Biogenesis of giant mitochondria during insect ... - Wiley Online Library

Eur. J. Biochem. 267, 11±17 (2000) q FEBS 2000

Biogenesis of giant mitochondria during insect flight muscle development in the locust, Locusta migratoria (L.) Transcription, translation and copy number of mitochondrial DNA Bettina Sogl1, Gerd Gellissen2 and Rudolf J. Wiesner3 1

Department of Physiology II, University of Heidelberg, Heidelberg, Germany, 2Rhein Biotech GmbH, DuÈsseldorf, Germany and Department of Vegetative Physiology, University of KoÈln, KoÈln, Germany

3

The biogenesis of giant mitochondria in flight muscle of Locusta migratoria (L.) was analyzed at the molecular level. During the 2 weeks between the beginning of the last larval stage and the imago capable of sustained flight, individual mitochondria have been shown to enlarge 30-fold and the fractional mitochondrial volume of muscle cells increases fourfold [Brosemer, R.W., Vogell, W. and BuÈcher, Th. (1963) Biochem. Z. 338, 854±910]. Within the same period, the activity of cytochrome c oxidase, containing subunits encoded on mitochondrial DNA, increased twofold. However, no significant change in mitochondrial DNA copy number, and even a threefold decrease in mitochondrial transcripts, was observed. Mitochondrial translation rate, measured in isolated organelles, was twofold higher in larval muscle, which can be explained only partly by the higher content of mitochondrial RNAs. Thus, rather unusually, in this system of mitochondrial differentiation, the mitochondrial biosynthetic capacity correlates with the rate of organelle biogenesis rather than the steady-state concentration of a marker enzyme. The copy number of mitochondrial DNA does not seem to play a major role in determining either mitochondrial transcript levels or functional mass. Keywords: copy number; development; gene expression; mitochondrial biogenesis; transcription.

Mitochondria are essential constituents of all eukaryotic cells, providing ATP for energy-consuming processes by oxidative phosphorylation (OXPHOS). In animals, the OXPHOS capacity can vary widely in different tissues of the body [1]. Thus, during development from a fertilized oocyte to a multicellular organism, mitochondrial biogenesis is regulated and OXPHOS capacity is adapted to the different energy demands of particular cell types. In addition, adult animal cells can also adjust OXPHOS capacity to changing energy demands under physiologically or pathologically changing conditions [2]. One of the most dramatic examples of developmental changes in mitochondrial biogenesis is observed during the maturation of insect flight muscle, which is the tissue with the highest maximal oxygen consumption identified to date in the animal kingdom [3]. A pronounced example is that of the migratory locust, Locusta migratoria (L.), where so-called giant mitochondria are formed during development. Sizing the organelles using quantitative electron microscopy has shown that the total mitochondrial functional mass increases by a factor of 60 during the 2 weeks between the beginning of the last larval stage and the imago capable of sustained flight [4]. Having the molecular tools at hand [5], this is an attractive system to study Correspondence to R. J. Wiesner, Institute of Vegetative Physiology, Robert-Koch-Str. 39, 50931 KoÈln, Germany. Fax: + 49 221 478 3538, Tel.: + 49 221 478 3610, E-mail: [email protected] Abbreviations: CO, cytochrome c oxidase; OXPHOS, oxidative phosphorylation; wwt, wet weight. Note: web page available at http://www.uni-koeln.de/med-fak/physiologie/ index.htm Received 18 June 1999, revised 11 October 1999, accepted 12 October 1999

the regulatory events that govern mitochondrial biogenesis at the molecular level. This process is complicated by the fact that two genomes, i.e. nuclear chromosomes contributing the vast majority of mitochondrial proteins and also the small mitochondrial genome (mtDNA), are involved. In all animals studied to date, including L. migratoria [5], mtDNA encodes 13 polypeptides, which are essential subunits of the large-inner membrane OXPHOS complexes, as well as the two ribosomal RNAs and all the tRNAs required for protein synthesis within the mitochondrial compartment. Increased transcription of mtDNA seems to be a hallmark of mitochondrial biogenesis as an adaptation to elevated energy demands in many instances [2]. In contrast, the rapid rise of OXPHOS capacity around birth in developing rat liver was attributed mainly to stimulation of translation, both in the cytosol and in the mitochondrial compartment [6]. Therefore, a concept of two alternative mechanisms has been proposed: mitochondrial proliferation, an increase in the number of organelles, is controlled mostly at the transcriptional level; mitochondrial differentiation, an augmentation of the OXPHOS capacity of pre-existing organelles, is controlled to a great extent by post-transcriptional mechanisms, as observed in rat liver development [6,7]. The maturation of insect flight muscle is obviously a dramatic example of the second mechanism, because an increased OXPHOS capacity results mainly from a remarkable enlargement of pre-existing mitochondria. Therefore, in the present study we tested whether these two concepts also hold true for the formation of giant locust mitochondria [4]. Moreover, our work was stimulated by the scarcity of data about the quantitative relations between mitochondrial volume, mitochondrial size and OXPHOS capacity on the one hand, and mtDNA copy number and mitochondrial transcript levels, respectively, on the other.

