 mM EGTA but not 150 mM (Figure
4B), although the total TMRM intensity of mitochondria was
slightly decreased at the higher EGTA concentration. The stimulation of pulsing activity with a broad range of compounds likely
indicates that a wide variety of stimuli can affect mitochondrial
function, which in turn impacts on pulsing indirectly. By contrast,
the strong inhibition of pulsing by lanthanum chloride, a general
competitive calcium channel inhibitor (Knight et al., 1996;
Bernardi, 1999), would be consistent with a more direct interaction with the pulsing machinery and points to a role for Ca2+
ion fluxes. The involvement of Ca2+ is further supported by the
inhibitory effect of EGTA on pulsing. MitoQ is a mitochondrial
antioxidant (Kelso et al., 2001, 2002) and also caused a significant decrease in pulsing (Figure 4B). Interestingly, other antioxidants, such as dithioerythritol and GSH, showed the
opposite effect. This may suggest that the impact of ROS is
specific to a certain chemical species or highly localized to
within the membrane, which is accessible to mitoQ to a greater
extent than to GSH or dithioerythritol (see Møller et al. [2007] for
a review of the diversity of cellular oxidative mechanisms).
Pulses Are Linked to Transient Matrix Alkalinization
We infer from the effector screening that pulsing may be due to
a specific ion flux that can be inhibited by lanthanum, rather
than the opening of a nonspecific pore, such as the mtPTP. A
powerful way of discriminating between the opening of a
nonspecific pore and a specific channel is to measure and
compare the effect on matrix pH. Opening of a nonspecific pore
would allow the equilibration of ions across the inner mitochondrial membrane. Because of the pH gradient across this
membrane, pore opening would cause a decrease in matrix pH.
By contrast, specific electrogenic influx of cations other than
protons (or specific efflux of anions) would cause an increase in
matrix pH. This relationship was first formalized by Peter
Mitchell (Mitchell, 1966): Electrogenic ion flux decreases the
membrane potential and thereby allows increased ETC activity
and increased proton pumping, decreasing proton concentration in the matrix.
Recently, we reported transient events of pronounced matrix
alkalinization (up to >1 pH unit) in single plant mitochondria
(Schwarzländer et al., 2011). This finding was the result of a
careful reassessment of the properties of a cpYFP that, although
originally proposed to be a specific reporter of superoxide (Wang
et al., 2008), is in fact extremely sensitive to pH changes in the
mitochondrial matrix (Schwarzländer et al., 2011). Since the
observed transient increases in cpYFP intensity were of a similar
frequency and duration as membrane potential pulses both in
vivo and in isolated mitochondria, we decided to investigate
possible links between the two phenomena. In root cells of
transgenic Arabidopsis expressing mitochondrially targeted
cpYFP, transient increases in cpYFP fluorescence exactly coincided with a pulse as measured by TMRM fluorescence (Figure
5A; see Supplemental Video 5 online). Temporal coincidence
between pulse and matrix alkalinization was confirmed in isolated mitochondria (Figure 5B; see Supplemental Video 6 online).
Indeed, there was a strict inverse correlation between TMRM
signal intensity and cpYFP signal intensity for each mitochondrion (within the technical limitations set by the maximal scan
speed of 0.5 s per frame and probe response times) (see
Supplemental Figure 2A online). The correlative changes in
fluorescence were not due to direct interaction between the
two fluorescent probes because the cpYFP transients were also
observed in the absence of TMRM (Schwarzländer et al., 2011).
The strict temporal coincidence of events suggests that membrane potential pulses and transient matrix alkalinization events
are manifestations of the same mitochondrial phenomenon.
