Clueless, a Conserved Drosophila Gene Required for Mitochondrial Subcellular Localization, Genetically Interacts with Parkin

clu encodes a Drosophila CluA ortholog

The Drosophila gene CG8443 produces a single annotated transcript encoding a 1448 aa predicted protein that is closely related to CluA from Dictyostelium and its orthologs (Fig. 1A, B). Consequently, we named this gene clueless (clu). Clu shows 53% identity to human Clu (KIAA0664) across the entire protein. Both proteins contain a region of tetratricopeptide repeats (TPR), as well as a putative “Clu domain” between amino acids 424-666 of Drosophila Clu, that is even more strongly conserved in metazoan Clu proteins (85% identity between Clu and KIAA0664, Fig. S1). N- and C-termini protein segments of Drosophila Clu were expressed and used to raise antibodies in guinea pigs (Fig. 1B, Methods). Both antibodies recognize a single band of apparent molecular weight 160 kd on Western blots (Fig. 1C) whose abundance is reduced in clu mutants (see below).

Flies mutant for clu display abnormal mitochondrial structure and function

To analyze clu function, we identified multiple strains bearing single insertions located within the clu transcription unit (Table 1, Fig. 1A). Flies homozygous for clu^d08713 or clu^f04554 lack detectable Clu protein on Western blots, while clu^d00713 or clu^EP0969 homozygotes contain reduced amounts (Fig. 1C). These clu mutant flies showed striking defects. Putative null clu^d08713 (Fig. 2A) or clu^f04554 homozygous flies (data not shown) are small, uncoordinated, sterile, and live only 3-7 days. They move slowly, do not fly, and frequently hold their wings up or down, a sign of flight muscle defects (Fig. 2A-B). The larval fat body fails to turn over within the first two days of adult life, another indication of a metabolic defect (Fig. 2C). Mutant flies bearing the weaker clu^d00713 or clu^EP0969 alleles are viable but male sterile. Flies bearing combinations of clu alleles or trans-heterozygous with a deficiency for the clu region, failed to complement, indicating that these defects are due to disruption of clu expression. Finally, the Clu protein trap line CA06604 (Buszczak et al. 2007), produced a Clu-fusion protein of the expected size (Fig. 1C) that is likely to be functional because CA06604 homozygotes are viable and fertile.

Flies mutant for park, pink1, or rho7, genes which encode mitochondrial proteins, exhibit many similar defects (Greene et al. 2003; Wang et al. 2006; McQuibban et al. 2006). To determine if changes in mitochondrial function underlie the abnormalities in clu mutant flies, we carried out electron microscopy on the flight muscles of 3-day old clu^d08713 adults. Nearly all the flight muscle mitochondria were swollen and showed severe abnormalities, especially vesiculation/loss of cristae (Fig. 2D, E). Mitochondria in the ovary showed similar but less severe effects, including reduced cristae and swelling (Fig. 2F, G). Mature sperm from sterile clu males were completely immobile (data not shown). In addition, the mitochondrial derivative in elongated spermatids was irregular and swollen (I) compared to wild type (Fig. 2H). We conclude that clu is required for normal mitochondrial structure and function.

To examine whether Drosophila clu is also needed for mitochondrial positioning, we analyzed mitochondria during oogenesis. Drosophila ovarian follicles arise from stem cells in the germarium of each ovariole, and subsequently develop in sequence (Fig. 3A). Changes in mitochondrial localization were observed in clu mutant germ cells beginning as early as the germline stem cells (GSCs). Normally, mitochondria in GSCs are enriched anteriorly, but are also found throughout the cytoplasm (Fig. 3B). In clu^d08713 mutant GSCs nearly all mitochondria occupy the anterior region of the cell (Fig. 3C). This increased clustering raises the question of whether during GSC division the daughter cystoblast and newly formed GSC inherit equal amounts of mitochondria. At mitosis in wild type GSCs and cystoblasts, mitochondria spread uniformly across the outer spindle and are inherited equally by the daughter cells (Fig. 3D; Cox and Spradling, 2003). In contrast, mitochondria remained clustered during mitosis in clu^EP0969 early germ cells (Fig. 3E, arrow), suggesting that inheritance may be unequal, and that one cell may have to make up for a deficit of mitochondria by increased mitochondrial replication (Cox and Spradling, 2006).