12 B. Sogl et al. (Eur. J. Biochem. 267)

M AT E R I A L S A N D M E T H O D S Animals, muscle preparation and determination of cytochrome c oxidase activity Fifth instar larvae (5 days after the last larval ecdysis) and adult locusts (2 weeks after imaginal ecdysis) were obtained from the animal breeding facility of the University of Konstanz, Germany. Animals were decapitated and the thoracic nerve cord was destroyed immediately, the abdomen was removed together with the gut and both longitudinal and transversal flight muscle tissue was collected from the opened thorax after removing fat body tissue. To analyze cytochrome c oxidase (CO) activity as a marker of OXPHOS capacity, a small piece of muscle was weighed (20±30 mg) and homogenized using a glass homogenizer and pestle in 1 mL of ice-cold NaK/Pi (100 mm, pH 7.0). Maximal enzyme activity was determined spectrophotometrically by measuring the rate of oxidation of reduced horse heart cytochrome c (Sigma), reflected by the change in absorbance at 550 nm [8]. The protein concentration of the homogenates was measured using the method of Bradford [9] with BSA as standard, which also yielded values for total tissue protein per gram wet weight (wwt). Enzyme activity was expressed as enzymatic units (mmol cytochrome c´min21´mg protein21), using the millimolar extinction coefficient of 29.5 for reduced horse heart cytochrome c, and was then converted to enzymatic units´g wwt21 to allow comparison with other parameters. The remainder of the tissue was frozen quickly and stored in liquid nitrogen. Extraction of nucleic acids and blotting procedures Isolation of RNA and DNA, blotting and hybridization were carried out using standard methods [10]. Tissue RNA was extracted by the acid guanidinium isothiocyanate procedure [11] from preweighed pieces of flight muscle pulverized under liquid nitrogen (< 50 mg of pooled tissue from 2 to 3 larvae and < 100 mg from one adult individual). DNA was isolated from preweighed, minced muscle pieces (< 30 mg) by proteinase K treatment in SDS-containing buffer, followed by RNAse digestion and phenol/chloroform extraction [10]. The concentrations of the resulting RNA and DNA solutions were determined spectrophotometrically at 260 nm and used to calculate contents of total tissue RNA´g wwt21 and total tissue DNA´g wwt21, respectively. RNA samples (6 mg) were separated on 1.5% formaldehyde agarose gels in Mops buffer and blotted onto nitrocellulose membranes by capillary transfer (Northern blot; 10). DNA (1 mg) was loaded onto a large 0.5% agarose gel and electrophoresed at 4 8C for 18 h in order to show mtDNA conformations. DNA gels were run in Tris/ acetate/EDTA buffer and blotted onto nitrocellulose membranes by capillary transfer (Southern blot; [10]). The separated DNA was visualized by ethidium bromide staining and gels were photographed before blotting. Negatives of these photographs were used to normalize DNA loading. Hybridization and evaluation of results The probes used were 1.5 kb and 7.5 kb EcoRI fragments of L. migratoria mtDNA [12] and a 1.4 kb gene fragment for human 28 S rRNA [1]. The isolated fragments were radiolabeled using the random priming method [10]. Prehybridization [2 h: 40% formamide, 5 NaCl/Cit (1 NaCl/Cit: 0.15 m NaCl, 0.015 m sodium citrate), 50 mmol´L21 NaCl/Pi pH 7.40, 10 Denhardt's solution, 0.2% SDS, 500 mg´mL21 of sheared,