The Mechanistic Link between Membrane Potential Pulses
and Matrix Alkalinization
The relationship between membrane potential pulses and matrix
alkalinization was dissected to gain further insight into the pulsing
mechanism. First, matrix pH was clamped by nigericin in the
presence of K+. Clamping of pH had no affect on membrane
potential pulses, but coincident changes in cpYFP signal were fully
abolished (see Supplemental Figures 2B and 2C online). Second,
the pH buffering capacity of the matrix was increased by loading
mitochondria with 5 mM phosphate, which strongly attenuated
the increase in cpYFP fluorescence during TMRM pulses, which
themselves were not changed (see Supplemental Figures 2D and
2E online). These experiments clearly show that the pH change is a
consequence, rather than cause, of the decrease in membrane
potential. It was then assessed if increased proton pumping by the
ETC is responsible for the increase in matrix pH. We used
malonate to partially inhibit the ETC at complex II and thereby
limit ETC capacity in isolated mitochondria respiring succinate. By
careful titration, it was possible to identify the highest malonate
concentration (1 mM) that did not affect the establishment of state
2 membrane potential (data not shown). Under these conditions,
membrane potential pulsing still occurred, but the associated
increase in pH was substantially attenuated (Figure 5C). This
experiment demonstrates that matrix alkalinization is caused by
increased proton pumping by the ETC, as a result of a decrease in
membrane potential. Pulses are therefore caused by an ion flux
Bioenergetic Pulses in Mitochondria
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Pulses Coincide with an Increase in Matrix Calcium
Figure 5. Mitochondrial TMRM Transients Are Coincident with an Increase in Matrix pH in Vivo and in Isolated Mitochondria.
(A) A single mitochondrion undergoing a pulse in a living Arabidopsis
root. Normalized fluorescence intensity (488-nm excitation) of mtcpYFP expressed in the mitochondrial matrix (green) and TMRM
intensity (red) are plotted. Confocal images captured at the three time
points are indicated by asterisks (right); green, mt-cpYFP; red, TMRM.
Bar = 2 mm.
(B) mt-cpYFP and TMRM fluorescence dynamics during pulses in each
of two individual mitochondria (1 and 2), energized with succinate (10
mM) in minimal medium.
(C) As in (B) but electron transport capacity was limited by the addition of
1 mM malonate. Representative events are shown.
without equilibration of protons. This means that membrane
potential pulsing cannot be mediated by mechanisms such as
mtPTP, uncoupling protein, or ATP-synthase and suggests that
pulsing is caused by transient opening of a channel specific for
ions other than protons.
We next sought to identify the ion responsible for pulsing. To
determine which cations are able to stimulate an increase in
matrix pH, the effect of different physiologically relevant cations
on cpYFP fluorescence was assessed in populations of energized mitochondria using fluorimetry under the same conditions
under which pulsing was analyzed (Figure 6). The divalent
cations Mg2+ and Ca2+ both caused an increase in cpYFP
fluorescence, whereas equal concentrations of the monovalent
cations K+ and Na+ did not (Figure 6A). This suggests that the
level of electrogenic influx of monovalent cations is insufficient to
alter matrix pH, making influx of monovalent cations an unlikely
cause of the pulsing phenomenon. By contrast, electrogenic
influx of either Mg2+ or Ca2+ can occur at a rate sufficient to
increase matrix pH. Furthermore, the increase in cpYFP signal
was due to alkalinization and not caused by direct interaction
between cpYFP and Mg2+ and Ca2+ since the response was
suppressed after increasing matrix pH buffer capacity by preloading mitochondria with phosphate (Figure 6B). To further
explore a link between Ca2+ and pulsing, we coloaded isolated
mitochondria with TMRM and the calcium sensor Fluo-4 (Gee
et al., 2000). The decrease in TMRM signal during a pulse
coincided with a small (up to approximately one-third) increase in
Fluo-4 fluorescence (Figure 7). The increase in Fluo-4 fluorescence was not due to pH changes of the magnitude estimated to
occur in the matrix during a pulse (see Supplemental Figures 3A
and 3B online) nor to changes in TMRM content of the matrix (see
Supplemental Figure 3C online), ruling out a direct effect of pH on
Fluo-4 or an artifact caused by interaction between Fluo-4 and
TMRM. The fact that Fluo-4 was retained in isolated mitochondria during pulses also provides additional evidence against the
involvement of a nonspecific pore, such as the putative plant
mtPTP. In animals, the pore allows nonspecific passage of
molecules smaller than ;1500 D molecular mass (Bernardi,
1999), and Fluo-4 (molecular mass 737 D) would therefore be
able to pass through. The retention of Fluo-4 during pulses was
independently confirmed using calcein (623 D; data not shown).
