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


    ABSTRACT
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

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 (Delta Psi ) when added to permeabilized rat hepatocytes (15%), rat fibroblasts (19%), and rabbit myocytes (10%). This large mtg-induced Delta Psi 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 Delta Psi and mitochondrial fusion. Accordingly, stereometric analysis shows that decreasing Delta Psi 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 Delta Psi changes upstream of AlF4-induced mitochondrial fusion, suggesting that GTPases exert Delta Psi -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


    INTRODUCTION
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

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 (Delta Psi ) is demonstrated. Transient Delta Psi 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 Delta Psi and mitochondrial fusion. The evidence presented here reveals a GTP-binding protein-mediated connection between energy-producing reactions and mitochondrial fusion.


    MATERIALS AND METHODS
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

Materials. Collagen (type VII, rat tail), GTP, guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S), guanosine 5'-O-(3-thiodiphosphate) (GDPbeta 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 (theta ; 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); theta  = 1 for a sphere, and theta  > 1 for ellipsoids. Mitochondrial ellipticity data are represented as theta  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 Delta Psi . Electrical Delta Psi 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 Delta Psi 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 Delta Psi (in mV) using a modified Nernst equation (6)
&Dgr;&PSgr; = −60 · log ([TMRM]<SUB>mt</SUB>/[TMRM]<SUB>ex</SUB>)
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 Delta Psi obtained for mitochondria vary between -140 and -200 mV (see Fig. 1); Delta Psi for the nuclear region is calculated as a control in each image, and it should be approximately -80 mV (6). Confocal images destined for Delta Psi 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 Delta Psi 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 Delta Psi measurements, we use Delta Psi 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 Delta Psi values (see Figs. 6 and 9). Variations of the Delta Psi 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 Delta Psi 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 Delta Psi ). To compare nonnormal Delta Psi 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 Delta Psi imaging conditions, windows smaller than 13 × 19 pixels gave essentially identical Delta Psi distributions (not shown). The standardized approach to Delta Psi measurements applied here reduces the error of this assay to <5%.


    RESULTS
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

Outer membrane-derived GTP-binding proteins affect Delta Psi . 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 Delta Psi values (see Methods), and Delta Psi 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 Delta Psi -dependent mitochondrial TMRM fluorescence (cf. Fig. 2, A and B). The average value of Delta Psi 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 Delta Psi (see Fig. 6A), and Delta Psi increased when this fraction was added to permeabilized hepatocytes, to an mtg-induced Delta Psi value (Delta Psi mtg) of -179.1 ± 6.1 mV (n = 45; see Figs. 2B and 6A). The absolute mtg-induced Delta Psi change varies between cells (see Figs. 6, A and B, and 9), but a 20- to 30-mV Delta Psi 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 Delta Psi (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 (Delta Psi ) by confocal microscopy. A: confocal image of a tetramethylrhodamine methyl ester (TMRM)-labeled intact rat hepatocyte. Bar, 10 µm. B: distribution histogram of Delta Psi values calculated from TMRM fluorescence intensities (n = 75 measurements).


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Fig. 2.   Hyperpolarizing effect on Delta Psi 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 Delta Psi (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 Delta Psi values for C-F. Bars, 10 µm.

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 (Delta Psi  = -156.5 ± 8.5 mV, n = 25; Fig. 2C) or adult rabbit myocytes (Delta Psi  = -153.3 ± 9.2 mV, n = 30; Fig. 2E) caused a Delta Psi increase (fibroblast Delta Psi mtg = -185.7 ± 9.7 mV, n = 25; myocyte Delta Psi mtg = -168.9 ± 6.2 mV, n = 30; Fig. 2, D and F). The smaller relative Delta Psi 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 Delta Psi 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 Delta Psi value of -36 mV was obtained from this fluorescence contribution, comparable to basal Delta Psi 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 Delta Psi 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 Delta Psi 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 Delta Psi 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).

Observation of mtg-treated, permeabilized hepatocytes over time reveals that the mtg-induced Delta Psi increase is transient, returning in 15-30 min to Delta Psi 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 Delta Psi ; Refs. 9, 32), the time course of mtg-induced Delta Psi changes was followed by Delta Psi 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 Delta Psi ) for a different cell each time. The same biphasic trend for Delta Psi values after mtg addition was observed for single-cell or multicell measurements, thus validating Delta Psi changes between Fig. 4, A and B, for a cell population. Nevertheless, the reversibility of mtg-induced Delta Psi changes suggests that proteins present in the mtg fraction affect mitochondrial reactions. To screen out extramitochondrial effects on Delta Psi (e.g., from glycolysis; Ref. 37), exogenous substrates were not added.


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Fig. 4.   Time-dependent stimulation of Delta Psi 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 Delta Psi stimulation by mtg fraction. Measurements of Delta Psi 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 (Delta Psi 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 Delta Psi measurement of cellular mitochondrion-free areas (open circle ) shows constancy of cytosolic Delta Psi (reference value = -36.0 ± 3.9; n = 20). Trends shown were similar for each individual time course experiment.

GTPase-specific modulators and inhibitors affect mtg-induced changes in Delta Psi . Addition of mtg fraction to control untreated cells did not affect Delta Psi , 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 Delta Psi . When added at concentrations (100-200 µM) lower than those present in hepatocytes (mM range; Ref. 30), GTP decreased the mtg-induced Delta Psi increase (Fig. 6A). Lower GTP concentrations (10 µM) did not affect mtg-induced Delta Psi 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 GTPgamma S (1 µM) and GDPbeta S (Fig. 6A). GDPbeta S behaves differently in the Delta Psi 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 Delta Psi changes precede mitochondrial fusion (see DISCUSSION).