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denatured salmon sperm DNA] and hybridization (16 h: 45% formamide, 3 NaCl/Cit, 10 mmol´L21 NaCl/Pi pH 7.40, 2 Denhardt's solution, 0.2% SDS, 170 mg´mL21 of sheared, denatured salmon sperm DNA) were performed at 42 8C. Blots were washed in 2 NaCl/Cit, 0.1% SDS followed by 0.1 NaCl/Cit, 0.1% SDS at 42 8C, twice for 15 min each. In order to reuse the membranes, probes were removed by gently shaking four times for 5 min in boiling 0.01 NaCl/Cit, 0.01% SDS. After washing, blots were exposed to X-ray film and autoradiograms were analyzed with a videocamera-based gel documentation and analysis system using the AIDA software (Raytest, Straubenhardt, Germany). Care was taken that signals were in the linear range of the film. The signal for 28 S rRNA was used for normalization of mtRNA levels on Northern blots, while ethidium bromide-stained, genomic DNA documented prior to blotting was used for normalization when estimating mtDNA levels. Measurement of mitochondrial translation capacity Mitochondria were isolated from pooled muscle (< 500 mg) by differential centrifugation. The tissue was gently homogenized by hand in 5 mL of buffer (300 mm sucrose, 1 mm EGTA, 1% BSA, 10 mm Tris/HCl pH 7.3) in a small glass homogenizer using a loosely fitting Teflon pestle. The homogenate was centrifuged at 600 g for 5 min, the supernatant was collected and centrifuged once more under the same conditions. Mitochondria were pelleted from the supernatant by centrifugation at 4500 g for 15 min, the supernatant was removed and the pellet was gently suspended in 0.5 mL of the same buffer. The protein content of this suspension was initially estimated spectrophotometrically at 225 nm using the method described by Waddell [13]. After the experiments, it was confirmed, using the Bradford method [9], that equal amounts of mitochondrial protein had been used in the assays. Mitochondria were incubated in 100 mL of a modified translation medium containing 50 mm KCl, 2 mm MgCl2, 1 mm Na2HPO4, 10 mm Hepes pH 7.0, 1 mm EGTA, 10 mm pyruvate, 3 mm malate, 3 mm ATP, 20 mm glutamate, 0.1 mg´mL21 cycloheximide and 5 mg´mL21 BSA. The medium was supplemented with 0.3 mm of each amino acid, except methionine, 20 mCi of [35S]methionine and 2 mg´mL21 of mitochondrial protein. In some experiments, 1 mg´mL21 chloramphenicol was added to the incubation mixture. Incubation was performed at room temperature on a rotating wheel, ensuring optimal suspension and oxygenation. After incubation for the indicated times, mitochondria were pelleted by centrifugation at 5000 g for 2 min and dissolved in SDS buffer [14]. Protein samples were separated on 12.5% SDS gels [14] and run at 10 V´cm21 gel length for 2 h. Gels were either treated for fluorography (Enhance, Dupont NEN), dried and exposed to X-ray film or proteins were stained with Coomassie Brilliant Blue and subsequently destained in order to reduce quenching. The protein-containing lanes were cut, blotted dry and dissolved individually in 1 mL of tissue solubilizer (NEN Dupont). Incorporated radioactivity was quantified by liquid scintillation counting. This method was found to be superior to precipitation of radioactive protein with trichloroacetic acid on glass fiber filters followed by liquid scintillation counting. Statistical analysis of data Data obtained for larvae were compared with those from adult animals using a two-tailed Student's t-test. A confidence level

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Mitochondria in insect flight muscle development (Eur. J. Biochem. 267) 13

Table 1. Total tissue DNA, RNA and protein concentration and cytochrome c oxidase activity in larval and adult locust flight muscle. Muscles were weighed, and nucleic acids were extracted, precipitated and quantitated spectrophotometrically. Enzyme activity was determined by a spectrophotometric assay.

Stage

DNA (mg´g wwt21)

RNA (mg´g wwt21)

Protein (mg´g wwt21)

Cytochrome c oxidase (enzymatic units´g wwt21)

Larvae Adults

1.82 ^ 0.67 0.52 ^ 0.07*

2.38 ^ 0.29 0.64 ^ 0.18*

122 ^ 23 231 ^ 17*

14 129 ^ 4141 26 277 ^ 5071*

Values are mean ^ SD, n = 5, *P , 0.05; wwt: wet weight.

of P , 0.05 was considered to be indicative of a statistically significant difference.

