Rat liver GTP-binding proteins mediate changes in
mitochondrial membrane potential and organelle fusion
Jorge Daniel
Cortese
Department of Cell Biology and Anatomy, University of North
Carolina, Chapel Hill, North Carolina 27599-7090
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ABSTRACT |
The variety of mitochondrial morphology in healthy and diseased
cells can be explained by regulated mitochondrial fusion. Previously, a
mitochondrial outer membrane fraction containing fusogenic, aluminum
fluoride (AlF4)-sensitive
GTP-binding proteins (mtg) was separated from rat liver (J. D. Cortese,
Exp. Cell Res. 240: 122-133,
1998). Quantitative confocal microscopy now reveals that mtg
transiently increases mitochondrial membrane potential (
) when added to permeabilized rat hepatocytes
(15%), rat fibroblasts (19%), and rabbit myocytes (10%). This large
mtg-induced 
increment is blocked by fusogenic GTPase-specific
modulators such as guanosine 5'-O-(3-thiotriphosphate),
excess GTP (>100 µM), and
AlF4, suggesting a linkage between

and mitochondrial fusion. Accordingly, stereometric analysis
shows that decreasing 
or ATP synthesis with respiratory inhibitors limits mtg- and
AlF4-induced mitochondrial fusion. Also, a specific G protein inhibitor
(Bordetella
pertussis toxin) hyperpolarizes
mitochondria and leads to a loss of
AlF4-dependent mitochondrial
fusion. These results place mtg-induced 
changes upstream of
AlF4-induced mitochondrial fusion,
suggesting that GTPases exert 
-dependent control of the fusion
process. Mammalian mitochondrial morphology thus can be modulated by
cellular energetics.
confocal microscopy; G protein; hepatocyte; pertussis toxin; signal
transduction; single-cell imaging
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INTRODUCTION |
MITOCHONDRIA EXHIBIT variable morphology in both
nonvertebrate and vertebrate cells, from spherical organelles to
tubular networks (4). The dynamics of this mitochondrial pleomorphism suggested that membrane fusion events maintain mitochondrial
morphology. Mitochondria will thus behave as a compartment in various
states of fragmentation, which is likely under specific cellular
regulation (25, 44). In Saccharomyces
cerevisiae, mitochondrial fusion has
been repeatedly shown since the pioneering study of Hoffmann and Avers
(27). Mitochondrial fusion can be detected during yeast mating (35) and
at the beginning of meiosis (33) and appears to be regulated by
metabolic growth conditions. A number of genes and gene products
associated with this process are being isolated (5). Some of these
proteins bind to the cytoskeleton (e.g., Mdm1p, an intermediate
filament-like protein that interacts with mitochondria; Ref. 16) or
relate to the expanding family of GTPases involved in controlling
membrane fusion (e.g., Mgm1p/Dnm1p is a dynamin-like protein that
maintains normal mitochondrial network morphology; Refs. 28, 41). Thus
the yeast mitochondrial compartment or "chondrioma" fragments and
reassociates in concert with cell cycle events, like the nuclear
membrane or endoplasmic reticulum. In higher organisms,
Drosophila possesses a 130-kDa GTPase
that participates in the formation of a fused mitochondrial aggregate
(Nebenkern) during stages of sperm development (21). Mitochondrial
fusion can also occur during type II pneumocyte development (49) and to
form the skeletal muscle mitochondrial reticulum (2), and it may be
involved in fast mitochondrial DNA redistribution observed in human
HeLa cells (24). Human liver disease or cirrhosis leads to pleomorphic
mitochondria and to the formation of megamitochondria, possibly through
a mechanism involving mitochondrial fusion (45).
I have shown that a protein fraction derived from rat liver outer
mitochondrial membranes (11) and containing a single 60-kDa G
protein-like molecule (mitochondrial GTP-binding protein fraction or
mtg; Ref. 9) induces extensive mitochondrial fusion under aluminum
fluoride (AlF4) stimulation.
These studies (9, 11) revealed that spheroidal rat liver mitochondria
have the potential to form a three-dimensional tubulovesicular network
resembling the giant yeast mitochondrion (9). They support the view of mitochondria as a virtual cellular compartment for mammalian cells and
suggest that members of the GTPase superfamily assume control of the
regulation of mitochondrial morphology.
Another aspect of mitochondrial fusion that emerged from these studies
was change in the intramembrane mitochondrial configuration (9) that is
consistent with increased utilization of mitochondrial electrochemical
potential (20). In the current study, the relationship between mtg
activities and cellular energetics is further explored. In
permeabilized cells, an effect of mtg on mitochondrial membrane potential (
) is demonstrated. Transient 
changes are
sensitive to guanine nucleotides and
AlF4, as were our in vitro (11)
and cell level (9) mitochondrial membrane fusion assays (MFA), suggesting the involvement of previously detected fusogenic GTP-binding proteins. As a confirmation of this dual role of mitochondrial GTPases,
treatment of cells with Bordetella
pertussis toxin (PTX) affects both

