InteractiveFly: GeneBrief

Autophagy-specific gene 1: Biological Overview | References

To survive starvation and other forms of stress, eukaryotic cells undergo a lysosomal process of cytoplasmic degradation known as autophagy. Autophagy has been implicated in a number of cellular and developmental processes, including cell growth control and programmed cell death. However, direct evidence of a causal role for autophagy in these processes is lacking, due in part to the pleiotropic effects of signaling molecules such as TOR that regulate autophagy. This study circumvents this difficulty by directly manipulating autophagy rates in Drosophila through the autophagy-specific protein kinase Atg1 (Atg signifies an autophagy-related gene). Overexpression of Atg1 is sufficient to induce high levels of autophagy, the first such demonstration among wild type Atg proteins. In contrast to findings in yeast, induction of autophagy by Atg1 is dependent on its kinase activity. Cells with high levels of Atg1-induced autophagy are rapidly eliminated, demonstrating that autophagy is capable of inducing cell death. However, this cell death is caspase dependent and displays DNA fragmentation, suggesting that autophagy represents an alternative induction of apoptosis, rather than a distinct form of cell death. In addition, this study demonstrates that Atg1-induced autophagy strongly inhibits cell growth, and that Atg1 mutant cells have a relative growth advantage under conditions of reduced TOR signaling. Finally, this study shows that Atg1 expression results in negative feedback on the activity of TOR itself. These results reveal a central role for Atg1 in mounting a coordinated autophagic response, and demonstrate that autophagy has the capacity to induce cell death. Furthermore, this work identifies autophagy as a critical mechanism by which inhibition of TOR signaling leads to reduced cell growth (Scott, 2007).

Under starvation conditions, eukaryotic cells recover nutrients via autophagy, a lysosome-mediated process of bulk cytoplasmic degradation. Through autophagy, long-lived proteins, organelles, and other components of the cytoplasm are non-selectively engulfed within specialized double-membraned vesicles known as autophagosomes. Subsequent fusion of the outer autophagosomal membrane with the lysosome results in a structure known as the autolysosome, in which the inner membrane and its cytoplasmic cargo are degraded. Breakdown products released from the autolysosome supply the cell with an internal source of nutrients that can support essential metabolic processes during starvation. Approximately 20 ATG (autophagy-related) genes specifically required for autophagy have been discovered in Saccharomyces cerevisiae (Klionsky, 2003), and many of these genes have functional homologs in metazoans (Levine, 2004; Scott, 2007 and references therein).

In addition to survival of starvation, autophagy has been implicated in many aspects of health and development, including aging, programmed cell death, pathogenic infection, stress responses, neurodegenerative and muscle disorders, cellular remodeling, cancer, and cell growth (Levine, 2004; Shintani, 2004). As in many of these processes, evidence for a role of autophagy in cell growth control is largely correlative. Autophagy is promoted by several tumor suppressor genes including PTEN, TSC1, TSC2, and beclin 1, and is inhibited by growth-promoting pathways such as type I PI3K and target of rapamycin (TOR) signaling. A variety of conditions that stimulate cell growth, such as growth factor addition, partial hepatectomy, and refeeding after starvation, also inhibit autophagy, while growth suppressive signals, such as contact inhibition and substrate detachment, induce autophagy (Newfeld, 2004). Together, these correlative findings are consistent with a model in which the catabolic effects of autophagy act as a brake on cell growth during development. However, this issue is complicated by the pleiotropic nature of the signaling pathways that regulate autophagy. For example, in addition to inhibiting autophagy, the TOR pathway controls cell metabolism and biosynthesis by promoting ribosome biogenesis, protein synthesis and nutrient uptake. The relevant contribution of each of these downstream effector pathways to net cell growth is poorly understood (Scott, 2007).

Autophagy is also known to be involved in programmed cell death, and distinguishes Type II (autophagic) from Type I (apoptotic) cell death. Autophagic cell death is characterized by an abundance of autophagosomes and autolysosomes in the dying cell and differs from apoptotic cell death in that dying cells are degraded by their own lysosomal enzymes, rather than by phagocytosis. It has been difficult, however, to establish whether autophagy plays a causal role in Type II cell death or represents a failed attempt at cell survival in cells undergoing programmed cell death. Death signals often induce features of both apoptosis and autophagy, and mutations that disrupt autophagy have been shown to suppress cell death in some cases (Shimizu, 2004; Veneault-Fourrey, 2006, Yu, 2006), and hasten it in others (see Lum, 2005). Thus, as in the case for cell growth, much of the support for a direct role for autophagy in cell death rests on correlative evidence (Scott, 2007).

One approach to addressing the potential role of autophagy in functions such as cell death and growth would be to induce autophagy directly, independent of the signaling pathways that normally control it. In yeast, multiple signaling pathways, including TOR, AMPK and Ras/PKA, converge on the Ser-Thr kinase Atg1 to regulate autophagy (Kamada, 2000; Wang, 2001; Budovskaya, 2005). In addition, Atg1 interacts with multiple components of the autophagic machinery, through direct association, phosphorylation, and/or through effects on intracellular localization (Kamada, 2000; Ptacek, 2005; Reggiori, 2004). Thus, Atg1 may represent a nodal point for controlling multiple steps in the autophagic process in response to various inductive cues (Scott, 2007).

Interestingly, the role of Atg1 kinase activity in yeast is the subject of some debate, and studies from different groups using ATP analog-sensitive and kinase-defective Atg1 mutants have reached conflicting conclusions. One study reported that Atg1 kinase activity is required for the autophagy-related biosynthetic cytoplasm to vacuole targeting (CVT) pathway but not for autophagy, and concluded that Atg1 plays a structural role in autophagy (Abeliovich, 2003). However, other studies using similar reagents found a requirement for Atg1 kinase activity in both CVT and autophagy (Kamada, 2000; Kabeya, 2005; Scott, 2007 and references therein).

It has been shown that the Drosophila homolog of Atg1 is required for autophagy in the larval fat body, an organ analogous to the vertebrate liver with roles in nutrient storage and mobilization. This study investigated the effects of Drosophila Atg1 loss of function and overexpression on autophagy induction, cell growth control, and cell death. The findings indicate that Atg1 expression is sufficient to effect a full autophagic response, resulting in a marked inhibition of cell growth and a rapid induction of apoptotic cell death (Scott, 2007).

Delivery of cytoplasmic components to the lysosome through autophagy involves multiple distinct steps including nucleation, expansion and closure of the autophagosome, and its subsequent fusion with the lysosome. These membrane trafficking events require the recruitment and subsequent retrieval of a large number of autophagy-specific Atg proteins, as well as general factors involved in vesicle trafficking. Given this complexity, the finding that overexpression of a single Atg protein is sufficient to accomplish all essential steps in this process is striking. As autophagy occurs constitutively at a basal rate in most eukaryotic cells, acceleration of a single rate-limiting step by overexpression of Atg1 may be sufficient to increase the overall rate of autophagy. Alternatively, the interaction of Atg1 with multiple Atg proteins suggests that Atg1 may function at multiple steps in the autophagic process. Identification of the relevant in vivo substrates of Atg1 will help to clarify this issue. A recent yeast proteomic microarray study (Ptacek, 2005) identified a number of in vitro substrates of Atg1, including Atg8 and Atg18, which function in autophagosomal expansion and retrieval of components of the autophagic machinery, respectively, as well as general factors involved in vesicle transport and vacuolar function (Scott, 2007).

The role of the kinase activity of yeast Atg1 in regulating autophagy is the matter of some debate, since different groups have drawn conflicting conclusions. A consensus view, however, may be that autophagy and the CVT pathway require different levels of Atg1 kinase activity, or that Atg1 has different substrates under differing nutrient conditions. In higher eukaryotes, the role of Atg1 kinase activity may differ from that in yeast, as the CVT pathway has not been observed in metazoan cells. Consistent with the findings of Ohsumi and colleagues (Kamada, 2000; Kabeya, 2005), however, the current results indicate that the kinase domain of Drosophila Atg1 is required for autophagy, since the kinase-inactive Atg1^K38Q mutant failed to restore starvation-induced autophagy to Atg1 mutants, and was unable to induce autophagy when overexpressed. However, the partial reduction in size and TOR activity observed upon expression of Atg1^K38Q, as well as its partial rescue of Atg1 mutants, indicate that Atg1 may have other, kinase-independent functions beyond regulating autophagy (Scott, 2007).

The mechanisms by which upstream signaling pathways regulate Atg1 are also likely to differ between yeast and multicellular organisms; essential components of the TOR-regulated Atg1 complex such as Atg13 and Atg17 are not readily identifiable in animal genomes. Nonetheless, the current results are consistent with a similar negative regulation of Atg1 by TOR, since activation of TOR signaling by Rheb overexpression or Tsc2 mutation suppresses the ability of Atg1 to induce autophagy. Increased Atg1 activity in response to loss of TOR signaling is likely to be critical for cell homeostasis and survival, since animals doubly mutant for Atg1 and Tor show a synthetic embryonic lethal phenotype (Scott, 2004). In addition, the results demonstrate an unexpected mode of signaling from Atg1 to TOR, in which increased Atg1 levels lead to downregulation of TOR kinase activity. It is unclear whether this reflects a direct effect of Atg1 on the activity of TOR or an upstream regulator, or an indirect consequence of the high level of autophagy in these cells, perhaps through increased turnover of TOR signaling components. The finding that a pool of TOR protein resides on intracellular vesicles and that TOR signaling is reduced in endocytic mutants suggests that TOR activity may respond to rates of vesicular trafficking, such as autophagy. Regardless of mechanism, these results suggest the existence of a self-reinforcing feedback loop, whereby increased Atg1 levels lead to downregulation of TOR activity, resulting in further activation of Atg1. In contrast, induction of autophagy in response to loss of TOR signaling is dampened by the resultant inactivation of S6K, which is required for normal autophagy (Scott, 2004). Thus TOR-mediated regulation of autophagy involves both positive and negative feedback (Scott, 2007).

