InteractiveFly: GeneBrief

stunted: Biological Overview | References


Gene name - stunted

Synonyms -

Cytological map position - 13F12-13F12

Function - peptide; subunit of mitochondrial ATP synthase

Keywords - a circulating insulinotropic peptide produced by fat cells - modulates physiological insulin levels in response to nutrients - ε-subunit of mitochondrial ATP synthase - required for normal spindle orientation during embryonic divisions

Symbol - sun

FlyBase ID: FBgn0014391

Genetic map position - chrX:15,847,859-15,849,258

Classification - Mitochondrial ATP synthase ε chain

Cellular location - secreted peptide & mitochondrial



NCBI links: Precomputed BLAST | EntrezGene
BIOLOGICAL OVERVIEW

Animals adapt their growth rate and body size to available nutrients by a general modulation of insulin-insulin-like growth factor signaling. In Drosophila, dietary amino acids promote the release in the hemolymph of brain insulin-like peptides (Dilps), which in turn activate systemic organ growth. Dilp secretion by insulin-producing cells involves a relay through unknown cytokines produced by fat cells. This study identified Methuselah (Mth) as a secretin-incretin receptor subfamily member required in the insulin-producing cells for proper nutrient coupling. Using genetic and ex vivo organ culture experiments, it was shown that the Mth ligand Stunted is a circulating insulinotropic peptide produced by fat cells. Therefore, Sun and Mth define a new cross-organ circuitry that modulates physiological insulin levels in response to nutrients (Delanoue, 2016).

Environmental cues, such as dietary products, alter animal physiology by acting on developmental and metabolic parameters like growth, longevity, feeding, and energy storage or expenditure. The systemic action of this control suggests that intermediate sensor tissues evaluate dietary nutrients and trigger hormonal responses. Previous work in Drosophila melanogaster established that a specific organ called the fat body translates nutritional information into systemic growth-promoting signals. The leptinlike Janus kinase-signal transducers and activators of transcription (JAK-STAT) ligand unpaired 2 and the CCHamid2 peptide are produced by fat cells in response to both sugar and fat and trigger a metabolic response. Dietary amino acids activate TORC1 signaling in fat cells and induce the production of relay signals that promote the release of insulin-like peptides (Dilps) by brain insulin-producing cells (IPCs). Two fat-derived peptides (GBP1 and GBP2) activate insulin secretion in response to a protein diet, although their receptor and neural targets remain uncharacterized. To identify critical components of this organ crosstalk, a genetic screen was conducted in Drosophila larvae. The gene methuselah (mth), which encodes a heterotrimeric GTP-binding protein (G protein)-coupled receptor belonging to the subfamily of the secretin-incretin receptor subfamily came out as a strong hit. Impairing mth function in the IPCs reduces larval body growth, whereas silencing mth in a distinct set of neurons or in the larval fat body had no impact on pupal volume. Larvae in which expression of the mth gene is reduced by RNA interference (RNAi), specifically in the IPCs (hereafter, dilp2>mth-Ri), present an accumulation of Dilp2 and Dilp5 in the IPCs, whereas dilp2 gene expression remains unchanged, a phenotype previously described as impaired Dilp secretion. Indeed, forced depolarization of the IPCs rescues pupal volume and Dilp2 accumulation upon IPC-specific mth depletion. Therefore, Mth is required for Dilps secretion and larval body growth (Delanoue, 2016).

Two peptides encoded by the stunted (sun) gene, SunA and SunB, serve as bona fide ligands for Mth and activate a Mth-dependent intracellular calcium response. Silencing sun in fat cells, but no other larval tissue, of well-fed larvae mimics the mth loss-of-function phenotype with no effect on the developmental timing. Conversely, overexpression of sun in the larval fat body (lpp>sun) partially rescues the systemic growth inhibition observed upon feeding larvae a diet low in amino acids or upon 'genetic starvation' [silencing of the slimfast (slif) gene in fat cells. This growth rescue is abolished in mth1 homozygous mutants. This shows that Sun requires Mth to control growth. However, sun overexpression has no effect in animals fed a normal diet. A modification of sun expression does not prevent fat body cells from responding to amino acid deprivation as seen by the level of TORC1 signaling, general morphology, and lipid droplet accumulation but affects the ability of larvae to resist to starvation (Delanoue, 2016).

