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1.  TIF-IA-Dependent Regulation of Ribosome Synthesis in Drosophila Muscle Is Required to Maintain Systemic Insulin Signaling and Larval Growth 
PLoS Genetics  2014;10(10):e1004750.
The conserved TOR kinase signaling network links nutrient availability to cell, tissue and body growth in animals. One important growth-regulatory target of TOR signaling is ribosome biogenesis. Studies in yeast and mammalian cell culture have described how TOR controls rRNA synthesis—a limiting step in ribosome biogenesis—via the RNA Polymerase I transcription factor TIF-IA. However, the contribution of TOR-dependent ribosome synthesis to tissue and body growth in animals is less clear. Here we show in Drosophila larvae that ribosome synthesis in muscle is required non-autonomously to maintain normal body growth and development. We find that amino acid starvation and TOR inhibition lead to reduced levels of TIF-IA, and decreased rRNA synthesis in larval muscle. When we mimic this decrease in muscle ribosome synthesis using RNAi-mediated knockdown of TIF-IA, we observe delayed larval development and reduced body growth. This reduction in growth is caused by lowered systemic insulin signaling via two endocrine responses: reduced expression of Drosophila insulin-like peptides (dILPs) from the brain and increased expression of Imp-L2—a secreted factor that binds and inhibits dILP activity—from muscle. We also observed that maintaining TIF-IA levels in muscle could partially reverse the starvation-mediated suppression of systemic insulin signaling. Finally, we show that activation of TOR specifically in muscle can increase overall body size and this effect requires TIF-IA function. These data suggest that muscle ribosome synthesis functions as a nutrient-dependent checkpoint for overall body growth: in nutrient rich conditions, TOR is required to maintain levels of TIF-IA and ribosome synthesis to promote high levels of systemic insulin, but under conditions of starvation stress, reduced muscle ribosome synthesis triggers an endocrine response that limits systemic insulin signaling to restrict growth and maintain homeostasis.
Author Summary
All animals need adequate nutrition to grow and develop. Studies in tissue culture and model organisms have identified the TOR kinase signaling pathway as a key nutrient-dependent regulator of growth. Under nutrient rich conditions, TOR kinase is active and stimulates metabolic processes that drive growth. Under nutrient poor conditions, TOR is inhibited and animals alter their metabolism to maintain homeostasis and survival. Here we use Drosophila larvae to identify a role for ribosome synthesis—a key metabolic process—in mediating nutrient and TOR effects on body growth. In particular, we show that ribosome synthesis specifically in larval muscle is necessary to maintain organismal growth. We find that inhibition of muscle ribosome synthesis leads to reduced systemic insulin-like growth factor signaling via two endocrine responses—decreased expression of brain derived Drosophila insulin-like peptides (dILPs) and increased expression of Imp-L2, an inhibitor of insulin signaling. As a result of these effects, body growth is reduced and larval development is delayed. These findings suggest that control of ribosome synthesis, and hence protein synthesis, in specific tissues can exert control on overall body growth.
doi:10.1371/journal.pgen.1004750
PMCID: PMC4214618  PMID: 25356674
2.  An investigation of nutrient-dependent mRNA translation in Drosophila larvae 
Biology Open  2014;3(11):1020-1031.
ABSTRACT
The larval period of the Drosophila life cycle is characterized by immense growth. In nutrient rich conditions, larvae increase in mass approximately two hundred-fold in five days. However, upon nutrient deprivation, growth is arrested. The prevailing view is that dietary amino acids drive this larval growth by activating the conserved insulin/PI3 kinase and Target of rapamycin (TOR) pathways and promoting anabolic metabolism. One key anabolic process is protein synthesis. However, few studies have attempted to measure mRNA translation during larval development or examine the signaling requirements for nutrient-dependent regulation. Our work addresses this issue. Using polysome analyses, we observed that starvation rapidly (within thirty minutes) decreased larval mRNA translation, with a maximal decrease at 6–18 hours. By analyzing individual genes, we observed that nutrient-deprivation led to a general reduction in mRNA translation, regardless of any starvation-mediated changes (increase or decrease) in total transcript levels. Although sugars and amino acids are key regulators of translation in animal cells and are the major macronutrients in the larval diet, we found that they alone were not sufficient to maintain mRNA translation in larvae. The insulin/PI3 kinase and TOR pathways are widely proposed as the main link between nutrients and mRNA translation in animal cells. However, we found that genetic activation of PI3K and TOR signaling, or regulation of two effectors – 4EBP and S6K – could not prevent the starvation-mediated translation inhibition. Similarly, we showed that the nutrient stress-activated eIF2α kinases, GCN2 and PERK, were not required for starvation-induced inhibition of translation in larvae. These findings indicate that nutrient control of mRNA translation in larvae is more complex than simply amino acid activation of insulin and TOR signaling.
doi:10.1242/bio.20149407
PMCID: PMC4232759  PMID: 25305039
Drosophila; TOR; Growth control; Insulin; mRNA translation; Nutrition
3.  Control of Drosophila endocycles by E2F and CRL4Cdt2 
Nature  2011;480(7375):123-127.