Autophagy is a catabolic process that inversely correlates with cell growth, suggesting that increased levels of autophagy observed in growth-restricted cells may contribute to their reduced growth rate. The results presented here provide genetic support for this model. The data indicate that clones of autophagy-defective cells have a growth advantage over wild type cells under physiological conditions that normally induce autophagy, including starvation and rapamycin treatment. These results are confirmed by the marked size reduction of cells overexpressing Atg1, further supporting the role of autophagy as a negative effector of growth in TOR signaling. Thus, autophagy is partly responsible for the growth restriction resulting from physiological inhibition of TOR signaling, and is capable of inhibiting growth independent of TOR signaling (Scott, 2007).

In contrast to the growth advantage observed in autophagy defective cells, previous studies using yeast or cultured mammalian cells reported that inhibition of autophagy results in rapid cell death in response to starvation. This difference is likely due to the mosaic nature of the current experiments, in which clones of Atg1 mutant cells are surrounded by autophagy-competent wild type cells, and may in effect parasitize nutrients liberated through the autophagic activity of their neighbors. This is likely to be particularly evident in fat body cells, which are specialized for nutrient storage and mobilization. It is noted that this genetic mosaicism is similar to the situation facing tumor cells within wild type tissues. Thus, the increased growth capacity resulting from disruption of autophagy may contribute to the tumorigenicity of cells mutant for tumor suppressors such as PTEN, TSC1 & 2, beclin 1/Atg6 and possibly LC3/Atg8 and Atg7 (Scott, 2007 and references therein).

Whereas autophagy had an inhibitory effect on cell growth under physiological conditions, this was not the case in cells with severely disrupted TOR signaling, such as in cells with null mutations in Pdk1 Tor, despite the strong correlation of reduced cell growth and increased autophagy in these cells. Disruption of autophagy had no effect on the growth of Pdk1 null cells, and actually led to a further decrease in cell size of Tor mutants. In the complete absence of TOR signaling, nutrient uptake is severely curtailed, and under these extreme conditions the metabolic benefits of nutrients liberated by autophagy may outweigh its potential growth-inhibitory catabolic effect. It is concluded that autophagy has context-dependent effects on cell growth, providing for a rudimentary level of metabolism and growth under conditions of severe nutrient deprivation, acting as a net inhibitor of growth under conditions of reduced TOR signaling, and strongly inhibiting cell growth when induced to high levels. These findings add autophagy to the growing list of effector pathways and cellular processes through which TOR signaling controls cell growth, including translation, transcription, nutrient import and endocytosis (Scott, 2007).

Previous studies in a number of experimental systems indicate that the role of autophagy in cell death is also likely to be context-dependent. For example, autophagy has been found to protect against cell death in cases of growth factor withdrawal, starvation, and neurodegeneration, but to be required for some cases of autophagic cell death. Thus, observations of autophagic structures in dying cells are equally consistent with a causal, neutral or even inhibitory role of autophagy in cell death. The ability to induce autophagy through Atg1 overexpression has enabled a direct test of the potential role of autophagy in promoting cell death. The results indicate that induction of autophagy is sufficient to induce cell death. It was found that death resulting from Atg1-induced autophagy is suppressed by caspase inhibition and is associated with caspase activation, DNA fragmentation, and cytoskeletal disruption, suggesting that high levels of autophagy result in apoptotic cell death. The connection between apoptosis and autophagy is further supported by the recent demonstration that overexpression of the anti-apoptotic protein Bcl-2 can inhibit autophagy by interacting with the Atg6 homolog Beclin 1 (Pattingre, 2005). Mutant versions of Beclin 1 that are unable to bind Bcl-2 stimulate autophagy and promote cell death, similar to the effects of Atg1 (Scott, 2007).

By what mechanisms might autophagy lead to cell death? The observation that starvation-induced autophagy is reversible and does not normally result in cell elimination suggests that starvation conditions may be protective against autophagy-induced death. Induction of autophagy is tolerated or even beneficial in cells with reduced biosynthetic activity, but may be detrimental in cells whose resources are devoted to continued growth. Self-limiting mechanisms also serve to prevent starvation-induced autophagy from proceeding at continuously high levels. During autophagic cell death, alternate activation of the autophagic machinery may circumvent these feedback mechanisms, resulting in high levels of sustained autophagy that are destructive to the cell. In addition, the level of Atg1 gene expression may be critical, since it is not upregulated under starvation conditions (Kirisako, 1999), but its levels peak at the beginning of the Drosophila pupal stage, when autophagic cell death is induced. Autophagic cell death may also result from the selective degradation of specific survival-promoting or death-inhibiting factors. In this regard, it was recently shown that the caspase-inhibitor zVAD results in targeted autophagic degradation of catalase, leading to accumulation of radical oxygen species and death (Scott, 2007).

In summary, these results demonstrate that constitutive induction of autophagy inhibits cell growth and leads to cell death, highlighting the importance of physiological mechanisms that restrain this process. In addition, the finding that Atg1 expression is sufficient to induce autophagy provides a new tool for experimental or therapeutic manipulation of autophagy. Compounds that target the kinase activity of Atg1 may lead to novel therapies for the wide range of diseases linked to autophagy (Scott, 2007).

An Atg1/Atg13 complex with multiple roles in Tor-mediated autophagy regulation

The TOR kinases are conserved negative regulators of autophagy in response to nutrient conditions, but the signaling mechanisms are poorly understood. This study describes a complex containing the protein kinase Atg1 and the phosphoprotein Atg13 that functions as a critical component of this regulation in Drosophila. Knockout of Atg1 or Atg13 results in a similar, selective defect in autophagy in response to TOR inactivation. Atg1 physically interacts with TOR and Atg13 in vivo, and both Atg1 and Atg13 are phosphorylated in a nutrient-, TOR- and Atg1 kinase-dependent manner. In contrast to yeast, phosphorylation of Atg13 is greatest under autophagic conditions and does not preclude Atg1-Atg13 association. Atg13 stimulates both the autophagic activity of Atg1 and its inhibition of cell growth and TOR signaling, in part by disrupting the normal trafficking of TOR. In contrast to the effects of normal Atg13 levels, increased expression of Atg13 inhibits autophagosome expansion and recruitment of Atg8/LC3, potentially by decreasing the stability of Atg1 and facilitating its inhibitory phosphorylation by TOR. Atg1/Atg13 complexes thus function at multiple levels to mediate and adjust nutrient-dependent autophagic signaling (Chang, 2009).

The remarkable conservation of autophagy-related proteins and processes between yeast and higher eukaryotes has contributed to a rapid advance in understanding of this process in multicellular animals. The data presented in this study extend this conservation to the mechanisms of autophagy regulation by nutrient-dependent TOR signaling. As in yeast, Drosophila Atg13 forms a complex with Atg1, stimulates its activity and is highly phosphorylated in a nutrient and TOR-dependent manner. Although the relevant substrates of Atg1 remain to be identified, these results suggest that this fundamental pathway from nutrient signal to autophagic induction has been largely retained between yeast and metazoans. This regulatory link between TOR and Atg1/Atg13 thus represents one of the most highly conserved TOR outputs described to date. Despite this conservation, critical differences were identified in the behaviors of these proteins, which likely stem in part from differences in the physiology of lower vs. higher eukaryotes. Chan (2009) reported an essential role in autophagy for human Atg13 using RNAi-mediated knockdown in cultured cell lines, and described interactions between Atg13 and Ulk1 and Ulk2 similar to those reported in this study. Hosokawa (2009) and Jung (2009) also describe related interactions between mammalian Atg13 and Ulk kinases. Together, these studies point to a model whereby Atg13 is phosphorylated and interacts with Atg1 under both fed and starved conditions in metazoans, in contrast to the growth-dependent phosphorylation and dissociation of Atg13/Atg1 observed in yeast. Although the greater nutrient buffering capacity of multi- vs. unicellular animals probably contributes to these differences, the requirement for significant basal rates of autophagy in the maintenance of large, long-lived metazoan cells may dictate that the Atg1-Atg13 complex remain partially active even under fed conditions. Furthermore, although Atg1 can clearly associate with phosphorylated Atg13, dephosphorylation of TOR-dependent sites on Atg13 may be masked by increased phosphorylation by Atg1, but may nonetheless contribute to regulation of Atg1-Atg13 interaction or activity. The observation that Atg1 kinase activity plays a role under both fed and starved conditions in supporting hyperphosphorylation of Atg13 suggests that rather than switching Atg1 kinase between off and on states, nutrient signals may instead affect its substrate specificity or accessibility (Chang, 2009).

Metazoan Atg1/Atg13 complexes have also accrued additional regulatory mechanisms that have not been described in yeast, including negative feedback from Atg1 to TOR, phosphorylation of Atg13 by Atg1, and nutrient-dependent effects on the stability of these proteins. These additional layers of regulation are in keeping with the elaboration of intracellular signaling pathways in metazoans. The observation that Atg13 phosphorylation is both TOR- and Atg1-dependent under fed conditions suggests a model whereby phosphorylation by one of these kinases may serve as a priming event for the other. In addition, the role of Atg1-dependent phosphorylation of Atg13 under starvation conditions remains an important question. Whereas the Atg1-independent localization of Atg13 to autophagosomes and its activation of Atg1 indicate that Atg13 functions upstream of Atg1, the finding that Atg13 acts as a substrate for Atg1-dependent phosphorylation raises the possibility that Atg13 may also act to transduce signals downstream of Atg1. Identification of additional Atg1 substrates and Atg13 interacting proteins may help clarify this issue (Chang, 2009).

