Reproductive Fitness and Quinone Content of Caenorhabditis elegans clk-1 Mutants Fed Coenzyme Q Isoforms of Varying Length*

Caenorhabditis elegans clk-1 mutants lack coenzyme Q9 and accumulate the biosynthetic intermediate demethoxy-Q9. A dietary source of ubiquinone (Q) is required for larval growth and development of the gonad and germ cells. We considered that uptake of the shorter Q8 isoform present in the Escherichia coli food may contribute to the Clk phenotypes of slowed development and reduced brood size observed when the animals are fed Q-replete E. coli. To test the effect of isoprene tail length, N2 and clk-1 animals were fed E. coli engineered to produce Q7, Q8, Q9, or Q10. Wild-type nematodes showed no change in reproductive fitness regardless of the Qn isoform fed. clk-1(e2519) fed the Q9 diet showed increased egg production; however, this diet did not improve reproductive fitness of the clk-1(qm30) animals. Furthermore, animals with the more severe clk-1(qm30) allele become sterile and their progeny inviable when fed Q7-containing bacteria. The content of Q7 in the mitochondria of clk-1 animals was decreased relative to Q8, suggesting less effective transport of Q7 to the mitochondria, impaired retention, or decreased stability. Additionally, regardless of E. coli diet, clk-1(qm30) animals contain a dysfunctional dense form of mitochondria. The gonads of clk-1(qm30) worms fed Q7-containing food were severely shrunken and disordered. The differential fertility of clk-1 mutant nematodes fed Q isoforms may result from changes in Q localization, altered recognition by Q-binding proteins, and/or potential defects in mitochondrial function resulting from the mutant CLK-1 polypeptide itself.

phobic isoprenoid tail. The number of isoprene units in the tail varies between organisms. For instance, Escherichia coli produce a tail with eight isoprene units (Q 8 ), Caenorhabditis elegans contain nine units (Q 9 ), and humans make Q 10 (2). The hydrophobic tail has long been thought to simply anchor Q to the membrane. It is unknown why different organisms produce quinones with different tail lengths.
In eukaryotes, Q is found primarily in the mitochondrial inner membrane, where it serves a critical role in respiration, moving electrons from Complex I or II to Complex III (1). However, Q is found in other intracellular membranes where it serves a variety of functions, including trans-plasma membrane electron transport (3), as a key cofactor in uridine synthesis (4), and as a lipid soluble antioxidant (5). Reduced Q (QH 2 ) can act either directly to scavenge lipid peroxyl radicals or indirectly by regenerating vitamin E.
Although Q can be taken as a supplement, most organisms synthesize Q de novo. Much of the Q biosynthetic pathway has been identified using Q-deficient E. coli and Saccharomyces cerevisiae mutants (6). In yeast, the COQ7 gene product was found to be responsible for the penultimate step in Q biosynthesis, the hydroxylation of demethoxy-Q (DMQ) to demethyl-Q (7)(8)(9). The predicted structure of Coq7p suggests that it is a di-iron carboxylate protein (10). C. elegans with mutations in the clk-1 gene, the COQ7 homologue, show slowed adult behaviors, including defecation cycles and pharyngeal pumping, slowed embryonic and post-embryonic development, and decreased brood sizes (11,12). These animals were found to accumulate DMQ 9 , the expected biosynthetic intermediate, and a small amount of Q 8 , incorporated from their E. coli diet (13,14). When clk-1 worms are fed GD1, a Q-less E. coli, they arrest at the L2 larval stage if fed from hatching or develop into sterile adults if fed GD1 as dauer larvae (14). The diet-derived Q 8 masks a Q auxotrophy in clk-1 animals. Unexpectedly, wild-type animals fed the Q-less E. coli have an extended adult life span (15). This suggests an interesting contrast in the requirements for Q; it is essential for development, yet exogenous Q 8 is detrimental to the longevity of adult animals.
It is not understood why the clk-1 animals remain abnormal when supplied a Q 8 -replete diet. The diet-derived Q 8 is present in mitochondria isolated from clk-1 mutant adults; however, it is important to note that the amount of Q 8 assimilated is far lower than the amount of Q 9 produced by N2 (16). We considered that uptake of the shorter Q 8 isoform derived from the diet may contribute to the Clk-1 phenotypes, because the worms normally synthesize the Q 9 isoform.