and mitochondrial fusion. The evidence presented here reveals a
GTP-binding protein-mediated connection between energy-producing
reactions and mitochondrial fusion.
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MATERIALS AND METHODS |
Materials.
Collagen (type VII, rat tail), GTP, guanosine
5'-O-(3-thiotriphosphate)
(GTP
S), guanosine
5'-O-(3-thiodiphosphate)
(GDP
S), PTX (from B.
pertussis), and streptolysin O (from
Streptococcus pyogenes) were purchased from Sigma
Chemical (St. Louis, MO). HEPES and collagenase D were from Boehringer
Mannheim (Indianapolis, IN), and tetramethylrhodamine methyl ester
(perchloric salt; TMRM) was from Molecular Probes (Eugene, OR).
Waymouth's MB 752/1 medium, DMEM, Joklik's modified MEM-medium 199, and FCS were obtained from GIBCO BRL Life Technologies (Gaithersburg, MD).
Mitochondrion-derived preparations.
Liver mitochondria were isolated from male Sprague-Dawley rats
according to standard protocols (12, 40). Solubilization of outer
membrane proteins and GTP affinity chromatography were used to separate
a protein fraction that is composed of a few mitochondrial GTP-binding
protein candidates and is thus named mtg fraction. (9, 11). A protein
mixture that does not bind to GTP agarose and lacks any immunoreactive
GTP-binding protein (9) was eluted as an initial (first) flow-through
fraction and is thus called G1 (11).
Cell culture.
Rat hepatocytes were prepared by collagenase digestion of perfused
liver (26), resuspended in Waymouth's MB-752/1 medium containing 27 mM
Na2CO3,
2 mM glutamine, 100 nM insulin, 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10 mM dexamethasone (medium
W), and plated at a density of
200,000 cells/ml as described by Cortese (9). Rat embryo fibroblasts
(REF-52) were grown at 37°C (5%
CO2 atmosphere) in DMEM
supplemented with 10% FCS, 50 U/ml penicillin, and 50 µg/ml
streptomycin (medium
D) to 50-75% confluence. Adult
rabbit cardiac myocytes were isolated by multiple enzyme digestion
(collagenase-hyaluronidase plus trypsin; Ref. 36), preserved in
nutrient medium (1:1 mixture of Joklik's modified MEM and medium 199)
supplemented with 0.05 U/ml insulin, 1 mM creatine, 1 mM octanoic acid,
1 mM taurine, 10 U/ml penicillin, and 10 µg/ml streptomycin
(medium
M), and used for experiments within
6 h. Rat myocytes did not tolerate permeabilization protocols well and
thus could not be used in our experiments.
Cell permeabilization and mitochondrial labeling.
Mitochondria were labeled with TMRM, a membrane-permeant cationic
fluorophore (6), as previously described by Cortese (9). Cells grown on
1% collagen-coated glass coverslips in medium
W (hepatocytes) or medium
D (rat embryo fibroblasts) were incubated with 0.6 µM
TMRM for 20 min at 37°C, placed in a 0.5-ml custom-made chamber,
and then mounted on the microscope stage in medium
W or medium D
containing 0.15 µM TMRM, thus maintaining an equilibrium distribution
of the fluorophore (6).
Permeabilization with streptolysin O was carried out by a modification
of standard methods (1) described by Cortese (9). FCS, which inhibits
the permeabilization reaction, was rinsed out of culture medium with a
permeabilization buffer containing 20 mM MOPS (pH 7.0), 250 mM
mannitol, 3 mM MgCl2, 3 mM ATP, 5 mM reduced glutathione, and 1 mM dithiothreitol. The optimal
concentration of streptolysin O was 25-50 U/ml. Permeabilization
kinetics were followed by phase-contrast microscopy, and cells with
apparent blebbing were not used for our assays. To stop the
permeabilization reaction, the cells were repeatedly washed and
incubated in FCS-containing medium W
or medium D (0.15 µM TMRM). During
the experiment, temperature was maintained using an air curtain
incubator. Rabbit myocytes were very sensitive to the addition of FCS
and streptolysin O, and thus they were permeabilized with a different
protocol. Cells were labeled in medium
M containing 0.6 µM TMRM for 20 min at 37°C,
rinsed with the permeabilization buffer described above diluted 1:1
with medium M for 2 min at 25°C,
and permeabilized using 10 U/ml streptolysin O. Permeabilization was
stopped by three washes with medium M
containing 0.15 µM TMRM. AlF4
complexes were generated in situ by incubating permeabilized cells for
5-10 min at 25°C in the appropriate medium containing 0.15 µM TMRM, 50 µM AlCl3, and 5 mM
KF (9).
Mitochondrial imaging and morphology analysis.
Digitized fluorescence images were acquired as described by Cortese
(9), using a Bio-Rad MRC-600 laser scanning confocal microscope
(argon-krypton laser) mounted on a Nikon Diaphot inverted microscope.
TMRM fluorescence with excitation at 568 nm was collected through a
595-nm long-pass barrier filter (0.3-1% neutral density filter).
For stereological measurements of mitochondrial morphology (9),
confocal images were processed using MetaMorph image analysis software
(Universal Imaging, West Chester, PA) to measure the ratio between the
major and minor diameters of mitochondria, i.e., mitochondrial
ellipticity (
; Ref. 9). This parameter is independent of the
magnification of the microscope and gives an estimate of true
mitochondrial fusion both for confocal and electron micrographs (9);
= 1 for a sphere, and
> 1 for ellipsoids. Mitochondrial ellipticity data are represented as
frequency distributions (see
Fig. 11), and their differences were statistically analyzed by
distribution-independent, nonparametric rank methods (8, 42). Levels of
significance were assigned for sets of two (Mann-Whitney test; Ref. 42)
or multiple (Kruskal-Wallis test; Ref. 8) independent samples, using
equations corrected for tied rank values and suitable for comparison of
differences between means or variances.
Determination of 
.
Electrical 
was estimated from the equilibrium distribution of
TMRM between mitochondria and bulk medium (6, 32). Our protocol uses
TMRM-labeled cells equilibrated with 0.15 µM TMRM, thus maintaining a
steady-state equilibrium distribution of fluorophore during the
measurements. TMRM then meets the requirements of the Nernst equation
(i.e., a thermodynamic potential-concentration relationship), and,
since the fluorescence of TMRM is proportional to dye concentration,
fluorescence intensities can be correlated with electric membrane
potentials. Because TMRM accumulates up to 10,000-fold in mitochondria
(32), we employed logarithmic amplifiers to acquire 
images with
a compressed grayscale (see Fig.
1A). For each image, obtained
against its own background set at a low pixel intensity (~0.5 units),
the average fluorescence intensity for the extracellular compartment
and cellular mitochondrion-containing regions was measured within a
very small area (9 × 14 pixels), optimized to be comparable in
size to a single mitochondrion. At least 25 sets of quantified areas
were averaged to estimate 
(in mV) using a modified Nernst
equation (6)
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where
[TMRM]mt is the
concentration of TMRM in the mitochondrial (intracellular) compartment,
a value proportional to its measured fluorescence and to the
logarithmic input voltage to the photomultiplier circuit (6).
[TMRM]ex is the
extracellular value, also expressed as a function of photomultiplier
readings. The values of 
obtained for mitochondria vary between
140 and
200 mV (see Fig. 1); 
for the nuclear
region is calculated as a control in each image, and it should be
approximately
80 mV (6). Confocal images destined for 
measurements were obtained under conditions (0.3% neutral density
filter, ×100 Nikon Fluor objective with numerical aperture = 1.3, zoom = 1.2, Kalman average with n = 4)
that preclude photobleaching for 12 averaged images. Long-term, free
radical-mediated photodynamic damage was not apparent from observation
of cells kept in culture for up to 1 h after imaging (not shown).
Sixteen averaged images can be collected from a single hepatocyte
without cellular photodamage (see Fig. 4 in Ref. 9). To display 
distributions, we use user-defined lookup tables that convert pixel
intensity to pseudocolor. The BASIC program that builds them for the
Bio-Rad COMOS software was kindly provided by Dr. J. J. Lemasters
(University of North Carolina, Chapel Hill, NC).
To represent results from 
measurements, we use 
distributions (see Figs. 1B and 7),
pseudocolor maps (see Fig. 2), fluorescence or pixel intensity
distributions (analogous to those obtained for cell populations by flow
cytometry; see Fig. 3), or numerical 
values (see Figs. 6
and 9). Variations of the 
absolute value originating in
variation of TMRM labeling between coverslips, or from the calibration
of logarithmic images, were avoided by presenting numerical values from
paired measurements before and after an experimental treatment. Because
the distribution of 
values is sometimes less Gaussian than that
of pixel intensities (cf. Figs. 1B and
3), we calculated standard deviations and confidence intervals for TMRM
fluorescence histograms based on pixel intensity values (later
converted to 
). To compare nonnormal 
distributions, we
used a nonparametric rank test (Wilcoxon-Mann-Whitney; Ref. 42) that
gives a level of significance P. Also,
the small observation window selected (9 × 14 pixels) minimized
the errors introduced by dark intermitochondrial areas. Under similar