Given its positive role in autophagy induction, the ability of Atg13 to inhibit autophagy when overexpressed was unexpected. This may in part reflect a dominant negative effect of Atg13 overexpression causing titration of Atg1 complexes or competition with other Atg1 substrates. However, the observation that Atg13 stimulates TOR-dependent phosphorylation of Atg1 and that Atg1 levels increase in Atg13 mutant cells suggests that Atg13 has both positive and negative roles in autophagy induction. These opposing activities of Atg13 are reminiscent of the mTOR complex I component Raptor, which plays an essential role in TOR signaling yet also inhibits TOR activity under starvation conditions. The results suggest that Atg13 may play an analogous role, switching between states of promoting or inhibiting autophagy, thereby sharpening the response to changes in nutrient conditions. These findings suggest that the relative ratio of Atg13 to Atg1 or to other components of the complex may play an important role in dictating its activity. In this regard, the differential regulation of Atg1 and Atg13 levels by TOR could provide an additional mechanism whereby TOR signaling can influence autophagic activity. Whereas the autophagy-defective phenotypes of Atg1 and Atg13 mutants reveals the dominant positive roles of these genes, disruption of other putative components of this complex leads to autophagy induction, suggesting some components may play primarily negative roles (Chang, 2009).

In conclusion, the results demonstrate that Atg1/Atg13 complexes play an essential, conserved role in promoting autophagy in response to TOR inactivation, with additional regulatory functions unique to metazoans. Further insight into the regulation of this complex and identification of its targets may lead to additional means of manipulating autophagy rates for therapeutic purposes (Chang, 2009).

Cell death during Drosophila melanogaster early oogenesis is mediated through autophagy

Autophagy is a physiological and evolutionarily conserved process maintaining homeostatic functions, such as protein degradation and organelle turnover. Accumulating data provide evidence that autophagy also contributes to cell death under certain circumstances, but how this is achieved is not well known. This study reports that autophagy occurs during developmentally-induced cell death in the female germline, observed in the germarium and during middle developmental stages of oogenesis in Drosophila. Degenerating germline cells exhibit caspase activation, chromatin condensation, DNA fragmentation and punctate staining of mCherry-DrAtg8a, a novel marker for monitoring autophagy in Drosophila. Genetic inhibition of autophagy, by removing atg1 or atg7 function, results in significant reduction of DNA fragmentation, suggesting that autophagy acts genetically upstream of DNA fragmentation in this tissue. This study provides new insights into the mechanisms that regulate cell death in vivo during development (Nezis, 2009).

The TUNEK assay was used ti measure cell death during Drosophila oogenesis. Such analysis revealed that cell death in the germarium occurred in 26% of the ovarioles, in young, well-fed flies. In contrast, the percentage of cell death during middle stages of oogenesis was 9.7% under the same conditions. In the germarium the degenerated germline cells are usually located in region 2 and, during middle stages of oogenesis, in egg chambers of developmental stages 7, 8 and 9. In order to detect caspase activity, immunolabeling was performed with the anti-active caspase-3 (cleaved caspase-3) antibody. The degenerated germaria revealed an intense staining in region 2, indicating the presence of activated caspase-3 proteases and therefore caspase-dependent cell death. A similar staining pattern is observed in the degenerating mid-stage egg chambers. Next whether autophagy occurs during cell death in the germarium and mid-stage egg chambers of Drosophila was investigated. GFP-tagged Atg8a or its vertebrate homologue LC3, has been extensively used to monitor autophagy as it labels the autophagic membranes. However, the pH sensitivity of GFP makes it impossible to follow GFP-Atg8a after the short-lived autophagosomes fuse with lysosomes. Fusion with late endosomes to create amphisomes may also lead to an environment where the fluorescence from GFP is quenched due to low pH. Therefore a transgenic fly was made in which DrAtg8a is fused to the acid-insensitive fluorescent protein mCherry that can be easily monitored into amphisomes and autolysosomes. Upon expression of mCherry-DrAtg8a in the germline using the germline-specific driver, nanos-VP16-Gal4 several mCherry-DrAtg8a puncta were detected in region 2 of the germarium and in the degenerating mid-stage egg chambers, indicating autophagic activity. When mCherry-DrAtg8a was coexpressed with GFP-LC3, it was observed that the number of mCherry-positive structures in degenerating mid-stage egg chambers was significantly higher compared to the number of GFP-positive structures. This observation reveals that mCherry-DrAtg8a is a very useful marker for monitoring autophagy in Drosophila, since it can be used for detecting both autophagosomes and autolysosomes (Nezis, 2009).

Examination of the ultrastructural morphology of degenerated germaria revealed that the cytoplasm of the germline cells indeed contained a variety of autophagosomes and autolysosomes. Autophagic compartments were filled with cytoplasm, numerous vesicles, dense masses and multi-lamellar membranes of various sizes. Additionally, while the nuclei in the normal non-degenerating germ line cysts were very irregular in shape with many invaginations and protrusions, the nuclei in the degenerated cysts had a round shape and condensed chromatin. Surprisingly, all these nuclei had irregular and extensive nuclear membrane dilation. Together, the above data demonstrate that autophagy occurs in caspase-dependent cell death during early oogenesis in Drosophila (Nezis, 2009).

To explore the potential role of autophagy in the cell death process of the germline cells in the germarium and in mid-stage egg chambers, germline mutant cells were generated for the core Drosophila autophagy genes atg1 and atg7 and scored for apoptotic cell death using the TUNEL assay in order to detect DNA fragmentation. Interestingly, cell death in the germarium of atg1 germline mutants was reduced to 8.4% (compared to 26% of the control) and to 3.5% for mid-stage egg chambers. atg7 germline mutants exhibited a similar reduction in apoptotic cell death. The percentage of germaria being positive for TUNEL was 13.8% and the percentage of midstage egg chambers being positive for TUNEL was 4.8%. The above data indicate that inhibition of autophagy reduces DNA fragmentation, suggesting that during Drosophila early oogenesis autophagy acts upstream of caspase activation and DNA fragmentation (Nezis, 2009).

How can autophagy promote cell death by acting upstream of caspase activation? One possible explanation is that proteins crucial for cell survival and organelles are degraded by autophagy, thus promoting cell death. Moreover, autophagy could also act in parallel and cooperatively with caspases for the most efficient degradation of the tissue. In future studies, molecular mechanisms that illuminate the contribution of autophagy in the cell death process will be identified (Nezis, 2009).

Genetic interactions between Drosophila melanogaster Atg1 and paxillin reveal a role for paxillin in autophagosome formation

Autophagy is a conserved cellular process of macromolecule recycling that involves vesicle-mediated degradation of cytoplasmic components. Autophagy plays essential roles in normal cell homeostasis and development, the response to stresses such as nutrient starvation, and contributes to disease processes including cancer and neurodegeneration. Although many of the autophagy components identified from genetic screens in yeast are well conserved in higher organisms, the mechanisms by which this process is regulated in any species are just beginning to be elucidated. In a genetic screen in Drosophila melanogaster, a link was identified between the focal adhesion protein paxillin and the Atg1 kinase, which has been previously implicated in autophagy. In mammalian cells, paxillin was found to redistributed from focal adhesions during nutrient deprivation, and paxillin-deficient cells exhibit defects in autophagosome formation. Together, these findings reveal a novel evolutionarily conserved role for paxillin in autophagy (Chen, 2008).

This paper reports genetic interactions between paxillin and components implicated in developmental autophagy, including Atg1 and the ecdysone receptor, during wing maturation. Intriguingly, in te newly emerged adults, autophagy is also observed at the time of wing spreading. It was also found that over-expression of DSRF, which suppresses Pax-induced wing blisters, rescues Atg1-induced wing defects, thus further supporting a role of Atg1 in wing morphogenesis. EP3348, which strongly suppresses Pax-induced blistered wings, harbors a single transposable element inserted in the 5' untranslated region of the Drosophila Atg1 gene. It is notable that EP3348 suppresses Pax-induced wing defects more effectively than does Atg1-RNAi. Since Atg1 mutants exhibit a pupal lethal phenotype, but neither EP3348 nor expression of Atg1-RNAi result in lethality, it is likely that neither of them cause complete knockdown of Atg1 expression. The reason that EP3348 suppresses dPax-induced wing defects better than Atg1-RNAi is most likely due to differences in their effects on Atg1 expression. However, such differences may be subtle, and spatially restricted, as it was not possible to distinguish their effects on Atg1 expression by RTPCR of whole tissues (Chen, 2008).

It was also found that overexpression of Atg1 induces cell death and an aberrant actin cytoskeleton. When paxillin is coexpressed with Atg1, these phenotypes were enhanced, leading to increased lethality and defects in arista patterning. A deletion mutation, Df(2L)Pr.A16, which uncovers the Pax gene suppressed both Atg1- and dPax-induced defects. However, since Df(2L)Pr.A16 contains a large deletion, the possibility that the suppression effect is due to disruption of a gene that is independent of dPax cannot be ruled out. Most significantly, a marked decrease was found in the number of autophagosomes in both Pax mutant flies and paxillin-deficient mouse fibroblasts upon nutrient deprivation, thus revealing a novel requirement for paxillin in autophagy. It was also demonstrated that Atg1 can directly phosphorylate paxillin in vitro, suggesting that Atg1 may regulate the activity of paxillin. Thus far, however, it has not been possible to demonstrate that a kinase-deficient form of Atg1 detectably affects paxillin localization in mammalian cells, suggesting that the interaction of these two proteins in vivo may be more complex. Moreover, the fact that no genetic interactions were observed between other autophagy mutants and paxillin in vivo leaves open the formal possibility that the interaction between Atg1 and paxillin in Drosophila may not reflect roles for these proteins in autophagy (Chen, 2008).