Enhanced clustering was also observed in older germ cells in the absence of normal Clu function. Mitochondria are dispersed in young wild type germline cysts (Fig. 3F) but were highly clustered in similarly aged cysts homozygous for clu^d08713 or clu^EP0969 where they associate with the tips of the fusome (Fig. 3G, H), an organelle that interconnects all the germ cells in a cyst. In growing ovarian follicles, mitochondria normally spread evenly throughout the nurse cell cytoplasm (Fig. 3I), but in similarly aged clu^d08713 follicles nearly all mitochondria cluster in a giant aggregate in each nurse cell (Fig 3J). clu mutations also caused mitochondrial clustering in some somatic follicle cells in the germarium (Fig. 3K, L).

Mitochondria cluster in park mutant cells

Many of the effects of clu mutation we observed resembled those described following disruption of park (Greene et al. 2003). Consequently, we examined mitochondria in park mutant follicles to see if they also form clusters. park¹ / Df females are sterile, but follicles develop far enough to reveal strong mitochondrial clustering in the nurse cells of previtellogenic follicles (Fig. 5A, B). As follicles age, mitochondrial clustering worsens (Fig. 5C, D). Examining these clustered mitochondria using light and electron microscopy revealed that some were doughnut shaped (Fig. 5E, F), and some grew larger than normal (Fig. 5F; compare Fig. 2F). park mutants also induced clustering in somatic follicle cells (Fig. 5G, H). During spermatogenesis, only one mitochondrial derivative forms in park¹ / Df mutant germ cells (Fig. 5P) as previously reported (Riparbelli and Callaini, 2007). clu^EP0969 forms two derivatives but they appear frayed (Fig. 5N-O). Despite this initial difference, during spermatid elongation the park¹ / Df mutant mitochondrial derivative becomes irregular and swollen like those in clu^EP0969 (Fig. 2I-J).

clu and park interact

Because of the similarities between clu and park, we investigated whether these two genes interact genetically. park mutant ovaries still contained normal amounts of Clu protein (Fig. 5I, J), so the clustering observed in these animals is not simply due to reduced Clu expression. The only difference appeared to be a reduction in the number of Clu particles in the park mutant cells. Females heterozygous for either clu or park alone were phenotypically wild type and mitochondria did not cluster in nurse cells from stage 5 follicles (Fig. 5K, L). In contrast, mitochondria were noticeably clustered in 100% of similarly aged follicles from park¹ / + ; clu^EP0969 / + females (Fig. 5M). Evidence of severe mitochondrial dysfunction was not observed, however, and both male and female trans-heterozygotes were fertile. We conclude that clu and park genetically interact to promote mitochondrial clustering.

Gene expression changes in clu mutant cells

To further analyze the roles played by Clu and Park we investigated if clu or park mutant animals contain differences in major mitochondrial proteins that are encoded in the cell nucleus. Western blots revealed both pyruvate dehydrogenase and Complex V-alpha chain levels were significantly reduced in null clu^d08713 homozygotes. This decrease was not observed in clu^EP0969 homozygotes, which retain a small amount of Clu protein. The observed reduction indicates that clu^d08713 mitochondria contain reduced amounts of these major mitochondrial proteins, or that the total number of mitochondria is reduced in clu^d08713 homozygotes.

To try and identify genes that are directly impacted by clu mutation, we analyzed stage 10B ovarian follicles bearing the weaker clu^EP0969 allele using microarrays. Such follicles appear normal and successfully complete oogenesis, however, Clu protein is significantly depleted (Fig. 1C), and mitochondria cluster early in germ cell development. These studies revealed a small number of high-level changes in gene expression between wild type and clu^EP0969 follicles (Table 2).