The transient increase in Fluo-4 fluorescence suggests that pulses
in the mitochondrial membrane potential are linked to an increase
in the free Ca2+ concentration in the mitochondrial matrix. Based
on these results, influx of small amounts of Ca2+ provides a robust
and consistent mechanistic explanation for pulsing.
DISCUSSION
Pulsing: A Novel Mode of Uncoupling
We observed an uncoupling phenomenon in single mitochondria
in vivo, which is manifested by a sporadic transient decrease in
the membrane potential of individual plant mitochondria.
The extent of the depolarization (measured as a reduction of
TMRM fluorescence in the mitochondria), as well as the duration,
were variable and in this respect are similar to the transient
membrane potential events reported previously (e.g., in toad
myocytes) (O’Reilly et al., 2003). Moreover, the membrane potential pulses are coincident with a substantial increase in matrix
8 of 14
The Plant Cell
pH (up to >1 unit based on the calibration by Schwarzländer et al.,
2011) and, therefore, a concomitant increase in the pH gradient
across the inner mitochondrial membrane, measured both in vivo
and in isolated mitochondria, which suggests specific electrogenic movement of ions other than protons are involved in pulsing.
Is Pulsing Caused by Calcium Flux?
An electrogenic cation influx while the inner membrane remains
impermeable to passive proton movement could cause the
observed decrease in membrane potential and the simultaneous
alkalinization of the matrix resulting from a stimulation of proton
pumping by the ETC. In support of this view, we provide several
lines of evidence that pulsing is linked to cation influx and show
that influx of Ca2+ ions can cause pulsing in isolated mitochondria. Nevertheless, pulsing was observed to continue in isolated
mitochondria in the complete absence of added exogenous
Figure 6. The Effect of Different Cations on cpYFP Fluorescence in
Isolated Mitochondria.
Isolated mitochondria from mt-cpYFP Arabidopsis seedlings in minimal
medium free of added metal ions (0.3 M Suc and 10 mM TES, pH 7.2
[Tris]) undergoing state 2 respiration (by provision with 10 mM Trissuccinate, pH 7.2) were supplemented with 100 mM or 1 mM NaCl, KCl,
MgCl2, or CaCl2. The response of mt-cpYFP fluorescence intensity
(excitation 485 nm/emission 535 nm) was determined in the absence (A)
and presence (B) of 5 mM Tris-phosphate, pH 7.2, which increases matrix
pH buffering capacity. n = 3, error bars = SE, *P # 0.05, and **P # 0.01
(t test). The experiment was repeated three times with similar results.
Figure 7. Membrane Potential Pulses Coincide with a Transient Increase
in Free Calcium Levels in Isolated Mitochondria.
Isolated Fluo-4 loaded wild-type Arabidopsis mitochondria were monitored by confocal microscopy upon energization in the presence of 100
nM TMRM. Intensities of Fluo-4 (green) and TMRM (red) signals from
three single mitochondria are shown. More than 40 mitochondria were
analyzed in three independent experiments, and consistent behavior was
observed. Representative events are shown.
Bioenergetic Pulses in Mitochondria
metal ions (data not shown). This is most likely due to the
presence of trace contaminants in the media constituents and
internal stores of cations that are retained in the mitochondria
during isolation and exported into the intermembrane space
upon energization of the mitochondria to then reenter the matrix
in an electrogenic fashion. Experimental estimations of free matrix
calcium by Fluo-4 were around the Kd of the probe (345 nM; data
not shown), which is consistent with the resting concentrations in
rosette leaf mitochondria at ;200 nM measured in vivo using
aequorin (Logan and Knight, 2003). This concentration range is
sufficient to explain changes in membrane potential of the
magnitude observed. In a simple model, a reduction of TMRM
signal to 10% of its baseline intensity can be caused by an
electrogenic influx of 1.5 3 10220 moles of Ca2+, corresponding
to ;9000 Ca2+ ions (assuming a capacitance of the inner membrane of 1 mF per cm2 [Cramer and Knaff, 1990], a spherical
mitochondrion of 1 mM diameter, and ideal Nernst behavior of
TMRM). The most parsimonious explanation for our current observations is that pulsing of isolated mitochondria involves calcium
cycling between the matrix and the intermembrane space via a
very low number of channels allowing electrogenic ion flux.
