In many metazoans, final adult size depends on the growth rate and the duration of the growth period, two parameters influenced by nutritional cues. In Drosophila, nutrition modifies the timing of development by acting on the prothoracic gland (PG), which secretes the molting hormone ecdysone. When activity of the Target of Rapamycin (TOR), a core component of the nutrient-responsive pathway, is reduced in the PG, the ecdysone peak that marks the end of larval development is abrogated. This extends the duration of growth and increases animal size. Conversely, the developmental delay caused by nutritional restriction is reversed by activating TOR solely in PG cells. Finally, nutrition acts on the PG during a restricted time window near the end of larval development that coincides with the commitment to pupariation. In conclusion, this study shows that the PG uses TOR signaling to couple nutritional input with ecdysone production and developmental timing. Previously studies have shown that the same molecular pathway operates in the fat body (a functional equivalent of vertebrate liver and white fat) to control growth rate, another key parameter in the determination of adult size. Therefore, the TOR pathway takes a central position in transducing the nutritional input into physiological regulations that determine final animal size (Layalle, 2008).
Previous experiments showed that insulin/IGF signaling controls basal levels of ecdysone synthesis in the PG. This, in turn, controls the larval growth rate without modifying the duration of larval growth. These data contrast with the present observations on the role of TOR signaling in the PG and indicate that PG cells discriminate between hormone-mediated activation of InR/PI3K signaling and the nutrient-mediated activation of TOR signaling for the control of ecdysone biosynthesis. Can TOR and InR/PI3K signaling pathways function separately in Drosophila tissues? It has been established both in cultured cells and in vivo that a gain of function for InR/PI3K allows for TORC1 activation through inhibition of TSC2 via direct phosphorylation by AKT/PKB. Such crosstalk between the InR and TOR signaling pathways has important functional implications in cancer cells in which inactivation of the PTEN tumor suppressor leads to an important increase in AKT activity. Nevertheless, the physiological significance of the crosstalk between AKT and TSC2 has been challenged by genetic experiments in Drosophila, leading to the notion that, in the context of specific tissues, TOR and insulin/IGF signaling can be part of distinct physiological regulations for the control of animal growth in vivo. Although not observe in standard conditions, strong InR/PI3K activation in the ring gland shortens larval developmental timing under conditions of food limitation. In light of the present data, this suggests that, in low-food conditions, providing high PI3K activity in PG cells allows for full activation of TOR through the AKT/PKB-mediated inhibitory phosphorylation of TSC2, thus modulating developmental timing. Inversely, a severe downregulation of InR/PI3K signaling in the PG extends larval timing by preventing early larval molts. However, it was observed that strong inhibition of the InR pathway compromises the growth of PG cells, therefore interfering with their capacity to produce normal levels of ecdysone for molting. Overall, previous works as well as the present work highlight the importance of studying signaling networks in the specific contexts (tissue, development) in which these pathways normally operate. This also illustrates that only mild manipulations of these intricate pathways are suitable to unravel the regulatory mechanisms that normally occur within the physiological range of their activities. In conclusion, it is proposed that the insulin/IGF system and TOR provide two separate inputs on PG-dependent ecdysone production: the insulin/IGF system controls baseline ecdysone levels during larval life, and TOR acts upon ecdysone peaks in response to PTTH at the end of larval development (Layalle, 2008).
Important literature describes intrinsic mechanisms controlling a growth threshold for pupariation in insects. After a critical size is attained, the hormonal cascade leading to ecdysone production initiates, and larvae are committed to pupal development, even when subjected to complete starvation. Recent findings in Drosophila by using temperature-sensitive mutants for dInR have revealed that reducing the larval growth rate before the critical size is attained postpones the attainment of this threshold, but has no effect on the final size. Conversely, reducing animals' growth rate after the critical size has been attained leads to strong reduction of the final size. This highlights an important period in the determination of final size, called the terminal growth period (TGP, also called interval to cessation of growth), which spans from the attainment of critical size to the cessation of growth. Due to its exponential rate, growth during that period makes an important contribution to the determination of final size. Interestingly, the duration of the TGP is not affected by the general insulin/IGF system, which explains why reduction of the insulin/IGF system during that period leads to short adults. The present data suggest that the duration of the TGP is an important parameter in the determination of final size that is controlled by TOR. By reducing the level of TOR activity specifically in the PG, neither the growth rate or the critical size for commitment to pupariation is affected. Therefore, the time to attainment of the critical size is not changed. The observation of the developmental transitions in P0206 > TSC1/2 larvae (ectopically expressing TSC1/2) indicate that, indeed, the timing of L1/L2 and L2/L3 molts are not modified. By contrast, the L3/pupa transition is severely delayed, indicating that the interval between attainment of critical size and the termination of growth, i.e., the TGP, is increased. Interestingly, activation of TOR in the PG of fasting larvae leads to a sensible (50%) reduction of the developmental delay induced by low nutrients, whereas it has no effect in normally fed animals. This indicates that the regulation of the TGP by TOR plays an important role in the adaptation mechanisms controlling the duration of larval development under conditions of reduced dietary intake. Other mechanisms, such as the delay to attainment of the critical size due to a reduced growth rate, also contribute to timing of larval development, giving a plausible explanation for the fact that PG-specific TOR activation only partially rescues the increase in larval development timing observed under low-nutrient conditions. Despite characterization in different insect systems, the mechanisms determining the critical size remain to be elucidated. The present study shows that inhibition of TOR signaling in the PG does not modify the minimum size for pupariation. This result is in line with previous findings indicating that nutritional conditions do not modify the critical size in Drosophila. Interestingly, animals depleted of PTTH present an important shift in critical size, indicating that PTTH might participate in setting this parameter. Therefore, mechanisms determining the critical size might reside in the generation or the reception of the PTTH signal, upstream of TOR function in the cascade of events leading to ecdysone production (Layalle, 2008).
What is the limiting step that is controlled by the TOR sensor during the process of ecdysone production? Results obtained by genetic analysis in vivo are reminiscent of in vitro work on dissected PG in the M. sexta model. In these previous studies, PTTH-induced ecdysone production in the PG was shown to induce the phosphorylation of ribosomal protein S6 and was inhibited by the drug rapamycin, later identified as the specific inhibitor of TOR kinase. Interestingly, rapamycin treatment blocked PTTH-induced, but not db-cAMP-induced, ecdysone production, indicating that the drug does not act by simply inhibiting general protein translation in PG cells, but, rather, by inhibiting a specific step controlling PTTH-dependent ecdysone production. More recently, many studies mostly carried out on large insects have started unraveling the response to PTTH in the PG, leading to ecdysone synthesis. No bona fide PTTH receptor is identified yet, and the previously identified response to PTTH is a rise in cAMP, leading to a cascade of activation of kinases, including PKA, MAPKs, PKC, and S6-kinase. S6-kinase-dependent S6 phosphorylation is currently being considered as a possible bottle-neck in the activation of ecdysone biosynthesis by PTTH. The present genetic analysis of ecdysone production in the Drosophila PG now introduces the TOR pathway, the main activator of S6-kinase, as a key controller of ecdysone production and therefore provides a plausible explanation for the rise of S6-kinase in PG cells following PTTH induction. The phenotypes obtained after TOR inhibition in the PG are remarkably similar to the phenotype obtained after ablation of the PTTH neurons. Moreover, ths study shows here that PTTH expression is not altered upon starvation, and that TOR inhibition in PTTH cells has no effect on the duration of larval development, suggesting that PTTH production is not modified by a nutritional stress. Taken together, these data suggest a model whereby limited nutrients induce a downregulation of TOR signaling in the PG, abolish the capacity of PG cells to respond to PTTH and produce ecdysone, and lead to an extension of the terminal growth period (Layalle, 2008).
In conclusion, this study illustrates how the TOR pathway can be used in a specific endocrine organ to control a limiting step in the biosynthesis of a hormone in order to couple important physiological regulations with environmental factors such as nutrition (Layalle, 2008).
The TOR and Jak/STAT signal pathways are highly conserved from Drosophila to mammals, but it is unclear whether they interact during development. The proline-rich Akt substrate of 40 kDa (PRAS40) mediates the TOR signal pathway through regulation of TORC1 activity, but its functions in TOR complex 1 (TORC1, a rapamycin-sensitive form of Tor in mice that consists of mTOR, raptor, and mLST8) proved in cultured cells are controversial. The Drosophila gene Lobe (L) encodes the PRAS40 ortholog required for eye cell survival. L mutants exhibit apoptosis and eye-reduction phenotypes. It is unknown whether L regulates eye development via regulation of TORC1 activity. This study found that reducing the L level, by hypomorphic L mutation or heterozygosity of the null L mutation, resulted in ectopic expression of unpaired (upd), which is known to act through the Jak/STAT signal pathway to promote proliferation during eye development. Unexpectedly, when L was reduced, decreasing Jak/STAT restored the eye size, whereas increasing Jak/STAT prevented eye formation. Ectopic Jak/STAT signaling and apoptosis are mutually dependent in L mutants, indicating that L reduction makes Jak/STAT signaling harmful to eye development. In addition, genetic data suggest that TORC1 signaling is downregulated upon L reduction, supporting the idea that L regulates eye development through regulation of TORC1 activity. Similar to L reduction, decreasing TORC1 signaling by dTOR overexpression results in ectopic upd expression and apoptosis. A novel finding from these data is that dysregulated TORC1 signaling regulates the expression of upd and the function of the Jak/STAT signal pathway in Drosophila eye development (Wang, 2009).
The target of rapamycin (TOR) and Jak/STAT signal pathways are highly conserved in animals and important in many developmental processes. Dysregulation of these pathways can lead to cancer formation. This study presents data showing that TOR regulates the function of Jak/STAT signaling during Drosophila eye development (Wang, 2009).
The gene unpaired (upd) encodes a ligand that activates Drosophila Jak/STAT signaling. It is expressed in the posterior margin of the dorsal/ventral (D/V) boundary, the posterior center (PC), in the larval eye imaginal disc at second and early third instar stages. Notch at the D/V boundary activates the transcription of eye gone (eyg), which activates upd expression at the PC. Expression of upd is also regulated by Hedgehog (Hh) signaling. The cells of Drosophila compound eyes are derived from the eye-antennal disc, which develops from ectoderm of the embryo and grows inside the larva. These cells proliferate rapidly during the first and second instar stage. In early third instar larvae, morphogenetic furrow (MF) that arise at the posterior margin progresses in a wave-like manner toward the anterior margin of the eye disc. Jak/STAT signaling is known to promote proliferation during eye development, and is required for MF initiation; a loss of Jak/STAT function results in reduced eyes. Therefore, Jak/STAT signaling is regulated by Notch/Eyg and the Hh signaling pathways, and plays positive roles in eye development (Wang, 2009).
TOR signaling is one of the downstream branches of insulin signal pathway. Insulin and insulin-like growth factor elicit a signal cascade involving phosphatidyl-inositol 3-kinase (PI3K) that stimulates PDK-mediated Akt phosphorylation. Phosphorylated Akt can activate TOR, which nucleates the TOR complex 1 (TORC1), allowing it to phosphorylate the downstream targets, the translational repressor eukaryotic initiation factor (4EBP) and the ribosomal protein S6 kinase (S6k). Phosphorylation of 4EBP and S6K promotes CAP-dependent translation and thereby increases protein synthesis. In addition, activation of TOR can also promote ribosome biogenesis via Myc. Loss of the Drosophila TOR (dTOR) function reduces eye size, indicating that TOR signaling is required for eye development (Wang, 2009).
PRAS40 mediates the insulin signal pathway from Akt to TORC1. Upon insulin stimulation, activated Akt phosphorylates PRAS40 and causes it to dissociate from TORC1, allowing TORC1 signaling to proceed. Thus, PRAS40 can apparently act as an inhibitor of TORC1. However, it has been reported that PRAS40 is required for TORC1 activity, and thus the interactions of PRAS40 with TORC1, based on studies in cultured cells are controversial. The effect of PRAS40 on TORC1 signaling in vivo is still unclear (Wang, 2009).
The Drosophila Lobe (L) protein shares high sequence conservation with PRAS40. L mutants have reduced adult eyes and exhibit ectopic apoptosis during eye development, indicating that L is required for eye development. But whether it regulates eye development via regulation of TORC1 activity is unknown (Wang, 2009 and references therein).
This study identified a new L allele, Lfee. Quantitative RT-PCR and genetic analysis revealed that Lfee is a hypomorphic allele. The eye defect was mediated by ectopic Jak/STAT signaling and cell apoptosis. In L mutants, the ectopic Jak/STAT signaling had a negative effect on eye development, but not a positive one as previously reported. It was also found that TORC1 signaling was hypoactivated in L mutants, suggesting that, like PRAS40, L is required for TORC1 activity. This study suggests that hypoactivated TORC1 signaling in L mutants result in ectopic Jak/STAT signaling and apoptosis, impairing eye development (Wang, 2009).
The spontaneous mutant fly, freaky eye (fee), is homozygously viable and has abnormal adult eyes. The eyes of most fee flies are smaller than those of wild type flies because of a nick at the anterior border of the eye. At the nicked region, extra hairs and/or rod-like tissues are usually present. Overgrowth of eye tissue occasionally occurs, resulting in eye enlargement. The eyes of fee flies were categorized into six classes depending on their size relative to the eyes of the wild type. The various eye-reduction phenotypes of fee flies were similar to those of L mutants. For example, the Lsi heterozygote exhibits slightly reduced eyes that are nicked near the anterior D/V boundary, similar to the major fee phenotype. In the Lsi homozygote, the ventral eye is absent, which is also reminiscent of the fee phenotype (Wang, 2009).
Whether fee is a mutant of L was investigated; the trans-heterozygotes for fee and the null mutant Lrev6-3 had smaller eyes than fee flies. In addition, fee could to be recombined with Lrev6-3, suggesting that fee is allelic to L. Quantitative RT-PCR showed that the L mRNA levels were highly reduced in fee flies, suggesting that fee is a L hypomorphic mutant; therefore these were designated Lfee (Wang, 2009).
This study shows that reduction of L phenocopies overexpression of dTOR. Overexpression of dTOR has been reported to produce phenotypes similar to that of loss of dTOR, because excess dTOR may titrate cofactors and thereby decrease TOR activity. This suggests that TOR signaling is downregulated by L reduction. Consistent with this, genetic analysis of L mutants and TOR signal pathway component suggest TORC1 hypoactivity in L mutants. PRAS40 has been proposed to function in the assembly of TORC1. It is possible that, in similar way to dTOR overexpression, reducing L impairs TORC1 assembly, thus decreasing TORC1 signaling. Reduction of L may disrupt eye development through downregulation of TORC1 signaling, supporting the idea that PRAS40 is required for TORC1 activity (Wang, 2009).
Drosophila eye development requires the TOR and Jak/STAT signal pathways, but it is not know whether an interaction between these two signal pathways occurs. Endogenous upd expression is present in the posterior center (PC) of the eye disc, but not in the interior eye disc. This study demonstrated that L reduction can induce ectopic upd expression in the interior eye disc, indicating that L is a negative regulator for upd expression. The data show that L reduction-mediated eye disruption is due to hypoactivation of TORC1 signaling, suggesting that hypoactivity of TORC1 is responsible for inducing upd expression (Wang, 2009).