imaging conditions, windows smaller than 13 × 19 pixels
gave essentially identical 
distributions (not shown). The
standardized approach to 
measurements applied here reduces the
error of this assay to <5%.
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RESULTS |
Outer membrane-derived GTP-binding proteins affect

.
Confocal images of TMRM-labeled hepatocytes allow easy distinction of
individual mitochondrial profiles (Fig.
1A).
The pixel fluorescence intensity of these images can be converted to

values (see Methods), and

distributions can be readily obtained for each cell (Fig.
1B). We found that the addition of
mtg fraction to permeabilized rat hepatocytes (i.e., semi-intact cells;
Ref. 1) substantially increases 
-dependent mitochondrial TMRM fluorescence (cf. Fig. 2,
A and
B). The average value of 
for control hepatocytes was
155.2 ± 5.2 mV
(n = 50); permeabilization with
streptolysin O did not affect this value (not shown). Addition of mtg
fraction to control nonpermeabilized cells does not change 
(see
Fig. 6A), and 
increased when
this fraction was added to permeabilized hepatocytes, to an mtg-induced

value (
mtg) of
179.1 ± 6.1 mV (n = 45; see
Figs. 2B and
6A). The absolute mtg-induced 
change varies between cells (see Figs. 6,
A and B, and 9), but a 20- to 30-mV 
increase occurred consistently. Addition of an outer membrane-derived
subfraction that does not contain GTP-binding protein candidates (G1
fraction; Refs. 9, 11) to permeabilized hepatocytes did not affect

(untreated,
143.6 ± 7.4, n = 34; G1 treated,
142.2 ± 3.4, n = 32).