Endocycles are variant cell cycles comprised of DNA Synthesis (S)- and Gap (G)- phases but lacking mitosis1,2. Such cycles facilitate post-mitotic growth in many invertebrate and plant cells, and are so ubiquitous that they may account for up to half the world’s biomass3,4. DNA replication in endocycling Drosophila cells is triggered by Cyclin E/Cyclin Dependent Kinase 2 (CycE/Cdk2), but this kinase must be inactivated during each G-phase to allow the assembly of pre-Replication Complexes (preRCs) for the next S-phase5,6. How CycE/Cdk2 is periodically silenced to allow re-replication has not been established. Here, using genetic tests in parallel with computational modeling, we show that Drosophila’s endocycles are driven by a molecular oscillator in which the E2F1 transcription factor promotes CycE expression and S-phase initiation, S-phase then activates the CRL4Cdt2 ubiquitin ligase, and this in turn mediates the destruction of E2F17. We propose that it is the transient loss of E2F1 during S-phases that creates the window of low Cdk activity required for preRC formation. In support of this model over-expressed E2F1 accelerated endocycling, whereas a stabilized variant of E2F1 blocked endocycling by de-regulating target genes including CycE, as well as Cdk1 and mitotic Cyclins. Moreover, we find that altering cell growth by changing nutrition or TOR signaling impacts E2F1 translation, thereby making endocycle progression growth-dependent. Many of the regulatory interactions essential to this novel cell cycle oscillator are conserved in animals and plants1,2,8, suggesting that elements of this mechanism act in most growth-dependent cell cycles.
doi:10.1038/nature10579
PMCID: PMC3330263  PMID: 22037307
4.  Controlling animal growth and body size – does fruit fly physiology point the way? 
The question of how growth and size are controlled has fascinated generations of biologists. However, the underlying mechanisms still remain unclear. The last year or so has seen a flurry of reports on the control of growth and body size in Drosophila, and a central theme to these papers is the idea of signaling between organs as a control mechanism for overall body growth and development. While this concept is obviously not new, these fly studies now open up the possibility of using a genetically tractable system to dissect in detail how organ-to-organ communication dictates body size.
doi:10.3410/B4-12
PMCID: PMC3369236  PMID: 22685490
5.  Nutritional control of gene expression in Drosophila larvae via TOR, Myc and a novel cis-regulatory element 
BMC Cell Biology  2010;11:7.
Background
Nutrient availability is a key determinant of eukaryotic cell growth. In unicellular organisms many signaling and transcriptional networks link nutrient availability to the expression of metabolic genes required for growth. However, less is known about the corresponding mechanisms that operate in metazoans. We used gene expression profiling to explore this issue in developing Drosophila larvae.
Results
We found that starvation for dietary amino acids (AA's) leads to dynamic changes in transcript levels of many metabolic genes. The conserved insulin/PI3K and TOR signaling pathways mediate nutrition-dependent growth in Drosophila and other animals. We found that many AA starvation-responsive transcripts were also altered in TOR mutants. In contrast, although PI3K overexpression induced robust changes in the expression of many metabolic genes, these changes showed limited overlap with the AA starvation expression profile. We did however identify a strong overlap between genes regulated by the transcription factor, Myc, and AA starvation-responsive genes, particularly those involved in ribosome biogenesis, protein synthesis and mitochondrial function. The consensus Myc DNA binding site is enriched in promoters of these AA starvation genes, and we found that Myc overexpression could bypass dietary AA to induce expression of these genes. We also identified another sequence motif (Motif 1) enriched in the promoters of AA starvation-responsive genes. We showed that Motif 1 was both necessary and sufficient to mediate transcriptional responses to dietary AA in larvae.
Conclusions
Our data suggest that many of the transcriptional effects of amino acids are mediated via signaling through the TOR pathway in Drosophila larvae. We also find that these transcriptional effects are mediated through at least two mechanisms: via the transcription factor Myc, and via the Motif 1 cis-regulatory element. These studies begin to elucidate a nutrient-responsive signaling network that controls metabolic gene transcription in Drosophila.
doi:10.1186/1471-2121-11-7
PMCID: PMC2827378  PMID: 20089194
6.  Drosophila TIF-IA is required for ribosome synthesis and cell growth and is regulated by the TOR pathway 
The Journal of Cell Biology  2007;179(6):1105-1113.