Ectopic upd expression is induced by reduction of L (Lfee and Lrev/+), but not by its complete loss (Lrev homozygous clones), suggesting that different L levels may cause distinct effects. As PRAS40 acts to transmit the Akt signal to TORC1, complete loss, but not reduction, of L could result in an uncoupling between Akt and TORC1. This would release the Akt-mediated inhibition of TORC1, resulting in increased TORC1 activity. Thus, complete loss of L or PRAS40 may increase TORC1 activity. It is possible that the opposite functions of PRAS40 reported in cultured cells could be due to different PRAS40 levels remaining after knockdown. Whether complete loss of L function inhibits or promotes TORC1 signaling in Drosophila eyes remains to be investigated (Wang, 2009).
Mosaic analysis data showed that dTOR homozygote clones did not induce ectopic upd expression, suggesting that complete loss of dTOR function has a different effect from that of L reduction. Overexpression of dMyc can completely restore the eye size in the Lfee flies, but only partially represses the eye defect of dTOR overexpression. These data support the idea that L reduction may not equate to loss of dTOR. It was reasoned that as TOR is involved in TORC1 and TORC2, its loss should eliminate the functions of both TORC1 and TORC2. Because L participates only in TORC1 signaling, reduction of L would affect TORC1 signaling only. The regulation of TORC1 and TORC2 signaling by L needs further investigation (Wang, 2009).
It was found that suppressing apoptosis can decrease ectopic upd expression upon L reduction, suggesting that apoptosis is a cause of ectopic upd expression. It has been reported that apoptosis can activate ectopic upd expression and Jak/STAT signaling via Notch signaling in apoptosis-induced compensatory proliferation. However, ectopic upd expression on L reduction is not likely to be mediated by Notch activity, and no ectopic proliferation occurs. Thus, apoptosis due to L reduction is different from apoptosis-induced compensatory proliferation. Further, TOR hypoactivation may trigger ectopic upd expression independent of apoptosis; suppression of apoptosis did not eliminate all ectopic upd expression. Further investigation of how hypoactivated TORC1 regulates upd expression is needed (Wang, 2009).
The Drosophila Upd acts through Jak/STAT signaling to promote proliferation during eye development. However, this study found that on L reduction, decreasing Jak/STAT signaling could restore the eye defect, whereas increasing the upd expression level could completely abolish eye development. Thus, an unexpected finding was that ectopic Jak/STAT signaling in L mutants is harmful to eye development (Wang, 2009).
The fact that decreasing Jak/STAT signaling can reduce apoptosis in L mutants indicates that the induction of ectopic Jak/STAT signaling is required for apoptosis. It was reasoned that the apoptosis-promoting ability of Jak/STAT is possibly due to its repression of Serrate (Ser) expression. Ser expression is inhibited by L mutation, and loss of Ser function during eye development causes apoptosis. The current data showed that heterozygosity for Ser can reduce eye size in Lfee heterozygotes, but not in the wild type, suggesting that decreased Ser expression may play a role in eye reduction. Whether Ser repression mediates the apoptosis remains to be investigated. In addition, because inhibition of apoptosis does not strongly restore the L eye defect, but decreasing Jak/STAT activity fully restores it (comparing ey > p35 and Stat92Ets), there is the possibility that the ectopic Jak/STAT activity affects eye development via an apoptosis-independent mechanism. Thus, a novel finding from the data is that Jak/STAT signaling can negatively regulate eye development (Wang, 2009).
An important issue is the control over the positive and negative roles of Jak/STAT signaling during eye development. Overexpression of upd driven by ey-GAL4 in the wild type produces adult with enlarged eyes, but it eliminates eye formation in L mutants. Because L reduction exhibits hypoactivation of TORC1 signaling, it is speculated that TORC1 signaling plays a role in controlling the balance between the opposing functions of Jak/STAT signaling (Wang, 2009).
In summary, reduction of the Drosophila PRAS40 L results in hypoactivation of TORC1 signaling. This leads to apoptosis and ectopic Jak/STAT activation, both of contribute to disruption of eye development. The data indicate that TORC1 signaling is able to regulate the expression and functions of the Jak/STAT signal pathway during eye development. Further studies using L mutants may uncover the mechanisms by which L regulates TORC1 signaling, and how TOR controls the Jak/STAT signal pathways. Also noteworthy is the report that decreasing PRAS40 can increase apoptosis of tumor cells, and it is therefore of interest to investigate whether PRAS40 and TORC1 can regulate the Jak/STAT signal pathway in tumors (Wang, 2009).
Many stem, progenitor and cancer cells undergo periods of mitotic quiescence from which they can be reactivated. The signals triggering entry into and exit from this reversible dormant state are not well understood. In the developing Drosophila central nervous system, multipotent self-renewing progenitors called neuroblasts undergo quiescence in a stereotypical spatiotemporal pattern. Entry into quiescence is regulated by Hox proteins and an internal neuroblast timer. Exit from quiescence (reactivation) is subject to a nutritional checkpoint requiring dietary amino acids. Organ co-cultures also implicate an unidentified signal from an adipose/hepatic-like tissue called the fat body. This study provides in vivo evidence that Slimfast amino-acid sensing and Target of rapamycin (TOR) signalling activate a fat-body-derived signal (FDS) required for neuroblast reactivation. Downstream of this signal, Insulin-like receptor signalling and the Phosphatidylinositol 3-kinase (PI3K)/TOR network are required in neuroblasts for exit from quiescence. Nutritionally regulated glial cells provide the source of Insulin-like peptides (ILPs) relevant for timely neuroblast reactivation but not for overall larval growth. Conversely, ILPs secreted into the haemolymph by median neurosecretory cells systemically control organismal size but do not reactivate neuroblasts. Drosophila thus contains two segregated ILP pools, one regulating proliferation within the central nervous system and the other controlling tissue growth systemically. These findings support a model in which amino acids trigger the cell cycle re-entry of neural progenitors via a fat-body-glia-neuroblasts relay. This mechanism indicates that dietary nutrients and remote organs, as well as local niches, are key regulators of transitions in stem-cell behaviour (Sousa-Nunes, 2011).
In fed larvae, Drosophila neuroblasts exit quiescence from the late first instar (L1) stage onwards. This reactivation involves cell enlargement and entry into S phase, monitored in this study using the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU). Reactivated neuroblast lineages (neuroblasts and their progeny) reproducibly incorporated EdU in a characteristic spatiotemporal sequence: central brain --> thoracic --> abdominal neuromeres. Mushroom-body neuroblasts and one ventrolateral neuroblast, however, are known not to undergo quiescence and to continue dividing for several days in the absence of dietary amino acids. This indicates that dietary amino acids are more than mere 'fuel', providing a specific signal that reactivates neuroblasts. However, explanted central nervous systems (CNSs) incubated with amino acids do not undergo neuroblast reactivation unless co-cultured with fat bodies from larvae raised on a diet containing amino acids. Therefore the in vivo requirement for a fat-body-derived signal (FDS) in neuroblast reactivation was tested by blocking vesicular trafficking and thus signalling from this organ using a dominant-negative Shibire dynamin (SHIDN). This strongly reduced neuroblast EdU incorporation, indicating that exit from quiescence in vivo requires an FDS. One candidate tested was Ilp6, known to be expressed by the fat body, but neither fat-body-specific overexpression nor RNA interference of this gene significantly affected neuroblast reactivation. Fat-body cells are known to sense amino acids via the cationic amino-acid transporter Slimfast (SLIF), which activates the TOR signalling pathway, in turn leading to the production of a systemic growth signal. Fat-body-specific overexpression of the TOR activator Ras homologue was shown to be enriched in brain (RHEB), or of an activated form of the p110 PI3K catalytic subunit, or of the p60 adaptor subunit, had no significant effect on neuroblast reactivation in fed animals or in larvae raised on a nutrient-restricted diet lacking amino acids. In contrast, global inactivation of Tor, fat-body-specific Slif knockdown or fat-body-specific expression of the TOR inhibitors Tuberous sclerosis complex 1 and 2 (Tsc1/2) all strongly reduced neuroblasts from exiting quiescence. Together, these results show that a SLIF/TOR-dependent FDS is required for neuroblasts to exit quiescence and that this may be equivalent to the FDS known to regulate larval growth (Sousa-Nunes, 2011).
Next, the signalling pathways essential within neuroblasts for their reactivation were investigated. Nutrient-dependent growth is regulated in many species by the interconnected TOR and PI3K pathways. In fed larvae, it was found that neuroblast inactivation of TOR signalling (by overexpression of TSC1/2), or PI3K signalling (by overexpression of p60, the Phosphatase and tensin homologue PTEN, the Forkhead box subgroup O transcription factor FOXO or dominant-negative p110), all inhibited reactivation. Conversely, stimulation of neuroblast TOR signalling (by overexpression of RHEB) or PI3K signalling [by overexpression of activated p110 or Phosphoinositide-dependent kinase 1 (PDK1)] triggered precocious exit from quiescence. RHEB overexpression had a particularly early effect, preventing some neuroblasts from undergoing quiescence even in newly hatched larvae. Hence, TOR/PI3K signalling in neuroblasts is required to trigger their timely exit from quiescence. Importantly, neuroblast overexpression of RHEB or activated p110 in nutrient-restricted larvae, which lack FDS activity, was sufficient to bypass the block to neuroblast reactivation. Notably, both genetic manipulations were even sufficient to reactivate neuroblasts in explanted CNSs, cultured without fat body or any other tissue. Together with the previous results this indicates that neuroblast TOR/PI3K signalling lies downstream of the amino-acid-dependent FDS during exit from quiescence (Sousa-Nunes, 2011).
To identify the mechanism bridging the FDS with neuroblast TOR/PI3K signalling, the role of the Insulin-like receptor (InR) in neuroblasts was tested. Importantly, a dominant-negative InR inhibited neuroblast reactivation, whereas an activated form stimulated premature exit from quiescence. Furthermore, InR activation was sufficient to bypass the nutrient restriction block to neuroblast reactivation. This indicates that at least one of the potential InR ligands, the seven ILPs, may be the neuroblast reactivating signal(s). By testing various combinations of targeted Ilp null alleles and genomic Ilp deficiencies, it was found that neuroblast reactivation was moderately delayed in larvae deficient for both Ilp2 and Ilp3 (Df(3L)Ilp2-3) or lacking Ilp6 activity. Stronger delays, as severe as those observed in InR31 mutants, were observed in larvae simultaneously lacking the activities of Ilp2, 3 and 5 [Df(3L)Ilp2-3, Ilp5] or Ilp1-5 [Df(3L)Ilp1-5]. Despite the developmental delay in Df(3L)Ilp1-5 homozygotes, neuroblast reactivation eventually begins in the normal spatial pattern -- albeit heterochronically -- in larvae with L3 morphology. Together, the genetic analysis shows that Ilp2, 3, 5 and 6 regulate the timing but not the spatial pattern of neuroblast exit from quiescence. However, as removal of some ILPs can induce compensatory regulation of others, the relative importance of each cannot be assessed from loss-of-function studies alone (Sousa-Nunes, 2011).
Brain median neurosecretory cells (mNSCs) are an important source of ILPs, secreted into the haemolymph in an FDS-dependent manner to regulate larval growth. They express Ilp1, 2, 3 and 5, although not all during the same development stages. However, this study found that none of the seven ILPs could reactivate neuroblasts during nutrient restriction when overexpressed in mNSCs. Similarly, increasing mNSC secretion using the NaChBac sodium channel or altering mNSC size using PI3K inhibitors/activators, which in turn alters body growth, did not significantly affect neuroblast reactivation under fed conditions. Surprisingly, therefore, mNSCs are not the relevant ILP source for neuroblast reactivation. Nonetheless, Ilp3 and Ilp6 messenger RNAs were detected in the CNS cortex, at the early L2 stage, in a domain distinct from the Ilp2+ mNSCs. Two different Ilp3-lacZ transgenes indicate that Ilp3 is expressed in some glia (Repo+ cells) and neurons (Elav+ cells). An Ilp6-GAL4 insertion indicates that Ilp6 is also expressed in glia, including the cortex glia surrounding neuroblasts and the glia of the blood-brain barrier (BBB) (Sousa-Nunes, 2011).
Next the ability of each of the seven ILPs to reactivate neuroblasts when overexpressed in glia or in neurons was assessed. Pan-glial or pan-neuronal overexpression of ILP4, 5 or 6 led to precocious reactivation under fed conditions. Each of these manipulations also bypassed the nutrient restriction block to neuroblast reactivation, as did overexpression of ILP2 in glia or in neurons, or ILP3 in neurons. In all of these ILP overexpressions, and even when ILP6 was expressed in the posterior Ultrabithorax domain, the temporal rather than the spatial pattern of reactivation was affected. Importantly, experiments blocking cell signalling with SHIDN indicate that glia rather than neurons are critical for neuroblast reactivation. Interestingly, glial-specific overexpression of ILP3-6 did not significantly alter larval mass. Thus, in contrast to mNSC-derived ILPs, glial-derived ILPs promote CNS growth without affecting body growth (Sousa-Nunes, 2011).
Focusing on ILP6, CNS explant cultures were used to demonstrate directly that glial overexpression was sufficient to substitute for the FDS during neuroblast exit from quiescence. In vivo, ILP6 was sufficient to induce reactivation during nutrient restriction when overexpressed via its own promoter or specifically in cortex glia but not in the subperineurial BBB glia, nor in many other CNS cells that were tested. Hence, cortex glia possess the appropriate processing machinery and/or location to deliver reactivating ILP6 to neuroblasts. Ilp6 mRNA is known to be upregulated rather than downregulated in the larval fat body during starvation and, accordingly, Ilp6-GAL4 activity is increased in this tissue after nutrient restriction. Conversely, it was found that Ilp6-GAL4 is strongly downregulated in CNS glia during nutrient restriction. Thus, dietary nutrients stimulate glia to express Ilp6 at the transcriptional level. Consistent with this, an important transducer of nutrient signals, the TOR/PI3K network, is necessary and sufficient in glia (but not in neurons) for neuroblast reactivation. Together, the genetic and expression analyses indicate that nutritionally regulated glia relay the FDS to quiescent neuroblasts via ILPs (Sousa-Nunes, 2011).
This study used an integrative physiology approach to identify the relay mechanism regulating a nutritional checkpoint in neural progenitors. A central feature of the fat-body --> glia --> neuroblasts relay model is that glial insulin signalling bridges the amino-acid/TOR-dependent fat-body-derived signal (FDS) with InR/PI3K/TOR signalling in neuroblasts. The importance of glial ILP signalling during neuroblast reactivation is also underscored by an independent study, published while this work was under revision (Chell, 2010). As TOR signalling is also required in neuroblasts and glia, direct amino-acid sensing by these cell types may also impinge upon the linear tissue relay. This would then constitute a feed-forward persistence detector, ensuring that neuroblasts exit quiescence only if high amino-acid levels are sustained rather than transient. This study also showed that the CNS 'compartment' in which glial ILPs promote growth is functionally isolated, perhaps by the BBB, from the systemic compartment where mNSC ILPs regulate the growth of other tissues. The existence of two functionally separate ILP pools may explain why bovine insulin cannot reactivate neuroblasts in CNS organ culture, despite being able to activate Drosophila InR in vitro. Given that insulin/PI3K/TOR signalling components are highly conserved between insects and vertebrates, it will be important to address whether mammalian adipose or hepatic tissues signal to glia and whether or not this involves an insulin/IGF relay to CNS progenitors. In this regard, it is intriguing that brain-specific overexpression of IGF1 can stimulate cell-cycle re-entry of mammalian cortical neural progenitors, indicating utilization of at least part of the mechanism identified by this study in Drosophila (Sousa-Nunes, 2011).