To further understand the effects of tail length on Q function and transport, we utilized E. coli strains that had been engineered to produce quinones with different tail lengths (17,18). The polyprenyldiphosphate synthase gene ispB was disrupted, and homologues from different organisms were introduced on plasmids, generating E. coli that produced Q 7 , Q 8 , Q 9 , or Q 10 . We examined the effects of feeding these different Q isoforms on reproductive fitness and progeny viability in both N2 and clk-1 animals. Although N2 animals showed no significant differences in brood size or morphology regardless of the Q fed, we found that clk-1(qm30) animals displayed a sterile phenotype when fed bacteria producing the shortest isoform, Q 7 . The gonads of these animals were shriveled and disordered. Lipid extracts from both the E. coli food and worms were analyzed to determine the levels of uptake and subcellular distribution of the varying Q isoforms within the worms. Although these animals assimilate Q 7 , it does not accumulate as efficiently within the mitochondria. Interestingly, the clk-1(e2519) animals have a very similar quinone profile to that of clk-1(qm30) animals, yet they produce viable progeny when fed the Q 7 -producing food. The gonadal morphology is also less affected. Mitochondria isolated from clk-1(qm30) mutants uniquely separate into two densities when purified on Nycodenz gradients, a functionally normal fraction and a less functional, denser fractions (termed the bottom band). Although this bottom band mitochondrial fraction is not observed in the clk-1(e2519) mutants fed Q 7 , it is present when this strain is fed a Q-less diet, which results in sterility. Together the data suggest that the Clk phenotypes result from several biochemical defects: a lack of endogenously produced Q, a decrease in bacterially derived mitochondrial Q content, poorly functioning mitochondria, and the specific mutation in the CLK-1 polypeptide itself.

EXPERIMENTAL PROCEDURES
Culture Conditions and Strains-Methods for C. elegans were standard (19). N2 (Bristol strain) was used as wild type, and the clk-1 alleles used in this study were e2519 and qm30 (12). E. coli strains used in this study are listed in Table I.
Brood Size Determination-The number of progeny was determined for animals fed the designated E. coli from the L1 stage and from the dauer larval stage. To obtain synchronous L1 larvae, gravid adult animals were treated with alkaline hypochlorite to isolate eggs as per Sulston and Hodgkin (19). The eggs were incubated in S Medium overnight (N2) or over two nights (clk-1) without E. coli to obtain synchronous starved L1 larvae. Worms were placed individually onto nematode growth medium plates with a thin lawn of E. coli and were moved once or twice daily to fresh plates during the egg laying period. Eggs and freshly hatched L1 larvae were counted (egg count). Plates were retained and observed until the larvae reached L4, as determined by the developing vulval morphology, or for 2 weeks. Larvae that reached L4 were removed and counted (progeny count).
Isolation and Quantification of Quinones-For the determination of E. coli quinone content, cultures were grown in Luria Broth at 37°C overnight and collected by centrifugation. Nematodes were cultured at 20°C in S Medium in the presence of OP50 until dauer larvae were obtained. To obtain large quantities of gravid adults, dauer larvae were isolated with 1% SDS treatment and allowed to recover in S Medium with OP50. Eggs were obtained from the gravid adults as above and were allowed to hatch overnight in S Medium without food. The synchronous L1 larvae were then fed the E. coli ispB mutants engineered to produce either Q 7 or Q 8 . When the animals had developed to young adults, as determined by vulval morphology, they were collected by centrifugation, separated from bacteria and debris by sucrose floatation, and allowed to clear bacteria from the gut for a minimum of 30 min in M9. Following another sucrose floatation, the samples were stored at Ϫ80°C until use. Prior to the lipid extraction, all samples were resus-pended in ϳ12 ml of water, and three 1-ml aliquots were moved to preweighed Eppendorf tubes. The small aliquots were centrifuged at maximum speed, and the wet weights of the pellets were determined. These samples were dried overnight using a heated speedvac, and the dry pellet weights were determined. The dried pellets were then resuspended in 1 N NaOH using ϳ1 ml per 2 mg dry weight. The samples were assayed for protein concentration by the bicinchoninic acid assay (Pierce). This allowed for direct comparisons between wet weight, dry weight, and amount protein. The remaining 9-ml samples were pelleted in tared glass tubes, and the wet pellet weights were determined. In general, ϳ0.55-g wet weight nematodes and 0.35-g wet weight E. coli was used per extraction. The pellets were resuspended in a small volume of water, and the internal standards (in ethanol) were added at a concentration of 20 -30 pmol per l of final resuspension volume. Q 10 was used as an internal standard for the nematode extractions and for the Q 7 -, Q 8 -, and Q 9 -producing bacterial strains. Q 6 was the internal standard for the Q 10 -producing bacteria. 9 ml of MeOH and 6 ml of petroleum ether was added to each tube, and the extraction proceeded with shaking overnight in the dark at 4°C. The tubes were centrifuged at 2000 rpm, 4°C for 10 min. The top petroleum ether layers were removed, and a second extraction was done with 4 ml of petroleum ether for one more hour. The petroleum ether extracts were combined and dried under nitrogen. The dried lipids were finally resuspended in 9:1 MeOH/EtOH. The nematode lipids were resuspended in 100 l, and the bacterial lipids were resuspended in 2 ml of 9:1 MeOH/EtOH. Quantification by HPLC linked with an electrochemical detector (ECD) was performed as described previously (16).