R E S U LT S The results on total tissue DNA, RNA and protein content in larval and adult flight muscle are summarized in Table 1. The content of DNA, indicative of the number of nuclei per gram or unit volume of muscle, is about four times higher in larval muscle. The same observation holds for total tissue RNA. In contrast, total tissue protein as well as the activity of CO, a key enzyme of OXPHOS, are about twice as high in adult muscle compared with larval muscle. Northern blots were probed consecutively with the 1.5 kb and the 7.5 kb EcoRI fragments of locust mtDNA (Fig. 1). The 1.5 kb probe contains sequences complementary to the mRNAs for subunits 2 and 3 of CO and the common mRNA for subunits 6 and 8 of ATP synthetase. The 7.5 kb fragment contains sequences complementary to the mRNAs for subunits 1, 2, 4,

4L and 6 of NADH dehydrogenase, cytochrome b as well as the large and small rRNAs. The same blot was finally probed with a human 28 S rRNA probe for normalization. Some RNAs are of very similar length, precluding a clear separation of the signals. Nevertheless the signals could easily be assigned to individual transcripts based on their length predicted from the locust mtDNA sequence [5]. Despite the limitations of RNA separation it is obvious that larval muscle contains rather more mitochondrial transcripts than adult muscle when the mtRNA signals are related to the 28 S rRNA signal. Arbitrary numbers obtained from these blots by densitometry were multiplied with the total RNA content per g wwt of individual muscles. In this way, it was found that larval muscle contains approximately three times the level of all mitochondrial transcripts compared with adult muscle, when expressed per g wwt (Table 2; P , 0.05). The tissue levels of the two rRNAs only slightly exceeded the levels of individual mRNAs, which is in accordance with previously published data on the stoichiometry of mitochondrial RNAs in differentiated mammalian tissues [15]. Differences in the translational capacity were investigated in isolated mitochondria. Incorporation of [35S]methionine into proteins was almost completely blocked by chloramphenicol under the experimental conditions (Fig. 2, left), showing that

Table 2. Levels of mitochondrial RNAs and mitochondrial DNA in larval and adult locust flight muscle. Values are arbitrary densitometric units derived from autoradiograms (Figs 1 and 3), normalized to the 28 S rRNA signal for mitochondrial RNAs or the ethidium bromide signal of genomic DNA for mtDNA, respectively. Values were then multiplied by the content of total tissue RNA or DNA (mg´g wwt21, Table 1), respectively, in order to obtain values representing the abundance of individual nucleic acids per g wet weight (wwt). Total mtDNA is the sum of supercoiled plus closed circular mtDNA (Fig. 3). Also given are the ratios of supercoiled to closed circular mtDNA.

Fig. 1. Mitochondrial RNA levels in larval and adult locust flight muscle. RNA isolated from flight muscles was separated by formaldehyde agarose gel electrophoresis, transferred to nitrocellulose (Northern blot) and probed consecutively with the 1.5 kb (top) and the 7.5 kb (middle) EcoRI fragments of locust mtDNA, respectively, as well as a human 28 S rRNA probe (bottom) for normalization. Each lane contains RNA from a different preparation (pooled muscles from larvae, individual muscles from adults). The autoradiogram was analyzed using a videocamera-based gel documentation and analysis system. Quantitative data are given in Table 2. ND, NADH dehydrogenase subunits; Cyt b, cytochrome b; CO, cytochrome c oxidase subunits; ATP, ATP synthetase subunits; l rRNA, s rRNA, large and small RNA subunit of mitochondrial ribosomes. Autoradiograms were scanned with a flat-bed scanner using photoshop, v 4.0, software; for layout and lettering, designer, v 7.0, software was used.

Sample

Larvae

mtRNAs Precursors ND4 l rRNA/Cyt b ND1/ND2 s rRNA/ND6 ATP 6/8 COIII/COII

0.66 0.89 1.16 0.87 2.08 1.05 1.95

mtDNA Total mtDNA level Ratio supercoiled/closed circular

3.74 ^ 1.47 0.19 ^ 0.13

^ ^ ^ ^ ^ ^ ^

Adults

0.39 0.33 0.38 0.30 0.64 0.23 0.30

0.26 0.33 0.42 0.33 0.76 0.31 0.56

^ ^ ^ ^ ^ ^ ^

0.09* 0.09* 0.11* 0.11* 0.23* 0.15* 0.24*

5.62 ^ 0.52 0.21 ^ 0.09

Values are mean ^ SD, n = 5 (n = 4 for larval DNA); *P , 0.05. For abbreviations, see Fig. 1.