The TOR protein kinases (TOR1 and TOR2 in yeast; mTOR/FRAP/RAFT1 in mammals) promote cellular proliferation in response to nutrients and growth factors, but their role in development is poorly understood. The Drosophila TOR homolog dTOR is required cell autonomously for normal growth and proliferation during larval development, and for increases in cellular growth caused by activation of the phosphoinositide 3-kinase (PI3K) signaling pathway. As in mammalian cells, the kinase activity of dTOR is required for growth factor-dependent phosphorylation of S6 kinase in vitro, and overexpression of S6k in vivo can rescue dTOR mutant animals to viability. Loss of dTOR also results in cellular phenotypes characteristic of amino acid deprivation, including reduced nucleolar size, lipid vesicle aggregation in the larval fat body, and a cell type-specific pattern of cell cycle arrest that can be bypassed by overexpression of the S-phase regulator cyclin E. These results suggest that dTOR regulates growth during animal development by coupling growth factor signaling to nutrient availability (Zhang, 2000).
The Drosophila genome encodes four members of the PIK-related family: mei41, which encodes an ATR/ATM homolog required for cell cycle arrest in response to DNA damage (Hari, 1995); CG6535, a second ATM-related gene of unknown function; CG4549, whose closest relative encodes the nonsense-mediated mRNA decay protein SMG-1 in C. elegans, and the dTOR. A fifth member of this family, the DNA-dependent protein kinase, is not found in Drosophila or C. elegans (Zhang, 2000).
To begin a mutational analysis of dTOR, the Berkeley Drosophila Genome Project P-element database was searched and two independent homozygous lethal lines bearing P insertions in the dTOR gene were identified (designated here as dTORP1 and dTORP2). Mobilization of these elements restores viability to each chromosome, indicating that the insertions are responsible for the associated lethality. Comparison of the insertion sites of dTORP1 and dTORP2 with the dTOR transcription unit reveal that the P-elements are inserted at 24 and 74 bases downstream of the dTOR transcription start site, respectively, and would likely interfere with normal dTOR expression (Zhang, 2000).
To generate additional dTOR alleles, a series of deletions spanning the dTOR gene was generated by imprecise mobilization of the P-elements. One such mutant, designated dTORDeltaP, was selected for further analysis. Sequence analysis reveals that the dTORDeltaP deletion originates at the dTORP2 insertion site and extends 3514 bp downstream, removing the dTOR translation start site and amino-terminal 902 codons, and thus likely represents a null allele of dTOR. This was confirmed by the absence of detectable dTOR protein in immunoblots of dTORDeltaP larval extracts. A 9.4-kb genomic rescue construct encompassing the dTOR gene and no other predicted transcription units restores full viability and fertility to dTORDeltaP homozygotes (Zhang, 2000).
dTORDeltaP homozygotes hatch at normal rates, but grow more slowly than normal and eventually arrest during larval development, reaching only 24% the mass of wild-type controls. Larvae homozygous for dTORP1 or dTORP2 alleles display a less severe phenotype, eventually growing to approximately 40% and 79% the mass of wild type, respectively, indicating that these alleles likely retain partial dTOR function. In each case, the mutants remain viable and active during an extended larval period of ~30 d, and eventually die without pupating. Larvae heterozygous for dTOR grow at a rate indistinguishable from wild-type controls under normal culture conditions, but are hypersensitive to low concentrations of rapamycin. Thus, dTOR encodes a rapamycin-sensitive protein required for normal growth during larval development (Zhang, 2000).
Overall growth of an organism is generally accompanied by increases in cell number (proliferation), cell size (hypertrophy), or both. To determine how mutations in dTOR inhibit growth, these parameters were examined in a number of tissues. The effect of dTOR on cell size was analyzed in marked clones of dTORDeltaP homozygous cells, which were generated by FLP/FRT-mediated mitotic recombination in dTORDeltaP heterozygous animals. Examination of adult cuticular structures reveal that dTOR homozygous mutant cells are markedly reduced in size. For example, bristles of the wing margin that lack dTOR were both thinner and shorter than adjacent wild-type cell. Area measurements of mutant clones in the wing epithelium show that dTORDeltaP mutant cells are approximately half (56%) the size of controls. Similar effects have been observed in the eye, abdomen, and notum (Zhang, 2000).
To determine whether loss of dTOR affects the size of actively proliferating cells, dTOR mutant clones were examined in the developing imaginal discs, epithelial primordia that proliferate mitotically to give rise to adult structures. Imaginal wing discs containing GFP-marked clones of dTORDeltaP homozygous cells were dissociated into single cells, which were then analyzed by flow cytometry. The mean forward light scatter value (a measure of cell size) of dTOR mutant cells is decreased by 30% compared to wild-type control cells from the same discs. This decrease in cell size is observed in all phases of the cell cycle. Thus, loss of dTOR causes a cell autonomous reduction in the size of both proliferating and postmitotic cells (Zhang, 2000).
Fluorescence-activated cell sorter (FACS) analysis has also revealed that the cell cycle phasing of dTOR cells differs significantly from that of controls, with relatively more cells in G1, and fewer in S and G2 phases. This is consistent with the ability of rapamycin to induce G1 arrest in yeast and in mammalian cell culture. To measure proliferation rates of dTOR mutant cells, the number of cells in dTORDeltaP clones were compared with that of their wild-type sister clones (twin spots). Clones of dTORDeltaP mutant cells are similar in size to their twin spots at 48 h after induction, but by 72-96 h they contain significantly fewer cells, indicating that loss of dTOR leads to a reduced rate of cell proliferation. In addition, lone twin spots lacking a corresponding mutant sister clone have occasionally been observed at 96 h after induction, indicating that at some frequency dTORDeltaP homozygous cells are eliminated from the disc epithelium. Because dTORDeltaP cells remain viable for weeks in the context of a homozygous mutant animal, the loss of dTORDeltaP mutant clones is likely the result of cell competition with adjacent wild-type cells, which is known to occur for cells with a growth disadvantage (Zhang, 2000).
Growth properties of cells in the salivary glands of homozygous dTORDeltaP larvae were also examined. The salivary gland is comprised of two cell types: polytene gland cells that undergo multiple rounds of endoreduplication to generate giant nuclei with a ploidy of up to 2048 C, and imaginal ring cells that remain diploid and cycle mitotically. Loss of dTOR affects both cell types. The endoreplicative cells in dTORDeltaP salivary glands undergo only four to five rounds of replication before entering quiescence, reaching a ploidy of 16-32C and a size ~10% that of wild type. The imaginal rings in dTORDeltaP larvae contain approximately fivefold fewer cells than wild type. Together, these results indicate that dTOR is required to promote cell cycle progression in both mitotic and endoreplicative cells, and acts primarily at the G1/S transition (Zhang, 2000).
The cell autonomous reduction in the size of dTOR mutant cells is reminiscent of mutations in components of the PI3K/S6K signaling pathway. Mutations in Pten, the fly homolog of the PTEN tumor suppressor, cause activation of this pathway, leading to increased cell growth. To determine whether dTOR is required for PI3K-dependent signaling, the growth properties of cells lacking both Pten and dTOR were examined. Clonal loss of Pten causes enlargement of imaginal and adult cells, and increases the percentage of cells in the S and G2 phases of the cell cycle. In contrast, cells carrying null alleles of both Pten and dTOR are indistinguishable from cells lacking dTOR alone, with a similar reduction in cell size and accumulation in G1. Loss of dTOR also prevents the increased proliferation caused by mutations in Pten. Cells mutant for weaker alleles of Pten and dTOR (MGH1 and P2, respectively) are intermediate in size, indicating that dTORP2 cells retain partial signaling function. It is concluded that dTOR is epistatic to Pten, and therefore, that dTOR functions at a step downstream of or in parallel to PI3K signaling (Zhang, 2000).
In further tests of dTOR's role in PI3K/S6k signaling, it was found that rapamycin inhibits the serum-dependent phosphorylation of Drosophila p70S6k (S6k) expressed in S2 cells. Dephosphorylation of S6k by rapamycin is prevented by cotransfection of a dTOR point mutant containing a Ser1956 to Thr substitution (dTORRR, which confers rapamycin resistance to mammalian and yeast TOR proteins. Expression of dTORRR carrying an additional point mutation in a residue crucial for kinase activity (dTORRRKD fails to protect S6k from rapamycin-induced dephosphorylation, indicating that the kinase function of dTOR is required to maintain S6k phosphorylation (Zhang, 2000).
To determine whether these biochemical interactions between dTOR and S6k are relevant to their functions in vivo, tests were performed for genetic interactions between them. Remarkably, constitutive overexpression of Drosophila S6k or human p70S6K1 is able to rescue dTORP2/P2 and dTORP1/P2 flies to viability. The greatest degree of rescue is provided by a mutant version of p70S6K1, in which four mitogen-induced phosphorylation sites are mutated to aspartate and glutamate residues (mutant D4). Expression of this construct allowed 74% of expected dTORP1/P2 progeny to survive to adulthood, whereas no dTORP1/P2 animals survived in the absence of S6k overexpression. dTOR flies rescued by S6k overexpression are slightly smaller than wild-type controls, but are fertile and develop at a similar rate as wild type. Although overexpression of S6k does not rescue dTORDeltaP null mutants to adulthood, it does enable them to progress to the pupal stage. Overexpression of S6k in wild-type larvae also confers significant resistance to rapamycin. Again, constitutively active p70S6K1 provides the greatest degree of rapamycin resistance. Together, these results indicate that a major function of dTOR is to maintain levels of active S6k sufficient for normal growth (Zhang, 2000).
Like dTOR mutants, wild-type larvae deprived of amino acids enter an extended larval period with little or no growth. Amino acid deprivation also causes a series of distinctive cellular phenotypes including a reduction in nucleolar area, changes in morphology of the larval fat body, and a cell type-specific cell cycle arrest. Since TOR proteins have been proposed to be regulated in response to amino acid levels, it was of interest to examine whether loss of dTOR mimics these cellular effects (Zhang, 2000).
The nucleolus is the major cellular site of ribosomal assembly, and its size has been shown to correlate with protein synthetic capacity and proliferation rate. To measure nucleolar size in wild-type and dTOR mutant cells, wing imaginal discs containing clones of dTORDeltaP homozygous cells were labeled with an antibody against the nucleolar protein fibrillarin, and examined by confocal sectioning. The nucleolar area in clones of dTOR mutant cells in the wing imaginal disc is approximately half that of surrounding wild-type cells, consistent with a role for dTOR in ribosome biogenesis (Zhang, 2000).
During metamorphosis or starvation, stores of protein, lipid, and glycogen are mobilized from adipose cells of the larval fat body and are used by other tissues as an energy source in place of dietary nutrients. These metabolic effects are visible as changes in appearance of fat body cells. The major visible change in fat body cells in larvae deprived of amino acids is an aggregation of lipid vesicles, and this effect is indistinguishable from that caused by loss of dTOR (Zhang, 2000).
Within 48-72 h of amino acid withdrawal, endoreplicative cells become quiescent, whereas mitotic neuroblasts of the central nervous system continue to cycle for at least 8 d in the absence of amino acids. This pattern of cell cycle responses is distinct from that caused by complete inhibition of protein synthesis, which causes all larval cells to arrest DNA synthesis. To determine whether loss of dTOR causes a cell cycle response similar to that elicited by starvation, cell cycle behavior of these cell types was examined in dTORDeltaP larvae at multiple stages of development. At 3-4 d after egg deposition (AED), both endoreplicative and mitotic tissues were found to cycle normally in dTORDeltaP homozygotes, as measured by incorporation of the nucleotide analog BrdU. In contrast, by 5-6 d AED all endoreplicative tissues including the gut, fat body, and salivary glands fail to incorporate BrdU, whereas neuroblasts continued to cycle. A similar pattern is observed at 10 d AED. Presumably the appearance of this cell cycle arrest at 4-5 d AED results from the perdurance of maternal stores of dTOR mRNA or protein until this time. The cell cycle arrest of dTOR endoreplicative cells can be bypassed by overexpression of the G1/S regulators cyclin E or dE2F/dDP, as has been demonstrated previously for endoreplicative cells arrested by amino acid withdrawal. Thus, amino acid insufficency and loss of dTOR each cause similar growth arrests, changes in cell morphology, and cell type-specific patterns of G1 arrest (Zhang, 2000).
In budding yeast, TOR proteins govern S-phase entry in response to nutrient levels by regulating translation of the G1/S regulator Cln3 (Barbet, 1996 and Polymenis, 1997). Drosophila cyclin E has been proposed to play a role analogous to Cln3, and its abundance increases in response to growth stimuli such as overexpression of activated Ras. Because cyclin E overexpression is able to bypass the cell cycle arrest in dTOR mutants, whether loss of dTOR affects cyclin E expression was examined. Immunoblot analysis of whole larval extracts has revealed that the level of cyclin E protein is reduced ~30-fold in dTORDeltaP mutants, as compared to wild-type larvae of similar stage (Zhang, 2000).
Clonal induction of cells lacking cyclin E in the wing imaginal disc results in a G1 arrest within one to two cell divisions. In contrast, cells mutant for dTOR continue to cycle slowly for several days, and give rise to clones containing multiple cells, indicating that dTOR mutant cells retain at least partial cyclin E activity. Accordingly, cyclin E protein is reduced but not eliminated in dTOR mutant clones in the wing disc. Although 72-h dTORDeltaP clones containing little or no detectable cyclin E immunoreactivity are often observed, many dTOR clones were found containing cells with apparently normal cyclin E levels. It is concluded that the observed reduction in cyclin E protein levels in dTORDeltaP larval extracts is due largely to reduced cyclin E levels in endoreplicating cells, which comprise the majority of larval mass, resulting in a G1 arrest in this cell type. In contrast, dTOR may be required in imaginal cells to maintain normal rates of cyclin E accumulation, rather than absolute levels, consistent with the reduced rate of cell division and extended G1 phase observed in dTOR mutant clones (Zhang, 2000).
dTOR mutants differ from the known PI3K pathway mutants in several important respects. (1) The growth defects caused by loss of dTOR are more severe than those arising from mutations in components of the PI3K signaling pathway. (2) Null mutations in the PI3K subunits Dp110 or p60 allow growth to the third instar larval stage, and chico (IRS-1) and S6k mutants survive to adulthood. At least in the case of chico and S6k, many cells are able to cycle normally throughout development. In contrast, animals lacking dTOR reach only the size of second instar larvae, at which point they undergo cell cycle arrest. (3) Whereas overexpression of Dp110, Akt, or S6k leads to increased growth rate and cell size, dTOR overexpression inhibits growth and reduces cell size. Although variations in genetic background may partially account for some of these phenotypic differences, these results are inconsistent with dTOR acting as an integral component of a linear PI3K/Akt/S6k pathway, and instead argue that dTOR may converge upon this pathway in response to a distinct set of cues (Zhang, 2000 and references therein).