Mitochondrial Isolation-Nematode subcellular fractionation and crude mitochondrial isolation were done as described previously (16) except a protease inhibitor mixture was added (catalog number 1873580; Roche Applied Science) to the isolation buffer (IB). The crude mitochondria were further purified over a linear Nycodenz gradient as described by Glick and Pon (20) with the following adjustments. The step gradient was prepared from a 60% Nycodenz stock in IB diluted to 30,25,20,15, and 10% in IB and layered. The brown band at the 20 -25% region contained the purified mitochondria, which were isolated as by Glick and Pon (20). The purified mitochondrial pellets were resuspended in a volume of IB similar to that originally loaded on the gradient. A lower, more dense band was seen in mitochondrial preparations from clk-1(qm30) animals at the 25-30% region. This band was also present in mitochondrial preparations of clk-1(e2519) animals fed GD1. These bands were separately extracted and identified as bottom bands. Protein concentrations were determined by the bicinchoninic acid assay. Lipid extractions and electrochemical detection were performed as above.
Enzyme Assays-Mannosidase II (Golgi) activity assays were adapted from Ren et al. (21) and were performed in a total volume of 0.2 ml containing 0.1 M sodium acetate, pH 6.0, 4 mM p-nitrophenyl-␣-Dmannopyranoside as substrate, and 100 -250 g of protein per assay. Glucose 6-phosphatase (ER) activity and 5Ј-nucleotidase (plasma membrane) activity were assayed as by Stephenson and Clarke (22) except the incubations took place at 30°C. Succinate-cytochrome c reductase activity was measured using 50 g of mitochondrial protein per assay in 40 mM sodium phosphate, pH 7.4, 20 mM sodium succinate, 500 M EDTA, pH 7.4, and 250 M potassium cyanide. Samples were incubated in this assay buffer for 15 min at 30°C. The reaction was initiated with the addition of 50 M horse heart cytochrome c and monitored for 3.5 min via spectrophoto-metric measurements of absorbance at 550 nm minus 540 nm. The reduction rate of cytochrome c was calculated using the extinction coefficient 18.5 mM Ϫ1 cm Ϫ1 .
Western Blotting-Western analysis utilizing the antibody against the ␤ subunit of the F 1 -ATPase was done as by Jonassen et al. (16). Similar methods were used with the following primary antibodies: hexokinase (1:1000 dilution) and actin (1:1000 dilution) except that 80 g of protein were loaded in each lane, and horseradish peroxidase- Image Acquisition-Dauer larvae were moved to nematode growth medium plates containing 1 g/ml tetramethylrhodamine, ethyl ester (TMRE; Molecular Probes) inoculated with either OP50 or the Q 7producing strain. The animals were allowed to develop to adulthood. The adults were picked to small droplets of 10 g/ml Hoechst 33342 (Sigma) on a 2% agarose pad and decapitated just below the pharyngeal bulb, allowing exposure and extension of the gonadal arm. Fluorescent images and DIC images were acquired using an Olympus Fluoview 300 confocal microscope with a 40ϫ oil objective (numerical aperture ϭ 1.35) and Cy3 filter, performed at the Optical Imaging Facility at University of Texas Health Science Center, San Antonio. Gonadal length was determined using Image J (National Institutes of Health: rsb.info.nih.gov/ij/).