Cycling of divalent cations has long been known, in principle, to
cause mitochondrial uncoupling (Mitchell, 1966). The calcium
channel blocker lanthanum increases coupling in our system
(see Supplemental Figure 4 online), providing independent evidence for a calcium cycling mechanism being active in isolated
mitochondria. Chelating calcium in the buffer with 300 mM EGTA
caused pulsing inhibition, but we were unable to test the effect of
even more rigorous chelation of mitochondrial calcium because
very high concentrations of chelator are required to completely
sequester cations in the mitochondrial matrix (Kushnareva et al.,
1999), which caused membrane instability and prevented the
establishment of a measurable membrane potential (data not
shown). Our data demonstrate that influx of Ca2+ is linked to
membrane potential pulsing in isolated mitochondria, but we
cannot rule out the involvement of other ions in vivo. Nevertheless,
we emphasize that the coincidence of the decrease in membrane
potential and increase in mitochondrial matrix pH in vivo provides
direct evidence for selective ion transport as the mechanism for
membrane potential pulsing. The recent identification of a calcium
uniporter of animal mitochondria (Baughman et al., 2011; De
Stefani et al., 2011) may provide a route to directly test the in vivo
involvement of electrogenic calcium transport in plant mitochondrial membrane potential pulsing. However, the lack of effect on
pulsing of Ruthenium Red and Ru360, inhibitors of the calcium
uniporter, may suggest the involvement of a different class of
mitochondrial transporter (Sparagna et al., 1995; Ryu et al., 2010)
that is still to be characterized in plants.
Pulsing May Be Triggered by ROS
Pulsing was induced under mitochondrial oxidative stress in vivo
and during electron transport modes associated with high ROS
release in isolated mitochondria. Furthermore, pulsing was significantly inhibited by preincubation with mitoQ, a mitochondrial
antioxidant (Kelso et al., 2001, 2002), raising the possibility that
ROS may induce pulsing, possibly by activating the ion channel
responsible. While other antioxidants increased pulsing rates,
9 of 14
the specificity and distribution of these compounds is likely to be
different, with mitoQ partitioning into the membrane, and this
might provide an explanation for the differential effects. It is
important to note that there is a possibility that photooxidative
ROS may be generated during excitation of the fluorescent
probes. Four recent studies have found that illumination itself can
influence pulsing-like phenomena in mammalian mitochondria
and bacterial cells (Hüser et al., 1998; Jacobson and Duchen,
2002; Falchi et al., 2005; Kralj et al., 2011), while others
observed no such effect (Wang et al., 2008). To minimize the
impact of photooxidation, we kept illumination to a minimum
and used TMRM concentrations lower than in most previous
studies (see Methods; Hüser et al., 1998; Falchi et al., 2005).
Moreover, membrane potential transients were consistently observed with independent probes [TMRM, DiOC6(3), and mtcpYFP] and different excitation wavelengths, arguing against
probe-specific photooxidative effects. The rarity of pulsing events
in vivo and its induction under stress conditions argues that the
stress treatment is the dominating stimulus under those conditions, rather than artifactual photooxidative effects. Additionally,
the pulsing inhibitor lanthanum increased coupling as measured
using an oxygen electrode, in which no photooxidation occurs.
Together, this suggests that technical photooxidation plays a
minor role in pulsing compared with biological effects.