Dilp2-containing secretion granules accumulate in the IPCs following starvation and are rapidly released upon refeeding. Mth is required in the IPCs to promote Dilp secretion after refeeding, and forced membrane depolarization of IPCs using a bacterial sodium channel (dilp2>NaChBac) is dominant over the blockade of Dilp2 secretion in dilp2>mth-Ri animals. This dominance indicates that Mth acts upstream of the secretion machinery. In addition, Dilp2 secretion after refeeding is abrogated in lpp>sun-Ri animals, and overexpression of sun in fat cells prevents Dilp2 accumulation upon starvation. Altogether, these findings indicate that Mth and its ligand Sun are two components of the systemic nutrient response controlling Dilp secretion (Delanoue, 2016).

Hemolymph from fed animals triggers Dilp2 secretion when applied to brains dissected from starved larvae. This insulinotropic activity requires the function of Mth in the IPCs and the production of Sun by fat body cells. Conversely, overexpressing sun in the fat body (lpp>sun) is sufficient to restore insulinotropic activity to the hemolymph of starved larvae. A 2-hour incubation with a synthetic peptide corresponding to the Sun isoform A (Sun-A) is also sufficient to induce Dilp secretion from starved brains. A similar effect is observed with an N-terminal fragment of Sun (N-SUN) that contains the Mth-binding domain but not with a C-terminal fragment (C-SUN) that does not bind Mth. The insulinotropic effect of N-SUN is no longer observed in brains from larvae of the mth allele, mth1 . This absence of effect indicates that N-SUN action requires Mth in the brain. In addition, preincubation of control hemolymph with antiserum containing Sun antibodies specifically suppresses its insulinotropic function. These results indicate that Sun is both sufficient and necessary for insulinotropic activity in the hemolymph of protein-fed animals (Delanoue, 2016).

To directly quantify the amount of circulating Sun protein, Western blot experiments wee performed on hemolymph using antibodies against Sun. A 6-kD band was detected in hemolymph collected from fed larvae, and size was confirmed using Schneider 2 (S2) cell extracts. The band intensity was reduced upon sun knockdown in fat body cells but not in gut cells. Therefore, circulating Sun peptide appears to be mostly contributed by fat cells, as suggested by functional experiments. The levels of circulating Sun are strongly reduced upon starvation. In line with this, sun transcripts are drastically reduced after 4 hours of protein starvation and start increasing after 1 hour of refeeding, whereas expression of the sun homolog CG31477 is not modified. sun transcription is not affected by blocking TORC1, the main sensor for amino acids in fat body cells. However, adipose-specific TORC1 inhibition induces a dramatic reduction of circulating Sun, indicating that TORC1 signaling controls Sun peptide translation or secretion from fat cells. PGC1-Spargel is a transcription activator, the expression of which relies on nutritional input. PGC1 was found to be required for sun transcription, and fat body silencing of PGC1 and sun induce identical larval phenotypes. Although PGC1 expression is strongly suppressed upon starvation, blocking TORC1 activity in fat cells does not reduce PGC1 expression. Conversely, knocking down PGC1 does not inhibit TORC1 activity. This finding suggests that PGC1 and TORC1 act in parallel. Therefore, Sun production by fat cells in response to nutrition is controlled at two distinct levels by PGC1 and TORC1 (Delanoue, 2016).

The Sun peptide is identical to the ε subunit of the mitochondrial F1F0-adenosine triphosphatase (F1F0-ATPase) synthase (complex V). Indeed, both endogenous Sun and Sun labeled with a hemagglutinin tag (Sun-HA) colocalize with mitochondrial markers in fat cells , and the Sun peptide cofractionates with mitochondrial complex V in blue native polyacrylamide gel electrophoresis. In addition, silencing sun in fat cells decreases mitochondrial Sun staining and the amounts of adenosine triphosphate (ATP). However, recent evidence indicates that an ectopic (ecto) form of the F1F0-ATP synthase is found associated with the plasma membrane in mammalian and insect cells. In addition, coupling factor 6, a subunit of complex V, is found in the plasma. Therefore, Stunted could participate in two separate functions carried by distinct molecular pools. To address this possibility, a modified form of Stunted carrying a green fluorescent protein (GFP) tag at its N terminus (GFP-Sun), next to the mitochondria-targeting signal (MTS), was used. When expressed in fat cells, GFP-Sun does not localize to the mitochondria, contrarily to a Sun peptide tagged at its C-terminal end (Sun-GFP). This suggests that addition of the N-terminal tag interferes with the MTS and prevents mitochondrial transport of Sun. However, both GFP-Sun and Sun-GFP are found in the hemolymph and rescue pupal size and Dilp2 accumulation in larvae fed a low-amino acid diet as efficiently as wild-type Sun (wt-Sun) and do so in a mth-dependent manner. This indicates that the growth-promoting function of Sun requires its secretion but not its mitochondrial localization and suggests the existence of one pool of Sun peptide located in the mitochondria devoted to F1F0-ATP synthase activity and ATP production and another pool released in the hemolymph for coupling nutrient and growth control. In this line, although fat body levels of Sun are decreased upon starvation, its mitochondrial localization is not reduced. This finding indicates that starvation affects a nonmitochondrial pool of Sun. In support of this, starved fat bodies contain normal levels of ATP and lactate, indicating that mitochondrial oxidative phosphorylation is preserved in fat cells in poor nutrient conditions. Last, other subunits from complex V (ATP5a) or complex I (NdufS3) were not detected in circulating hemolymph. Therefore, the release of Sun in the hemolymph relies on a specific mechanism (Delanoue, 2016).