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Fig. 3. Mitochondrial DNA levels and conformations in larval and adult locust flight muscle. DNA isolated from flight muscles was separated by agarose gel electrophoresis, transferred to nitrocellulose and probed with the 7.5 kb fragment of locust mtDNA (Southern blot). Each lane contains DNA from a different preparation. The upper part shows the autoradiogram, while the lower part shows the same gel stained with ethidium bromide before blotting. The position of a 17 kb molecular mass marker is indicated on both panels. Both the autoradiogram and the negative of the ethidium bromide picture were analyzed using a videocamera-based gel documentation and analysis system. Quantitative data are given in Table 2. Autoradiograms were scanned with a flat-bed scanner using photoshop v 4.0, software; for layout and lettering, designer v 7.0, software was used.

Fig. 2. Translation products of isolated locust flight muscle mitochondria and incorporation of radioactivity. Mitochondria were isolated from locust flight muscle and incubated in the presence of [35S]methionine. (A) Mitochondrial protein was subjected to SDS/PAGE, the gel was treated for fluorography, dried and exposed to X-ray film. Where indicated, chloramphenicol was added to the incubation medium. The position of molecular mass markers and mitochondrial translation products identified according to their molecular mass are given. (B) Incorporation of [35S]methionine (c.p.m.´mg protein21) into mitochondrial translation products was quantitated by liquid scintillation counting of solubilized gel lanes as shown in the upper panel. (X) Adult muscle; (W) larval muscle. Values are mean ^ SD, n = 6, *P , 0.05. Autoradiograms were scanned with a flat-bed scanner using the Photoshop, Version 4.0, software; for layout and lettering, the Designer, Version 7.0, software was used.

mitochondrial translation was indeed assessed. According to their apparent molecular masses, the 13 mitochondrial translation products could be identified unequivocally. The translational capacity per mg of protein was approximately twice as high in mitochondria from larval muscle than adult muscle (Fig. 2, right). For this estimation, the values for 15 min of incubation were used (1704 ^ 822 c.p.m.´mg protein21 in adults versus 4163 ^ 1433 c.p.m.´mg protein21 in larvae; P , 0.05), because incorporation was linear only for up to 30 min. Differential expression of mitochondrial genes could, in principle, also be due to different amounts of mtDNA as well as changes in the ratio of its various physical conformations. Thus we assessed the content, as well as the ratio, of supercoiled versus relaxed circular mtDNA by probing Southern blots of native muscle DNA with the 7.5 kb EcoRI fragment. The analysis showed that these two are indeed the major conformations of mtDNA in the muscle tissues (Fig. 3, upper). The upper band, migrating considerably more slowly than a 17 kb linear DNA marker, corresponds to closed circular,

relaxed molecules, while a fast moving band close to the position of a 10-kb marker represents supercoiled molecules. High molecular mass bands, which were only seen in adult muscle, probably represent catenated molecules. Because we found no linearized mtDNA indicative of artefacts occurring during the isolation procedure, not even after long overexposure of the blots (not shown), we propose that the ratio between these two species reflects the in vivo situation. Fragmentation of mtDNA by digestion with EcoRI led to the disappearance of all these bands and the appearance of one single signal at 7.5 kb, when probed with the 7.5-kb EcoRI fragment, unequivocally identifying all bands as mtDNA (not shown). The intensity of the autoradiographic signals was analyzed densitometrically and normalized to genomic DNA quantitated by ethidium bromide staining (Fig. 3, lower). Adult muscle was found to contain 5.3-fold more mtDNA/genomic DNA than larval muscle (Fig. 3). However, it has to be taken into account that larval muscle had 3.5-fold more total tissue DNA than adult muscle (Table 1). Thus, after multiplication arbitrary numbers were obtained (Table 2), showing that adult muscle contains only < 1.5-fold more mtDNA per unit volume or gram than larval muscle (difference not significant). No difference in the ratio between supercoiled and relaxed circular mtDNA was observed between adult and larval muscle.