Several of the most strongly altered genes protect cells from reactive oxygen species that are generated during mitochondrial metabolism. For example, both CG12896 and GstE1, genes thought to counteract reactive oxygen damage to thiols and to lipids, respectively (Sawicki et al. 2003), are strongly decreased by clu mutation. Several putative DNA repair proteins, including CG10534, a 5′3′ exonuclease, CG5181 (SSB1), a single-stranded DNA binding protein, and phr6-4, a 6-4 photolyase, are all strongly lowered. Apopotosis regulators such as the Rep1 caspase inhibitor (Mukae et al. 2000) and the putative SALL4-homolog CG18446, are reduced as much as tenfold. Several genes involved in mitochondrial metabolism are also changed, including SCAP, which regulates fatty acid production (reviewed in Rawson, 2003). In Drosophila, SCAP and its partner SREBP sense 16-carbon fatty acids and control the transcription of target genes involved in fatty acid biosynthesis in mitochondria. Perhaps in response to altered lipid metabolism, transcripts of several other candidate metabolic genes are also altered, including CG3424 encoding an amino acid/fatty acid transporter, CG11880 encoding a putative choline transporter, U26 encoding a putative aminoadipate-semialdehyde dehydrogenase, and others. Thus, the changes in transcript levels we observe in relatively mild clu mutant follicles suggest that multiple cellular functions linked to mitochondria have been compromised. The results suggest that clu mutation reduces mitochondrial function, enhances oxidative damage and decreases repair or apoptosis.

clu encodes a highly conserved protein required for mitochondrial localization and function

Our studies identify a novel Drosophila gene, clu, that is essential for the normal cytoplasmic localization and function of cellular mitochondria. Clu-related proteins are highly conserved, especially within a novel 243 amino acid region (the “Clu domain”) which is 85% identical (0 gaps) between Drosophila and human Clu. Clu function also appears to be remarkably conserved because mutations in orthologs in Dictyostelium, Saccharomyces, and Arabidopsis all cause mitochondria to cluster into a restricted cytoplasmic region (Zhu et al. 1997; Fields et al. 1998; Dimmer et al. 2002; Logan et al. 2003). In Drosophila, the Clu pathway is also essential to maintain mitochondrial integrity, while Dictyostelium and yeast cells lacking CluA or Clu1 have no detected functional defects.

park interacts with clu

Disruptions in a conserved process of mitochondrial maintenance involving the park and pink1 genes underlies many cases of Parkinson's disease (Abou-Sleiman et al. 2006). We find that Drosophila clu mutations cause phenotypes that strongly resemble those caused by park mutations. Moreover, we showed that mitochondria in park mutants cluster in ovarian cells in a very similar manner to their behavior in clu mutants. In addition, park and clu genetically interact; compound heterozygotes have a mitochondrial clustering phenotype not seen in either heterozygote alone. These observations argue that Clu and Park function in the same or closely related pathways, and that the human Clu ortholog KIAA0664 might play a role in Parkinson's disease. Furthermore, these findings suggest that changes in mitochondrial localization may be of significance in this condition.

Mitochondria cluster at microtubule plus ends in the early germ cells of clu mutants

Mitochondria are often positioned within cells by motor-dependent movement along microtubules (reviewed in Hollenbeck and Saxton, 2005). Clu protein appears to contact mitochondria, especially in the large Clu particles, many of which are located adjacent to microtubules. Our experiments suggest that clu-induced mitochondrial aggregates are caused by changes in microtubule-based mitochondrial transport. In both the Drosophila brain and ovary, mitochondria associate with transport complexes containing the adaptor protein Milton that are linked to both plus end-directed motors such as Kinesin and minus-end directed motors such as Dynein (Glater et al. 2006; Cox and Spradling, 2006). During oogenesis, loss of Khc, or of one of the Milton isoforms that interacts with Khc, cause mitochondria to cluster in cellular regions rich in microtubule “−“ ends, presumably because countervailing movement toward “+” ends has been lost (see Fig. 5 in Cox and Spradling, 2006). Strikingly, mitochondria in clu mutant ovaries accumulate at predicted sites of microtuble “+” ends. These include the proximal region of germline stem cells (Fig. 3C) and against the fusome in early cysts (Fig. 3G, H). Thus, clu mutations may act by interfering with minus-end directed mitochondrial movement along microtubules in some situations. Whether Clu particles correspond to mitochondrial transport complexes that mediate these changes, or whether Clu acts indirectly on mitochondrial positioning could not be determined with certainty from our studies.