In conclusion, this study has provided evidence for a molecular cross-talk between fat cells and brain IPCs involving the ligand Stunted and its receptor Methuselah. Stunted is a moonlighting peptide present both in the mitochondria as part of the F1F0-ATP synthase complex and as an insulinotropic ligand circulating in the hemolymph. The mechanism of Stunted release remains to be clarified. The beta subunit of the ectopic form of F1F0-ATP synthase is a receptor for lipoproteins, which serve as cargos for proteins and peptides. In addition, Drosophila lipid transfer particle-containing lipoproteins were shown to act on the larval brain to control systemic insulin signaling in response to nutrition. This suggests that Sun could be loaded on lipoproteins for its transport. Given the role of insulin-insulin-like growth factor (IGF) signaling in aging, the current findings could help in understanding the role of Sun/Mth in aging adult flies (Delanoue, 2016).

The same genetic screen previously identified the fly tumor necrosis factor α Eiger (Egr) as an adipokine necessary for long-term adaptation to protein starvation, and recent work pointed to other adipose factors, illustrating the key role of the larval fat body in orchestrating nutrient response. The multiplicity of adipose factors and their possible redundancy could explain the relatively mild starvation-like phenotype obtained after removal of only one of them. Overall, these findings suggest a model whereby partially redundant fat-derived signals account for differential response to positive and negative valence of various diet components, as well as acute versus long-term adaptive responses (Delanoue, 2016).

The ε-subunit of mitochondrial ATP synthase is required for normal spindle orientation during the Drosophila embryonic divisions

This study describes the maternal-effect and zygotic phenotypes of null mutations in the Drosophila gene for the epsilon-subunit of mitochondrial ATP synthase, stunted (sun). Loss of zygotic sun expression leads to a dramatic delay in the growth rate of first instar larvae and ultimately death. Embryos lacking maternally supplied sun (sun embryos) have a sixfold reduction in ATP synthase activity. Cellular analysis of sun embryos shows defects only after the nuclei have migrated to the cortex. During the cortical divisions the actin-based metaphase and cellularization furrows do not form properly, and the nuclei show abnormal spacing and division failures. The most striking abnormality is that nuclei and spindles form lines and clusters, instead of adopting a regular spacing. This is reflected in a failure to properly position neighboring nonsister centrosomes during the telophase-to-interphase transition of the cortical divisions. This study is consistent with a role for Sun in mitochondrial ATP synthesis and suggests that reduced ATP levels selectively affect molecular motors. As Sun has been identified as the ligand for the Methuselah receptor that regulates aging, Sun may function both within and outside mitochondria (Kidd, 2005).

This study describes the maternal and zygotic effects of null mutations in the sun locus. sun encodes the ε-subunit of the mitochondrial ATP synthase, the universal enzyme for cellular ATP synthesis. In yeast the ε-subunit is a nonessential gene required for dimerization and oligomerization of ATP synthase and is involved in generating the inward foldings of the inner mitochondrial membrane, the cristae. The ε-subunit is also a potential binding site or target of the natural inhibitor protein IF1 that serves to prevent ATP hydrolysis. The ε-subunit appears necessary for the maximum efficiency of the ATP synthase complex and it has been proposed to be a molecular clutch regulating the coupling of ATP synthesis to proton flow. Expression of the bovine ε-subunit can rescue the growth defect of S. cerevisiae carrying a deletion of the ε-subunit gene. This result suggests that the molecular function of the ε-subunit within the mitochondrial ATP synthase complex has been conserved throughout eukaryotic evolution. Thus the sun mutant phenotypes are interpreted to be a consequence of reduced levels of ATP. This is consistent with the increased severity of the defects seen at higher temperatures in the sun mutants, because ATP requirements and oxygen consumption increase at higher temperatures. Reducing ATP levels in the embryo by inhibiting oxidative phosphorylation with cyanide or azide induces a cell cycle arrest. This study saw no evidence of cell cycle arrests in sun maternal-effect embryos (Kidd, 2005).