RESULTS
Profile of Quinones in Engineered E. coli-Before determining the effects of feeding the engineered E. coli ispB mutant strains to the nematodes, the quinone content of these bacterial strains was quantified with a sensitive electrochemical detection system linked with HPLC. Although the bacteria primarily produced the designated Q isoform, each strain also produced other isoforms (Fig. 1). The total amounts of the primary isoforms for the Q 8 -producing strains were highest, at 6371 Ϯ 176 pmol Q 8 per mg protein for OP50 and 5207 Ϯ 124 pmol Q 8 per mg protein for K0229:pKA3 (Q 8 diet). K0229:pMN18 (Q 7 diet) accumulated 4733 Ϯ 120 pmol Q 7 per mg protein, but it also produced a significant amount of Q 6 , at 1205 Ϯ 44 pmol Q 6 per mg protein. The total quinone levels in the longer chain Q 9 and Q 10 -producing strains were 57 and 35% of OP50 levels, respectively. K0229:pSN18 (Q 9 diet) accumulated 3024 Ϯ 63 pmol Q 9 coli strains engineered to produce distinct isoforms of Q, from Q 7 to Q 10 . An asterisk (*) indicates statistical significance (p Ͻ 0.05 by Student's two-tailed t test) when compared with the same nematode strain fed OP50. A pound symbol (#) indicates statistical significance when compared with the same nematode strain on the Q 8 -producing E. coli K0229:pSN18. L1, worms were fed the designated diets commencing as L1 larvae; dauer, clk-1(qm30) mutant dauer larvae were placed on the designated diets.
FIG. 3. Assimilation of Q 7 and Q 8 from the E. coli diet is similar in the clk-1 mutants as compared with N2. Starved L1 larvae of the N2, clk-1(e2519), and clk-1(qm30) strains were fed the E. coli ispB mutant strain engineered to produce either Q 7 or Q 8 and were allowed to develop to adulthood. HPLC/ECD was used to quantify the quinone levels in terms of pmol quinones per mg protein. A displays total quinones, and B shows an expanded graph displaying bacterially derived quinones. Error bars represent standard deviation from three separate injections of the same sample, and data shown are representative of three independent experiments. per mg protein, and K0229:pLD23 (Q 10 diet) only accumulated 1733 Ϯ 8 pmol Q 10 per mg protein. Interestingly, unlike OP50, the engineered ispB mutants did not contain detectable levels of menaquinone (MK 8 , vitamin K 2 ), an isoprenoid naphthoquinone required by some prokaryotic electron transport chains (23).
Brood Size of clk-1 Mutants Is Affected by the Distinct Q n Diets-Once the lipid profiles of the bacterial foods had been analyzed, the effects of the distinct Q n isoforms on the reproductive fitness of N2 and clk-1 animals were determined. N2 animals fed OP50 laid ϳ300 eggs per individual ( Fig. 2A). There was no significant difference in the number of eggs laid by N2 animals fed the different strains of E. coli containing predominantly Q 7 , Q 8 , or Q 10 . There was a statistically significant increase in the number of eggs laid when the animals were fed the Q 9 diet; however, the number of progeny that developed to L4 larvae did not vary significantly on any food source tested (Fig. 2B).
The clk-1(e2519) animals showed a 40% decrease in egg laying when fed the Q 7 diet as compared with the Q 8 -replete diets. These mutants displayed a statistically significant increase in egg production when fed Q 9 -or Q 10 -producing E. coli, with 25-30% more viable progeny than those hermaphrodites fed OP50. The worms fed the Q 7 -containing food developed slightly more slowly than their siblings fed the Q 8 -producing food; their generation time, from egg to first egg laid, was about one and a half days longer. The generation time of mutants fed the Q 9 -or Q 10 -producing bacteria was about half a day faster than those fed the Q 8 diet.