In yeast, TOR1 and TOR2 regulate cell growth directly in response to levels of nutrients such as amino acids, rather than in response to intercellular signals. Similarly, human mTOR may also be regulated by nutrient levels. These considerations prompted a comparison of the phenotypes of dTOR mutant animals with the physiological changes caused by nutrient deprivation. By the three criteria examined -- nucleolar size, fat body vesicle formation, and endoreplicative cell cycle arrest -- loss of dTOR precisely mirrors the effects of starvation. An efficient explanation of these results is that dTOR is required for normal responses to changes in nutrient levels. This would be consistent with a model in which full activation of growth targets such as S6k requires two distinct inputs: growth factor-mediated intercellular signals through PI3K, and nutrient-sensing signals through TOR. In this view, TOR proteins may act as part of a checkpoint to attenuate growth factor signaling when local conditions are unfavorable for cell growth (Zhang, 2000).
How TOR proteins might be regulated by nutrient levels is unclear. In the case of amino acids, recent evidence suggests that the primary signal may be uncharged tRNA, which increases in abundance when amino acid levels are low. Interestingly, other members of the PIK-related kinase family such as ATM and DNA-PK also act as checkpoint proteins that are regulated by specific nucleic acid structures. In addition, a recently described member of this family, SMG-1, is involved in the degradation of aberrant mRNAs containing inappropriate nonsense codons. Thus, regulation by nucleic acid may be a common feature of this kinase family (Zhang, 2000 and references therein).
Activation of p70S6K is a common response to virtually all mitogenic stimuli. Phosphorylation of the target of p70S6K, ribosomal protein S6, leads to the selective increase in translation of a subset of transcripts that contain an oligopyrimidine tract at their 5' termini (5' TOP). This class of messages encodes ribosomal proteins and translation elongation factors, and thus p70S6K activation leads to increased ribosomal biogenesis. The demonstration in this study that overexpression of S6k can rescue dTOR mutants to viability indicates that S6k is a critical effector of dTOR, and that one essential function of dTOR is to regulate the activity of S6k (Zhang, 2000).
Despite the central role of S6k in mediating the functions of dTOR, several lines of evidence indicate that dTOR has additional S6k-independent roles. (1) dTOR mutant phenotypes are more severe than those of S6k; lack of dTOR results in growth arrest at the second instar larval stage, whereas S6k mutant animals survive to adulthood, albeit with a delayed development and decreased body size. (2) Although S6k overexpression suppresses the lethality of dTOR mutants, this is only the case for hypomorphic dTOR allelic combinations; dTOR null animals expressing S6k advance only to the pupal stage. (3) dTOR flies rescued by S6k do not grow to the full size of wild-type controls. Thus, some dTOR functions are not fully rescued by S6k. As noted above, in mammalian cells mTOR also stimulates translation through phosphorylation and inactivation of 4E-BP1, an inhibitory binding factor of the translation initiation factor eIF4E. Drosophila eIF4E mutants display a severe growth arrest phenotype, and thus aspects of dTOR function that are not rescued by S6k may reflect diminished eIF4E activity. However, neither overexpression of eIF4E nor mutations in 4E-BP1 detectably alleviate dTOR mutant phenotypes (Zhang, 2000).
In addition to effects on translation, studies in budding yeast have found that inactivation of TOR modulates the level of a number of growth-related or nutrient-regulated transcripts, including those encoding ribosomal proteins (Barbet, 1996; Zaragoza, 1998; Beck, 1999; Cardenas, 1999; Hardwick, 1999; Powers, 1999). A transcriptional role for TOR in higher eukaryotes has been reported as well. Moreover, recent studies have found that mTOR can interact with a number of additional signaling factors including STAT3, protein kinase C, c-Abl, and 14-3-3 (Parekh, 1999; Kumar, 2000a and b; Mori, 2000; Yokogami, 2000). Rapamycin has also recently been shown to disrupt microtubule assembly and function in yeast, independent of its effects on translation (Choi, 2000). Thus, the inability of S6k overexpression to fully supplant dTOR may be due to a requirement for dTOR in multiple cellular functions (Zhang, 2000).
The reduced growth of dTOR mutants reflects reductions in both cell size and cell number. Because direct inhibition of proliferation increases rather than decreases cell size, it is proposed that the primary function of dTOR is to promote cell growth, and that the decreased proliferation of dTOR mutant cells is a secondary effect in response to their reduced rate of growth. The accumulation of dTOR mutant cells in the G1 phase of the cell cycle suggests that the G1/S transition is particularly sensitive to growth rate. This is consistent with previous observations that stimulation of cell growth by overexpression of dMyc, PI3K, or activated Ras promotes progression through the G1/S but not G2/M transition (Zhang, 2000 and references therein).
A factor likely to be involved in coupling growth and division rates is the G1/S regulator cyclin E. Cyclin E is rate limiting for G1/S progression in imaginal discs, and its levels increase by a post-transcriptional mechanism in response to stimulation of cell growth. The demonstration that dTOR is required for normal accumulation of cyclin E suggests that translational control is likely to play an important role in cyclin E regulation. In budding yeast, translation of the G1 cyclin Cln3 is regulated by a leaky scanning mechanism involving a small upstream ORF (Polymenis, 1997). In this regard, it is interesting to note that the 5'-untranslated region of the Drosophila cyclin E message contains multiple upstream ORFs. Thus, the mechanisms connecting G1/S progression to cell growth may be conserved between yeast and multicellular organisms (Zhang, 2000).
Precise body and organ sizes in the adult animal are ensured by a range of signaling pathways. Rheb (Ras homolog enriched in brain), a novel, highly conserved member of the Ras superfamily of G-proteins, promotes cell growth. Overexpression of Rheb in the developing fly causes dramatic overgrowth of multiple tissues: in the wing, this is due to an increase in cell size; in cultured cells, Rheb overexpression results in accumulation of cells in S phase and an increase in cell size. Rheb is required in the whole organism for viability (growth) and for the growth of individual cells. Inhibition of Rheb activity in cultured cells results in their arrest in G1 and a reduction in size. These data demonstrate that Rheb is required for both cell growth (increase in mass) and cell cycle progression; one explanation for this dual role would be that Rheb promotes cell cycle progression by affecting cell growth. Consistent with this interpretation, flies with reduced Rheb activity are hypersensitive to rapamycin, an inhibitor of the growth regulator target of rapamycin(TOR), a kinase required for growth factor-dependent phosphorylation of ribosomal S6 kinase (S6K). In cultured cells, the effect of overexpressing Rheb was blocked by the addition of rapamycin. These results imply that Rheb is involved in TOR signaling (Patel, 2003). Additional studies show that Rheb functions downstream of the tumor suppressors Tsc1 (tuberous sclerosis 1)-Tsc2, with Tsc2 functioning as a GAP for Rheb (Saucedo, 2003; Zhang, 2003), and that a major effector of Rheb function in controlling growth is, in fact, ribosomal S6 kinase (Stocker, 2003). It is still not clear, however, how Rheb signals to TOR (Zhang, 2003).
Given the similarities between Rheb and mutants in the InR and TOR signalling pathways, it is conceivable that Rheb represents a novel component of one of these growth control pathways. To test this possibility, a detailed epistasis analysis was performed. Examined first was whether the negative regulators of InR and TOR signalling (PTEN and Tsc1-Tsc2, respectively) could counteract the effects of Rheb overexpression. All overexpression experiments were performed in the eye using the GMR-Gal4 driver line. Expression of either PTEN or Tsc1-Tsc2 alone results in a very similar size reduction of the ommatidia when compared with control ommatidia. However, whereas expression of PTEN has no influence on the increase in ommatidial size caused by Rheb overexpression, co-expression of Tsc1-Tsc2 results in ommatidia of approximately wild-type size, indicating that the activities of Rheb and Tsc1-Tsc2 can counteract each other. Next, the enlarged ommatidia phenotype of GMR-Rheb was assayed in a number of mutant backgrounds. Reducing the activity of Drosophila protein kinase B (PKB) has no effect on ommatidial size. Similar results were obtained with hypomorphic mutations in InR and Dp110, respectively. In contrast, ommatidial size is dominantly reduced by a mutation in TOR (TOR2L1), and a suppression to wild-type size is observed in a S6K mutant background. Thus, the Rheb overexpression phenotype is dependent on TOR and S6K function, but is independent of InR signal strength. Finally, the behaviors of Rheb PTEN and Rheb Tsc1 double mutants were examined. The phenotypic consequences were assayed in mosaic animals using the ey-Flp method. As expected, the Rheb PTEN double-mutant tissue clearly displays a Rheb phenotype. The Rheb Tsc1 mutant tissue also resembles Rheb single mutants, indicating that Rheb is epistatic over (functions downstream of) Tsc1 (Stocker, 2003).
In response to starvation, eukaryotic cells recover nutrients through autophagy, a lysosomal-mediated process of cytoplasmic degradation. Autophagy is known to be inhibited by TOR signaling, but the mechanisms of autophagy regulation and its role in TOR-mediated cell growth are unclear. Signaling through TOR and its upstream regulators PI3K and Rheb is necessary and sufficient to suppress starvation-induced autophagy in the Drosophila fat body. In contrast, TOR's downstream effector S6K promotes rather than suppresses autophagy, suggesting S6K downregulation may limit autophagy during extended starvation. Despite the catabolic potential of autophagy, disruption of conserved components of the autophagic machinery, including ATG1 and ATG5, does not restore growth to TOR mutant cells. Instead, inhibition of autophagy enhances TOR mutant phenotypes, including reduced cell size, growth rate, and survival. Thus, in cells lacking TOR, autophagy plays a protective role that is dominant over its potential role as a growth suppressor (Scott, 2004).
Autophagy likely evolved in single-cell eukaryotes to provide an energy and nutrient source allowing temporary survival of starvation. In yeast, Tor1 and Tor2 act as direct links between nutrient conditions and cell metabolism. These proteins sense nutritional status by an unknown mechanism, and effect a variety of starvation responses including changes in transcriptional and translational programs, nutrient import, protein and mRNA stability, cell cycle arrest, and induction of autophagy. Autophagy thus occurs in the context of a comprehensive reorganization of cellular activities aimed at surviving low nutrient levels (Scott, 2004).
In multicellular organisms, TOR is thought to have retained its role as a nutrient sensor but has also adopted new functions in regulating and responding to growth factor signaling pathways and developmental programs. Thus in a variety of signaling, developmental, and disease contexts, TOR activity can be regulated independently of nutritional conditions. In these cases, autophagy may be induced in response to downregulation of TOR despite the presence of abundant nutrients and may potentially play an important role in suppressing cell growth rather than promoting survival. Identification of the tumor suppressors PTEN, and TSC1 and TSC2 as positive regulators of autophagy provides correlative evidence supporting such a role for autophagy in growth control. Alternatively, since TOR activity is required for proper expression and localization of a number of nutrient transporters, inactivation of TOR may lead to reduced intracellular nutrient levels, and autophagy may therefore be required under these conditions to provide the nutrients and energy necessary for normal cell metabolism and survival (Scott, 2004).
The results presented here provide genetic evidence that under conditions of low TOR signaling, autophagy functions primarily to promote normal cell function and survival, rather than to suppress cell growth. This conclusion is based on the finding that genetic disruption of autophagy does not restore growth to cells lacking TOR, but instead exacerbates multiple TOR mutant phenotypes. It is important to note that mutations in TOR do not disrupt larval feeding, and thus disruption of autophagy is detrimental in TOR mutants despite the presence of ample extracellular nutrients. The finding that autophagy is critical in cells lacking TOR further supports earlier studies suggesting that inactivation of TOR causes defects in nutrient import, resulting in an intracellular state of pseudo-starvation (Scott, 2004).
Can the further reduction in growth of TOR mutant cells upon disruption of autophagy be reconciled with the potential catabolic effects of autophagy? TOR regulates the bidirectional flow of nutrients between protein synthesis and degradation through effects on nutrient import, autophagy, and ribosome biogenesis. When TOR is inactivated, rates of nutrient import and protein synthesis decrease, resulting in a commensurate reduction in mass accumulation and cell growth. In addition, autophagy is induced to maintain intracellular nutrient and energy levels sufficient for normal cell metabolism. When autophagy is experimentally inhibited in cells lacking TOR, this reserve source of nutrients is blocked, leading to a further decrease in energy levels, protein synthesis, and growth. It is noted that autophagy may have additional functions in cells with depressed TOR signaling, including recycling of organelles damaged by the absence of TOR activity, or selective degradation of cell growth regulators, analogous to the regulatory roles of ubiquitin-mediated degradation (Scott, 2004).
Autophagy is required for normal developmental responses to inactivation of insulin/PI3K signaling in the nematode C. elegans. In response to starvation or disruption of insulin/PI3K signaling, C. elegans larvae enter a dormant state called the dauer. Autophagy has been observed in C. elegans larvae undergoing dauer formation: disruption of a number of ATG homologs interfers with normal dauer morphogenesis. Importantly, simultaneous disruption of insulin/PI3K signaling and autophagy genes results in lethality, similar to the results presented in this study. Thus despite significant differences in developmental strategies for surviving nutrient deprivation, autophagy plays an essential role in the starvation responses of yeast, flies, and worms (Scott, 2004).
The prevailing view that S6K acts to suppress autophagy was founded on correlations between induction of autophagy and dephosphorylation of rpS6 in response to amino acid deprivation or rapamycin treatment. However, the genetic data presented in this study argue strongly against a role for S6K in suppressing autophagy: unlike other positive components of the TOR pathway, null mutations in S6K do not induce autophagy in fed animals. It is suggested that the observed correlation between S6K activity and suppression of autophagy is due to common but independent regulation of S6K and autophagy by TOR. Thus, autophagy suppression and S6K-dependent functions such as ribosome biogenesis represent distinct outputs of TOR signaling (Scott, 2004).
How might TOR signal to the autophagic machinery, if not through S6K? In yeast, this is accomplished in part through regulation of Atg1 kinase activity and ATG8 gene expression (Kamada, 2000 and Kirisako, 1999). The demonstration of a role for Drosophila ATG1 and ATG8 homologs [see TG8a (CG32672) and ATG8b (CG12334)] in starvation-induced autophagy, and the genetic interaction observed between ATG1 and TOR, are consistent with a related mode of regulation in higher eukaryotes. However, it is noted that other components of the yeast Atg1 complex such as Atg17 and Atg13, whose phosphorylation state is rapamycin sensitive, do not have clear homologs in metazoans, indicating that differences in regulation of autophagy by TOR are likely (Scott, 2004).
In addition to excluding a role for S6K in suppression of autophagy, these results reveal a positive role for S6K in induction of autophagy. S6K may promote autophagy directly, through activation of the autophagy machinery, or indirectly through its effects on protein synthesis. The latter possibility is consistent with previous reports that protein synthesis is required for expansion and maturation of autophagosomes. Interestingly, despite being required for autophagy, S6K is downregulated under conditions that induce it, including chronic starvation and TOR inactivation. Consistent with this, it was found that lysotracker staining is significantly weaker in chronically starved animals or in TOR mutants than in wild-type animals starved 3-4 hr. Furthermore, expression of constitutively activated S6K has no effect in wild-type, but restores lysotracker staining in TOR mutants to levels similar to those of acutely starved wild-type animals. It is suggested that downregulation of S6K may limit rates of autophagy under conditions of extended starvation or TOR inactivation and that this may protect cells from the potentially damaging effects of unrestrained autophagy (Scott, 2004).