DISCUSSION A vast amount of detailed information exists on the mechanisms of transcription and replication of mtDNA at the molecular level [16,17]. In contrast, little is known in vivo about the quantitative relationships between mitochondrial transcript levels and the copy number of mitochondrial DNA on the one hand, and functional content of OXPHOS complexes, mitochondrial number and mass on the other hand. Thus, a comprehensive collection of such data is indispensible for understanding the regulatory mechanisms underlying the

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adaptation of OXPHOS capacity to changing energy demands. Probably the most thoroughly studied system with respect to these questions is the development of rat liver [6]. Here, the number of mitochondria increases throughout fetal life. This is accompanied by increases of both mtDNA copy number and mitochondrial transcript levels. In contrast, during the first hour of extrauterine life, a rapid rise in OXPHOS capacity is observed without changes in mitochondrial number, or copy number of mtDNA or mitochondrial transcripts. Rather, this is achieved by stimulation of mitochondrial translation on the one hand [6], and stabilization and an augmentation of the intrinsic translational efficiency of nuclear-encoded mRNAs for mitochondrial proteins on the other hand [7,18]. Consequently, these authors have postulated that two alternative biological programs, termed mitochondrial proliferation and mitochondrial differentiation, respectively, lead to an increase in functional mitochondrial mass by two different mechanisms. Other examples that sustain this concept of alternative programs are reviewed briefly below. When different rat tissues were compared, a strong, positive correlation was found between the abundance of a mitochondrial mRNA for CO and maximal enzyme activity [1,19]. In contrast, such a relation was not found either between mtDNA copy number and CO activity or between mtDNA copy number and mitochondrial fractional volume, respectively [20]. Moreover, it has been calculated that differentiated cells contain a large excess of mtDNA molecules that are not transcribed at a given time [15,21]. Thus, in the steady-state, the rate of transcription from a subpopulation of mitochondrial DNA seems to determine the available amount of mitochondrially encoded mRNAs and, consequently, mitochondrial proteins, whereas the copy number of mtDNA does not seem to be of major importance. Less is known about the coordination between expression of mtDNA and that of the hundreds of nuclear genes encoding mitochondrial proteins. However, a similar positive correlation between CO activity and levels of the nuclear-encoded mRNAs for CO subunit VIc [1,19] and Va (Wiesner, unpublished data) was observed in the same set of rat tissues. This indicates that transcription also regulates the number of nuclear-encoded OXPHOS subunits available and that both genomes are expressed in a coordinated fashion, at least in the steady-state. Transcription factors binding to highly homologous cis-acting elements have been described, being good candidates for the regulation and coordination of such genes [22,23]. In addition, in many instances of mitochondrial biogenesis stimulated by various physiological challenges, increased tissue levels of mRNAs for both nuclear and mitochondrially encoded OXPHOS subunits are also found, emphasizing even more the important role of transcription regulation. Examples are endurance training of skeletal muscle [24,25], hyperthyroidism in liver and skeletal muscle [26] or hyperglucocorticoidism in colon epithelium [27]. In contrast, during cold adaptation in brown adipose tissue [28] and during heart hypertrophy induced by thyroid hormone [29,30], the increase in functional mitochondrial mass is brought about not only by elevated levels of such transcripts, but at the same time by an additional, specific stimulation of mitochondrial translational capacity. A noteworthy hallmark of these two models is that the number of mitochondria remains fairly constant [31], but the size of individual organelles increases, thus representing examples of mitochondrial differentiation. It remains to be shown whether such a stimulation of mitochondrial translation also occurs during the adaptive processes mentioned above, in which mitochondria increase in both number and size (training of skeletal muscle, [24]) or size