clk-1(qm30) animals exhibited a dramatic phenotype when fed the Q 7 -producing bacteria from the L1 larval stage; some of these worms were completely sterile, whereas the rest laid very few eggs, with a range of 0 to 32 eggs per hermaphrodite. The average number of eggs was 14, but the average number of viable larvae produced from these eggs was less than one. The length of time it took for these eggs to hatch and develop was also variable and extended. It took up to 1 week for these hatchlings to develop to the L4 larval stage, and much of this time was spent in embryonic development. After 2 weeks, all remaining eggs were considered nonviable. The generation time, from egg to first egg laid, was 10 days for mutants fed the Q 7 diet, as compared with 6 days when the animals were fed OP50. When fed E. coli producing Q 8 , Q 9 , or Q 10 , the clk-1(qm30) nematodes produced similar numbers of eggs as when fed OP50. Although the number of eggs laid when the animals were fed the Q 9 -and Q 10 -producing bacteria was similar to that laid with Q 8 -replete food, far fewer eggs developed into L4 larvae. The dead eggs were examined, revealing embryos at different stages of development. Thus, it appears that there is a mild embryonic lethality associated with the clk-1(qm30) mutant and the Q 9 and Q 10 diets.
When clk-1(qm30) dauer larvae were recovered on the Q 7 diet, the brood sizes were significantly lowered, but the effect was not as striking as when the animals were fed this diet commencing as L1 larvae. The average number of eggs laid was 91 Ϯ 38 per individual; however, most of these eggs were nonviable. The average number of progeny developing to the L4 stage was 30 Ϯ 25. The numbers of eggs produced when the animals were fed the Q 8 , Q 9 , or Q 10 diets were similar to those fed OP50, although the viability of the eggs laid when the nematodes were fed Q 9 or Q 10 diets was lower. Therefore, with all diets, the clk-1(qm30) animals produced more eggs and more viable progeny when developing from the dauer stage than when developing from L1 larvae on the designated food.
Assimilation of Dietary Q 7 and Q 8 by Nematodes-Considering the lack of viable progeny from the clk-1(qm30) animals fed the Q 7 -producing strain, it was important to determine whether these worms could assimilate this shorter isoform. N2, clk-1(e2519), and clk-1(qm30) animals were fed the Q 7 -producing bacteria or the isogenic Q 8 -producing strain from starved L1 larvae until adulthood. The lipids were extracted from these animals, and the quinones were quantified. There was no significant difference in the number of pmol quinones per mg protein of endogenous Q 9 or RQ 9 produced in the N2 animals whether they were fed the Q 7 -or Q 8 -producing bacteria (Fig. 3). There was slightly more Q 8 than Q 7 assimilated from the diet in the N2 animals, 101 Ϯ 6 pmol Q 8 per mg protein versus 65 Ϯ 5 pmol Q 7 per mg protein. The mutant strains clk-1(e2519) and clk-1(qm30) similarly accumulated Q 7 and Q 8 from their respective diets, in addition to the endogenously synthesized DMQ 9 and RQ 9 . In three separate experiments, the levels of DMQ 9 did not show a consistent trend when the animals were fed either food.
Characterization of Subcellular Fractions-Although Q 7 could be assimilated by all strains tested, the question remained whether it could be successfully transported to the mitochondria where it could function in mitochondrial respiration. Therefore, purified mitochondria were isolated from N2, clk-1(e2519), and clk-1(qm30) adults that had been fed different E. coli foods as starved L1 larvae. In addition to the expected purified mitochondrial band in the Nycodenz gradient, a denser band at the 25-30% region of the gradient was observed in all mitochondrial preparations from clk-1(qm30) animals. This band was also identifiable in mitochondrial preparations of clk-1(e2519) animals fed the Q-less E. coli strain GD1 (data not

clk-1 Fed Varying Q Isoforms: Fertility and Quinone Analysis
shown). These bands were separately extracted and identified as bottom bands.