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Fig. 1.
Measurements of mitochondrial membrane potential ( ) by confocal
microscopy. A: confocal image of a
tetramethylrhodamine methyl ester (TMRM)-labeled intact rat hepatocyte.
Bar, 10 µm. B:
distribution histogram of  values calculated from TMRM
fluorescence intensities (n = 75 measurements).
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Fig. 2.
Hyperpolarizing effect on  of outer mitochondrial
membrane-derived protein fraction containing putative mitochondrial
GTP-binding proteins (mtg protein fraction).
A and
B: confocal images of a TMRM-labeled,
streptolysin O-permeabilized rat hepatocyte before
(A) and after
(B) treatment with mtg fraction (250 µg/ml). Pseudocolor bar shows values of  (in mV) corresponding
to colors in both digitized images. C
and E: confocal images of
TMRM-labeled, streptolysin O-permeabilized rat fibroblasts
(C) and rabbit myocyte
(E).
D and
F: permeabilized rat fibroblasts
(D) and rabbit myocyte
(F) shown in
C and
E were treated with 250 µg/ml mtg
fraction. Pseudocolor bar shows  values for
C-F.
Bars, 10 µm.
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The mtg-induced hyperpolarization was also apparent for cultured
cells derived from tissues other than liver (Fig. 2,
C-F). Addition of mtg fraction to control permeabilized rat embryo
fibroblasts (
=
156.5 ± 8.5 mV,
n = 25; Fig.
2C) or adult rabbit myocytes (
=
153.3 ± 9.2 mV, n = 30;
Fig. 2E) caused a 
increase
(fibroblast 
mtg =
185.7 ± 9.7 mV, n = 25;
myocyte 
mtg =
168.9 ± 6.2 mV, n = 30; Fig. 2,
D and
F). The smaller relative 
effect of the mtg fraction in rabbit myocytes (10%), compared with
19% for rat fibroblasts and 15% for rat hepatocytes, may be related
to interspecies differences or the additional constraints on diffusion of mtg proteins posed by the actomyosin muscle cytoskeleton. Conditions for treatment with streptolysin O were also different for myocytes, as
their plasma membrane was easily damaged, leading to compromised cell viability.
The effect of the mtg fraction on 
can be ascribed to the
mitochondrial compartment (Fig. 3).
Comparison of pixel intensity distributions between control
permeabilized hepatocytes and mtg-treated permeabilized hepatocytes
(Fig. 3) showed that TMRM fluorescence originating in the nuclear
region remains constant after treatment with mtg fraction, whereas TMRM
fluorescence from cytosolic mitochondrion-containing regions increases
after mtg addition. Also, treating permeabilized cells with mtg
fraction does not lead to changes in cytosolic TMRM fluorescence (see
Fig. 5), which likely originates from nonspecific binding of TMRM to
cellular membranes. A low 
value of
36 mV was obtained
from this fluorescence contribution, comparable to basal 
values
for uncoupler-treated, depolarized mitochondria (not shown). Taken
together, our results indicate that the shift in fluorescence observed
upon mtg treatment is not a background artifact. Other possibly
nonspecific actions of the mtg fraction and buffer on 
can be
ruled out because different buffers were used for each cell type
assayed, the mitochondrial G1 fraction of comparable origin to mtg
lacks this effect, and mtg effectiveness in the 
assay is greatly
diminished by repeated freezing and thawing or by extended incubation
at temperatures >4°C (not shown). The mtg-induced shift of
TMRM-dependent mitochondrial fluorescence was statistically significant
(P = 0.01; Fig. 3). Analysis of a
20-experiment set revealed a consistent, significant 
change (16 were significant at P = 0.01 and 4 at
P = 0.05).

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Fig. 3.
Specific effect of mtg fraction on mitochondrial TMRM fluorescence.
Confocal images of TMRM-labeled, permeabilized rat hepatocytes before
(open symbols) and after (solid symbols) treatment with mtg fraction
(250 µg/ml) were analyzed to obtain frequency distribution histograms
of fluorescence intensity for nuclear (squares) and mitochondrial
(circles) areas. Difference between mitochondrial fluorescence
distributions is statistically significant
(P = 0.01).
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Observation of mtg-treated, permeabilized hepatocytes over time reveals
that the mtg-induced 
increase is transient, returning in
15-30 min to 
values similar to those of an untreated cell (Fig. 4). Because photobleaching limits the
number of confocal images that can be taken from a single cell (see
Determination of

; Refs. 9,
32), the time course of mtg-induced 
changes was followed by

imaging of permeabilized hepatocytes at specific times after
addition of mtg fraction (Fig. 5). This
alternative approach precludes photobleaching or photodamage by
obtaining confocal images (and 
) for a different
cell each time. The same biphasic trend for 
values after mtg
addition was observed for single-cell or multicell measurements, thus
validating 
changes between Fig. 4,
A and
B, for a cell population.
Nevertheless, the reversibility of mtg-induced 
changes suggests
that proteins present in the mtg fraction affect mitochondrial
reactions. To screen out extramitochondrial effects on 
(e.g.,
from glycolysis; Ref. 37), exogenous substrates were not added.