In other cells, such as nurse cells, clu mutations also cause pronounced mitochondrial clustering (Fig. 3J), but that clustering is not mimicked by mutations in genes encoding microtubule motor proteins or Milton (Cox and Spradling, 2006). Other systems of mitochondrial localization may predominate in such cells, but still be subject to regulation by the Clu pathway. For example, in plant cells, Clu was postulated to control mitochondrial localization by regulating the choice between microtubule-dependent and microfilament-dependent transport (Logan et al. 2003). In particular, the TPR domain of Clu was proposed to repress the interaction of mitochondria with microtubules by competing for binding between a similar TPR region in kinesin light chain, thereby allowing transport along actin to predominate. The loss of this postulated interaction via clu mutation predicts that mitochondria would accumulate at microtubule minus ends in these cells. Although the default position where mitochondria accumulate in the absence of Clu might vary depending on the particular cell type and transport systems involved, the use of a Clu-dependent pathway to control mitochondrial subcellular location appears to be widespread.

Clu and Park may function in a novel pathway affecting mitochondrial physiology

Our experiments show that mitochondria require Clu to maintain their subcellular location and structural integrity. In the presence of even a relatively mild clu mutation, transcripts from genes involved in mitochondrial function and in protection from oxidative damage are reduced. Consequently, we propose that the normal role of Clu is to function in a pathway that controls the location and activity of mitochondria within the cell. Frequently, mitochondria may need to move to a different subcellular location in order to maximize access to substrates or to mitigate damage from oxidative metabolism. For example, in neurons, microtubule-dependent mitochondrial transport is modulated based on the level of respiratory activity (Miller and Sheetz, 2004). Clu protein would participate in a pathway that senses the internal physiological state of individual mitochondria and transduces this information into homeostatic changes in its position and metabolic activity. When the Clu pathway is impaired, mitochondria would not move or operate normally, and might consequently suffer damage, a scenario consistent with the changes we observed in the inner mitochondrial membranes of Clu mutants, with their reduced levels of mitochondrial enzymes, and with the observed changes in nuclear gene expression. The greater severity of clu mutations in Drosophila compared to Dictyostelium and yeast clu might be due to an intrinsically greater requirement for dynamic mitochondrial positioning in the specialized cells of complex metazoans. This model may provide a rationale for the unexpected effects of microtubule inhibitors on mitochondrial function that were reported recently (Wagner et al. 2008).

Fig. 7 illustrates how the clu and park pathway(s) might link microtubule-based mitochondrial transport, mitochondrial physiology and oxidative damage. Under normal conditions, the clu and park pathway(s) would sense the physiological state of mitochondria and activate appropriate levels of minus-end directed mitochondrial movement along microtubules. For example, mitochondria low in respiratory substrates might move to cellular regions where these substrates are abundant. Mitochondria in need of repair might move close to the nucleus where appropriate repair genes would be induced. In conjunction with active plus end-directed motors, the result would be that mitochondria move dynamically to locations throughout the cell appropriate to their physiological state. However, without clu and park pathway(s) function or if the local level of toxic metabolic products exceeded a threshold, the mitochondria in question would cease minus-end directed transport and undergo concerted plus-end directed movement. In many cells, the major locus of microtubule minus ends is found near the nucleus so that plus end-directed movement would increase the distance between reactive oxygen production and the nuclear DNA.

Clu and mitochondrial inheritance

During early germ cell development in many organisms, a subset of mitochondria within the germ cell cyst is transported and accumulates in a concerted fashion to form the Balbiani body (Cox and Spradling, 2003; Kloc et al, 2004). It has been postulated that Balbiani body formation is a selective process that rids the germline of mitochondria with defective mitochondrial genomes (Pepling et al. 1999; Cox and Spradling, 2003). In Drosophila, mitochondrial movement leading up to Balbiani body formation takes place by minus-end directed transport along microtubules (Cox and Spradling, 2003; 2006). Consequently, the Clu pathway might contribute to mitochondrial screening. However, Balbiani bodies of normal size were observed to form in clu mutants, suggesting that Clu function is not utilized or is redundant at this critical stage of germ cell development.

The Clu pathway and Parkinson's disease

Our studies have several implications for understanding Parkinson's disease. According to our model, the Clu pathway would contribute strongly to the ability of mitochondria to remain functional during aging, despite the high metabolic requirements and environmental stresses experienced by many tissues. This may be particularly important in neural cells such as those that are compromised in Parkinson's disease. In a cell whose Clu pathway is compromised, mitochondria would operate less efficiently, suffer more damage and wear out faster because they would spend more time in cellular locations that are not appropriate to their metabolic state. The number of mitochondria producing elevated levels of ROS would rise, increasing reactive oxygen damage to the nucleus and other cellular components, thereby leading to greater cell death. Consequently, the level of Clu pathway function within an individual might influence their susceptibility to sporadic Parkinson's disease and to other late-onset neurological disorders.