Western analysis was used to verify that mitochondrial components were enriched in the purified mitochondrial fractions and to assist in identifying the bottom band fraction. The ␤ subunit of the F 1 -ATPase, a marker of the inner mitochondrial membrane, was found to be clearly present in the crude and purified mitochondrial fractions from both N2 and clk-1 animals and in the bottom band fraction from clk-1(qm30) animals (data not shown). Additionally, Western analysis verified the lack of hexokinase, a cytosolic marker, in both crude and puri-fied mitochondrial fractions, and the absence of actin, a cytoskeletal marker, in purified mitochondria (data not shown). Unfortunately, antibodies generated against polypeptides localized to various other membranous organelles in other species failed to cross-react with expected homologues in C. elegans. Therefore, to determine the extent of contamination of mitochondria by other organelles, mannosidase II (Golgi), glucose 6-phosphatase (ER), and 5Ј-nucleotidase (plasma membrane) activities were each assayed in all subcellular fractions of clk-1(e2519) ( Table II). Although the crude mitochondrial fractions showed an enrichment of the Golgi and ER membrane   FIG. 4. Q 7 content in isolated mitochondria from clk-1(e2519) and clk-1(qm30) adults is decreased relative to Q 8 and to Q 7 in wild-type pure mitochondria. Starved L1 larvae of the N2 (A and B), clk-1(e2519) (C and D), and clk-1(qm30) (E and F) strains were fed the E. coli ispB mutant strain engineered to produce either Q 7 or Q 8 . When the animals reached adulthood, they were homogenized and separated into subcellular fractions: post-nuclear supernatant (pns), post-mitochondrial supernatant (pms), crude mitochondria (crude), Nycodenz-purified mitochondria (pure), and, in the case of clk-1(qm30), the bottom band fraction (bb). Lipids were extracted from each fraction, and quinones were quantified in terms of pmol quinones per mg protein by HPLC/ECD. A, C, and E display total quinones; B, D, and F show an expanded graphs displaying bacterially derived quinones. Error bars represent standard deviation from three separate injections of the same sample, and data shown are representative of two independent experiments per strain.

clk-1 Fed Varying Q Isoforms: Fertility and Quinone Analysis
marker enzymes per mg protein compared with the post-nuclear supernatants, the purified mitochondrial fractions showed a substantial decrease in mannosidase II and glucose 6-phosphatase activities and an elimination of 5Ј-nucleotidase activity.
Respiratory Activity of Mitochondrial Fractions-To further characterize the mitochondrial fractions isolated from the nematodes, succinate-cytochrome c reductase activities were measured on the mitochondrial fractions isolated from clk-1(qm30) animals fed either the Q 7 or Q 8 diets (Table III). For each sample tested, the purified mitochondria consistently had higher levels of activity than the crude mitochondria. The bottom band fractions had statistically lower levels of activity than the pure mitochondria, indicating that, although there are mitochondrial proteins present in this fraction, the bottom band is dysfunctional. This band may represent damaged or improperly formed mitochondria accumulating in mutants with the more severe allele, qm30, and in the clk-1(e2519) animals fed the least favorable Q-less diet GD1. RQ 9 has been demonstrated to be present in higher quantities in clk-1 dauer larvae (14). The contribution made by RQ 9 to this assay is unknown.
Analysis of Q 7 and Q 8 Content in Subcellular Fractions-Lipids extracted from each subcellular fraction were analyzed by HPLC/ECD. N2 animals showed enrichment in the amount of Q 9 per mg protein in the crude and purified mitochondrial preparations as compared with the post-nuclear and post-mitochondrial supernatant samples (Fig. 4A). There were no significant differences in the endogenously produced quinones when the N2 animals are fed either strain of bacteria. The amount of Q 7 or Q 8 that accumulated in the mitochondria when the animals were fed the Q 7 or Q 8 -enriched food was similar, 45 Ϯ 3 pmol Q 7 per mg protein versus 32 Ϯ 4 pmol Q 8 per mg  5. clk-1 mutants show defects in in vivo gonadal morphology. N2 (A-D), clk-1(e2519) (E-H), and clk-1(qm30) (I-L) dauer larvae were fed either the Q 7 -producing bacteria or OP50. Adults were decapitated to allow extension of the gonadal arm and visualization by confocal microscopy. For all differential interference contrast images, the yellow bar represents 50 m. The TMRE images display the area designated by the blue boxes. The yellow bar in panel B applies to all TMRE images and represents 10 m. protein in the crude mitochondria, respectively (Fig. 4B).
In contrast, both the clk-1(e2519) and the clk-1(qm30) animals failed to transport Q 7 as efficiently to the mitochondria as Q 8 (Fig. 4, D and F). They accumulated less than 20 pmol Q 7 per mg in the purified mitochondrial fractions. These mutants accumulated over 200 pmol Q 8 per mg protein in their crude fractions. The amount of endogenously produced DMQ 9 was similar regardless of the food source provided, and significant enrichment was seen in both the crude and purified mitochondria as compared with post-nuclear and post-mitochondrial fractions (Fig. 4, C and E).
clk-1(qm30) animals had one distinguishing feature, the presence of the bottom band fraction regardless of food type. They contain a significant amount of DMQ 9 and RQ 9 (Fig. 4E). However, the bottom band fraction from worms fed the Q 7 diet contained very little bacterially derived quinone, specifically 19 Ϯ 3 pmol Q 7 per mg protein and 17 Ϯ 3 pmol Q 8 per mg protein (Fig. 4F).