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Fig. 4.
Time-dependent stimulation of  by mtg fraction. Confocal images
of TMRM-labeled, permeabilized rat hepatocytes before
(A), 1 min after
(B), and 15 min after
(C) treatment with mtg fraction (250 µg/ml). Bar, 10 µm.
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Fig. 5.
Time course of  stimulation by mtg fraction. Measurements of
 were carried out at different times after treatment of
TMRM-labeled, permeabilized rat hepatocytes with mtg fraction
[250 µg/ml; ; reference value at
time
0 ( t=0),
159 ± 5.1 mV, n = 20]. A single confocal image (average no. = 4) was taken from
individual cultured cells at specific times, thus avoiding
photobleaching by repetitive observation. Results are shown as means ± SD of 3 independent time course experiments, each with
n = 20. A spline regression curve is
included to help in discerning biphasic trend. Simultaneous 
measurement of cellular mitochondrion-free areas ( ) shows constancy
of cytosolic  (reference value = 36.0 ± 3.9;
n = 20). Trends shown were
similar for each individual time course experiment.
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GTPase-specific modulators and inhibitors affect mtg-induced changes
in 
.
Addition of mtg fraction to control untreated cells did not affect

, compared with the effect of the protein fraction on permeabilized cells (Fig.
6A; cf.
control with permeabilized). GTP and its analogs modulated the
mtg-induced effect on 
. When added at concentrations
(100-200 µM) lower than those present in hepatocytes (mM range;
Ref. 30), GTP decreased the mtg-induced 
increase (Fig.
6A). Lower GTP concentrations (10 µM) did not affect mtg-induced 
changes (not shown). As shown
for mitochondrial MFA, this concentration of GTP stimulates membrane
and content mixing between mitochondrial membranes (11). Similar
inhibitory effect was detected at very low concentrations of GTP
S (1 µM) and GDP
S (Fig. 6A). GDP
S
behaves differently in the 
assay than when used in mitochondrial
MFA (11), in which it did not modify mtg-stimulated membrane mixing.
The behavior of guanine nucleotides in MFA can be interpreted as
supporting a model in which 
changes precede mitochondrial fusion
(see DISCUSSION).

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Fig. 6.
Stimulation of  by mtg fraction is modulated by guanine
nucleotides and AlF4.
A:  measurements for rat
hepatocytes before and after addition of mtg fraction (250 µg/ml).
Experiments were carried out for untreated control (C) and
permeabilized (P) cells, and also for permeabilized cells in presence
of 100 or 200 µM GTP, 10 µM guanosine
5'-O-(3-thiotriphosphate)
(GTP S), and 100 µM guanosine
5'-O-(3-thiodiphosphate)
(GDP S). Results are shown as means ± SD
(n = 25 measurements).
* P = 0.05;
** P = 0.01. B:  measurements for control
permeabilized rat hepatocytes subjected to first
(1) and second
(2) treatments with mtg fraction
and/or AlF4 as indicated
(n = 25 measurements).
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Also suggestive of a linkage between the effect of mtg on 
and
mitochondrial fusion, sequential addition of
AlF4 to permeabilized hepatocytes
treated with mtg reverses mtg-induced 
changes (Fig. 6B). Whether
AlF4 is added before mtg or
together with mtg (Fig. 6B), it
blocks the effect of mtg on 
. This experiment suggests that
stimulation of a G protein-dependent pathway by
AlF4 affects the energetic state
of mtg-treated mitochondria. We did not detect a significant effect on

of F
or
Al3+ when added separately or from
ATP addition (not shown). ADP and ATP
S were poor controls for these
experiments, affecting respiratory control indexes (12).
Because addition of AlF4 and some
guanine nucleotides decreased mtg-induced mitochondrial
hyperpolarization, the GTP-binding proteins contained in the mtg
fraction may be decreasing 
when activated. This depolarizing
(
-consuming) activity is likely suppressed by our addition of
excess inactive (GTP-depleted) mtg proteins, leading to
hyperpolarization (Figs. 2-6). Thus inactivation of cellular mtg
proteins should lead to an increase in 
. This rationale is
supported by the observed activity of a known inhibitor of liver G
proteins, PTX (17). Addition of 200 ng/ml PTX to rat hepatocytes (10 h)
led to mitochondrial hyperpolarization (Fig.
7). Under these conditions, PTX completely
ADP-ribosylates Gi subunits from
cultured hepatocytes (48). We were unable to duplicate this finding
with Vibrio
cholerae toxin, a more narrowly targeted G protein inhibitor (not shown). The PTX-mediated 
increase was larger that the one reversibly induced by mtg fraction and
close to the maximal 
value attainable with rat liver
mitochondria (approximately
220 mV; Ref. 23). The effect of PTX
was not reversed by AlF4 addition
(not shown). This experiment narrows the set of 
- and
fusion-active candidates contained in the mtg fraction to PTX-sensitive
proteins.

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Fig. 7.
Hyperpolarizing effect of pertussis toxin (PTX) on  . Confocal
images of TMRM-labeled, intact rat hepatocytes from control ( ) and
PTX-treated ( ) populations were analyzed to obtain histograms of
frequency distribution for  (n = 325 measurements/distribution). Cells were treated with PTX (200 ng/ml)
6 h after plating and during an overnight incubation (10 h;
medium W).
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The 
affects mtg-stimulated
mitochondrial fusion.
Changes in 
induced by the PTX-sensitive mtg fraction can be
related to its ability to stimulate mitochondrial fusion (9). Cortese
(9) showed that addition of AlF4
or of mtg and AlF4 (mtg/AlF4) to
permeabilized rat hepatocytes induced fast changes in mitochondrial
morphology consistent with true mitochondrial fusion.
AlF4 also penetrates intact cells
in long-term incubation (>3 h; not shown), leading to mitochondrial
fusion (Fig. 8,
right). However, addition of PTX
blocks AlF4-stimulated fusion
(Fig. 8, left). This result suggests
that PTX-sensitive mtg proteins are responsible for
AlF4-stimulated mitochondrial
fusion, in addition to their effect on 
.