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Fig. 6.   Stimulation of Delta Psi by mtg fraction is modulated by guanine nucleotides and AlF4. A: Delta Psi 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) (GTPgamma S), and 100 µM guanosine 5'-O-(3-thiodiphosphate) (GDPbeta S). Results are shown as means ± SD (n = 25 measurements). * P = 0.05; ** P = 0.01. B: Delta Psi 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).

Also suggestive of a linkage between the effect of mtg on Delta Psi and mitochondrial fusion, sequential addition of AlF4 to permeabilized hepatocytes treated with mtg reverses mtg-induced Delta Psi changes (Fig. 6B). Whether AlF4 is added before mtg or together with mtg (Fig. 6B), it blocks the effect of mtg on Delta Psi . 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 Delta Psi of F- or Al3+ when added separately or from ATP addition (not shown). ADP and ATPgamma 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 Delta Psi when activated. This depolarizing (Delta Psi -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 Delta Psi . 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 Delta Psi increase was larger that the one reversibly induced by mtg fraction and close to the maximal Delta Psi 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 Delta Psi - 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 Delta Psi . Confocal images of TMRM-labeled, intact rat hepatocytes from control () and PTX-treated (open circle ) populations were analyzed to obtain histograms of frequency distribution for Delta Psi (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).

The Delta Psi affects mtg-stimulated mitochondrial fusion. Changes in Delta Psi 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 Delta Psi .


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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.

The relationship between mtg-dependent effects on Delta Psi and mitochondrial fusion was studied by manipulating Delta Psi with respiratory inhibitors (Figs. 9-11). Addition of rotenone to inhibit mitochondrial complex I (NADH-dehydrogenase, phosphorylation site 1) increased Delta Psi (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 Delta Psi by depleting cytochrome c-mediated electron transport to complex IV. Interestingly, addition of rotenone plus antimycin A led to a Delta Psi 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 Delta Psi 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 Delta Psi . Measurements of Delta Psi 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 (theta ) values for control permeabilized rat hepatocytes (open circle ) and those treated with mtg fraction (250 µg/ml) + AlF4 in presence of no additions (A; control; , n = 270 measurements), rotenone (B; triangle ; 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 theta  histograms before and after mtg/AlF4 addition. Changes in theta  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 theta  distribution of untreated hepatocytes (P = 0.01, Fig. 11A; Ref. 9). Addition of rotenone alone led to a theta  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 Delta Psi 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 theta  distribution with a greater fraction of almost spherical mitochondria (Figs. 10 and 11D). The theta  distribution of untreated cells was not affected by the addition of respiratory inhibitors; the Kruskal-Wallis test did not detect significant differences between these theta  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 Delta Psi or restricting the coupling between Delta Psi and ATP phosphorylation limits the change in mitochondrial morphology elicited by the mtg/AlF4 treatment.


    DISCUSSION
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

Changes in Delta Psi induced by mtg. The mtg fraction affected Delta Psi 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 Delta Psi 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 Delta Psi 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 Delta Psi 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 Delta Psi . Treatment with a specific covalent inhibitor of G proteins (PTX) hyperpolarizes mitochondria, suggesting the presence of Delta Psi -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 Delta Psi after mtg addition and stimulates extensive mitochondrial fusion (9), whereas treatment with PTX blocks the effects of AlF4 both on Delta Psi and mitochondrial fusion.

One possible activity of mtg proteins that could lead to their effects on Delta Psi 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 (Delta Psi ). 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 Delta Psi .

Change in Delta Psi 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 (GTPgamma S) or stabilize GDP-bound conformations (GDPbeta S). This indicates that the effect of mtg on Delta Psi occurs after a GTP-binding protein binds GTP (by displacement of bound GDP; GDPbeta S-inhibited step) and before GTP is hydrolyzed (GTPgamma 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 Delta Psi by increasing the mtg-dependent rate of GTP hydrolysis.

AlF4 mimics the activated state of the gamma -phosphate group bound to G protein alpha -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 Delta Psi is AlF4 sensitive. Addition of AlF4 before or together with mtg fraction prevents the characteristic Delta Psi increase. Also, when Delta Psi is manipulated with respiratory inhibitors, the effect of AlF4 on mitochondrial fusion is restricted. When Delta Psi is decreased by addition of antimycin A, mtg/AlF4-induced mitochondrial fusion is restricted. The Delta Psi -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 Delta Psi implies that the mtg-sensitive Delta Psi 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 Delta Psi 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 Delta Psi 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 GTPgamma S stimulated intermitochondrial membrane mixing, and GDPbeta 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 Delta Psi ) 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 GTPgamma 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 Delta Psi . The linkage between Delta Psi and mitochondrial fusion could take several forms. First, there could be a direct coupling between Delta Psi and mitochondrial fusion. Mitochondria have been shown to fuse under electric fields (38), and localized Delta Psi increases can trigger mitochondrial fusion directly or through effects on molecules with Delta Psi -sensitive conformation (3). This does not agree with the lack of mitochondrial fusion observed in PTX-treated hepatocytes exhibiting high Delta Psi . Second, coupling could occur through the utilization of Delta Psi 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, Delta Psi 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 Delta Psi changes and mitochondrial fusion, thus arguing for a direct biochemical connection mediated by ATP-dependent reactions.

Also, a requirement of Delta Psi 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 Delta Psi in cells. Data presented here do not support such a mechanism, since addition of respiratory inhibitors that modify Delta Psi 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 Delta Psi -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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