Synthesis of ribosomal RNA (rRNA) is a key step in ribosome biogenesis and is essential for cell growth. Few studies, however, have investigated rRNA synthesis regulation in vivo in multicellular organisms. Here, we present a genetic analysis of transcription initiation factor IA (TIF-IA), a conserved RNA polymerase I transcription factor. Drosophila melanogaster Tif-IA−/− mutants have reduced levels of rRNA synthesis and sustain a developmental arrest caused by a block in cellular growth. We find that the target of rapamycin (TOR) pathway regulates TIF-IA recruitment to rDNA. Furthermore, we show that the TOR pathway regulates rRNA synthesis in vivo and that TIF-IA overexpression can maintain rRNA transcription when TOR activity is reduced in developing larvae. We propose that TIF-IA acts in vivo as a downstream growth–regulatory target of the TOR pathway. Overexpression of TIF-IA also elevates levels of both 5S RNA and messenger RNAs encoding ribosomal proteins. Stimulation of rRNA synthesis by TIF-IA may therefore provide a feed-forward mechanism to coregulate the levels of other ribosome components.
doi:10.1083/jcb.200709044
PMCID: PMC2140016  PMID: 18086911
7.  Rheb-TOR signaling promotes protein synthesis, but not glucose or amino acid import, in Drosophila 
BMC Biology  2007;5:10.
Background
The Ras-related GTPase, Rheb, regulates the growth of animal cells. Genetic and biochemical tests place Rheb upstream of the target of rapamycin (TOR) protein kinase, and downstream of the tuberous sclerosis complex (TSC1/TSC2) and the insulin-signaling pathway. TOR activity is regulated by nutritional cues, suggesting that Rheb might either control, or respond to, nutrient availability.
Results
We show that Rheb and TOR do not promote the import of glucose, bulk amino acids, or arginine in Drosophila S2 cells, but that both gene products are important regulators of ribosome biogenesis, protein synthesis, and cell size. S2 cell size, protein synthesis, and glucose import were largely insensitive to manipulations of insulin signaling components, suggesting that cellular energy levels and TOR activity can be maintained through insulin/PI3K-independent mechanisms in S2 cell culture. In vivo in Drosophila larvae, however, we found that insulin signaling can regulate protein synthesis, and thus may affect TOR activity.
Conclusion
Rheb-TOR signaling controls S2 cell growth by promoting ribosome production and protein synthesis, but apparently not by direct effects on the import of amino acids or glucose. The effect of insulin signaling upon TOR activity varies according to cellular type and context.
doi:10.1186/1741-7007-5-10
PMCID: PMC1847425  PMID: 17371599
8.  Controlling cell division in yeast and animals: does size matter? 
Journal of Biology  2003;2(1):5.
In yeast, cell-size checkpoints coordinate cellular growth with cell-cycle progression. Now, evidence has been provided that such checkpoints probably do not exist in mammalian cells. These findings highlight an important difference between how yeast and animal cells proliferate in response to extracellular cues.
doi:10.1186/1475-4924-2-5
PMCID: PMC156596  PMID: 12733996
9.  Role of Phosphoinositide 3-Kinase and Endocytosis in Nerve Growth Factor-Induced Extracellular Signal-Regulated Kinase Activation via Ras and Rap1 
Molecular and Cellular Biology  2000;20(21):8069-8083.
Neurotrophins promote multiple actions on neuronal cells including cell survival and differentiation. The best-studied neurotrophin, nerve growth factor (NGF), is a major survival factor in sympathetic and sensory neurons and promotes differentiation in a well-studied model system, PC12 cells. To mediate these actions, NGF binds to the TrkA receptor to trigger intracellular signaling cascades. Two kinases whose activities mediate these processes include the mitogen-activated protein (MAP) kinase (or extracellular signal-regulated kinase [ERK]) and phosphoinositide 3-kinase (PI3-K). To examine potential interactions between the ERK and PI3-K pathways, we studied the requirement of PI3-K for NGF activation of the ERK signaling cascade in dorsal root ganglion cells and PC12 cells. We show that PI3-K is required for TrkA internalization and participates in NGF signaling to ERKs via distinct actions on the small G proteins Ras and Rap1. In PC12 cells, NGF activates Ras and Rap1 to elicit the rapid and sustained activation of ERKs respectively. We show here that Rap1 activation requires both TrkA internalization and PI3-K, whereas Ras activation requires neither TrkA internalization nor PI3-K. Both inhibitors of PI3-K and inhibitors of endocytosis prevent GTP loading of Rap1 and block sustained ERK activation by NGF. PI3-K and endocytosis may also regulate ERK signaling at a second site downstream of Ras, since both rapid ERK activation and the Ras-dependent activation of the MAP kinase kinase kinase B-Raf are blocked by inhibition of either PI3-K or endocytosis. The results of this study suggest that PI3-K may be required for the signals initiated by TrkA internalization and demonstrate that specific endocytic events may distinguish ERK signaling via Rap1 and Ras.
PMCID: PMC86417  PMID: 11027277

Results 1-9 (9)