A Role of Pulsing in Maintenance of Mitochondrial Function
and Quality Control: A Model
Based on our analysis of the pulsing mechanism, and our
knowledge of what triggers pulsing, we propose that one role of
pulsing is to provide a means to avoid excessive mitochondrial
ROS production. We developed a simple model that integrates
abiotic stress signaling with pulsing to avoid excessive generation of mitochondrial ROS (Figure 8). Damage to, or imbalance of,
the ETC leads to increased reduction of ETC complexes and
ROS production. In turn, this triggers the opening of a channel or
transporter capable of electrogenic cation transport (either a
uniporter or an electrogenic exchanger). Electrogenic influx of
cations into the matrix decreases the membrane potential and
simultaneously leads to matrix alkalinization due to increased
ETC proton pumping. In this state, the mitochondrial ETC will
become more oxidized, reducing the rate of ROS production,
and the channel will close allowing the reestablishment of a high
membrane potential. If the fault in the ETC is not repaired, the
pulsing cycle will repeat. This process could counteract excessive ROS production and subsequent oxidative damage to the
organelle. In principle, the influx of calcium into the matrix during
a pulse could also act as a signal. An alternative framework for
signaling is provided by the conditional nature of matrix alkalinization in response to a pulse that could act as a quality control
mechanism for individual mitochondria during stress. The low
matrix phosphate concentration and high ETC capacity of a
healthy, fully functional mitochondrion will lead to pronounced
matrix alkalinization as a result of a pulse. However, in an unhealthy mitochondrion with a low rate of ATP synthesis due to
ETC or ATP-synthase dysfunction, alkalinization would be less
pronounced due to phosphate buffering of pH (phosphate will
accumulate if the rate of its incorporation into ATP is low). pH
10 of 14
The Plant Cell
mitochondrial ETC and thereby increase the frequency of pulsing.
Abiotic stress is also known to trigger release of Ca2+ from intracellular stores, such as the endoplasmic reticulum (Knight et al.,
1996), which may facilitate Ca2+ uptake into individual mitochondria.
A Unified Framework for Pulsing in Plants and Animals
Figure 8. A Model of Mitochondrial Pulsing.
(A) A high membrane potential and a low pH gradient contribute to the
motive force. The rate of electron transport is thermodynamically limited
by proton motive force, leading to ROS release.
(B) A channel opens in an ETC-dependent fashion (possibly due to ROS),
allowing influx of divalent cations, such as calcium, into the matrix. The
charge influx uncouples the mitochondrion, lowers the membrane potential, and removes the constraint on the rate of electron transport,
thereby decreasing ROS release. The rate of electron transport
increases, as does the rate of proton pumping, leading to increased
matrix pH.
(C) The channel closes and cations are electroneutrally exported from
the matrix in exchange for protons (directly or indirectly), reestablishing
matrix pH and membrane potential. Note that charge stoichiometry is
not considered in the schematic representation.
transients could be sensed by a pH-sensitive signaling protein
(e.g., a kinase) mounting a constitutive signal in response to a
short lived pH transient. Under stress, this signal could provide a
mechanism for functional mitochondria to adjust to their
changed environment by synthesis of mitochondrially encoded
ETC subunits that are required for acclimation. This would
represent an alternative mechanism to the redox control of
mitochondrial genome expression that is predicted by the
colocation for redox regulation hypothesis (Allen, 2003). Abiotic
stress would increase the likelihood of faults developing in the
Mitochondrial membrane potential pulses have also been observed in animal mitochondria. However, several contradictory
explanations have been reported to account for the phenomenon. Pulsing has been linked to ATP-synthase activity (Buckman
and Reynolds, 2001), mitochondrial anion transport (O’Rourke,
2000), and calcium and sodium flux (Duchen et al., 1998; Vergun
and Reynolds, 2004, 2005; Poburko et al., 2011). However, most
frequently, pulsing in animal mitochondria is attributed to opening of the mtPTP (Hüser et al., 1998; Hüser and Blatter, 1999;
Zorov et al., 2000; Collins et al., 2002). Although it is entirely
conceivable that pulses could be caused by different and mechanistically independent underlying events, we believe that the
data presented here resolve a number of the current uncertainties. We simultaneously monitored the dynamics of both
components of the proton motive force, membrane potential,
and pH gradient (measured as matrix pH) in single mitochondria.
This allowed the observation of a strong dynamic interdependency between the two parameters (see Supplemental Figure 2A
online), which becomes particularly apparent during pulsing. As
a result, greater bioenergetic detail is now available to reconstruct the mechanism as an important step toward a unifying
concept of pulsing.