Despite the identification of many autophagy-associated genes, the mechanism of autophagosome biogenesis, including the origin of membranes and how they are transported for vesicle formation, remains unclear. It has been proposed that autophagosomes form by the addition of membranes derived from ER or Golgi complex in the form of vesicle transport. Alternatively, autophagosomes could form by de novo synthesized membrane (Chen, 2008).

The precise function of Atg1 in this process is still unclear. In yeast two=hybrid screens, several proteins that directly interact with Atg1 have been identified, including Syntenin, a Rab5 GTPase.interacting protein, VAB-8, a kinesin subfamily like molecule, GABA receptor associated protein (GABARAP), and the Golgi-associated ATPase enhancer of 16kDa (GATE16). Significantly, several of these Atg1-interacting proteins are involved in membrane dynamics and vesicle trafficking, suggesting a conserved role for Atg1 in the regulation of membrane trafficking. Indeed, recent findings have implicated the mouse Atg1 ortholog, Ulk1/2, in endocytotic processes during neurogenesis (Chen, 2008).

Paxillin is a multidomain protein and functions as an essential regulatory molecule that couples integrins to the actin cytoskeleton in focal adhesions. Several paxillin-associated proteins are involved in regulation of integrin-mediated signaling associated with cell adhesion to extracellular matrix, motility and growth factor responses. The association of paxillin with Arf GAPs regulates paxillin localization, and the Arf small GTPases play a central role in membrane trafficking and cytoskeletal dynamics. While a role was found for paxillin in the regulation of membrane trafficking, whether this is related to its role in autophagy is not clear. Interestingly, the results indicate that integrin-mediated signaling is not required for paxillin's role in autophagy, suggesting that paxillin's role in the formation of autophagosomes is independent of signals from extracellular matrix. However, this aspect of the paxillin.autophagy relationship has only been examined thus far in Drosophila melanogaster, and so it remains to be determined whether the role of paxillin in autophagasome formation in mammalian cells is similarly integrin-independent (Chen, 2008).

Growth arrest and autophagy are required for salivary gland cell degradation in Drosophila

Autophagy is a catabolic process that is negatively regulated by growth and has been implicated in cell death. This study finds that autophagy is induced following growth arrest, and precedes developmental autophagic cell death of Drosophila salivary glands. Maintaining growth by expression of either activated Ras or positive regulators of the class I phosphoinositide 3-kinase (PI3K) pathway inhibits autophagy and blocks salivary gland cell degradation. Developmental degradation of salivary glands is also inhibited in autophagy gene (atg) mutants. Caspases are active in PI3K-expressing and atg mutant salivary glands, and combined inhibition of both autophagy and caspases increases suppression of gland degradation. Further, induction of autophagy is sufficient to induce premature cell death in a caspase-independent manner. These results provide in vivo evidence that growth arrest, autophagy, and atg genes are required for physiological autophagic cell death, and that multiple degradation pathways cooperate in the efficient clearance of cells during development (Berry, 2007).

These studies indicate that arrest of PI3K-dependent growth is an important determinant of autophagic cell death of salivary glands during Drosophila development. Maintenance of growth by expression of either activated Ras, Dp110, or Akt in salivary glands is sufficient to inhibit salivary gland degradation. It is possible that the larger Dp110-, Akt- and Ras^V12-expressing salivary glands simply have more material to degrade, and this is why they persist. It is suspected that this is not the case, however, since Dp110-expressing glands are larger than Ras^V12-expressing glands, yet Ras^V12-expressing glands are less degraded. Although PI3K-dependent growth inhibits autophagy, growth could influence other downstream targets. However, the Atg1-induced suppression of the Dp110 persistent salivary gland phenotype and the persistence of vacuolated salivary gland cell fragments in atg loss-of-function mutants support the conclusion that growth arrest and autophagy are required for proper salivary gland degradation (Berry, 2007).

Ras and class I PI3K signaling are complex, and cross-talk occurs between these pathways. Although the data indicate that both activated Ras and PI3K have similar effects on salivary gland cell growth and inhibition of autophagy, it is observed that Ras-expressing cells were more intact 24 hours apf. Caspase activity is detected in Dp110-and Akt-expressing glands, and it is speculated that part of the degradation observed in Dp110 and Akt glands was due to caspases. Indeed, combining Dp110 expression with caspase inhibition resulted in intact salivary glands. This additive phenotype indicates that multiple degradation pathways are involved in autophagic cell death in vivo. Caspases were also active in Ras-expressing glands that were predominantly intact; thus activated Ras likely influences factors separate from caspases and the PI3K pathway. Ras regulates PI3K-independent pathways including MAPK and the cell cycle. Proliferating cells usually double in size prior to division, and because of this, cell growth and division are often considered synonymous. These studies demonstrate that although expression of either Myc or CyclinD with Cdk4 is sufficient to induce nuclear size, they do not inhibit salivary gland degradation. These data support the conclusion that growth arrest, but not cell cycle arrest, is an important determinant of salivary gland autophagic cell death. While many studies have defined relationships between cell cycle arrest and cell death, this study defines a unique relationship between cell growth arrest and cell death (Berry, 2007).

Given autophagy’s well established function in cell survival, a role for autophagy in cell death seems paradoxical. The discovery that caspases function in cells dying with a Type II autophagic morphology led to speculation that all programmed cell death is regulated by apoptosis factors. Further, the preponderance of in vitro evidence shows a role for autophagy in cell death when caspases or apoptosis factors are inhibited. This study found that reduced function of any one of seven atg genes inhibits salivary gland degradation. The incomplete degradation of salivary glands in multiple atg loss-of-function mutants provides the first in vivo evidence that autophagy and atg genes are required for proper degradation of cells during developmental cell death. Caspase activity and caspase-dependent DNA fragmentation occurs in these atg mutants, indicating that autophagy is a caspase-independent degradation pathway required for complete cell degradation in autophagic cell death during development. Further, induction of autophagy by Atg1 expression leads to premature caspase-independent salivary gland degradation. The data do not exclude a role for caspases in autophagic cell death. Either inhibition of caspases by p35 or reduced atg gene function result in delayed and incomplete degradation of salivary glands, and the combined inhibition of caspases with reduced atg function results in increased persistence of this tissue. These data suggest that autophagy and caspases function in parallel pathways during salivary gland cell death, and that both independently contribute to cell destruction. Further, the presence of both autophagy and caspases appears to be more typical of autophagic cell death that occurs under physiological conditions. Autophagic cell death models of mammary lumen formation and embryonic cavitation, as well as amphibian developmental cell death, all involve both processed caspase-3 and autophagy (Berry, 2007).

The designations of type I apoptotic death and type II autophagic death are based on morphological criteria. The current studies indicate that cell morphology likely reflects difference in the factors that are used to activate cell death and degrade the dying cell. The degradation of salivary glands in caspase mutants indicates that caspase-independent factors are involved in autophagic cell death. The presence of autophagosomes in dying salivary glands led to an investigation of cell death in atg gene mutants; stronger defects were observed in salivary gland degradation with perturbed atg gene function than with drice, ark, and dronc mutants. These data indicate that cell morphology is informative, given that it suggested autophagy is involved in the death of salivary glands. However, it is important to note that cell death classification that is based on morphology can be misleading, since salivary glands clearly use both caspases and autophagy, degradation mechanisms that had been speculated to be strictly associated with a single morphological form of cell death (Berry, 2007).

Now that it is clear that autophagy participates in cell death under some circumstances, it will be critical to determine how autophagy participates in cell killing and removal. A recent study showed that autophagy is required to generate the energy needed to promote phagocytosis signaling in an in vitro model of embryonic cavitation (Qu, 2007). This is not believed to be the same as the role of autophagy in salivary glands, since no phagocytosis is observed during salivary gland death. Alternatively, autophagy may be used to recruit and degrade factors that promote cell survival, such as the degradation of cytoplasmic catalase in mouse L929 cells. Finally, extreme levels of autophagy may be sufficient to cause a metabolic catastrophe by degrading substrates and mitochondria that are needed for energy. The latter possibility does exist in salivary glands, as expression of the Atg1 kinase is sufficient to induce the death of fat (Scott, 2007) and salivary gland cells. Unlike fat cells, elevated autophagy does not induce caspase-dependent DNA fragmentation in salivary gland cells, and expression of p35 does not inhibit Atg1-induced death (Berry, 2007).

The prevalence of apoptosis and the potent killing potential of caspases raise the question of why autophagy participates in developmental cell death. In the context of Drosophila and other insects, larval cells have a modified endoreplication cell cycle that results in the production of gigantic cells. The number and size of cells may prohibit engulfment and digestion by phagocytes, and autophagy may be necessary for self-degradation. Further, the life history of the organism may informative as to why autophagy participates in the destruction of tissues. Drosophila do not feed during the 3 day period of metamorphosis. Thus, the differentiation and morphogenesis of the entire adult occurs in the absence of food, and the resources to build the adult fly must come from reserves that are set aside during larval development. One important source of these resources is the fat that exhibits elevated levels of autophagy at the onset of metamorphosis. Several other large larval tissues are destroyed by autophagic cell death during metamorphosis including the midgut and salivary glands. It is speculated that like fat, catabolism of these tissues by autophagy provides resources that are needed to construct the adult. Similarly, it is speculated that the large number of autophagosomes observed in dying amphibian cells may serve to recycle nutrients during metamorphosis when these animals do not feed (Berry, 2007).