In Vivo Morphology of Gonadal Arms-Fertility is affected, so the mitochondria in the gonads were visualized microscopically for N2, clk-1(e2519), and clk-1(qm30) animals fed either OP50 or KO229:pMN18, the Q 7 -producing strain. The mitochondrial dye TMRE, whose accumulation is driven by membrane potential, was placed in the media, and the worms incorporated it while growing from dauer larvae to adulthood. The gonadal structure, visualized by microscopy, of N2 animals fed either food was similar (Fig. 5, A-D). The gonads were large, the syncytial nuclei were ordered, and cellularization was clearly evident. The clk-1(e2519) (Fig. 5, E-H) and clk-1(qm30) (Fig. 5, I-L) gonads were more disordered than wild type, but oocyte formation was evident when the animals were fed OP50. The lengths of the clk-1 mutant gonads were significantly shorter than wild type (Table IV). When clk-1(e2519) dauer larvae were raised to adulthood on the Q 7 -producing strain, the resulting gonads appeared more disordered. The gonads from clk-1(e2519) animals fed OP50 were about 1.3 times longer than those from animals fed the Q 7 food. The most striking phenotype was seen when clk-1(qm30) nematodes were fed the Q 7 -producing strain. These gonads were only a quarter the size of gonads from wild-type animals fed the same food. They were shrunken in length and width, and there was no cellularization (oocytes) evident. The nuclei in these gonads were extremely disordered. Supplementation with OP50 led to increased gonad length, 2.3 times that of those fed the Q 7 food, but it was still less than wild type. Interestingly, the lengths of the clk-1(qm30) gonads fed OP50 were the same as clk-1(e2519) animals fed the Q 7 diet. DISCUSSION clk-1 mutants can synthesize only DMQ 9 and RQ 9 and must rely on dietary Q 8 for development and fertility (14,16). However, even when consuming Q 8 -replete bacteria, the developmental timing is slower, and brood sizes are lower than wild type (12). This work describes the effects of feeding distinct Q n isoforms, with both longer and shorter tail lengths than the natural C. elegans isoform, to both wild-type worms and to clk-1 mutants.
Reproductive fitness of the N2 nematodes, as assessed by brood size and progeny viability, was not affected by providing Q 7 , Q 8 , Q 9 , or Q 10 in the diet. Lipid analysis demonstrated that N2 animals could incorporate Q 7 and Q 8 from the diet and transport both isoforms to the mitochondria in similar quantities. Gonadal morphology of N2 nematodes was normal upon feeding of either Q 7 or Q 8 diets.
Provision of Q 8 , Q 9 , or Q 10 in the food allows all clk-1(qm30) mutant nematodes to develop and be fertile. However, the shortest Q isoform tested, Q 7 , was incapable of rescuing the sterility defect in the clk-1(qm30) mutant strain when raised on this diet from L1 larvae. These animals were nearly sterile. This severe phenotype was recently independently verified (24). Even when fed the Q 7 diet as dauer larvae, their gonads displayed a strikingly abnormal phenotype, shrunken in size and disordered. For wild-type animals, ablation of germline increases life span whereas whole gonad ablation does not change life span (25). This would imply that the germline provides a life-shortening signal in N2 animals. Similarly, germline ablation further extends life span of clk-1(qm30) animals (26). However, whole gonad ablation, instead of having no effect, shortens the lifespan; gonad ablated clk-1(qm30) animals have a wild-type life span. This is apparently unique to the long-lived clk-1 mutant, because the longer life span of animals subjected to RNAi of a cytochrome c oxidase subunit is unaffected by gonad ablation (26). One interesting possibility is that clk-1(qm30) animals live longer, because their germ cell nuclei and mitochondria are disorganized and dysfunctional such that they do not provide an N2-like life-shortening signal.