The observation that matrix alkalinization is attenuated by the
buffering effect of free phosphate may also resolve the contradictory explanations that have been put forward for mt-cpYFP
flashes in animal mitochondria (Fang et al., 2011; Ma et al., 2011;
Wei et al., 2011). When characterizing mt-cpYFP flashes in living
cardiomyocytes, Wang et al. (2008) and Fang et al. (2011)
observed that each flash coincided with a membrane potential
pulse. This was interpreted as a transient opening of the mtPTP,
causing a burst of superoxide and resulting in a cpYFP flash.
However, our recent finding that cpYFP is highly pH sensitive in
the mitochondrial matrix (Schwarzländer et al., 2011) cast into
doubt the ability of cpYFP to report mitochondrial superoxide
flashes (although dynamic mitochondrial ROS and redox
changes during pulses remain possible and even likely). Instead,
matrix alkalinization is the predominant feature reported by
mt-cpYFP. The amplitude (and therefore the detected frequency)
of mt-cpYFP flashes is strongly dependent on the pH buffering
capacity of the matrix and the proton-pumping capacity of the
ETC. We have shown that free phosphate in the matrix strongly
buffers against the pH change during a pulse. It is therefore
relevant that several effectors of the mtPTP (such as bonkrekic
acid or atractyloside) are actually inhibitors of the adenine
nucleotide carrier, postulated to be one of the mtPTP components. The effect of these inhibitors on mt-cpYFP pulsing observed by Wang et al. (2008) could be explained by pH buffering,
as a disruption of ATP/ADP homeostasis in the matrix will
necessarily impact on matrix phosphate levels. Variability in the
coincidence of pulses and flashes in vivo therefore probably
reflects variation in matrix buffering and ETC capacity between
Bioenergetic Pulses in Mitochondria
mitochondria. A mitochondrion with highly active electron transport and ATP-synthesis will show significant matrix alkalinization
upon a pulse, while mitochondria with dysfunctional ETC and
ATP-synthesis will show little pH response due to a relatively high
matrix phosphate concentration and a relatively slow rate of
proton pumping. Our discovery that single plant mitochondria
can undergo transient and pronounced matrix alkalinization
events (Schwarzländer et al., 2011) has now been matched by
experiments using astrocytes (Azarias and Chatton, 2011) and a
different pH sensing protein, SypHer, which is also based on a
cpYFP (Poburko et al., 2011). The dynamic pH events in astrocytes
also coincided with a transient decrease in membrane potential.
This suggests that specific electrogenic ion flux across the mitochondrial inner membrane as a causative principle for membrane
potential pulsing is conserved between plants and animals.
Conclusions and Perspectives
Pulsing of the membrane potential in individual mitochondria
provides an autonomous uncoupling mode. We present a mechanistic framework to explain how those dramatic bioenergetic
events occur and suggest a role for pulsing in maintaining mitochondrial functionality and quality control. Beyond its occurrence
in plants, electrochemical pulsing also occurs in animal mitochondria and has recently been demonstrated in bacteria (Kralj et al.,
2011). This latter observation raises the possibility that pulsing
may have already been a characteristic of the prokaryotic endosymbiont and has remained conserved in modern mitochondria
across the eukaryotic domain.
METHODS
Chemicals and Plant Material
Chemicals and inhibitors were purchased from Sigma-Aldrich. TMRM,
DiOC6(3), Fluo-4 acetoxymethyl ester (AM), and calcein AM were bought
from Invitrogen. Arabidopsis thaliana Columbia-0 was the background for
all transgenic lines: mito-GFP (Logan and Leaver, 2000), mt-roGFP1
(Schwarzländer et al., 2008), and mt-cpYFP (Schwarzländer et al., 2011).
For in vivo microscopy of roots, seedlings were cultivated for 7 to 10 d on
vertical half-strength Murashige and Skoog (MS) medium (Murashige and
Skoog, 1962) agar plates under long-day conditions. Epidermal cells from
the upper elongation zone and lower differentiation zone were imaged.
For mitochondrial isolations, seedlings were cultured in hydroponic pots
for 14 d as described before (Sweetlove et al., 2007).