Co-culture and conditioned media experiments have shown that the Drosophila fat body is a source of diffusible mitogens. The fat body has also been shown to act as a nutrient sensor through a TOR-dependent mechanism and to regulate organismal growth through effects on insulin/PI3K signaling. The results in this study extend these findings by showing that this endocrine response is accompanied by the regulated release of nutrients through autophagic degradation of fat body cytoplasm. Preventing this reallocation of resources, either through constitutive activation of PI3K or through inactivation of ATG genes, results in profound nutrient sensitivity. Thus, in response to nutrient limitation, the fat body is capable of simultaneously restricting growth of peripheral tissues through downregulation of insulin/PI3K signaling and providing these tissues with a buffering source of nutrients necessary for survival through autophagy (Scott, 2004).
Reducing insulin/IGF signaling allows for organismal survival during periods of inhospitable conditions by regulating the diapause state, whereby the organism stockpiles lipids, reduces fertility, increases stress resistance, and has an increased lifespan. The Target of Rapamycin (TOR) responds to changes in growth factors, amino acids, oxygen tension, and energy status; however, it is unclear how TOR contributes to physiological homeostasis and disease conditions. This study shows that reducing the function of Drosophila TOR results in decreased lipid stores and glucose levels. Importantly, this reduction of TOR activity blocks the insulin resistance and metabolic syndrome phenotypes associated with increased activity of the insulin responsive transcription factor, FOXO. Reduction in TOR function also protects against age-dependent decline in heart function and increases longevity. Thus, the regulation of TOR activity may be an ancient 'systems biological' means of regulating metabolism and senescence, that has important evolutionary, physiological, and clinical implications (Luong, 2006).
The major cause of metabolic syndrome (defined as a cluster of metabolic abnormalities such as elevated glucose and lipid levels, related to a state of insulin resistance) and diabetes in humans is reduction of insulin signaling, but the underlying pathways and mechanisms are not completely understood. Likewise, caloric excess can lead to nutrient toxicity and desensitization of insulin signaling. Thus, dysregulation of energy homeostasis can lead to metabolic disturbances and predisposition to a variety of endocrine diseases including diabetes, cardiovascular disease, and cancer (Luong, 2006).
One major system that regulates energy homeostasis in higher metazoa is the insulin/IGF pathway. The functionally conserved components of the insulin/IGF pathway like insulin, the insulin receptor (InR), insulin receptor substrate (IRS), phosphoinositide 3-kinase (PI3K), protein kinase B (PKB, a.k.a. Akt) and the forkhead transcription factor FOXO have been shown to be involved in glucose and lipid homeostasis. Loss of insulin signaling in the periphery and in pancreatic β cells can lead to hyperglycemia and diabetes. For example, disruption of the InsR gene in the pancreatic β cells reduces islet size and insulin secretion. IRS1 knockout mice are hyperglycemic, but their pancreatic β cells hypertrophy to compensate for increased peripheral insulin resistance. In contrast, IRS2 knockout mice are diabetic because their pancreatic β cells are absent due to increased cell death. Additionally, systemic loss of insulin signaling in metazoans leads to elevated lipids as seen in the Daf-2 mutant worms, Chico/IRS mutant flies, and IRS2 ablated mice (Luong, 2006 and references therein).
Many of these insulin/IGF-mediated metabolic effects depend on the winged helix transcription factor, FOXO. FOXO was first identified in the worm, C. elegans as Daf-16, a mutation that can suppress the increased lipid levels and longevity caused by loss of Daf-2, the worm InR ortholog. There is a single evolutionarily conserved Drosophila FOXO ortholog and three mammalian FOXO genes (FOXO1, FOXO3a, and FOXO4). FOXO1 controls glucose homeostasis in both peripheral tissues and pancreatic β cells. For example, expression of a constitutively activated FOXO1 (resistant to insulin/IGF-mediated inactivation) in liver and pancreatic β cells causes hepatic insulin resistance and loss of pancreatic β cells via increased apoptosis, whereas reduction of FOXO1 function can reverse the loss of pancreatic β cells and hyperglycemia seen in the IRS2 ablated mice. Thus, FOXO is a critical mediator of insulin signaling in both insulin sending and receiving tissues (Luong, 2006).
The Tuberous Sclerosis Complex (TSC1-2)/Target of Rapamycin (TOR) pathway responds to changes in insulin/IGF levels, amino acid levels, energy charge, lipid status, mitochondrial metabolites, and oxygen tension by adjusting cell growth. In addition to its well-defined role in controlling cell growth, the TSC1-2/TOR pathway may also potentially be a critical regulator of glucose and lipid homeostasis as TSC1-2/TOR signaling functionally interacts with the insulin/IGF pathway. A role for TOR signaling in glucose and lipid homeostasis in mammalian systems is demonstrated by the S6K1 knockout mice. These mice are hyperglycemic caused by diminished insulin secretion due to reduced pancreatic β cell mass. This result is in keeping with studies that show that rapamycin treatment leads to decreased levels of translation, growth, and survival of pancreatic. However, the mS6K1 mutant mice have low lipid levels because of adipocytes that have increased fatty acid β-oxidation. Additionally, the mS6K1 mutant mice show enhanced glucose uptake upon exogenous insulin addition due to insulin hypersensitivity in peripheral tissues via loss of a negative feedback loop on IRS. Thus, TOR signaling via S6K can modulate insulin sensitivity by altering Ser307 and Ser636/639 phosphorylation and IRS protein levels (Luong, 2006).
There are additional levels where TSC1-2/TOR signaling may positively and negatively regulate insulin signaling. There are data that suggest that the IRS Ser302 site is required for signaling to TOR and S6K. Thus, ser/thr phosphorylation of the IRS proteins may mediate both positive and negative signals for energy homeostasis. Furthermore, Akt/PKB activity may also be directly regulated by the nutrient-sensitive TOR pathway. Although the insulin/IGF pathway can signal to the TSC1-2/TOR pathway, recent evidence suggests that TOR may directly control Akt/PKB function because Akt/PKB activation depends on TORC2 complex-specific TOR Ser473 phosphorylation of Akt/PKB. Thus, these studies suggest that dysregulation of TSC1-2/TOR signaling may contribute to the pathological progression of metabolic syndrome and diabetes, yet the direct role and function of TOR is unclear in this context (Luong, 2006).
Another functionally conserved energy homeostatic pathway is the AMP-activated protein kinase (AMPK) pathway (see AMP-activated protein kinase). This pathway responds to altered energy states caused by cellular stresses like mitochondrial dysfunction, anti-diabetic drugs, and exercise. Activation of the energy sensing AMPK pathway by activated AMPK as well as metformin or AICAR treatment results in decreased lipogenesis and gluconeogenesis via both central. Activated AMPK can phosphorylate TSC2, which inhibits TOR signaling, while loss of AMPK activity causes an increase in TOR signaling. However, the requirement of TOR function for the AMPK energy response is not known. These effects may also be mediated by targets including glycogen synthase, hormone-sensitive lipase, acetyl-CoA carboxylase-2, HMG-CoA reductase, p300, and p53; the different roles of these proteins in the AMPK-mediated low energy response are not well known. Furthermore, activation of AMPK leads to IRS Ser-789 phosphorylation and enhancement of insulin signaling, which suggests that the AMPK response can act separately from the TOR pathway to enhance insulin signaling. Clearly, there is a great need to understand the regulation of TSC1-2/TOR signaling as it relates to the maintenance of energy homeostasis because TOR function is implicated in both insulin/IGF and AMPK signaling (Luong, 2006).
Although TOR occupies a central node that governs catabolic or anabolic responses to different nutritional and energy states, the resultant metabolic effects of altering TOR function in a metazoan are incompletely and poorly understood. This study examines in detail the function of Drosophila TOR in terms of energy homeostasis and senescent responses. Reduction of TOR function is show to result in decreased glucose and lipid levels with concomitant increase of DILP2 from the insulin producing cells. A reduction of TOR function can block activated FOXO-mediated insulin resistance and metabolic syndrome phenotypes. Taken together, these data indicate that TOR function is required for the maintenance of energy homeostasis and organismal senescence. The additional ramifications of this study are that reduction of TOR function may have clinical utility for treating metabolic syndrome and insulin resistance (Luong, 2006).
In contrast to the elevated lipid levels caused by reduction of systemic insulin signaling, the dTOR7/P mutant (containing a P-element insert into TOR) does not show increased lipid levels. Instead, the dTOR7/P mutant shows decreased lipid levels of the fat body that depend on the function of a lipase involved in lipid metabolism. Elevated ketone bodies were observed in the hypoglycemic dTOR7/P mutant, which is indicative of the increased utilization of lipids. Studies in mammalian cardiac tissue have shown that ketone bodies provide their high energy electrons directly to complex I, the NADH dehydrogenase multienzyme complex, of the mitochondrial electron transport chain. Thus, the altered lipid levels show that TOR has a critical role in determining the fate of fats (Luong, 2006).
It has also been shown that 4EBP is involved in lipid metabolism because the increased lipid levels caused by rapamycin treatment are blocked by a 4EBP mutant. Furthermore, loss of the melted mutant has lower lipid levels, due to lowered triglyceride production. This effect is due to increased 4EBP protein levels via FOXO activation in the fatbody. However, there is no change in glucose levels. In this respect, the melted mutant resembles the FIRKO mouse because it shows decreased triglyceride levels without a change in glucose levels. The dTOR7/P mutant has a different lipid phenotype than the one caused by rapamycin treatment, which suggests that rapamycin alters TOR function in a different manner than the dTOR7/P mutant. Additionally, a novel hypomorphic dTOR FAT domain allele in combination with the dTORP allele also shows low glucose and lipid levels, which suggests that partial reduction of TOR activity represents a unique phenotypic class of TOR metabolic effects versus an allele specific phenotype. Although rapamycin affects TORC1 directly, TORC2 may be altered indirectly via TOR depletion and blocking of TORC2 assembly. Thus, it is not currently clear if the effects of rapamycin are due to inhibiting TORC1 and/or TORC2. It is known that rapamycin can impair pancreatic β cell function because it causes decreased growth and survival. Thus, rapamycin treatment can lead to elevated glucose and lipid levels, possibly as a result of systemic insulin loss (Luong, 2006).
Evidence shows that DILP2 levels are increased in the IPCs of the dTOR7/P mutant and the dTOR7/P mutant has lowered glucose levels. Thus, reduction of TOR function can lead to increased DILP2 levels and a reduction of glucose levels. Recent studies with the Drosophila miRNA-278 mutant also showed elevated DILP levels, yet displayed fatbody-mediated insulin resistance as shown by elevated d4EBP and glucose levels. It is believed that the dTOR7/P mutant represents an insulin-sensitized state because the dTOR7/P mutant shows decreased levels of the insulin resistance marker 4EBP, the dTOR7/P mutant shows decreased glucose levels, and, as discussed below, the dTOR7/P mutant blocks activated FOXO-mediated insulin resistance phenotypes. Thus, the dTOR mutant phenotype resembles a whole-animal 'insulin-sensitized' state that can function below the level of constitutive FOXO activity (Luong, 2006).
Overexpression of activated FOXO in peripheral and IPC tissues results in elevated glucose and lipid levels. Although TOR signaling can alter insulin signaling upstream of FOXO, reducing TOR function is able to reverse these effects. Thus, the results show that reduction of TOR activity can block the activated FOXO-mediated insulin resistance and metabolic syndrome phenotypes. These results suggest that strategies to dampen, reduce, or block TOR signaling may be able to overcome insulin resistance (i.e., hyperglycemia and hypertriglyceridemia) below the level of increased FOXO activity in mammalian systems (Luong, 2006).
Although FOXO has >100 potential targets that might contribute to the metabolic phenotype, this study identified Fatty acid synthase (FAS) and DILP2 as candidate mediators of the TOR effect on the FOXO metabolic phenotypes. The effect on FAS is interesting because it is upregulated by FOXO overexpression and in an IRS/chico mutant and may be an important determinant of the lipid levels. It has also been shown that activation of daf-16/FOXO can decrease the mRNA levels of a worm insulin gene, ins-7 . This result is consistent with results showing that DILP2 mRNA levels are decreased and reducing TOR activity can reverse this FOXO-mediated reduction of DILP2. These results might have parallels with a role for FOXO and TOR in the regulation of insulin levels in mammals (Luong, 2006).
A selective and unexpected regulation of TOR effectors is also seen: loss of 4EBP protein and a mild effect on S6K Ser389 phosphorylation. It has been recently shown that the 4EBP gene is a target of FOXO in Drosophila and thus may represent one of the TOR targets responsible for contributing to the FOXO-mediated metabolic phenotypes. It has also been shown that daf-15/Raptor is a target of daf-16/FOXO in C. elegans and may also contribute to the TOR metabolic and senescent phenotypes. Raptor may also account for the selective difference in the regulation of 4EBP and S6K function by TOR because Raptor binds to both S6K and 4EBP and loss of 4EBP may allow for more S6K binding to Raptor for TOR-mediated phosphorylation. Thus, these results suggest that reduction of TOR function may have selective effects on translation (Luong, 2006).
Reduction of TOR function does not provide resistance against acute stresses or cause sterility. This result is in contrast to the yeast TOR1 mutant, which shows elevated stress resistance, and the d4EBP mutant, which shows stress and starvation sensitivity. Nevertheless, the dTOR7/P mutant has an increased lifespan. This result is in keeping with the yeast, worm and fly studies that show that loss of TOR signaling can increase lifespan, as a major mediator of caloric restriction. Thus, alterations of TOR signaling contribute to the regulation of lifespan (Luong, 2006).
It is also seen that reduction of TOR activity prevents age-dependent functional decline of heart performance. It is not currently clear how TOR is regulating these organ and organismal responses, but the altered lipid metabolism may underlie these changes. For example, changes in lipid metabolism can both autonomously and non-autonomously affect heart function. Thus, reduction of TOR function may reallocate energy stores preferentially for the control of ‘long-term’ responses such as lifespan and organ maintenance. Importantly, there are many potential links between changes in energy homeostasis with alterations in aging and organ senescence. Channelling diverse stimuli like amino acids, growth factors, oxygen tension, and energy charge into the TOR pathway may be an economic method to mobilize fuel stores like lipids to counteract these fluctuations (Luong, 2006).
The conservation of basic mechanisms between Drosophila and mammals is well established. It has been shown that disruption of insulin signaling in non-mammalian systems like Drosophila results in altered glucose and lipid levels. Reducing TOR function can reverse activated FOXO-mediated insulin resistance phenotypes induced in both insulin producing and insulin receiving tissues, and thus this study provides the first direct evidence that reducing TOR function may have a clinical benefit to counter insulin resistance, metabolic syndrome, and/or diabetes. Furthermore, altering TOR signaling may underlie the benefits of various diet and nutritional regimens. These results demonstrate the utility of using the powerful genetics of this system to unravel the complex pathways involved in maintaining glucose and lipid homeostasis. In unraveling the complex genetic network of TOR and InR signaling, although far from completion, the Drosophila model has been indispensable in finding critical components and uncovering functionally important genetic interactions between these two pathways. Thus, the basic mechanisms controlling glucose and lipid homeostasis, including mechanisms by which the TSC1-2/TOR pathway influences insulin signaling as well as the influence of TSC1-2/TOR signaling on peripheral tissue and IPC physiology, are also functionally conserved (Luong, 2006).