only (hyperthyroid state of liver and muscle, [26]). With the exception of some forms of skeletal muscle training [25] and heart hypertrophy induced by thyroid hormone [30], the copy number of mtDNA was found to be unchanged under most conditions, emphasizing that up-regulation of mtDNA copy number is also of minor importance for adaptive stimulation of mitochondrial biogenesis [21]. Here we tried to validate the concept of two different biological programs of mitochondrial biogenesis by studying the development of insect flight muscle in L. migratoria (L.), which is probably the most dramatic example of OXPHOS up-regulation in the animal kingdom. During the 2 weeks between the beginning of the last larval stage and the adult animal capable of sustained flight, the total mitochondrial mass rises by a factor of 60, when the net growth by a factor of 7 of the flight muscles is also taken into account [4]. Morphometric measurements show that the fractional mitochondrial volume increases from 8 to 32%. If locust muscle mitochondria are assumed to be rod-shaped cylinders, it can be calculated from electron-microscopy that the volume of individual mitochondria increases some 30-fold. Thus, the number of individual organelles must have decreased by a factor of 8. Therefore, the development of insect flight muscles seems to be a perfect model system for studying mitochondrial differentiation, the development of large mitochondria from small preexisting organelles. Assuming for simplicity that wet weight is approximately equivalent to unit volume of muscle, we can conclude the following about the molecular mechanisms underlying this massive production of mitochondria: larval muscle is quite obviously in a stage of intense myofibrillogenesis and mitochondriogenesis, increasing both its net mass and the fractional content of these two major muscle cell constituents [4]. The total DNA content, i.e. the density of nuclei supplying a unit volume of muscle with RNAs, is fourfold higher in larval than in adult muscle (Table 1). As an obvious consequence, the amount of total RNA, mainly consisting of cytosolic ribosomes, is also fourfold higher in larval muscle (Table 1). At the end of the developmental process, when adult muscle has reached its final cellular architecture and protein composition, both DNA and RNA contents are lower than in larval muscle. Obviously, fewer nuclei, as well as ribosomes, are necessary per unit volume to maintain the new steady-state in mature muscle. Interestingly, total muscle protein and CO activity increase by the same factor of two and it has been reported that the activities of other mitochondrial marker enzymes also increase by factors of 2±4 [4]. Because it has recently been shown that CO activity very closely reflects maximal in vivo carbon and O2 flux rates in insect flight muscle [3], CO is probably a good marker for OXPHOS capacity in general. This means that myofibrils and mitochondria, the major constituents of flight muscle cells, have been synthesized during development in perfectly constant proportions. However, mitochondria have enlarged considerably more than predicted from the mere increase of CO activity. The concentration of mitochondrial RNAs per unit volume of tissue is about threefold higher in larval muscle (Table 2). Thus, in contrast to the steady-state [1] as well as in the abovementioned examples of stimulated mitochondrial biogenesis in mammals [2], the abundance of mitochondrial RNAs does not correlate with the abundance of the mitochondrial marker protein CO, but rather with its rate of synthesis. This has also been reported for the synthesis of b-F1ATPase in developing rat liver [18], but not for mitochondrial biogenesis during rat kidney development [32]. The higher protein synthetic capacity


of isolated larval mitochondria (Fig. 2, right) can largely, but not totally, be explained by their higher RNA content. The data in Tables 1 and 2 allow us to calculate that larval mitochondria contain < 50% more RNA than adult mitochondria. Thus, a higher mitochondrial translational capacity, which has been found, for example, in cold-adaptated brown adipose tissue [28], is obviously not involved in the maintenance of high CO levels in the adult muscle. Therefore, we must conclude that stabilization of mitochondrial proteins is probably the major mechanism for the maintenance of a high OXPHOS capacity in giant mitochondria of adult flight muscle, in the presence of low levels of mitochondrial transcripts as well as a low mitochondrial translational capacity. In vivo labeling of mitochondrial proteins together with pulse±chase protocols would be the appropriate method to support our proposal. The differentiation of giant mitochondria thus resembles mitochondrial differentiation during rat liver development around birth in many aspects, albeit on a much longer time scale, however, with a much more impressive increase in organelle size. As discussed above, mtDNA copy number does not seem to play a major role in determining either the levels of mitochondrial transcripts or the functional mitochondrial mass in both the steady-state and under most conditions of stimulated mitochondrial biogenesis. Development of locust flight muscles clearly emphasizes this point: a moderate increase in mtDNA by a factor of 1.5 in adult muscle (Table 2, not significant) is accompanied by a threefold decrease in mitochondrial transcripts and an enlargement of individual mitochondria by a factor of 30! So far, the interpretation does not provide an answer to the question of which factor(s) is responsible for the elevated transcription of mtDNA in larval muscles. As a second possibility besides copy number control, we analyzed the putative role of different physical conformations of mtDNA, because supercoiled molecules are thought to be the actively transcribed conformation, as suggested by studies in Xenopus eggs [33,34], However, no difference in the ratio of supercoiled to relaxed conformations was observed (Table 2.). Unfortunately, analyzing Western blots of locust muscle with antisera against human or mouse mitochondrial transcription factor A (mtTFA), the best described modulator of mitochondrial transcription and replication described to date in animal systems [17], did not yield interpretable results. Further work is necessary to test the involvement and functional role of a putative locust mtTFA.

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ACKNOWLEDGEMENT This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG Wi 889/3-2).

21. 22.

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