Isolation of Mitochondria
Mitochondria were freshly isolated from 14-d-old Arabidopsis seedling
cultures as described before (Sweetlove et al., 2007). Coupling was
measured in a Clark-type oxygen electrode (Oxygraph; Hansatech Instruments) at 208C as described before (Sweetlove et al., 2002) either in
basic incubation medium (0.3 M Suc, 5 mM KH2PO4, 10 mM TES, 10 mM
NaCl, 2 mM MgSO4, 0.1% [w/v] fatty acid–free BSA, pH 7.2 [KOH]) for
quality control or in minimal medium (0.3 M Suc, 10 mM TES, pH 7.2,
adjusted with KOH or Tris) for pulsing assays.
11 of 14
solution under the same conditions. All treatments were performed in the
presence of TMRM. For Fluo-4 loading, isolated mitochondria were
incubated in 20 mM Fluo-4 AM in minimal medium for 60 min at room
temperature (to allow sufficient accumulation and internal hydrolytic
cleave of the AM-ester groups to release free dye in the matrix) and
washed twice. Mitochondria were immobilized onto round glass cover
slides by centrifugation (2000g, 5 min, 48C) in minimal medium. Immediately before imaging, mitochondria were energized by addition of medium
containing succinate as respiratory substrate (if not stated otherwise) and
labeled with 100 nM TMRM. Effectors were added as indicated and
samples were imaged using conditions described before (Schwarzländer
et al., 2011). Microscopy was performed using a Zeiss LSM510 META
confocal microscope (Carl Zeiss MicroImaging) equipped with laser lines
for 405-, 458-, 488-, and 543-nm excitation. Excitation and emission
wavelengths were selected for the different probes as follows: TMRM,
543/565 to 615 nm; mito-GFP, 458/505 to 530 nm; roGFP1, 405,488/505
to 530 nm; cpYFP, 488 and 405/505 to 530 nm; DiOC6(3) and Fluo-4, 488/
505 to 530 nm. Imaging was performed with a 363 lens (Zeiss PlanApochromat 363, numerical aperture = 1.4, oil) and a 340 lens for
roGFP1 (Zeiss C-Apochromat 340, numerical aperture = 1.2, water) in
multitrack mode with line switching between channels. The pinhole
diameter was 2 airy units. Laser output was kept under 2% of maximal
power equaling <2.5 mW for any wavelength as measured in the
defocused beam at the backplane of the objective. Pixel spacing was
typically 50 to 100 nm, pixel dwell time was 1 to 2 ms, and image size was
512 3 512 pixels. Time series were collected with time intervals between
2 and 5 s if not indicated otherwise.
Data Analysis
CLSM time series imaging data of mitochondria were analyzed using a
custom Matlab program (available from M.D.F. on request). For in vivo
imaging, data spatial regions corresponding to single mitochondria were
manually selected, and average values from a small (typically 5-pixel radius)
circular region extracted for each time point following background subtraction. Analysis of mt-roGFP1 images was performed as described before
(Morgan et al., 2008; Schwarzländer et al., 2008, 2009). The percentage of
oxidized roGFP was calculated using in vivo calibration. For time series of
isolated mitochondria, the positions of individual mitochondria were automatically identified as local maxima following convolution with a 2-D
Mexican Hat kernel, scaled to match the typical mitochondrial size. Average
fluorescence intensities for each channel were sampled from a circular
region corresponding to half the radius of the mitochondrion from this point
to generate time course traces for a large number of mitochondria (>30) per
time series. CVs (the ratio of signal standard deviation to signal mean) were
recorded for each mitochondrial time series. The mean CV of a large
number of individual mitochondria was recorded as a summary statistic
for its degree of pulsing. Signal traces from frequently and strongly
pulsing mitochondria had high CVs (as a result of repeated large
fluctuations). Significance was tested using a two-sample unpaired t
test for the ETC screen and a one-sample location test (z-test), against
unity, of the ratio of treated mean CV to control mean CV for the
pharmacological screen. Significance values were Bonferroni corrected
for the multiple comparisons performed. Meaningful error bars were
derived for highly skewed pulsing frequency data by appropriate transformations (Figure 2A, square-root transformation; Figure 4B, logarithmic
transformation), and the back-transformed mean and 95% confidence
bands are shown.