This study has described a new use for reducing TOR activity to block insulin resistance, metabolic syndrome, and diabetic-like phenotypes downstream of activated FOXO, underlining the utility of the Drosophila model to identify and analyze components and compounds that block insulin resistance and metabolic syndrome phenotypes as well as pathological aspects of aging and organ senescence (Luong, 2006).
Determining how growth and differentiation are coordinated is key to understanding normal development, as well as disease states such as cancer, where that control is lost. Growth and neuronal differentiation are coordinated by the insulin receptor/target of rapamycin (TOR) kinase (InR/TOR) pathway. The control of growth and differentiation diverge downstream of TOR. TOR regulates growth by controlling the activity of S6 kinase (S6K) and eIF4E. Loss of s6k delays differentiation, and is epistatic to the loss of tsc2, indicating that S6K acts downstream or in parallel to TOR in differentiation as in growth. However, loss of eIF4E inhibits growth but does not affect the timing of differentiation. This study shows that there is crosstalk between the InR/TOR pathway and epidermal growth factor receptor (EGFR) signaling. InR/TOR signaling regulates the expression of several EGFR pathway components including pointedP2 (pntP2). In addition, reduction of EGFR signaling levels phenocopies inhibition of the InR/TOR pathway in the regulation of differentiation. Together these data suggest that InR/TOR signaling regulates the timing of differentiation through modulation of EGFR target genes in developing photoreceptors (McNeill, 2008).
Tight coordination of growth and differentiation is essential for normal development. InR/TOR signaling controls the timing of neuronal differentiation in the eye and leg in Drosophila. This study demonstrates that the InR/TOR pathway regulates neuronal differentiation in an S6K-dependent, but 4EBP/eIF4E-independent manner. It has previously been impossible to determine whether InR/TOR signaling was acting downstream or in parallel to the EGFR/MAPK pathway. Using argos and rho as reporters this study shows that the InR/TOR pathway is able to regulate EGFR/MAPK signaling downstream of MAPK. Moreover, pntP2 expression is up- and downregulated by activation or inhibition of InR/TOR signaling, respectively, and InR/TOR and EGFR pathways interact through pntP2. Taken together these data suggest that temporal control of differentiation by the InR/TOR pathway is achieved by modulation of EGFR pathway transcriptional targets in differentiating PRs (McNeill, 2008).
TOR is part of two multimeric complexes (TORC1 and TORC2) and is a core component of the InR pathway. TORC1 activity is regulated by nutrient and energy levels providing a conduit for hormonal and catabolic cellular inputs. Growth is regulated by two downstream targets of TORC1: S6K and 4EBP. The current data demonstrate that upstream of TORC1, differentiation and growth are regulated by the same factors. Downstream of TORC1, differentiation and growth differ significantly in that loss of s6k, but not eIF4E (or overexpression of 4EBP) affects differentiation. eIF4E regulates 7-methyl-guanosine cap-dependent translation and is the rate-limiting factor in translation initiation. The finding that eIF4E does not affect differentiation suggests that the temporal control of differentiation is not based on a translation initiation-dependent mechanism. Strikingly, loss of s6k blocks the precocious differentiation induced by loss of tsc2. Given the relatively weak effects of loss of s6k this may seem surprising. However, the degree of suppression is similar to the effect of loss of s6k on the overgrowth phenotype caused by loss of tsc2, namely, tsc2, s6k double-mutant cells are the same size as wild-type cells. Although loss of eIF4E has no affect on differentiation it may act redundantly with another factor, such as s6k. Testing this hypothesis though is technically challenging since the Drosophila genome contains eight different eIF4E isoforms. It will be interesting in future to test whether any of these isoforms regulate differentiation or alternatively whether eIF4E and s6k act redundantly. Although further work is required to determine the precise relationship between S6K and the InR/TOR pathway, the data point to a critical role of S6K in coordinating neuronal differentiation and growth (McNeill, 2008).
As in other neuronal systems, differentiation of PRs in the Drosophila eye occurs in a stereotyped manner. The advantage of the Drosophila retina as an experimental system is that the PRs differentiate spatiotemporally. Using this feature, as well as a series of cell-type-specific antibodies, this study has demonstrated that InR/TOR signaling is selective in the cell-types that it affects. The differentiation of PRs 2/5, 3/4, and 8 are unaffected by perturbations in InR/TOR signaling, whereas PRs 1, 6, and 7 and cone cells are dependent on this pathway for temporal control of differentiation. Interestingly the affected cells all differentiate after the second mitotic wave. However, regulators of the cell cycle do not affect the temporal control of differentiation. Why then are PRs 1, 6, and 7 and cone cells specifically affected? In cells with increased InR/TOR signaling, the expression of argos, rho, and pntP2 is precocious and increased throughout the clone, suggesting that the upregulation of EGFR signaling occurs in all cells. However, decreasing EGFR activity using a hypomorphic pntP2 allele specifically affects the differentiation of PRs 1, 6, and 7 and cone cells. Interestingly, pntP2 expression in differentiated cells is also restricted to PRs 1, 6, and 7 and cone cells. These observations suggest that differentiation of PRs 1, 6, and 7 and cone cells is critically dependent on EGFR levels signaling through pntP2. Therefore, although activation of InR/TOR signaling causes upregulation of EGFR transcriptional targets in all cells as they differentiate, the phenotypic effect is seen only in PRs 1, 6, and 7 and cone cells since these cells are highly sensitive to EGFR activity signaling through pntP2. This possibility is supported by the fact that precocious differentiation caused by overexpression of Dp110 can be suppressed by the simultaneous reduction of pntP2 levels. The complete suppression of the Dp110 differentiation phenotype by simultaneous reduction of pntP2 strongly suggests that pntP2 acts downstream of Dp110 and InR/TOR signaling in a pathway that regulates the temporal control of differentiation. It has been suggested that later differentiating PRs require higher levels of EGFR activity than their earlier differentiating neighbors. In particular, the activation of PR 7 requires both EGFR and Sevenless RTKs. In the case of InR/TOR pathway activation it may be that, through its regulation of EGFR downstream targets, the 'second burst' of RTK activity is enhanced causing PRs 1, 6, and 7 and cone cells to differentiate precociously. There may also be other as yet unidentified factors through which the InR/TOR pathway controls the expression of Aos and rho in PRs 2-5 and 8 (McNeill, 2008).
Activation of insulin and insulin-like growth factor receptors in mammalian systems is well known to elicit a response via the Ras/MAPK pathway. However, loss of the InR in the Drosophila eye does not result in a loss of PRs, a hallmark of the Ras pathway, nor does mutation of the putative Drk binding site in chico affect the function of the Drosophila IRS. In accordance with these data no change is seen in dpERK staining when the InR/TOR pathway is activated in the eye disc. Rather than a direct activation of Ras signaling by the InR, the data suggest that in the developing eye crosstalk between these pathways occurs at the level of regulation of the expression of EGFR transcriptional outputs. The most proximal component of the EGFR pathway that is regulated by InR/TOR signaling is pntP2. However, the data suggest that temporal control of PR differentiation requires concerted regulation of EGFR transcriptional outputs, since overexpression of pntP2 alone is not sufficient to cause precocious differentiation, whereas overexpression of activated EGFR is sufficient. Interestingly, microarray analyses of Drosophila and human cells have shown that the InR/TOR pathway regulates the expression of hundreds of genes. The mechanism by which this transcriptional control is exerted has yet to be elucidated. It will be interesting in future to determine the extent of transcriptional crosstalk between InR/TOR and EGFR pathways in developing neurons (McNeill, 2008).
Cell growth arrest and autophagy are required for autophagic cell death in Drosophila. Maintenance of growth by expression of either activated Ras, Dp110, or Akt is sufficient to inhibit autophagy and cell death in Drosophila salivary glands, but the mechanism that controls growth arrest is unknown. Although the Warts (Wts) tumor suppressor is a critical regulator of tissue growth in animals, it is not clear how this signaling pathway controls cell growth. This study shows that genes in the Wts pathway are required for salivary gland degradation and that wts mutants have defects in cell growth arrest, caspase activity, and autophagy. Expression of Atg1, a regulator of autophagy, in salivary glands is sufficient to rescue wts mutant salivary gland destruction. Surprisingly, expression of Yorkie (Yki) and Scalloped (Sd) in salivary glands fails to phenocopy wts mutants. By contrast, misexpression of the Yki target bantam is able to inhibit salivary gland cell death, even though mutations in bantam fail to suppress the wts mutant salivary gland-persistence phenotype. Significantly, wts mutant salivary glands possess altered phosphoinositide signaling, and decreased function of the class I PI3K-pathway genes chico and TOR suppressed wts defects in cell death. Although it has been shown that salivary gland degradation requires genes in the Wts pathway, this study provides the first evidence that Wts influences autophagy. These data indicate that the Wts-pathway components Yki, Sd, and bantam fail to function in salivary glands and that Wts regulates salivary gland cell death in a PI3K-dependent manner (Dutta, 2008).
Wts was identified as a protein that is expressed during autophagic cell death of Drosophila larval salivary glands with a high-throughput proteomics approach. This was surprising, given that wts RNA was not detected with DNA microarrays. Therefore, this study investigated whether Wts is present in salivary glands, and it was determined to be constitutively expressed at stages before and after the rise in ecdysone that triggers autophagic cell death. Animals that are homozygous for the hypomorphic wtsP2 allele, which is caused by a P element insertion, are defective in salivary gland cell death (Martin, 2007). Significantly two forms of Hpo are expressed during stages preceding salivary gland cell death, suggesting that phosphorylated Hpo is present in these cells and that this signaling pathway is activated (Dutta, 2008).
These studies indicate that Wts and other core components of this tumor-suppressor pathway are required for autophagic cell death of Drosophila salivary glands. wts is required for cell growth arrest and for proper regulation of caspases and autophagy, which contribute to the destruction of salivary glands. Although it is well known that cell division, cell growth, and cell death are important regulators of tissue and tumor size, it has been unclear whether a mechanistic relationship exists between cell growth and control of cell death (Dutta, 2008).
It is possible that wts and associated downstream growth-regulatory mechanisms could suppress cell death in other animals and cell types. Autophagic cell-death morphology has been reported in diverse taxa, but little is known about the mechanisms that control this form of cell death, and this lack of understanding is probably related to the limited investigation of physiologically relevant models of this process (Dutta, 2008).
This study used steroid-activated autophagic cell death of salivary glands as a system to study the relationship between cell growth and cell death. It is logical that cell growth influences cell death in salivary glands, given that autophagy is known to be regulated by class I PI3K signaling, which contributes to the death of these cells (Berry, 2007). It is unclear whether growth arrest is a determinant of autophagic cell death in other cell types and animals, and this question is important to resolve because of the importance of growth and autophagy in multiple disorders, including cancer. wts mutant salivary gland cells fail to arrest growth at the onset of puparium formation, and this suppresses the induction of autophagy. The inhibitor of apoptosis DIAP1 influences salivary gland cell death and is one of the best-characterized target genes of the Wts signaling pathway, but DIAP1 levels are not altered in wts mutant salivary glands. Significantly, the data provide the first evidence that Wts regulates autophagy and support previous studies indicating that caspases and autophagy function in an additive manner during autophagic cell death. Given the importance of both the Wts pathway and autophagy in human health, it is critical to determine whether this relationship exists in other cells (Dutta, 2008).
Cell growth and division are often considered to be synonymous, even though they are controlled by independent mechanisms. The Wts signaling pathway must influence cell growth, but most studies have emphasized the influence of this pathway on cell division and death. bantam is the only previously studied gene that is regulated by the Wts pathway and that is known to regulate cell growth. However, the mechanism of bantam action remains obscure. The current studies suggest the possibility that Wts may regulate growth via different mechanisms and that the nature of this regulation may depend on cell context. It is premature to conclude that bantam regulates a completely novel cell growth program, but the fact that misexpression of bantam stimulates cell growth in the absence of changes in a phosphoinositide marker and that chico and TOR fail to suppress the bantam-induced salivary gland-persistence phenotype minimally suggests that this microRNA regulates genes downstream of TOR. Significant progress has been made in the identification of microRNA targets, and future studies should resolve the mechanism underlying bantam regulation of cell growth (Dutta, 2008).
Recent studies of Wts signaling in Drosophila have identified a linear pathway that terminates with Yki and Sd regulation of effector genes that influence cell growth, cell division, and cell death. These studies indicate that the Wts pathway may not always regulate downstream effector genes via Yki and Sd, given that Yki expression was not able to phenocopy the wts mutant salivary gland destruction and expression of Sd induced premature degradation of salivary glands. Although bantam expression is sufficient to induce growth and inhibit cell death in salivary glands, bantam function is not required for the wts mutant phenotype. wts mutant salivary glands possess altered markers of PI3K signaling, and their defect in cell death is suppressed by chico and TOR. Combined, these results indicate that Wts regulates cell growth and cell death via a PI3K-dependent, and Yki- and Sd-independent, mechanism. Future studies will determine whether Wts regulates cell growth in a PI3K-dependent manner in other cells and animals (Dutta, 2008).
Stem cells depend on intrinsic and local factors to maintain their identity and activity, but they also sense and respond to changing external conditions. Germline stem cells (GSCs) and follicle stem cells (FSCs), the latter being somatic stem cells, in the Drosophila ovary respond to diet via insulin signals. Insulin signals directly modulate the GSC cell cycle at the G2 phase, but additional unknown dietary mediators control both G1 and G2. Target of rapamycin, or TOR, is part of a highly conserved nutrient-sensing pathway affecting growth, proliferation, survival and fertility. This study shows that optimal TOR activity maintains GSCs but does not play a major role in FSC maintenance, suggesting differential regulation of GSCs versus FSCs. TOR promotes GSC proliferation via G2 but independently of insulin signaling, and TOR is required for the proliferation, growth and survival of differentiating germ cells. TOR also controls the proliferation of FSCs but not of their differentiating progeny. Instead, TOR controls follicle cell number by promoting survival, independently of either the apoptotic or autophagic pathways. These results uncover specific TOR functions in the control of stem cells versus their differentiating progeny, and reveal parallels between Drosophila and mammalian follicle growth (LaFever, 2010).
The Drosophila ovary houses stem cells in the germarium, the anterior-most portion of each ovariole. Two or three germline stem cells (GSCs) in a specialized niche self-renew and produce cystoblasts, which divide four times with incomplete cytokinesis to form germline cysts containing one oocyte and fifteen nurse cells. Follicle stem cells (FSCs) self-renew and produce follicle cells that envelop each cyst to form an egg chamber, or follicle. After leaving the germarium, each follicle develops through fourteen stages, forming a mature oocyte. As the cyst grows, follicle cells divide mitotically until stage 7, when they begin endoreplicating. Yolk uptake or vitellogenesis initiates at stage 8. The control of distinct stem cell populations and their differentiating progeny can thus be probed in this system (LaFever, 2010).