These studies have indicated that it is necessary to be cautious when considering autophagy to be either a cell survival or cell death process. Perhaps it is useful to consider autophagy for what it is; a catabolic process that contributes to many cellular and biological processes. This is not that different from the caspase proteases that are widely considered to be apoptosis proteases, as it is now clear that caspases also function in cell differentiation. Future studies are likely to show that autophagy functions in many cell types, and that its contribution to cell survival and cell death are dependent on the type and physiological context of the cell (Berry, 2007).

ATG1, an autophagy regulator, inhibits cell growth by negatively regulating S6 kinase

It has been proposed that cell growth and autophagy are coordinated in response to cellular nutrient status, but the relationship between them is not fully understood. This study characterized the fly mutants of Autophagy-specific gene 1 (ATG1), an autophagy-regulating kinase, and it was found that ATG1 is a negative regulator of the target of rapamycin (TOR)/S6 kinase (S6K) pathway. The Drosophila studies have shown that ATG1 inhibits TOR/S6K-dependent cell growth and development by interfering with S6K activation. Consistently, overexpression of ATG1 in mammalian cells also markedly inhibits S6K in a kinase activity-dependent manner, and short interfering RNA-mediated knockdown of ATG1 induces ectopic activation of S6K and S6 phosphorylation. Moreover, ATG1 specifically inhibits S6K activity by blocking phosphorylation of S6K at Thr 389. Taken together, these genetic and biochemical results strongly indicate crosstalk between autophagy and cell growth regulation (Lee, 2007).

Previous studies have demonstrated that homozygous EP3348 flies, in which a single P element is inserted into the 5' untranslated region of DmATG1, show larval/pupal lethality. However, after the genomic background of the EP3348 line was cleared by four backcrosses with w1118 flies, about 30% of homozygous mutants were found to develop to adults. To confirm that the P-element insertion in EP3348 hampers transcription of DmATG1 (Scott, 2004), quantitative real-time reverse transcriptase-PCR (qRT-PCR), was performed. This showed highly reduced DmATG1 expression in the mutant. Therefore, this cleared EP3348 allele was named DmATG1¹ (Lee, 2007).

The mitotic phenotypes in the larval brain and imaginal discs of DmATG1 mutants, DmATG1¹ and DmATG1^Δ3d (a null allele of DmATG1 were examined. However, no notable mitotic defects could be found in the mutants compared with the wild-type control (w1118. Consistently, DmATG1 mutants had no gross chromosomal abnormalities in the neuroblast cells of third instar larval brains (Lee, 2007).

Next, defects in autophagy in DmATG1¹ flies were examined using toluidine blue-azure II staining and transmission electron microscopy (TEM) analyses. As expected, DmATG1¹ flies showed marked defects in the induction of autophagy under conditions of starvation. This is highly consistent with the previous study using DmATG1^Δ3d (Scott, 2004). These results strongly supported that DmATG1¹ is a new hypomorphic mutant allele of DmATG1 (Lee, 2007).

To understand further the in vivo roles of ATG1 in Drosophila, double-mutant lines were generated for dTOR^P1 (a loss-of-function mutant allele of dTOR and DmATG1 mutants (DmATG1¹, DmATG1^Δ3d and EP3348). Homozygous dTOR^P1 mutants showed growth arrest in the second/early third instar larval stage and markedly delayed growth. Surprisingly, homozygous dTOR^P1 larvae with a heterozygous genetic background of DmATG1¹ or DmATG1^Δ3d not only grew faster than homozygous dTOR^P1, but also extended their developmental stage to the mid-late third instar larval stage. Furthermore, the double-homozygous mutants between dTOR^P1 and various DmATG1 mutants survived up to the mid-late third instar larval stage, which is inconsistent with the previous results (Scott, 2004; Lee, 2007).

In addition, it was found that another phenotype of dTOR^P1 mutants, lipid vesicle aggregation in the fat body, was also suppressed by a reduction of the gene dosage of DmATG1. These results implicated that ATG1 negatively mediates the developmental and physiological roles of TOR in Drosophila (Lee, 2007).

Since dTOR regulates cell growth in a cell-autonomous manner, whether DmATG1 is also involved in this role of dTOR was examined. The cell and nuclear sizes of the salivary gland cells were markedly reduced in dTOR^P1 larvae. Intriguingly, the heterozygous genetic background of DmATG1¹ or DmATG1^Δ3d partly rescued the reduced cell and nuclear size phenotype of dTOR^P1, strongly implicating that DmATG1 mediates the crucial function of dTOR in cell growth regulation. This is further supported by recent results, that DmATG1-null cells have a relative growth advantage over wild-type cells when treated with rapamycin, a specific inhibitor of dTOR (Lee, 2007).

To examine the possibility that the suppression of dTOR^P1 phenotypes by DmATG1 mutation resulted from altered auto-phagic activities, the genetic interactions were investigated between dTOR and other autophagy-related genes, such as ATG6 (Beclin1) and UVRAG (UV radiation resistance associated gene), which are known to form a complex regulating autophagosome formation (Liang, 2006). As a result, ATG6 and UVRAG mutations did not suppress the developmental delay and cell growth defects of dTOR mutants, showing that the interaction between dTOR and DmATG1 is not caused indirectly by uncontrolled regulation of autophagy (Lee, 2007).

To determine the functional interaction between ATG1 and S6K, a downstream effector of TOR, double-mutant lines were generated between DmATG1¹ and dS6K mutants -- a hypomorphic allele, dS6K⁰⁷⁰⁸⁴, and a null allele, dS6K^l−1. Using TEM analyses, it was observed that a reduction of dS6K gene dosage did not rescue the defects in autophagosome formation in starved DmATG1¹ homozygous larvae. However, surprisingly, the reduced gene dosage of dS6K increased the eclosion rate of homozygous DmATG1¹ in a dS6K gene dosage-dependent manner. Although the possibility that S6K promotes autophagy as reported previously (Scott, 2004) cannot be excluded, these data indicate that dS6K has an important role in DmATG1-dependent developmental processes (Lee, 2007).

Next, a biochemical analysis was conducted to examine the effect of loss of DmATG1 on dS6K activation. As a result, it was found that dS6K was markedly activated (~threefold increase) in homozygous DmATG1¹ larvae and pupae, measured by the phosphorylation of dS6K at the Thr 398 site, compared with the wild-type controls. Notably, the increased level of dS6K phosphorylation in DmATG1¹ mutants was about one-third of that in flies overexpressing Rheb. Consistent with this, DmATG1 overexpression almost completely inhibited dS6K Thr 398 phosphorylation in Drosophila (Scott, 2007). These results strongly suggest an important role of ATG1 in the regulation of S6K (Lee, 2007).

To extend these findings to the mammalian system and also to investigate further the molecular mechanism of the interaction between ATG1 and TOR/S6K, the effect of ATG1 on the activity of S6K was examined in mammalian cells. There are two isoforms of ATG1 in mammals, which are named UNC-51-like kinase (ULK) 1 and 2 (Zhou, 2007). However, according to the agreement on gene nomenclature made by researchers in the field of autophagy, they were renamed ATG1α and ATG1β, respectively. Nutrient deprivation of HEK293T cells abolished the phosphorylation of S6K at both Thr 229 and Thr 389 sites, which represents the activation status of S6K . When nutrients including amino acids and glucose (DMEM) were added back to the cells, the phosphorylation of both sites in S6K was strongly induced. However, co-expression of wild-type mouse ATG1α (ATG1α WT) strongly inhibited S6K activity induced by DMEM. On the contrary, a kinase-dead form of ATG1α (ATG1α KI) was not able to block the nutrient-induced activation of S6K, showing that ATG1α inhibits S6K in a kinase activity-dependent manner. Consistently, epidermal growth factor (EGF)-stimulated S6K activation was also inhibited by ATG1α. Furthermore, ATG1β, another isoform of ATG1, has the same inhibitory effect on S6K phosphorylation as ATG1α. These data strongly suggest that ATG1 regulates the activities of upstream kinases or phosphatases of S6K, which affect both Thr 229 and Thr 389 phosphorylation (Lee, 2007).

As ATG1 is a crucial regulator of autophagy in yeast and Drosophila, whether overexpression of ATG1 can induce autophagy in mammalian cells was tested. Nutrient deprivation was able to induce autophagy in MCF-7 cells, whereas overexpression of ATG1 did not induce autophagy in MCF-7 and HEK 293T cells, indicating that the inhibition of TOR/S6K by ATG1 is not an indirect consequence of an ectopic induction of autophagy. This was further supported by the observation that overexpression of ATG6 and UVRAG did not inhibit the phosphorylation of S6K , which is also highly consistent with the above Drosophila data (Lee, 2007).

Then, short interfering RNA (siRNA) targeting was used for ATG1α and ATG1β messenger RNA to confirm that ATG1 inhibits S6K activity. The efficacy of siRNA was verified by qRT–PCR using ATG1-specific primers. Transfection of ATG1α and ATG1β siRNA to HEK 293T cells led to increased phosphorylation of S6K Thr 389 and S6 (the only proven in vivo substrate of S6K) Thr 235/236. This result was further supported by immunocytochemistry by using phosphospecific S6 antibody; ATG1 siRNA transfection alone induced phosphospecific immunostaining of S6 in starved cells. Taken together, these results clearly demonstrate that ATG1 inhibits S6K and S6 in vivo (Lee, 2007).

Notably, the level of S6K activation by ATG1 siRNA was about 5% of that by nutritional stimulation. This weak activation of S6K resulted from partial gene knockdown by RNAi. Consistent with this conclusion, more pronounced activation of dS6K was observed in DmATG1 mutants in Drosophila, which contains only a single orthologue of ATG1 (Lee, 2007).