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Fig. 8.
Blockage of AlF4-stimulated
mitochondrial fusion by PTX. Confocal images of TMRM-labeled, intact
rat hepatocytes from control (C) and
AlF4-treated and/or
PTX-treated primary cell cultures.
Left: cells treated with PTX as
described in Fig. 6. Right: no PTX.
Top: no
AlF4.
Middle and
bottom:
AlF4 treatment.
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The relationship between mtg-dependent effects on 
and
mitochondrial fusion was studied by manipulating 
with
respiratory inhibitors (Figs.
9-11).
Addition of rotenone to inhibit mitochondrial complex I
(NADH-dehydrogenase, phosphorylation site 1) increased 
(Fig. 9),
most likely due to a larger involvement of intramitochondrial substrates in phosphorylation sites 2 and 3. Addition of antimycin A to
block complex III (cytochrome
c-reductase; phosphorylation site 2)
decreased 
by depleting cytochrome
c-mediated electron transport to complex IV. Interestingly, addition of rotenone plus antimycin A led to a 
value intermediate between those obtained with individual inhibitor additions, regardless of the addition order
(Fig. 8). With the two inhibitors, the ability of mitochondria to
synthesize ATP is greatly diminished by restriction of electron transport (14); cells maintain 
and are still viable for
short-term experiments (<1 h). The effects of oligomycin on ATP
synthesis were somewhat toxic and concentration dependent (15), thus
"clamping" electron transport with rotenone and antimycin A
provided a less deleterious treatment.