Ovarian stem cells and their progeny respond to diet. On a protein-rich diet, GSCs and FSCs proliferate rapidly and their descendents divide and grow robustly. On a protein-poor diet, proliferation and growth are slowed, early germline cysts die and vitellogenesis is blocked. Insulin signaling is required for all responses, except early cyst viability. Insulin-like peptides directly promote GSC division, cyst growth and vitellogenesis and indirectly control GSC maintenance. Insulin-like peptides promote GSC G2 progression through PI3 kinase and FOXO; however, additional diet mediators control both G1 and G2 (LaFever, 2010 and references therein).
This study reveals specific Tor roles in Drosophila GSCs versus FSCs. Although Tor is required for proper proliferation of GSCs and FSCs, it plays a major role in GSC, but not FSC, maintenance. TOR also differentially regulates stem cells versus their progeny. Tor is necessary for early cyst proliferation, growth and survival by preventing apoptosis. By contrast, TOR does not regulate follicle cell proliferation and controls follicle cell growth and survival independently of apoptosis or autophagy. Follicle cell TOR activity also affects underlying cyst growth. Finally, TOR regulates these processes via insulin-dependent and -independent mechanisms. These studies uncover specific roles for TOR in the control of stem cells and their differentiating progeny in the Drosophila ovary. TOR is a known nutrient sensor in many systems; it is therefore speculated that TOR is part of a broadly conserved mechanism that ties stem cell maintenance and function, and the survival, proliferation and growth of their descendents, to diet-dependent factors (LaFever, 2010).
This study has reveal strikingly specific effects of Tor on GSCs, FSCs and their progeny. Coupled to studies showing the conserved role of TOR as a nutrient sensor, these results address how specific effects of a nutrient-responsive factor might contribute to the coordination between different stem cell populations and their descendents (LaFever, 2010).
Both insulin signals and TOR are nutrient-sensing factors that converge on G2 to regulate Drosophila GSC proliferation. G2 regulation in response to diet/insulin signals also occurs in Drosophila male GSCs and in Caenorhabditis elegans germline precursors. Starvation promotes deleterious mutations during Saccharomyces cerevisae division, and cancer cells form repair foci during a delayed G2 upon DNA damage. The multitude of GSC G2 regulators might reflect a mechanism to ensure genomic integrity under poor dietary conditions (LaFever, 2010).
Although TOR regulates the G1-S transition, it also modulates G2-M in S. cerevisae, Schizosaccharomyces pombe and mammalian cells. Combined with the Tor role in GSC G2, the increased phosphorylation of 4E-BP specifically during M suggests that a TOR activity increase might precede the G2-M transition. Interestingly, activated TOR is highly enriched at the mitotic spindles of rat ovarian granulosa cells and TOR inhibition by rapamycin impairs their proliferation. Marked increases in S6K activity and 4E-BP1 phosphorylation in M occur in HeLa cells, further suggesting TOR activity cell cycle regulation as part of a conserved mechanism to tie G2-M to nutrient availability (LaFever, 2010).
Although both GSCs and FSCs require Tor for normal proliferation, only GSC maintenance requires optimal TOR activity. These distinctions do not reflect a fundamental difference between germline and somatic stem cells because TOR appears to control the maintenance of several, although probably not all, mammalian somatic stem cell types. In hematopoietic stem cells, Tsc1 or PTEN loss results in increased TOR signaling and short-term expansion, but also progressive stem cell depletion. TOR activation downstream of Wnt1 overexpression leads to transiently increased hair follicle proliferation followed by stem cell loss. By contrast, PTEN mutant ovarian granulosa cells do not become depleted, despite elevated TOR activity. Because granulosa cells might derive from stem cells, it is tempting to speculate that maintenance of these stem cells might not require precise TOR regulation, similar to Drosophila FSCs (LaFever, 2010).
Ovarian stem cells and their progeny respond to TOR differently. Reduced Tor activity leads to apoptosis of 8- and 16-cell cysts, but Tor mutant GSCs do not appear to undergo apoptotic or autophagic death. The niche might conceivably prevent GSC death. Indeed, there are reports of GSC cell death within their in vivo niche. Consistent with the niche promoting GSC survival, GSCs die at higher rates when separated from somatic cells in culture. Laser ablation of the single apical niche cell causes death of Locusta migratoria male GSCs. This model, however, does not account for a normal number of Tor mutant cystoblasts and 2-cell cysts. Perhaps a combination of niche displacement and growth defects leads to Tor mutant 8- and 16-cell cyst death (LaFever, 2010).
Reduced Tor activity slows FSC proliferation, but has no effect on the cell cycle of follicle cells. This striking difference suggests that follicle cell proliferation might be largely insensitive to direct effects of diet. Follicle cells might instead receive their primary cue to divide from the underlying germline, perhaps via the actomyosin cytoskeleton. Consistent with this idea, when germline cyst growth is slowed down by InR or Myc mutation, surrounding wild-type follicle cells adjust their numbers accordingly, although it remains to be determined if this reflects changes in follicle cell proliferation per se (LaFever, 2010).
Cell competition can occur when cell populations with different growth capacities coexist. It has been proposed that a cell senses the translational capacity of their neighbors and thus distinguishes 'winner' versus 'loser' cells. The 'losers' undergo apoptosis and secrete factors that stimulate 'winner' proliferation. Although Tor regulates growth and translation, Tor mutant follicle cells do not exhibit apoptosis, but are instead extruded from mosaic monolayers. Apoptosis-independent extrusion of cells with compromised Decapentaplegic (DPP, a bone morphogenetic protein family member) signaling has been reported in mosaic Drosophila wing disc epithelia. This similarity suggests a possible connection between DPP signaling and TOR (LaFever, 2010).
TOR can be activated downstream of insulin signaling but also receives additional inputs. Insulin signaling controls germline growth via TOR, whereas insulin (via FOXO) and TOR signaling regulate GSC proliferation in parallel. Tor-null ovarian cell defects are also more severe than InR-null defects, implying that TOR receives additional inputs during oogenesis (LaFever, 2010).
Amino acid transport activates TOR signaling in Drosophila and mammals. The Drosophila genome predicts approximately 40 amino acid transporters and recent evidence suggests that methionine is a key dietary amino acid for oogenesis in Drosophila (Grandison, 2009). Further studies should investigate how various classes of amino acid transporters affect ovarian TOR signaling and amino acid requirements for specific oogenesis processes (LaFever, 2010).
4E-BP, encoded by Thor, represses cap-dependent translation via eIF4E inhibition. TOR phosphorylates and inhibits 4E-BP, leading to translation de-repression. 4E-BP, however, does not mediate Tor ovarian phenotypes, suggesting that TOR probably acts through S6K or MYC. Indeed, S6K overexpression partially restores Tor mutant growth, viability and fertility, whereas MYC loss causes germline growth phenotypes similar to Tor defects (LaFever, 2010).
Whether or not 4E-BP is required in any other tissues to mediate the effects of reduced TOR activity remains unclear. Although overexpression of eIF4E increases cell growth rates and overexpression of 4E-BP results in smaller cell size, loss of 4E-BP does not phenocopy eIF4E overexpression. Furthermore, Thor mutation has no obvious phenotype in Drosophila except for increased sensitivity to stress and impaired innate immunity. Although Thor is required for dietary restriction effects on lifespan, no reports of Tor Thor double mutants exist in the literature (LaFever, 2010).
The results bring to light interesting parallels between the role of TOR in Drosophila and mammalian ovaries. Insulin and TOR signaling are active in mammalian ovaries and rapamycin inhibits follicle growth in cultured mouse ovaries, suggesting similar regulation of oocyte growth and follicle cell numbers between Drosophila and mammals. Although adult mammalian ovaries do not contain GSCs, overexpression of either insulin or TOR signaling in mouse primordial germ cells leads to premature ovarian failure caused by the hyperactivation and subsequent depletion of the primordial germ cell pool, a phenotype that is arguably reminiscent of the rapid loss of Tsc1 mutant GSCs (LaFever, 2010).
Alarcon, C. M., Heitman, J. and Cardenas, M. E. (1999). Protein kinase activity and identification of a toxic effector domain of the target of rapamycin TOR proteins in yeast. Mol. Biol. Cell 10: 2531-2546. 10436010
Bai, X., et al. (2007). Rheb activates mTOR by antagonizing its endogenous inhibitor, FKBP38. Science 318(5852): 977-80. PubMed citation: 17991864
Barbet, N. C., Schneider, U., Helliwell, S. B., Stansfield, I., Tuite, M. F. and Hall, M. N. (1996). TOR controls translation initiation and early G1 progression in yeast. Mol. Biol. Cell 7: 25-42. PubMed Citation: 8741837
Beck, T. and Hall, M. N. (1999). The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors. Nature 402: 689-692. PubMed Citation: 10604478
Berry, D. L., and Baehrecke, E. H. (2007). Growth arrest and autophagy are required for salivary gland cell degradation in Drosophila. Cell 131: 1137-1148. PubMed Citation: 18083103
Beugnet, A., Wang, X. and Proud, C. G. (2003). The TOR-signaling and RAIP motifs play distinct roles in the mTOR-dependent phosphorylation of initiation factor 4E-binding protein 1 in vivo. J. Biol. Chem. 12912989
Brown, E. J., Albers, M. W., Shin, T. B., Ichikawa, K., Keith, C. T., Lane, W. S., and Schreiber, S. L. (1994). A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature 369: 756-758. 8008069
Bryk, B., Hahn, K., Cohen, S. M. and Teleman, A. A. (2010). MAP4K3 regulates body size and metabolism in Drosophila. Dev. Biol. 344(1): 150-7. PubMed Citation: 20457147
Boehlke, C., et al. (2010). Primary cilia regulate mTORC1 activity and cell size through Lkb1. Nat. Cell Biol. 12(11): 1115-22. PubMed Citation: 20972424
Brugarolas, J., et al. (2004). Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex. Genes Dev. 2004 18(23): 2893-904. 15545625
Bryk, B., Hahn, K., Cohen, S. M. and Teleman, A. A. (2010). MAP4K3 regulates body size and metabolism in Drosophila. Dev. Biol. 344(1): 150-7. PubMed Citation: 20457147
Budanov, A. V. and Karin, M. (2008). p53 target genes sestrin1 and sestrin2 connect genotoxic stress and mTOR signaling. Cell 134: 451-60. PubMed Citation: 18692468
Cardenas, M. E., et al. (1999). The TOR signaling cascade regulates gene expression in response to nutrients. Genes Dev. 13: 3271-3279. PubMed Citation: 10617575
Chang, Y.Y. amd Neufeld, T. P. (2009). An Atg1/Atg13 complex with multiple roles in tor-mediated autophagy regulation. Mol. Biol. Cell. 20(7): 2004-14. PubMed Citation: 19225150
Chell, J. M. and Brand, A. H. (2010). Nutrition-responsive glia control exit of neural stem cells from quiescence. Cell 143: 1161-1173. PubMed Citation: 21183078
Chen, C. C., et al. (2010). FoxOs inhibit mTORC1 and activate Akt by inducing the expression of Sestrin3 and Rictor. Dev. Cell 18(4): 592-604. PubMed Citation: 20412774
Choi, J. H., et al. (2000). TOR signaling regulates microtubule structure and function. Curr. Biol. 10: 861-864. PubMed Citation: 10899009
Choi, K. M., McMahon, L. P. and Lawrence, J. C. (2003). Two motifs in the translational repressor PHAS-I required for efficient phosphorylation by mammalian target of rapamycin and for recognition by raptor. J. Biol. Chem. 278(22): 19667-73. 12665511
Chou, M.M. and Blenis, J. 1995. The 70 kDa S6 kinase: Regulation of a kinase with multiple roles in mitogenic signaling. Curr. Opin. Cell Biol. 7: 806-814. 8608011
Coelho, C. M. and Leevers, S. J. (2000}. Do growth and cell division rates determine cell size in multicellular organisms? J. Cell Sci. 113: 2927-2934. 10934032
Colombani, J., et al. (2003). A nutrient sensor mechanism controls Drosophila growth. Cell 114: 739-749. Medline abstract: 14505573
Cutler, N. S., Heitman, J. and Cardenas, M. E. (1999). TOR kinase homologs function in a signal transduction pathway that is conserved from yeast to mammals. Mol. Cell Endocrinol. 155: 135-142. 10580846
Cutler, N. S., Pan, X., Heitman, J. and Cardenas, M. E. (2001). The TOR signal transduction cascade controls cellular differentiation in response to nutrients. Mol. Biol. Cell 12(12): 4103-13. 11739804
Dan, H. C., et al. (2008). Akt-dependent regulation of NF-kappaB is controlled by mTOR and Raptor in association with IKK. Genes Dev. 22(11): 1490-500. PubMed Citation: 18519641
Dennis, P. B., Fumagalli, S. and Thomas, G. (1999). Target of rapamycin (TOR): Balancing the opposing forces of protein synthesis and degradation. Curr. Opin. Genet. Dev. 9: 49-54. 10072357
Dutta, S. and Baehrecke, E. H. (2008). Warts is required for PI3K-regulated growth arrest, autophagy, and autophagic cell death in Drosophila. Curr. Biol. 18(19): 1466-75. PubMed Citation: 18818081
Findlay, G. M., Yan, L., Procter, J., Mieulet, V. and Lamb, R. F. (2007). A MAP4 kinase related to Ste20 is a nutrient-sensitive regulator of mTOR signalling. Biochem. J. 403(1): 13-20. PubMed Citation: 17253963
Gao, X., et al. (2002). Tsc tumour suppressor proteins antagonize amino-acid-TOR signalling. Nat. Cell Biol. 4(9): 699-704. 12172555
Giordano, E., Peluso, I., Senger, S. and Furia, M. (1999). minifly, a Drosophila gene required for ribosome biogenesis. J. Cell Biol. 144: 1123-1133. Medline abstract: 10087258
Grandison, R. C., Piper, M. D. and Partridge, L. (2009). Amino-acid imbalance explains extension of lifespan by dietary restriction in Drosophila. Nature 462: 1061-1064. PubMed Citation: 19956092
Guertin, D. A., et al. (2006). Functional genomics identifies TOR-regulated genes that control growth and division. Curr. Biol. 16(10): 958-70. Medline abstract: 16713952