Dye Loading and CLSM
Fluorimetry
Whole seedlings were equilibrated in 20 nM TMRM (in 0.53 MS medium)
for $15 min before confocal imaging of roots bathed in fresh probe
Mitochondria were suspended in minimal medium in 96-well plates. The
total assay volume was 200 mL. A Beckman Coulter DTX880 multimode
12 of 14
The Plant Cell
detector was used with filters for 485-nm (cpYFP, Fluo-4) or 535-nm
(TMRM) excitation and 535-nm (cpYFP, Fluo-4) or 595-nm (TMRM)
emission, respectively. Data were background-subtracted and processed in Microsoft Excel.
Received February 2, 2012; revised February 2, 2012; accepted February
16, 2012; published March 6, 2012.
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Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. DiCO6(3) Is a Dynamic Membrane Potential
Probe Enabling the Visualization of Pulses in Single Mitochondria.
Supplemental Figure 2. Mt-cpYFP Dynamics during Pulses, at
Clamped Matrix pH and Increased Matrix pH Buffering Capacity.
Supplemental Figure 3. Effect of pH and TMRM on Fluo-4 Fluorescence.
Supplemental Figure 4. The Effect of Lanthanum on Oxygen Uptake
of Isolated Arabidopsis Mitochondria.
Supplemental Table 1. A Screen of Respiratory Substrates and
Inhibitors to Determine the Influence of ETC Mode on Pulsing.
Supplemental Table 2. A Pharmacological Screen of Mitochondrial
Inner Membrane Channel Inhibitors and Redox-Active Compounds to
Determine Their Effect on Pulsing.
Supplemental Video 1. Pulses of Single Mitochondria in an Arabidopsis Root Epidermal Cell.
Supplemental Video 2. Pulsing in Isolated Arabidopsis Mitochondria.
Supplemental Video 3. Pulsing in Isolated Arabidopsis Mitochondria
in the Presence of 1 mM LaCl3.
Supplemental Video 4. Pulsing in Isolated Arabidopsis Mitochondria
in the Presence of 1 mM mitoQ.
Supplemental Video 5. Coincidence of TMRM Pulse, Change in
Shape, and Increase in mt-cpYFP Fluorescence in a Single Mitochondrion in an Arabidopsis Root Epidermal Cell.
Supplemental Video 6. Coincidence of TMRM Pulse and Increase in
mt-cpYFP Fluorescence in Isolated Arabidopsis Mitochondria.
ACKNOWLEDGMENTS
We thank Mike Murphy (Medical Research Council Mitochondrial Unit,
Cambridge, UK) for excellent experimental advice and Andrew Halestrap (University of Bristol, Bristol, UK), Steve Roberts (University of
Oxford, Oxford, UK), and George Ratcliffe (University of Oxford, Oxford,
UK) for stimulating discussions that significantly strengthened this work.
I.G.J. and N.S.J. acknowledge funding by the Biotechnology and
Biological Science Research Council (BB/D020190/1) and D.C.L. by
the Natural Sciences and Engineering Research Council of Canada.
M.S. was funded by New College Oxford through a Weston Junior
Research Fellowship.
AUTHOR CONTRIBUTIONS
M.S. designed and performed the research, analyzed the data, and
cowrote the article. D.C.L. coperformed and cosupervised research
and helped to write the article. I.G.J. and N.S.J. provided analytic and
computational tools and analyzed pulsing screen data. A.J.M. cosupervised research and helped to write the article. M.D.F. cosupervised
research, contributed the custom imaging analysis software, and
consulted on the data analysis. L.J.S. codesigned and supervised
research and cowrote the article.
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Pulsing of Membrane Potential in Individual Mitochondria: A Stress-Induced Mechanism to
Regulate Respiratory Bioenergetics in Arabidopsis
Markus Schwarzländer, David C. Logan, Iain G. Johnston, Nick S. Jones, Andreas J. Meyer, Mark D.
Fricker and Lee J. Sweetlove
Plant Cell; originally published online March 6, 2012;
DOI 10.1105/tpc.112.096438
This information is current as of June 14, 2017
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