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Fig. 9.
Effects of respiratory inhibitors on  . Measurements of  for
control permeabilized rat hepatocytes (C) subjected to first
(1) and second
(2) treatments with rotenone (R; 4 µM final concentration) and antimycin A (A; 1 µg/ml final
concentration). Rotenone or antimycin A was added first, followed by
the other inhibitor. Results are shown as means ± SD for 3 independent experiments (n = 50). All
differences between measurements in
R(1),A(2)
and
A(1),R(2)
groups were significant by two-mean hypothesis test at
P = 0.01, except difference between
rotenone and rotenone-antimycin A additions in
R(1),A(2),
which was significant at P = 0.05.
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Fig. 10.
Stimulation of mitochondrial fusion induced by mtg fraction and
AlF4 in presence of respiratory
inhibitors. Right: confocal images of
permeabilized rat hepatocytes treated with mtg fraction (250 µg/ml)
and AlF4.
Left: no
mtg/AlF4 treatment.
Top: no rotenone or antimycin A. Bottom: effect of
mtg/AlF4 in cells treated with
rotenone and antimycin A as in Fig. 6 (R/A). Bar, 10 µm.
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Fig. 11.
Morphological changes induced by mtg fraction and
AlF4 in presence of respiratory
inhibitors. Frequency distribution histograms of mitochondrial
ellipticity ( ) values for control permeabilized rat hepatocytes
( ) and those treated with mtg fraction (250 µg/ml) + AlF4 in presence of no additions
(A; control; ,
n = 270 measurements), rotenone
(B; ;
n = 270), antimycin A
(C; ;
n = 160), and rotenone + antimycin A (D; ;
n = 160). Each experiment is shown
with its respective control cell population
(n = 120).
|
|
Addition of rotenone and antimycin A decreased
mtg/AlF4-stimulated fusion (Fig.
10, right). The effect of these
inhibitors was also demonstrated by analysis of
histograms before
and after mtg/AlF4 addition.
Changes in
allow quantitative visualization of changes in
mitochondrial morphology in cell populations, and modeling has revealed
that mtg/AlF4 treatment leads to
mitochondrial fusion (9). The
mtg/AlF4-treated control
experiment showed a significant rightward shift from the
distribution of untreated hepatocytes
(P = 0.01, Fig.
11A; Ref. 9). Addition of rotenone alone led to a
distribution similar to that of the control
(P > 0.05) but with
greater variance (P = 0.01; cf. Fig.
11, A and B). Therefore, this treatment does
not appear to block the fusogenic effect of adding
mtg/AlF4. Antimycin A addition
decreased the effect of mtg/AlF4
treatment (P = 0.01; Fig.
11C), leading to a distribution
closer to that of the untreated control. Addition of rotenone plus
antimycin A also blocked the effect of
mtg/AlF4 treatment, while
maintaining physiological 
values (Fig. 9). Statistical analysis
revealed that the rotenone- and antimycin A-treated sample is
significantly left shifted with respect to the control
(P = 0.05) and that its left tail
variance is greater than that of the control sample
(P = 0.01; cf. Fig. 11,
A and
D). Thus this dual inhibitor
treatment leads to a
distribution with a greater fraction of almost
spherical mitochondria (Figs. 10 and 11D). The
distribution of
untreated cells was not affected by the addition of respiratory
inhibitors; the Kruskal-Wallis test did not detect significant
differences between these
distributions. Effects shown in Fig. 11
for mtg/AlF4-treated samples occur
within minutes, well before cytosolic ATP is depleted, and are
insensitive to ATP supplementation (data not shown). Overall, the
experiment indicates that decreasing 
or restricting the coupling
between 
and ATP phosphorylation limits the change in
mitochondrial morphology elicited by the
mtg/AlF4 treatment.
 |
DISCUSSION |
Changes in 
induced by mtg.
The mtg fraction affected 
when added to permeabilized cells.
These changes were statistically significant (15-20% increase) and specific to mitochondria, since TMRM-dependent fluorescence changes
were only detected in mitochondrial regions. Changes in 
were
dependent on preservation of mtg activity in mitochondrial MFA (11) and
were buffer independent. The degree of mitochondrial hyperpolarization
(i.e., 20-30 mV) is in a range that can have effects on cellular
energetics, as its value in pH units (~0.5 units) approaches that of
the pH component of liver mitochondrial electrochemical potential (13).
The effect of mtg on 
was not limited to liver, and mtg-induced
mitochondrial hyperpolarization occurred when rat liver proteins were
incubated with permeabilized rat fibroblasts or rabbit myocytes. Such
general interaction between mtg proteins and semi-intact cells suggests
that mtg-induced 
changes are triggered by a conserved
mitochondrial mechanism rather than an activity associated with
metabolic liver functions. It also suggests that similar proteins could
be widely distributed in eukaryotic cells.
Evidence presented here points to a G protein-like molecule as the mtg
component that affects 
. Treatment with a specific covalent
inhibitor of G proteins (PTX) hyperpolarizes mitochondria, suggesting
the presence of 
-consuming activities in PTX-sensitive proteins.
This would explain the mtg-induced hyperpolarization in permeabilized
cells, inasmuch as dilution of cellular mtg proteins with inactive,
exogenous mtg fraction (depleted in guanine nucleotides after GTP-free
affinity chromatography) will quench the activity of endogenous mtg
proteins. Addition of guanine nucleotides or AlF4 to activate GTP-binding
proteins led to a decrease in the hyperpolarization response. Also
consistent with an active G protein being present in mtg,
AlF4 normalizes 
after mtg
addition and stimulates extensive mitochondrial fusion (9), whereas
treatment with PTX blocks the effects of
AlF4 both on 
and
mitochondrial fusion.
One possible activity of mtg proteins that could lead to their effects
on 
is the activation of ATP-dependent downstream reactions. An
indication that mtg proteins can have metabolic effects in mitochondria
comes from the observation that, in addition to stimulating
mitochondrial fusion, mtg and AlF4
induce changes in mitochondrial configuration. Mitochondrial
ultrastructure changes from the normal orthodox configuration (state
with distended matrix and small intermembrane space) to a more
condensed configuration (condensed matrix, distended cristae, and large
intermembrane space; Ref. 19). This can occur as a consequence of
exacerbated ATP production/consumption, which will decrease the
electrochemical gradient during state 3 respiration (10, 12, 19).
However, this mitochondrial configuration change cannot account for the effect of mtg on TMRM-dependent fluorescence (
).
AlF4, which condensed the
mitochondrial matrix (9), does not significantly affect TMRM
fluorescence (Fig. 6B). Irrespective
of the precise mechanism, our data establish that GTP-binding proteins
contained in mtg affect mitochondrial fusion and 
.
Change in 
as an intermediate to
mitochondrial fusion.
The sequence of events that involve proteins contained in mtg and lead
to mitochondrial fusion is affected by nucleotides that modulate
GTPase-dependent reactions. Mitochondrial hyperpolarization induced by
mtg can be blocked by nonhydrolyzable GTP analogs that inhibit GTP
hydrolysis (GTP
S) or stabilize GDP-bound conformations (GDP
S).
This indicates that the effect of mtg on 
occurs after a
GTP-binding protein binds GTP (by displacement of bound GDP; GDP
S-inhibited step) and before GTP is hydrolyzed (GTP
S-inhibited step). The GTP-bound form of GTPases can have different effects; for small-molecular-weight GTPases, formation of the GTP-bound form
becomes the rate-limiting step of vesicular fusion (39), and for G
proteins this form interacts with the terminal effector until GTP
hydrolysis (7). Also, GTP effects were concentration dependent, thus
suggesting that excess nucleotide affects 
by increasing the
mtg-dependent rate of GTP hydrolysis.
AlF4 mimics the activated state of
the
-phosphate group bound to G protein
-subunits, locking them
in an active conformation by avoiding deactivation through GTP
hydrolysis (18). AlF4 also bypasses receptor-mediated activation (7). We have already established
that adding mtg fraction and AlF4
to permeabilized cells triggers mitochondrial fusion (9) and that the
effect of mtg fraction on 
is
AlF4 sensitive. Addition of
AlF4 before or together with mtg
fraction prevents the characteristic 
increase. Also, when 
is manipulated with respiratory inhibitors, the effect of
AlF4 on mitochondrial fusion is
restricted. When 
is decreased by addition of antimycin A,
mtg/AlF4-induced mitochondrial fusion is restricted. The 
-dependent changes in mitochondrial fusion do not correlate with cytosolic ATP depletion, thus suggesting a
fusogenic role for intramitochondrial ATP. This control of
mitochondrial fusion by 
implies that the mtg-sensitive 
change precedes and may directly or indirectly influence the critical
membrane fusion event activated by
AlF4. We hypothesize that
AlF4 acts fusogenically beyond
mtg-induced 
changes, blocking G protein deactivation by GDP-GTP
exchange and allowing continuous GTPase-dependent stimulation that
activates one or more fusogenic reactions. The effect of PTX also
supports this linkage between the 
event and mitochondrial
fusion; PTX-treated cells exhibited
AlF4-insensitive mitochondrial
polarization, and their mitochondria did not fuse when exposed to
AlF4.
Results with in vitro mitochondrial MFA (9, 11) also support the
proposed sequence for the activities of guanine nucleotides and
AlF4. True in vitro mitochondrial
fusion, characterized by increases in both membrane and content mixing
(11), occurred only with excess GTP (250 µM) and
AlF4. GTP and GTP
S stimulated intermitochondrial membrane mixing, and GDP
S had no effect on the
addition of mtg fraction. These assays are also insensitive to
extramitochondrial ATP (11). As suggested above, addition of mtg will
lead to an early event that requires metabolic energy (thus affecting