The sun maternal effect is most dramatic during the late cortical cycles, presumably reflecting a greater energetic load at these stages. Lack of sun activity disrupts alignment of neighboring spindles and formation of the metaphase and cellularization furrows. A direct consequence of this is fusions of sister nuclei. Computational studies of these mitoses indicate that the even spacing results from interactions of each nucleus with its neighbors. These interactions most likely arise from centrosome-based astral microtubules repelling one another. The force of their repulsion is inversely proportional to the distance between neighboring centrosomes. The abnormal arrangement of spindles in sun embryos is not due to a failure to form astral microtubules, as no difference was seen between wild-type and sun astral microtubules in the light microscope (Kidd, 2005).

One interpretation of the sun phenotype is that the activity of the motor proteins during the cortical divisions places extra energetic demands on the embryo. In sun embryos, while the reduced ATP levels are sufficient for the early divisions, they are insufficient for the cortical divisions. On the basis of the sun maternal-effect phenotype, it is proposed that maintaining regular spacing of the closely packed nuclei is the most ATP-demanding process in the syncytial embryo. The inability to keep nuclei apart leads to inappropriate microtubule interactions, nuclear fusion, and dropping of nuclei back toward the yolk (Kidd, 2005).

The repulsive force between anti-parallel microtubules is generated by microtubule-based motor proteins. Given the number of molecular motors already described, it is highly likely that several contribute to spindle positioning. The sun mutant phenotype could be a failure of the motor proteins to provide this repulsive force. For example, the motor protein KLP61F acts on anti-parallel microtubules to maintain separation of sister centrosomes. In addition, embryos lacking the motor protein Ncd display centrosomal defects and microtubule spurs between mitotic spindles shows three aligned spindles reminiscent of the sun maternal effect. A further candidate motor is the Drosophila homolog of MKLP1 kinesin-like protein, which transports oppositely oriented microtubules relative to one another (Kidd, 2005).

One might have expected decreased levels of ATP to have highly pleiotropic effects, and so it is surprising that sun has such a specific effect on the syncytial mitoses. Many other mutations affecting the syncytial mitoses turn out to be centrosomal or key regulators of the cell cycle, e.g., nuclear fallout. The sun locus is distinctive by encoding a component of a central metabolic enzyme. Mutations in another essential metabolic enzyme, Glutamine synthetase 1 (Gs1), also specifically affect the syncytial mitoses. Gs1 catalyzes the amination of glutamate in an ATP-dependent manner to produce glutamine, which is required for amino acid, purine, and pyrimidine biosynthesis. Analysis of Gs1 mutants suggests that delays in syncytial cell cycle progression lead to nuclei being discarded due to a reduction in amino acid and nucleotide availability. Surprisingly, given the requirement of Gs1 for ATP, the sun and Gs1 mutant phenotypes are almost reciprocal, with Gs1 affecting nuclear events during S phase and sun affecting cytoplasmic events during M phase, with little or no effect on DNA segregation (Kidd, 2005).

Why are the sun and Gs1 mutant phenotypes so reciprocal? Gs1 may function at lower ATP concentrations than required for the molecular motors to maintain spindle separation. In the sun mutant, there may be sufficient ATP to carry out the functions of Gs1, but not those of the microtubule-associated motors. There may also be distinct biochemical pools from which Gs1 and the motors obtain ATP. Under most conditions, intracellular circulation keeps the ATP concentration perfectly homeostatic, meaning the concentration does not change even when ATP-dependent work is being performed. The sun mutant syncytium may resemble a fatigued cell, with local differences in intracellular circulation of ATP to the metabolic pools containing molecular motors and biosynthetic enzymes (Kidd, 2005).