Mitochondrial mutations may fall into two classes based on their phenotype, and on their relationship to the Clu pathway. Some mutations, such as those affecting mitochondrial ribosomal proteins (Royden et al. 1987), or certain metabolic pathways (Zhang et al. 1999), cause bang-sensitivity, but differ phenotypically from clu mutation (Fergestad et al. 2006). In contrast, mutations in park (Greene et al. 2003), pink1 (Wang et al. 2006), rho7 (McQuibban et al. 2006), mitochondrial ATP6 (Celotto et al. 2006), and mitochondrial cytochrome oxidase (Xu et al. 2008) resemble clu mutations in causing general defects in movement, flight, muscle or nerve degeneration, male fertility and longevity. The effects of this latter class may be too severe to be compensated by Clu pathway operation, leading to mitochondrial mis-positioning and increased production of reactive oxygen species. Consistent with this, expression of anti-oxidants partially suppresses the affects of pink1 on neurodegeneration (Wang et al. 2006). Mutations in some of these genes, in addition to park, may also cause mitochondrial clustering and genetically interact with clu. Clearly, further studies of the Clu pathway will deepen our understanding of how mitochondria are maintained in cells and why they sometimes become damaged with age.

Drosophila strains

All symbols are as described in FlyBase. Park[1] (Cha et al, 2005) was obtained from Dr. Kyoung Sang Cho, and park[Z472] (Greene et al, 2003) was obtained from Leo Pallanck. WT flies were CantonS, except for Fig. 3C, which is OregonR. All other stocks were obtained from Bloomington or Harvard. All stocks were cultured at 22-25°C on standard food.

Antibody production

Antibodies were raised to amino acids 1-104 and 1301-1448 of Clu using the protocol described in Buszczak and Spradling (2006).

Immunofluorescene Microscopy

Fattened females and males were dissected and fixed following the protocol in Cox and Spradling (2003). Primary antibodies were diluted at follows: mouse Complex V, alpha subunit (1:500, MitoSciences), mouse 1B1 (1:100, Developmental Studies Hybridoma Bank), ginea pig Clu-C (1:2000). The following secondaries were used: goat anti-mouse IgG2b and goat anti-mouse AlexaFluor488, goat anti-mouse IgG1 AlexaFluor 568, goat anti-ginea pig AlexaFluor 568 (1:400, Molecular Probes). For microtubule labeling, fattened ovaries were fixed for fifteen minues in 4% paraformaldehyde in Graces with 1mM EGTA. Primary antibodies were a 1:1:1 mix of E7, AA 12.1, AA 4.3 (1:50, Developmental Studies Hybridoma Bank). For DNA labeling, DAPI was added 5 ug/ml for 10 minutes prior to adding VectoShield (Vector Labs, Inc.). Confocal analysis was done using a 63× (NA 1.32) PlanApo lens and Leica TCS SP2 or SP5 confocal microscopes, or a Zeiss LSM510 Pascal.

Electron microscopy

Adult flight muscles and fattened ovaries were dissected in Grace's media (Sigma), then processed and imaged as described in Cox and Spradling (2006).

Western blotting

Western blotting was done as described in Wilhelm et al (2000) with the following modifications. To isolate protein, adults were ground with a pestle in Laemmli buffer + BME. Sample was boiled seven minutes before loading on a 4-15% acrylamide gel. The following primary antibodies were used: goat α-Clu-N was used 1:15,000, mouse α-actin (JLA-20, 1:200, Sigma), mouse α-Pyruvate dehydrogenase E1 α-subunit (PDF, 1:500, Mitosciences), mouse α-Complex V, α-subunit (CVα, 1:10,000, Mitosciences)

Microarray analyses

RNA was extracted from 100 stage 10B ovarian follicles as described in Allen and Spradling (2008). Microarray analyses were performed in duplicate with RNA samples isolated on separate days, and greater than 90% of the genes that changed significantly were reproducible.