Guertin, D. A., et al. (2007). Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCa, but not S6K1. Dev. Cell. 11: 859-871. Medline abstract: 17141160
Hara, K., et al. (2002). Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell 110(2): 177-89. 12150926
Hardwick, J. S., et al. (1999). Rapamycin-modulated transcription defines the subset of nutrient-sensitive signaling pathways directly controlled by the Tor proteins. Proc. Natl. Acad. Sci. 96(26): 14866-70. PubMed Citation: 10611304
Hari, K. L., et al. (1995). The mei-41 gene of D. melanogaster is a structural and functional homolog of the human ataxia telangiectasia gene. Cell 82: 815-821. PubMed Citation: 7671309
Heitman, J., Movva, N. R. and Hall, M. N. (1991). Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science 253: 905-909. 1715094
Hennig, K.M. and Neufeld, T.P. (2002). Inhibition of cellular growth and proliferation by dTOR overexpression in Drosophila. Genesis 34: 107-110. 12324961
Hennig, K. M., Colombani, J. and Neufeld, T. P. (2006). TOR coordinates bulk and targeted endocytosis in the Drosophila melanogaster fat body to regulate cell growth. J. Cell Biol. 173(6): 963-74. Medline abstract: 16785324
Huang, S., et al. (2003). Sustained activation of the JNK cascade and rapamycin-induced apoptosis are suppressed by p53/p21Cip1. Mol. Cell 11: 1491-1501. 12820963
Inoki, K., Li, Y., Zhu, T., Wu, J. and Guan, K. L. (2002). TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat. Cell Biol. 4(9): 648-57. 12172553
Isotani, S., et al. (2000). Immunopurified mammalian target of rapamycin phosphorylates and activates p70 S6 kinase alpha in vitro. J. Biol. Chem. 274(48): 34493-8. PubMed Citation: 10567431
Jia, K. Chen, D. and Riddle, D. L. (2004). The TOR pathway interacts with the insulin signaling pathway to regulate C. elegans larval development, metabolism and life span. Development 131: 3897-3906. 15253933
Juhasz, G., et al. (2008). The class III PI(3)K Vps34 promotes autophagy and endocytosis but not TOR signaling in Drosophila. J. Cell Biol. 181: 655-666. PubMed Citation: 18474623
Kaeberlein, M., et al. (2005). Regulation of yeast replicative life span by TOR and Sch9 in response to nutrients. Science 310(5751): 1193-6. Medline abstract: 16293764
Kamada, Y., Funakoshi, T., Shintani, T., Nagano, K., Ohsumi, M. and Ohsumi, Y. (2000). Tor-mediated induction of autophagy via an Apg1 protein kinase complex. J. Cell Biol. 150: 1507-1513. 10995454
Kapahi, K., et al. (2004). Regulation of lifespan in Drosophila by modulation of genes in the TOR signaling pathway. Curr. Biol. 14: 885-890. 15186745
Khurana, V., et al. (2006). TOR-mediated cell-cycle activation causes neurodegeneration in a Drosophila tauopathy model. Curr. Biol. 16: 230-241. 16461276
Kim, D. H., et al. (2002). mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 110(2): 163-75. 12150925
Kim, D. H., et al. (2003). GbetaL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR. Mol. Cell 11(4): 895-904. 12718876
Kirisako, T., Baba, M., Ishihara, N., Miyazawa, K., Ohsumi, M., Yoshimori, T., Noda, T. and Ohsumi, Y. (1999). Formation process of autophagosome is traced with Apg8/Aut7p in yeast. J. Cell Biol. 147: 435-446. 10525546
Kumar, V., et al. (2000a). Functional interaction between RAFT1/FRAP/mTOR and protein kinase cdelta in the regulation of cap-dependent initiation of translation. EMBO J. 19: 1087-1097. PubMed Citation: 10698949
Kumar, V., et al. (2000b). Regulation of the rapamycin and FKBP-target 1/mammalian target of rapamycin and cap-dependent initiation of translation by the c-Abl protein-tyrosine kinase. J. Biol. Chem. 275(15): 10779-87. PubMed Citation: 10753870
LaFever, L., Feoktistov, A., Hsu, H. J. and Drummond-Barbosa, D. (2010). Specific roles of Target of rapamycin in the control of stem cells and their progeny in the Drosophila ovary. Development 137(13): 2117-26. PubMed Citation: 20504961
Lawrence, J. C. and Abraham, R. T. (1997). PHAS/4E-BPs as regulators of mRNA translation and cell proliferation. Trends Biochem. Sci. 22: 345-349. 9301335
Lee, J. H., et al. (2010). Sestrin as a feedback inhibitor of TOR that prevents age-related pathologies. Science 327: 1223-1228. PubMed Citation: 20203043
Luong, N., et al. (2006). Activated FOXO-mediated insulin resistance is blocked by reduction of TOR activity. Cell Metab. 4(2): 133-42. Medline abstract: 16890541
Layalle, S., Arquier, N., Léopold, P. (2008). The TOR pathway couples nutrition and developmental timing in Drosophila. Dev. Cell 15(4): 568-77. PubMed Citation: 18854141
Mao, J. H., et al. (2008). FBXW7 targets mTOR for degradation and cooperates with PTEN in tumor suppression. Science 321(5895): 1499-502. PubMed Citation: 18787170
Martin, D. E., Soulard, A. and Hall, M. N. (2004). TOR regulates ribosomal protein gene expression via PKA and the Forkhead transcription factor FHL1. Cell 119: 969-979. 15620355
Martin, D. N., Balgley, B., Dutta, S., Chen, J., Rudnick, P., Cranford, J., Kantartzis, S., DeVoe, D. L., Lee, C. and Baehrecke, E. H. (2007). Proteomic analysis of steroid-triggered autophagic programmed cell death during Drosophila development. Cell Death Differ. 14: 916-923. PubMed Citation: 17256009
Matsuo, T., Kubo, Y., Watanabe, Y. and Yamamoto, M. (2003). Schizosaccharomyces pombe AGC family kinase Gad8p forms a conserved signaling module with TOR and PDK1-like kinases. EMBO J. 22(12): 3073-83. 12805221
Mayer, C., Zhao, J., Yuan, X. and Grummt, I. (2004). mTOR-dependent activation of the transcription factor TIF-IA links rRNA synthesis to nutrient availability. Genes Dev. 18: 423-434. 15004009
McNeill, H., Craig, G. M. and Bateman, J. M. (2008). Regulation of neurogenesis and epidermal growth factor receptor signaling by the Insulin receptor/Target of rapamycin pathway in Drosophila. Genetics 179: 843-853. PubMed Citation: 18505882
Nicklin, P., et al. (2009). Bidirectional transport of amino acids regulates mTOR and autophagy. Cell 136(3): 521-34. PubMed Citation: 19203585
Migeon, J. C., Garfinkel, M. S. and Edgar, B. A. (1999). Cloning and characterization of peter pan, a novel Drosophila gene required for larval growth. Mol. Biol. Cell 10: 1733-1744. Medline abstract: 10359593
Mori, H., et al. (2000). 14-3-3tau associates with a translational control factor FKBP12-rapamycin-associated protein in T-cells after stimulation by pervanadate. FEBS Lett. 467: 61-64. PubMed Citation: 10664457
Nojima, H., et al. (2003). The mammalian target of rapamycin (mTOR) partner, raptor, binds the mTOR substrates p70 S6 kinase and 4E-BP1 through their TOR signaling (TOS) motif. J. Biol. Chem. 278(18): 15461-4. 12604610
Oldham, S., Boehni, R., Stocker, H., Brogiolo, W., and Hafen, E. (2000a). Genetic control of size in Drosophila. Phil. Trans. R. Soc. Lond. B 355: 945-952. 11128988
Oldham, S., et al. (2000b). Genetic and biochemical characterization of dTOR, the Drosophila homolog of the target of rapamycin. Genes Dev. 14: 2689-2694. 0523807
Parekh, D., et al. (1999). Mammalian TOR controls one of two kinase pathways acting upon nPKCdelta and nPKCepsilon. J. Biol. Chem. 274: 34758-34764. PubMed Citation: 10574945
Patel, P. H., et al. (2003). Drosophila Rheb GTPase is required for cell cycle progression and cell growth. J. Cell Sci. 116(Pt 17): 3601-10. 12893813
Pelkmans, L., Fava, E., Grabner, H., Hannus, M., Habermann, B., Krausz, E. and Zerial, M. (2005). Genome-wide analysis of human kinases in clathrin- and caveolae/raft-mediated endocytosis. Nature 436: 78-86. Medline abstract: 15889048
Peterson, T. R., et al. (2009). DEPTOR is an mTOR inhibitor frequently overexpressed in multiple myeloma cells and required for their survival. Cell 137(5): 873-86. PubMed Citation: 19446321
Polymenis, M. and Schmidt, E. V. (1997). Coupling of cell division to cell growth by translational control of the G1 cyclin CLN3 in yeast. Genes Dev. 11: 2522-2531. PubMed Citation: 9334317
Powers, T. and Walter, P. (1999). Regulation of ribosome biogenesis by the rapamycin-sensitive TOR-signaling pathway in Saccharomyces cerevisiae. Mol. Biol. Cell 10: 987-1000. PubMed Citation: 10198052
Powers, R. W., et al. (2005). Extension of chronological life span in yeast by decreased TOR pathway signaling. Genes Dev. 20(2): 174-84. Medline abstract: 16418483
Resnik-Docampo, M. and de Celis, J. F. (2011). MAP4K3 is a component of the TORC1 signalling complex that modulates cell growth and viability in Drosophila melanogaster. PLoS One. 6(1): e14528. PubMed Citation: 21267071
Sabatini, D.., et al. (1994). RAFT1: A mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs. Cell 78: 35-43. 7518356
Sarbassov, D. D., et al. (2004), Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr. Biol. 14: 1296-1302. 15268862
Sarbassov, D., Guertin, D. A., Ali, S. M. and Sabatini, D. M. (2005a). Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307: 1098-1101. Medline abstract: 15718470
Sarbassov, D. D. and Sabatini, D. M. (2005b). Redox regulation of the nutrient-sensitive raptor-mTOR pathway and complex. J. Biol. Chem. 280(47): 39505-9. Medline abstract: 16183647
Sarbassov, D. D., et al. (2006). Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol. Cell 22(2): 159-68. Medline abstract: 16603397
Saucedo, L. J., et al. (2003). Rheb promotes cell growth as a component of the insulin/TOR signalling network. Nat. Cell Biol. 5(6):566-71. 12766776
Schalm, S. S. and Blenis, J. (2002). Identification of a conserved motif required for mTOR signaling. Curr. Biol. 12(8): 632-9. 11967149
Schalm, S. S., Fingar, D. C., Sabatini, D. M. and Blenis, J. (2003). TOS motif-mediated Raptor Binding regulates 4E-BP1 multisite phosphorylation and function. Curr. Biol. 13(10): 797-806. 12747827
Scott, P. H., et al. (1999). Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway. Proc. Natl. Acad. Sci. 95: 7772-7777. 9636226
Scott, R. C., Schuldiner, O. and Neufeld, T. P. (2004). Role and regulation of starvation-induced autophagy in the Drosophila fat body. Dev Cell. 7: 167-178. 15296714
Scott, R. C., Juhász, G. and Neufeld, T. P. (2007). Direct induction of autophagy by Atg1 inhibits cell growth and induces apoptotic cell death. Curr. Biol. 17(1): 1-11. PubMed Citation: 17208179
Shah, O.J., Wang, Z. and Hunter. T. (2004). Inappropriate activation of the TSC/Rheb/mTOR/S6K cassette induces IRS1/2 depletion, insulin resistance, and cell survival deficiencies. Curr. Biol. 14: 1650-1656. Medline abstract: 15380067
Sheaffer, K. L., Updike, D. L. and Mango, S. E. (2008). The Target of Rapamycin pathway antagonizes pha-4/FoxA to control development and aging. Curr. Biol. 18(18): 1355-64. PubMed Citation: 18804378
Sofer, A., Lei, K., Johannessen, C. M. and Ellisen, L. W. (2005). Regulation of mTOR and cell growth in response to energy stress by REDD1. Mol. Cell. Biol. 25(14): 5834-45. 15988001
Soukas, A. A., Kane, E. A., Carr, C. E., Melo, J. A. and Ruvkun, G. (2009). Rictor/TORC2 regulates fat metabolism, feeding, growth, and life span in Caenorhabditis elegans. Genes Dev. 23(4): 496-511. PubMed Citation: 19240135
Sousa-Nunes, R., Yee, L. L. and Gould, A. P. (2011). Fat cells reactivate quiescent neuroblasts via TOR and glial insulin relays in Drosophila. Nature 471(7339): 508-12. PubMed Citation: 21346761
Stocker, H., et al. (2003). Rheb is an essential regulator of S6K in controlling cell growth in Drosophila. Nat. Cell Biol. 5(6):559-65. 12766775
Tang, S. J., et al. (2002). A rapamycin-sensitive signaling pathway contributes to long-term synaptic plasticity in the hippocampus. Proc. Natl. Acad. Sci. 99(1): 467-72. 11756682
Tee, A. R., et al. (2003). Tuberous sclerosis complex gene products, Tuberin and Hamartin, control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb. Curr. Biol. 13(15): 1259-68. 12906785
Teleman, A. A., Chen, Y. W. and Cohen, S. M. (2005). Drosophila Melted modulates FOXO and TOR activity. Dev Cell 9(2): 271-81. 16054033
Thomas, G. and Hall, M. N. (1997). TOR signaling and control of cell growth. Curr. Opin. Cell Biol. 9: 782-787. 9425342
Thomas, G. (2002). The S6 kinase signaling pathway in the control of development and growth. Biol. Res. 35: 305-313. 12415748
Topalidou, I., Papamichos-Chronakis, M. and Thireos, G. (2003). Post-TATA binding protein recruitment clearance of Gcn5-dependent histone acetylation within promoter nucleosomes. Mol. Cell. Biol. 23(21): 7809-17. 14560024
Tsang, C. K., Bertram, P. G., Ai, W., Drenan, R. and Zheng, X. F. (2003). Chromatin-mediated regulation of nucleolar structure and RNA Pol I localization by TOR. EMBO J. 22(22): 6045-56. 14609951
Wang, L, Fraley, C. D., Faridi, J., Kornberg, A. and Roth. R. A. (2003). Inorganic polyphosphate stimulates mammalian TOR, a kinase involved in the proliferation of mammary cancer cells. Proc. Natl. Acad. Sci. 100(20) :11249-54. 12970465
Wang, Y. H. and Huang, M. L. (2009). Reduction of Lobe leads to TORC1 hypoactivation that induces ectopic Jak/STAT signaling to impair Drosophila eye development. Mech. Dev. 126(10): 781-790. PubMed Citation: 19733656
Wittmann, C. W., et al. (2001). Tauopathy in Drosophila: neurodegeneration without neurofibrillary tangles. Science 293:. 711-714. 11408621
Yokogami, K., et al. (2000). Serine phosphorylation and maximal activation of STAT3 during CNTF signaling is mediated by the rapamycin target mTOR. Curr. Biol. 10: 47-50. PubMed Citation: 10660304
Zaffran, S., et al. (1998). A Drosophila RNA helicase gene, pitchoune, is required for cell growth and proliferation and is a potential target of d-Myc. Development 125: 3571-3584. Medline abstract: 9716523
Zaragoza, D., et al. (1998). Rapamycin induces the G0 program of transcriptional repression in yeast by interfering with the TOR signaling pathway. Mol. Cell. Biol. 18: 4463-4470. PubMed Citation: 9671456
Zeng, Z., et al. (2006). Rapamycin derivatives reduce mTORC2 signaling and inhibit AKT activation in AML. Blood 109(8): 3509-12. Medline abstract: 17179228
Zhang, H., et al. (2000). Regulation of cellular growth by the drosophila target of rapamycin dTOR. Genes Dev. 14(21): 2712-24. PubMed Citation: 11069888
Zhang, Y., et al. (2003). Rheb is a direct target of the tuberous sclerosis tumor suppressor proteins. Nat. Cell Biol. 5(6): 578-81. 12771962
Zinke, I., et al. (2002). Nutrient control of gene expression in Drosophila: microarray analysis of starvation and sugar-dependent response. EMBO J. 21: 6162-6173. Medline abstract: 12426388
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