S. cerevisiae deleted for the ε-subunit grow slowly on medium with glycerol as the carbon source, indicating that the ε-subunit is not an essential gene. In contrast to S. cerevisiae, the ε-subunit of ATP synthase is essential for survival of Drosophila. As the bovine ε-subunit can rescue the yeast ε-deletion mutant, it is believed that the phenotypic differences originate in differences in ATP homeostasis between unicellular and multicellular organisms. No other eukaryotic ε-subunit mutants have been published. Phenotypic and biochemical observations indicate that ATP levels are reduced but not eliminated, supporting the hypothesis that the ε-subunit is required for maximal efficiency of ATP synthase. The presence of maternal ATP in sun mutants allows growth until an energetically demanding process is encountered. In the early embryo, the first defects are seen in the cortical divisions, but the embryos continue to grow and cellularize, albeit abnormally. Embryos lacking maternal sun fail to gastrulate, suggesting the dynamic cell movements are incompatible with the reduced ATP levels. In the larva, the energetically demanding processes of DNA replication and protein synthesis normally drive a 200-fold increase in mass over 4 days. The absence of zygotic sun causes a larval growth arrest before any significant growth has occurred. Interestingly, the same phenotype is seen for a mutation, colibri, in the α-subunit of ATP synthase. The α-subunit, known as bellwether in Drosophila, should be absolutely required for ATP synthesis; a series of alleles have been characterized as recessive lethal, but the exact lethal phase was not determined. The colibri allele of bellwether is a P-element insertion in an intron and is most probably a hypomorphic allele allowing some synthesis of ATP in a manner similar to that of sun alleles. Mutant clones of colibri in the wing show a severe size reduction whereas mutant clones in the eye survive well, suggesting different energetic requirements in the two tissues. It is believed that ε-subunit mutations will be essential in all multicellular organisms, but the effects will vary from tissue to tissue (Kidd, 2005).

Sun was unexpectedly identified as the ligand for the Drosophila G-protein-coupled receptor (GPCR), Methuselah (Mth; Cvejic, 2004). Mutations in methuselah (mth) extend life span, and the protein is required in motor neurons where it regulates neurotransmitter release. A Mth-GFP fusion localizes to the plasma membrane of the presynaptic terminals, although it has not conclusively been shown to be exposed to the extracellular environment. Analysis of life span in sun mutants (using the alleles generated in this study) revealed extended life span (Cvejic, 2004; Kidd, 2005 and references therein).

This diverse function might indicate that the sun gene does not encode a genuine homolog of mitochondrial ATP synthase ε-subunits from other species. However, this view appears unlikely. The results from the current study strongly suggest Sun indeed participates in ATP synthesis. Rather, the genetic and biochemical analyses suggest that Sun is a bifunctional protein. Such a dual function is reminiscent of another mitochondrial protein, cytochrome C, which functions within the respiratory electron transport chain and is released from the mitochondria to participate in apoptosis. A better understanding of Sun function awaits the development of antibody reagents to visualize Sun localization, particularly in Drosophila models of aging. For the moment, there are some intriguing hints as to how Sun might be regulated: sun transcription has been shown to be downregulated by oxidative stress, which is thought to limit life span in multicellular organisms. Sun has been shown to bind the regulatory subunit of cAMP protein kinase, suggesting it may be a substrate for phosphorylation (Pka-R1), although it remains to be confirmed in vivo (Kidd, 2005).

The endogenous ligand Stunted of the GPCR Methuselah extends lifespan in Drosophila

Many extracellular signals are transmitted to the interior of the cell by receptors with seven membrane-spanning helices that trigger their effects by means of heterotrimeric guanine-nucleotide-binding regulatory proteins (G proteins). These G-protein-coupled receptors (GPCRs) control various physiological functions in evolution from pheromone-induced mating in yeast to cognition in humans. The potential role of the G-protein signalling system in the control of animal ageing has been highlighted by the genetic revelation that mutation of a GPCR encoded by methuselah extends the lifespan of adult Drosophila flies. How Methuselah functions in controlling ageing is not clear. A first essential step towards the understanding of Methuselah function is to determine the ligands of Methuselah. This study reports the identification and characterization of two endogenous peptide ligands of Methuselah, designated Stunted A and B. Flies with mutations in the gene encoding these ligands show an increase in lifespan and resistance to oxidative stress. It is concluded that the Stunted-Methuselah system is involved in the control of animal ageing (Cvejic, 2004).


REFERENCES

Search PubMed for articles about Drosophila Stunted

Cvejic, S., Zhu, Z., Felice, S. J., Berman, Y. and Huang, X. Y. (2004). The endogenous ligand Stunted of the GPCR Methuselah extends lifespan in Drosophila. Nat Cell Biol 6(6): 540-546. PubMed ID: 15133470

Delanoue, R., Meschi, E., Agrawal, N., Mauri, A., Tsatskis, Y., McNeill, H. and Leopold, P. (2016). Drosophila insulin release is triggered by adipose Stunted ligand to brain Methuselah receptor. Science 353: 1553-1556. PubMed ID: 27708106

Kidd, T., Abu-Shumays, R., Katzen, A., Sisson, J. C., Jimenez, G., Pinchin, S., Sullivan, W. and Ish-Horowicz, D. (2005). The ε-subunit of mitochondrial ATP synthase is required for normal spindle orientation during the Drosophila embryonic divisions. Genetics 170(2): 697-708. PubMed ID: 15834145


Biological Overview

date revised: 5 December 2016

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