Neither the fertility nor the behavioral defects are simply due to improper transport of Q 7 to the mitochondria, because the amount of Q 7 present in purified mitochondrial is very similar between the sterile clk-1(qm30) animals and the fertile clk-1(e2519) adults. The distinguishing feature that separates the two mutant phenotypes is the presence of the bottom band fraction in the clk-1(qm30) animals fed either Q 7 -or Q 8 -replete bacteria. Although these dense bottom band fractions contain mitochondrial protein, they have low complex II ϩ III activity, indicating they are dysfunctional. The presence of the bottom band fraction is not strictly associated with sterility, as the clk-1(qm30) animals fed the Q 8 diet are fertile. However, these dysfunctional mitochondria are associated with the slower behaviors of the clk-1(qm30) animals as compared with the clk-1(e2519) nematodes, which lack the bottom band when fed Q-replete food. Other studies of mitochondria isolated from rat brain have shown that heavy mitochondrial fractions contain higher levels of lipid peroxides and low respiratory enzyme activity and suggest that heavy mitochondria are damaged (27). In another study, mitochondrial defects have been demonstrated to produce defects in brood size and abnormal gonadal morphology. RNAi against genes encoding the prohibitin complex, which has been shown in yeast to play a role in stabilization of newly synthesized mitochondrial respiratory subunits, resulted in decreased brood sizes and progeny viability and in abnormal gonadal morphology (28). Additionally, it was found that mitochondrial DNA amplification is specifically linked to germline development and maturation, specifically to oocyte production (29). Although the distribution of the clk-1(qm30) bottom band abnormal mitochondria in various tissues has not been determined, they may play a significant role in the slowed behaviors in these animals.
The quinone head group of Q provides the redox property required for electron transport. The hydrophobic tail has long been thought to simply link Q to the membrane. Data support the model where Q is located in the membrane midplane, with the polar head group oscillating across the membrane (30,31). However, there is some evidence to indicate that tail length may affect Q function. Jemiota-Rzeminska et al. (32) demonstrated that Q with a tail length consisting of four isoprene units is incorporated into a phospholipid bilayer at an efficiency greater than 95%. Q with less than four isoprene units demonstrated noticeably lower incorporation. The longer isoprenoid side chains resulted in stronger hydrophobic properties. Q 10 has been shown to enter into a highly stable folded conformation (33). The cut-off for the ability to fold into this structure is at four isoprene units. Interestingly, Q 10 H 2 was found to have a stronger tendency to be oxidized than Q 9 H 2 (34). This group also demonstrated a difference in electron transport activity, depending on the tail length. Shorter isoforms of Q displayed less activity in Complex II; Q 4 showed only 10 -25% of the activity of Q 10 , and Q 7 displayed 80% of the Q 10 activity. It has been suggested that the reason different organisms produce different isoforms of Q is that there may be a specific affinity between the lipid composition of the membrane of an organism and its specific Q tail length (35). Although tail length can have an effect on the physiological properties of Q, supplementation with the non-endogenously produced isoform is common; rats and mice primarily produce Q 9 , yet studies frequently employ oral supplementation with Q 10 (36,37).
Our model to explain the more severe phenotypes seen for the clk-1(qm30) allele as compared with the clk-1(e2519) allele is that there is a combination of lipid and CLK-1 protein effects. Although the crude mitochondria have similar quinone profiles between the strains, the pure mitochondrial fraction isolated from clk-1(qm30) and clk-1(e2519) mutants contains less Q 7 than the N2 pure mitochondrial fraction. However, the clk-1(qm30) animals are unique in that they have another fraction that probably consists of damaged or abnormal mitochondria. An additional contribution to phenotypic differences derive from the characteristics of the mutant polypeptides. The e2519 allele encodes a missense E148K mutation. This should interfere with iron binding because of a disruption of the required EXXH motif. The resulting polypeptide is predicted to be enzymatically inactive, but the overall structure could remain intact. In contrast, the qm30 allele results in an early stop codon. The C-terminal 37 amino acids are not translated. This polypeptide could therefore be missing essential binding contacts with other key proteins that stabilize mitochondrial complexes. Thus, a combination of effects may result in a more severe phenotype in the clk-1(qm30) animals.
There are some intriguing links between mitochondrial function, gonad development, and longevity. Further work is needed to identify the specific biochemical pathways linking these processes, and C. elegans should prove to be an outstanding model system for such studies.