S6K is in the AGC kinase family, which also includes Akt and p90 ribosomal S6 kinase (RSK). These kinases are regulated by a similar mechanism in which both phosphorylation at their activation loop and a hydrophobic motif next to the kinase domain are required for full activation. 3-Phosphoinositide-dependent kinase 1 (PDK1) is a kinase responsible for phosphorylation at the activation loop of AGC kinases. In the case of S6K, PDK1 directly phosphorylates the Thr 229 residue at the activation loop of S6K, which is strictly dependent on the previous phosphorylation of Thr 389 at the hydrophobic motif. These motifs are well conserved among the family members in different species. Therefore, whether ATG1α also affects the phosphorylation of Akt and RSK was examined. Interestingly, the phosphorylation of Akt and RSK was not affected by ATG1α, with or without stimulation by insulin and EGF. These data indicate that ATG1α specifically modulates S6K activity (Lee, 2007).

Next, to understand the molecular mechanism of the specific regulation of S6K by ATG1, whether ATG1 affects the phosphorylation of Thr 229 in S6K was investigated by using an S6K mutant that specifically mimics the phosphorylated form of S6K, Thr 389 Glu. As a result, Thr 229 phosphorylation of the S6K Thr 389 Glu mutant was not affected by wild-type ATG1. Because Akt was not inhibited by ATG1, it is unlikely that ATG1 regulates Thr 389 phosphorylation of S6K by inhibiting the PDK1/Akt signalling module. Therefore, it is believed that ATG1 modulates S6K activity by affecting S6K Thr 389-specific kinases or phosphatases (Lee, 2007).

In summary, under nutrient-rich conditions, activation of TOR leads to inhibition of ATG1, which facilitates S6K Thr 389 phosphorylation and the subsequent phosphorylation of Thr 229 by PDK1 to activate S6K fully. Consequently, activated S6K promotes cell growth. However, under conditions of starvation, TOR becomes quiescent and ATG1 can inhibit S6K by blocking Thr 389 phosphorylation. This nutrient-dependent signalling switch operated by TOR and ATG1 is highly consistent with that in yeast. The observations described in this study clearly show the presence of crosstalk between ATG1 and S6K signalling, in which ATG1 specifically inhibits S6K. This study also showed that this is evolutionarily conserved between Drosophila and mammals. It is believed that these biochemical data and the fly system will be useful in future studies that address the detailed molecular mechanism of crosstalk between the two nutrition-dependent physiological processes -- autophagy and cell growth (Lee, 2007).

Role and regulation of starvation-induced autophagy in the Drosophila fat body

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 (see Drosophila Tor), 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).

C. elegans UNC-51/ATG1 kinase regulates axonal transport by mediating motor-cargo assembly

Axonal transport mediated by microtubule-dependent motors is vital for neuronal function and viability. Selective sets of cargoes, including macromolecules and organelles, are transported long range along axons to specific destinations. Despite intensive studies focusing on the motor machinery, the regulatory mechanisms that control motor-cargo assembly are not well understood. This study shows that UNC-51/ATG1 kinase regulates the interaction between synaptic vesicles and motor complexes during transport in Drosophila. UNC-51 binds UNC-76, a kinesin heavy chain (KHC) adaptor protein. Loss of unc-51 or unc-76 leads to severe axonal transport defects in which synaptic vesicles are segregated from the motor complexes and accumulate along axons. Genetic studies show that unc-51 and unc-76 functionally interact in vivo to regulate axonal transport. UNC-51 phosphorylates UNC-76 on Ser(143), and the phosphorylated UNC-76 binds Synaptotagmin-1, a synaptic vesicle protein, suggesting that motor-cargo interactions are regulated in a phosphorylation-dependent manner. In addition, defective axonal transport in unc-76 mutants is rescued by a phospho-mimetic UNC-76, but not a phospho-defective UNC-76, demonstrating the essential role of UNC-76 Ser(143) phosphorylation in axonal transport. Thus, these data provide insight into axonal transport regulation that depends on the phosphorylation of adaptor proteins (Toda, 2008).

This study demonstrates that loss of unc-51 function affects the transport of several axonal cargoes, including SV and mitochondria. In unc-51 mutants, SV transport is severely attenuated and many SV aggregates are found along the larval SGN axons. SV aggregation in unc-51 mutants is similar to that observed in Khc mutants. However, unlike Khc mutants, SV aggregates in unc-51 mutants do not contain mitochondria, suggesting that the aggregated SVs in unc-51 mutants do not cause overall 'steric hindrance,' in which the impaired cargo interferes with the transport of other cargoes as a secondary effect. In unc-51 mutants, Rab5-positive membranes also exhibited a pattern of aggregation different from that for SVs, supporting the idea that loss of unc-51 results in defective axonal transport in a membrane type-dependent manner. This view is further supported by a recent analysis of unc-51 mutants in C. elegans that showed that only a subset of cargoes are selectively mislocalized, whereas a majority of other cargoes are not affected (Toda, 2008).

SVs are one of the most severely affected axonal cargoes in unc-51 mutants. Two anterograde motors, kinesin-1 and kinesin-3, have been implicated in SV transport in Drosophila. A recent study addressed an essential role of unc-104 (imac, kinesin-3) in SV transport (Pack-Chung, 2007). Virtually all SVs fail to enter axons and accumulate in neuronal cell bodies during the embryonic period. In contrast, mutations in Khc, a catalytic component of kinesin-1, and also mutations in Klc, an accessory component of kinesin-1, cause SV accumulations within axons of larval SGNs. These studies suggest that, although kinesin-3 is primarily responsible for SV transport, kinesin-1 plays a role in SV transport in a manner distinct from that for kinesin-3, or the two motors may have complementary roles in SV transport at the larval stage. The unc-51 mutant phenotypes, as well as the biochemical evidence that UNC-51 forms a complex with UNC-76/KHC, are most consistent with the idea that UNC-51 functions in SV transport through a kinesin-1-dependent pathway (Toda, 2008).

In unc-51 mutants, SVs accumulate within axons at sites distant from cell bodies, suggesting that SVs could partially transport along axons in the absence of unc-51. It is possible that maternally deposited unc-51 contributes to partial transport of SVs into axons, as suggested for defective SV transport in Khc mutants. It is also possible that there are specific subcellular locations (e.g., axon hillock) or earlier developmental periods where SV transport does not require unc-51 activity. The axonal SV accumulation in unc-51 mutants could be a result of spatially distinct requirement of unc-51 activity for maintaining SV-motor integrity during transport. In addition, kinesin-3 likely contributes to SV transport in unc-51 mutants, resulting in translocation of a subset of SVs into axons and to synapses. SVs that are localized to unc-51 mutant NMJs may reflect a subpopulation of those that were carried by kinesin-3 and did not need unc-51 activity for their transport. In summary, the cooperative action of multiple pathways, including unc-51, unc-76, kinesin-1 and kinesin-3, may be necessary for complete SV transport (Toda, 2008).

Previous in vitro studies that addressed a role of phosphorylation in regulating organelle motility have remained unclear and controversial. A series of kinases, including PKA, PKC, and PKG, were shown to have no effect on kinesin-dependent axonal transport, whereas phosphorylation of kinesin by PKA was proposed to inhibit fast axonal transport and kinesin binding to membrane organelles. Glycogen synthase kinase 3β (GSK3β) phosphorylates KLC and kinesin-based motility is inhibited by perfusion of active GSK3β into squid axons. An inhibitory effect of CaMKII in disrupting KIF17-Mint1 association in vitro has recently been reported. However, it was not until recently that the physiological role of phosphorylation in axonal transport was addressed in vivo (Horiuchi, 2007), in which JNK-mediated phosphorylation inhibits the kinesin-1-JIP1 adaptor interaction (Toda, 2008).

This study has revealed a critical role of UNC-76 phosphorylation during axonal transport, which is likely elicited at the motor-cargo interface. Several lines of evidence support this notion. First, unc-51 genetically interacts with the kinesin-1 adaptor unc-76 in axonal transport in vivo. Second, biochemical experiments show that the association of UNC-76 and Syt-1 is mediated by UNC-51-dependent phosphorylation of UNC-76. Third, FRET analysis confirms the direct association between UNC-76 and Syt-1 in cells, which is attenuated by inhibiting UNC-51 kinase activity. Finally, the SV transport defects of unc-76 mutants are rescued in vivo by phosphomimetic UNC-76, but not by phosphodefective UNC-76 (Toda, 2008).

Based on these findings, a model is proposed in which adaptor phosphorylation is a key regulatory step that maintains motor-cargo association within axons and leads to efficient SV transport. Upon phosphorylation by UNC-51 kinase, UNC-76 displays an increased affinity to SV membrane proteins such as Syt-1. Attenuation of UNC-51 kinase activity would reduce the affinity of UNC-76 for SV membrane proteins and cause the dissociation of SV cargoes from the motor complexes. In this model, motor-cargo affinity could also be reduced by dephosphorylation of UNC-76, although such regulatory factor is yet to be identified. Attenuation of UNC-51 activity or activation of phosphatase activity could explain the mechanism of SV cargo detachment from the kinesin motors. Additional work is needed to determine how UNC-51 kinase activity is spatially and temporally controlled to regulate axonal transport. It is notable that both UNC-51 and UNC-76 are undetectable at NMJ of the wild-type third instar larvae, suggesting a spatial control of motor-cargo dissociation at the axonal termini (Toda, 2008).