) and increases contact between mitochondrial membranes. True
mitochondrial fusion will be specifically initiated by GTP hydrolysis,
with GTP and AlF4 driving these
reactions forward and GTP
S only inducing progress to the point at
which membranes are brought in contact. Although this
first appraisal of a GTP-binding protein-mediated mechanism for
mitochondrial fusion needs refinement and verification of each step, it
logically shows a sequential relationship between events involving GTP
hydrolysis and mitochondrial fusion, which could be connected by
ATP-dependent reactions. Our results also suggest that one or more
GTP-dependent components of the mtg fraction are involved in the
fusogenic and energetic effects detected. One such candidate for a rat
liver fusogenic GTP-binding protein has already been identified in the
mtg fraction (9).
GTPases, mitochondrial morphology, and

.
The linkage between 
and mitochondrial fusion could take several
forms. First, there could be a direct coupling between 
and
mitochondrial fusion. Mitochondria have been shown to fuse under
electric fields (38), and localized 
increases can trigger mitochondrial fusion directly or through effects on molecules with

-sensitive conformation (3). This does not agree with the lack of
mitochondrial fusion observed in PTX-treated hepatocytes exhibiting
high 
. Second, coupling could occur through the utilization of

by ATP synthesis. Newly produced ATP would be used by a fusogenic protein to change its conformation, thus activating fusion.
The N-ethylmaleimide-sensitive factor,
a protein involved in endocytic fusion, undergoes such ATP-driven
conformational changes (47). Third, 
changes could reflect the
activation of ATP-consuming reactions but be unrelated to the sequence
of events leading to mitochondrial fusion. Our data reveals a linkage between guanine nucleotide- and mtg-dependent 
changes and
mitochondrial fusion, thus arguing for a direct biochemical connection
mediated by ATP-dependent reactions.
Also, a requirement of 
for mitochondrial fusion suggests a
connection between mitochondrial morphology and cell energetics. Three
types of relations are possible. 1)
With strict coupling between mitochondrial morphology and function,
mitochondrial fusion may occur as a result of modifications in
organelle activity. Cable theories assimilate mitochondria to a
conducting network (43), suggesting that highly active mitochondria
will fuse with inactive mitochondria to redistribute 
in cells.
Data presented here do not support such a mechanism, since addition of
respiratory inhibitors that modify 
did not cause morphological
changes in untreated cells (Fig. 11).
2) Alterations of the molecular
mitochondrial structure may lead to changes in mitochondrial
morphology, e.g., different protein expression levels may make
mitochondria more or less prone to fuse. In support of this model,
cytochrome-c oxidase-deficient
fibroblasts present a mitochondrial reticulum suggestive of extensive
fusion (22). 3) Mitochondrial
morphology adapts to cellular energetics. For example, mitochondrial
morphology varies between the centroacinar and periportal regions of
the liver acini, where concentrations of oxygen, redox state, and hormone concentrations differ (29). Here, changes in stimulation by
cellular or extracellular effectors can lead to enlarged mitochondria through fusion events. This may explain distortions of mitochondrial shape in liver and muscle disease (45) or under short-term exhaustive exercise (31). Interestingly, our experiments in which antimycin A
decreases mtg/AlF4-induced
mitochondrial fusion parallel the effects of diazepam, a drug that acts
as a respiratory inhibitor in kidney cells, causing mitochondrial
fragmentation (46).
Evidence presented here and in previous articles (9, 11) makes a strong
case for energy-dependent, GTP-binding protein-stimulated fusion
between rat liver mitochondria. Together with the finding of a
developmentally regulated Drosophila
GTPase that is expressed coincidentally with the formation of a
mitochondrial aggregate (21) and with discovery of a yeast homologue of
this protein (41), this research suggests that members of the GTPase
protein superfamily regulate mitochondrial morphology and may
substantially affect 
-dependent reactions. They could be
components of a signal transduction pathway that relays
cellular or extracellular information with effects on mitochondrial
morphology or exert such control by regulating protein expression
levels. The formation of a mitochondrial tubulovesicular network
mediated by GTPase signaling (9) may force us to reassess the
physiological significance of a virtual mitochondrial compartment or
chondrioma (34).
 |
ACKNOWLEDGEMENTS |
I thank B. Herman (University of Texas, San Antonio) and M. Colombini (University of Maryland at College Park) for valuable suggestions regarding this paper and C. R. Hackenbrock
[University of North Carolina at Chapel Hill (UNC-CH)] for
providing support in the early phase of this research. I am also
grateful to L. Voglino (Duke University) for expert technical advice
regarding confocal imaging and to L. Romer and D. Trollinger (UNC-CH)
for the generous gift of primary cell cultures. Provision of rat
hepatocytes by the Advanced Cell Technologies Core (UNC-CH) is also
greatly appreciated.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: J. D. Cortese, Dept. of Cell Biology and
Anatomy, University of North Carolina at Chapel Hill, 117 C Taylor
Hall, Chapel Hill, NC 27599-7090.
Received 5 August 1998; accepted in final form 1 December 1998.
 |
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