The proposed mechanism can explain the transport of a subset of SVs. Only ~20% of SVs appear to colocalize with UNC-76 in wild-type segmental nerves (SGNs), suggesting that an additional motor/adaptor system, such as kinesin-3, is responsible for carrying the rest of the SVs at the larval stage. Indeed, a subpopulation of SVs successfully reaches the synapses in unc-51 mutant NMJs, although these synaptic boutons are smaller in size and fewer in number. It is also likely that UNC-51/UNC-76/kinesin-1 activity is dispensable for loading SVs onto the motor complexes at cell bodies, because SV aggregates are found within axons and distant from cell bodies in these mutants. Thus, the UNC-51/UNC-76/kinesin-1 complex seems important for maintaining motor-cargo association within axons rather than being responsible for initial cargo loading. Alternatively, maternally deposited unc-51 or Khc may contribute to partial transport of SVs into axons, and the potential role of UNC-51/UNC-76/kinesin-1 complex in initial cargo loading may be masked in the analysis of unc-51 mutants (Toda, 2008).

Although this study clearly demonstrates that phosphorylation of UNC-76 by UNC-51 kinase is critical for SV transport, the phosphomimetic UNC-76 transgene failed to rescue defective SV transport in unc-51 mutants. This implies that phosphorylation of UNC-76 is not sufficient for SV transport, and suggests that additional targets of UNC-51 phosphorylation are necessary for proper SV transport. Both KHC and KLC are phosphoproteins, and unc-51 interacts genetically with Klc. Therefore, additional transport components, including KHC and KLC, need to be tested as candidate substrates for UNC-51 kinase in order to understand the whole picture of unc-51-mediated axonal transport machinery (Toda, 2008).

It is unclear how loss of unc-51 results in aggregation of the kinesin motor complex. Biophysical studies show that cargo binding to the kinesin tail domain is required to unfold kinesin molecules and to activate their motor function on MTs, suggesting that a failure of motor-cargo assembly could cause stalling and aggregation of kinesin motors. With respect to the kinesin motor activation, a recent report has addressed a novel role for UNC-76/FEZ1 in kinesin motor unfolding and thus activation (Blasius, 2007). In this model, two scaffolding/adaptor proteins, JIP1 and UNC-76/FEZ1, induce a step-wise conformational change in kinesin-1, leading to its full activation as a motor in vitro. In good agreement with this model, loss of unc-76 results in disorganized localization of SVs and KHC in vivo. Taken together with the finding that the phosphomimetic UNC-76 is capable of rescuing axonal transport defects in unc-76 mutants, it is possible that UNC-76/FEZ1 not only serves as a motor-cargo linker, but also functions as a kinesin-1 activator in a phosphorylation-dependent manner. Thus, this phosphorylation-dependent regulation of adaptors may address an additional mechanism for controlling kinesin-1 activity that is essential for axonal transport (Toda, 2008).

Although the unc-51 and unc-76 pathways could cooperatively activate kinesin-1 activity, it is unlikely that loss of unc-51 leads to complete loss of Khc activity. In unc-51 mutants, mitochondrial transport is partially attenuated, but the majority of mitochondria are still transported, suggesting that the overall activity of KHC, a major motor for mitochondrial transport, is preserved. This view is supported by immunohistochemical evidence, which shows that a subpopulation of KHC is present in aggregates, whereas the rest of KHC was distributed throughout the axons. This suggests that a part of KHC activity may be unaffected in unc-51 mutants, and that the UNC-76-independent population of KHC is functionally active and participate in the transport of other cargoes. Although UNC-51 forms a complex with KHC via UNC-76 and KHC distribution is altered in unc-51 mutants, which strongly suggests a functional interaction between UNC-51 and KHC, it is possible that UNC-51 regulation of kinesin-1 activity is mediated through UNC-76 or an additional factor such as KLC, and the effect of UNC-51 on the KHC motor may be indirect. In this regard, it is suggestive that unc-76 and unc-51 show a clear genetic interaction, whereas Khc and unc-51 do not exhibit an apparent genetic interaction (Toda, 2008).

This model of phosphorylation-dependent regulation of motor-cargo assembly could be extended to include additional adaptors or cargo vesicles. UNC-14, a protein that interacts with UNC-51, has recently been reported to play a role in kinesin-1-dependent axonal transport in C. elegans (Sakamoto, 2005). UNC-14 might serve as an adaptor for the kinesin motor complex to regulate motor-cargo affinity in an UNC-51-dependent manner. In addition, unc-51 mutations also result in aggregation of vesicles positive for UNC-5, a Netrin/UNC-6 receptor (Ogura, 2006). Again, affinity between the UNC-5-positive vesicles and their respective motor complexes might be regulated by UNC-51-dependent phosphorylation (Toda, 2008).

Previous studies in worms and mice, as well as this study, addressed a role of unc-51 in axon formation. It remains to be studied whether this model could be extended to explain the regulation of membrane components necessary for axon formation, in which the assembly of axonal membranes with the corresponding motors may be mediated via unc-51-dependent phosphorylation (Toda, 2008).

Recent studies identified unc-51 as a homolog of atg1, which plays a role in autophagy, a catabolic cellular process responsible for bulk degradation of proteins and organelles, particularly when cells are under nutrient-deprived conditions. Disruption of the autophagy genes atg5 or atg7 in mouse brains results in neuronal cell death, which accompanies intracellular accumulation of ubiquitin-positive aggregates. Neither ubiquitin-positive aggregates nor symptoms of cellular death were observed in unc-51 mutant SGNs, suggesting that the role of unc-51/atg1 in axonal transport is distinct from its role in autophagy, which is induced under nutrient-deficient conditions. Although autophagy critically depends on intracellular vesicle transport, and UNC-51 kinase activity seems to be required for autophagy induction, a link between axonal transport and autophagy remains to be studied (Toda, 2008).

In conclusion, this study identifies a novel regulatory step for axonal transport that depends on the UNC-51 kinase-mediated phosphorylation of a kinesin adaptor. Further studies on the regulation of unc-51 activity will provide a better understanding of axonal transport, as well as dynamic neuronal control of synaptic development and plasticity (Toda, 2008).

Autophagy promotes synapse development in Drosophila

Autophagy, a lysosome-dependent degradation mechanism, mediates many biological processes, including cellular stress responses and neuroprotection. This study demonstrates that autophagy positively regulates development of the Drosophila larval neuromuscular junction (NMJ). Autophagy induces an NMJ overgrowth phenotype closely resembling that of highwire (hiw), an E3 ubiquitin ligase mutant. Moreover, like hiw, autophagy-induced NMJ overgrowth is suppressed by wallenda (wnd) and by a dominant-negative c-Jun NH2-terminal kinase (bsk^DN). Autophagy promotes NMJ growth by reducing Hiw levels. Thus, autophagy and the ubiquitin-proteasome system converge in regulating synaptic development. Because autophagy is triggered in response to many environmental cues, these findings suggest that it is perfectly positioned to link environmental conditions with synaptic growth and plasticity (Shen, 2009).

Autophagy involves multiple steps, including induction, autophagosome formation, fusion of autophagosomes with lysosomes, and recycling of autophagy components. Disrupting any of these steps impairs autophagy. Several highly conserved ATG genes encoding core components of the autophagy machinery have been identified in yeast. Mutations in genes, including atg1, -2, -6, and -18, have also been isolated and characterized in Drosophila. To assess the role of autophagy in NMJ development, the effects were examined of mutations in atg genes, whose normal functions span the entire process: atg1 is defective in autophagy induction, atg6 is defective in autophagosome formation, and atg2 and -18 are defective in retrieval of other ATG proteins from autophagosomes. Regardless of the step impaired, all of these atg mutants exhibited significant reduction in NMJ size. These results demonstrate that a basal level of autophagy is required to promote NMJ development (Shen, 2009).

Overexpression of atg1⁺ is sufficient to induce high levels of autophagy in larval fat bodies and salivary glands. If autophagy is a positive regulator of NMJ development, an increase in autophagy might enhance synaptic growth. Consistent with previous studies, panneuronal overexpression of UAS-atg1⁺ under the control of C155-Gal4 or elav-Gal4 drivers induced high levels of autophagy in the nervous system, as indicated by increased staining with LysoTracker, an acidophilic dye which has been used to assess autophagy by labeling acidic structures, including lysosomes. Under these conditions, bouton number increased more than twofold. To further verify that this NMJ overgrowth was caused by elevated autophagy rather than to some other effect of atg1⁺ overexpression, whether mutations in other atg genes suppress this phenotype was examined. For this purpose, a null allele of atg18 (atg18^δ) was generated. Removal of one copy of atg18⁺ had no affect on NMJ growth in an otherwise wild-type background but significantly suppressed NMJ overgrowth caused by atg1⁺ overexpression. Removal of both copies of atg18⁺ conferred almost complete suppression. Therefore, NMJ overgrowth caused by atg1⁺ overexpression is primarily caused by elevated levels of autophagy (Shen, 2009).

As a further test, NMJ morphology was examined after feeding larvae with rapamycin, which induces autophagy by inhibiting TOR (target of rapamycin), the key negative regulator of autophagy. Wild-type larvae fed rapamycin exhibited striking NMJ overgrowth similar to that caused by overexpressing atg1⁺. Rapamycin-induced NMJ overgrowth was completely suppressed by mutations in atg18. Collectively, these results demonstrate that autophagy is a key positive regulator of NMJ growth (Shen, 2009).

Wairkar (2009) observed NMJ undergrowth in atg1 mutants but did not see overgrowth with atg1⁺ overexpression. This discrepancy likely results from the use of different UAS-atg1⁺ transgenes. For example, Wairkar was able to obtain only partial (~50%) rescue of NMJ undergrowth in atg1 mutants by overexpression of their UAS-atg1^rescue construct, whereas this study obtained complete rescue of this phenotype (Shen, 2009).

Atg1 has several functions unrelated to autophagy. It was found that axonal transport is disrupted in atg1-null mutants, which is a result also recently reported by Toda (2008) and Wairkar (2009). In addition, Atg1 suppresses translation by inhibiting the S6K kinase (Lee, 2007; Scott, 2007) and controls active zone density by inhibiting extracellular signal-regulated kinase (ERK) signaling (Wairkar, 2009). However, several lines of evidence indicate that these functions of Atg1 are not responsible for the NMJ phenotypes observed when Atg1 activity was altered. (1) atg2 or -18 mutants exhibited similar NMJ undergrowth but did not have defects in axonal transport. Thus, in agreement with Toda (2008), it is concluded that Atg1's role in axonal transport is distinct from its function in autophagy and NMJ growth. (2) Blocking or activating translation by overexpressing a dominant-negative S6K transgene or constitutively activated S6K transgenes by elav-Gal4 driver had little affect on NMJ growth. Moreover, coexpression of any of the three constitutively activated S6K transgenes failed to suppress NMJ overgrowth caused by atg1⁺ overexpression. Thus, the role of Atg1 in S6K-dependent translation does not contribute to the NMJ phenotypes associated with manipulations of Atg1. (3) An ERK mutation does not affect NMJ growth. Although this ERK mutation suppresses the deficit in active zone density in atg1 mutants, it does not suppress atg1's NMJ undergrowth phenotype (Wairkar, 2009), indicating that it is not mediated by the ERK pathway. Collectively, these results demonstrate that altered levels of autophagy are primarily responsible for the effects of Atg1 on NMJ development (Shen, 2009).

NMJ overgrowth induced by autophagy is distinctive and offers potential clues about pathways that may be involved. Formation of multiple long synaptic branches containing many small diameter boutons without any hyperbudding or satellite boutons most closely resembles the hiw phenotype, suggesting that autophagy and Hiw may function through the same pathway. Recent evidence indicates that Hiw inhibits NMJ growth by down-regulating Wnd, which in turn activates a Jun kinase encoded by bsk (basket). NMJ overgrowth in hiw is suppressed by mutations of wnd and by a dominant-negative mutation of bsk (bsk^DN; Collins, 2006). If the phenotypic similarity between hiw and increased autophagy reflects convergence on a common pathway, autophagy-induced NMJ overgrowth should also be suppressed by wnd and bsk^DN. Indeed, this is what was observed in this study. These results strongly support the idea that autophagy and Hiw converge on a Wnd-dependent MAPK signaling pathway to regulate NMJ development (Shen, 2009).

If autophagy and Hiw act via a common pathway, where do they converge? As a positive regulator of NMJ growth, autophagy could promote degradation of a negative regulator. An intriguing possibility is that Hiw is the negative regulator affected by autophagy. If a decrease in Hiw levels is responsible for NMJ overgrowth when autophagy is elevated, restoration of Hiw should suppress overgrowth. This possibility was tested by coexpressing wild-type Hiw with Atg1; Atg1-mediated NMJ overgrowth was found to be significantly suppressed. This suppression is not simply an indirect consequence of the dilution of GAL4 caused by addition of a second UAS element because coexpression of UAS-nwk⁺ did not suppress such NMJ overgrowth. This result also shows that Nwk (Nervous wreck), another negative regulator of NMJ growth, is not an apparent target of autophagy, as predicted by differences in phenotypes. Thus, autophagy appears to regulate NMJ growth through its effects on particular presynaptic proteins, and Hiw represents a key downstream effector (Shen, 2009).

To further test whether autophagy promotes NMJ growth by limiting Hiw, one copy of hiw⁺ was eliminated to determine whether this further decrease in Hiw levels enhanced the effects of atg1⁺ overexpression. In an otherwise wild-type background, loss of one copy of hiw⁺ had no affect, but it significantly enhanced atg1⁺-induced NMJ overgrowth. The phenotype of hiw homozygotes overexpressing atg1⁺ was no more extreme than hiw homozygote alone. The absence of any additive or synergistic effects further supports the hypothesis that autophagy promotes NMJ development by down-regulating Hiw (Shen, 2009).

Because Hiw antibodies do not work for immunohistochemistry, Hiw was visualized using a fully functional GFP-tagged construct to test directly whether abundance of Hiw is affected by autophagy. In an otherwise wild-type background, Hiw-GFP was strongly expressed in neurons throughout the ventral ganglion and brain lobes driven by C155-Gal4, as detected by anti-GFP staining. However, in larvae co-overexpressing atg1⁺, the GFP signal was reduced by ~60% relative to anti-HRP staining. This result was confirmed by Western blot analysis. Reduction of Hiw-GFP is not caused by the dilution of GAL4 by the presence of a second UAS element because coexpression of UAS-myr-RFP did not affect abundance of Hiw-GFP. These results further indicate that autophagy promotes NMJ growth by down-regulating Hiw (Shen, 2009).

These results indicate that NMJ overgrowth caused by elevated autophagy is primarily caused by reduction in Hiw. Is the converse also true? Is NMJ undergrowth in atg mutants caused by elevated levels of Hiw? To address these questions, Hiw-GFP was expressed in neurons using C155-Gal4 in various backgrounds. Hiw-GFP levels were significantly elevated in atg1 and -6 mutants compared with the controls, consistent with the idea that Hiw is down-regulated by autophagy. If this increase in Hiw is a primary cause of NMJ undergrowth in atg loss-of-function mutants, eliminating Hiw should prevent this undergrowth; i.e., mutations in hiw should be epistatic to atg mutations. Thus, NMJ morphology was examined in hiw; atg2 and hiw; atg18 double mutants, and it was found that hiw was completely epistatic, demonstrating the role of elevated levels of Hiw in NMJ undergrowth of atg mutants (Shen, 2009).

A more direct test is to determine whether overexpression of Hiw can reduce NMJ size. However, this experiment is complicated because overexpression of Hiw by a relatively weak neuronal driver (elav-Gal4) does not affect NMJ size, whereas overexpression of Hiw by a strong neuronal driver (Elav-GeneSwitch) has a modest dominant-negative effect. To determine whether increased levels of Hiw can limit NMJ growth, it appears necessary to overexpress Hiw at an intermediate level. Therefore, NMJs were examined in larvae overexpressing UAS-hiw⁺ via C155-Gal4. C155-Gal4/+; UAS-hiw⁺/+ female larvae exhibited very mild NMJ undergrowth. Stronger undergrowth was observed in C155-Gal4/Y; UAS-hiw⁺/+ male larvae. This difference is consistent with higher levels of C155-Gal4 expression in males than in females, owing to dosage compensation. No differences were observed in NMJ growth between C155-Gal4/+ female and C155-Gal4/Y male larvae, indicating that the undergrowth phenotypes are dependent on the levels of Hiw overexpression and not on differences in gender or expression of GAL4 alone. Thus, moderate increases in Hiw levels result in NMJ undergrowth. Furthermore, the modest NMJ undergrowth in C155-Gal4/+; UAS-hiw⁺/+ larvae was enhanced when one copy of atg1⁺, -2⁺, or -6⁺ was removed. Together, these results indicate that elevated levels of Hiw account for most of the NMJ undergrowth in atg mutants. However, excess Hiw cannot fully explain NMJ undergrowth in atg mutants because NMJ undergrowth caused by Hiw overexpression is less severe than that of atg1 and -18 mutants. Thus, when autophagy is impaired, additional negative regulators may accumulate to depress NMJ growth. It is also likely that elevated levels of Hiw target proteins other than Wnd to limit synaptic growth because loss-of-function mutations of wnd do not affect NMJ development (Shen, 2009).

Because autophagy is generally thought of as a nonselective bulk degradation process, the idea that autophagy regulates NMJ growth primarily through its effects on Hiw levels seems difficult to understand at first. However, recent studies demonstrate that autophagy can also operate in a substrate-selective mode in regulating specific developmental events (Rowland, 2006; Zhang, 2009). For example, in Caenorhabditis elegans, when postsynaptic cells fail to receive presynaptic contact, GABA_A receptors selectively traffic to autophagosomes (Rowland, 2006). However, the detailed mechanism of such selectivity is unknown. Zhang identified SEPA-1 as a bridge that mediates the specific recognition and degradation of P granules by autophagy in C. elegans. Thus, one possibility is that Hiw is specifically targeted to autophagosomes via a mechanism that remains to be elucidated. It is also possible that many presynaptic proteins besides Hiw are degraded by autophagy, but it is the reduction in Hiw that primarily affects NMJ size. Moreover, although the idea that autophagy regulates Hiw directly is favored, the possibility cannot be ruled out that autophagy promotes degradation of Hiw through an indirect mechanism involving the proteasome or other pathway (Shen, 2009).

In principle, autophagy could be acting on either side of the NMJ to regulate its development. Because atg1⁺ overexpression in muscle results in lethality at the first larval instar, it was not possible to assess whether this affects NMJ growth. Although a postsynaptic role of autophagy in NMJ development cannot be ruled out, several results suggest that the effects of autophagy are primarily presynaptic: neuronal expression of UAS-atg1⁺ is sufficient to completely rescue the NMJ undergrowth in atg1 mutants, the Hiw-Wnd pathway functions presynaptically (Wu, 2005; Collins, 2006), and hiw is completely epistatic to autophagy for NMJ growth (Shen, 2009).

Autophagy is of particular interest as a regulator of synaptic growth because it is triggered in response to many environmental cues. These results demonstrate that decreasing or increasing autophagy from basal levels results in corresponding effects on synaptic size. Thus, autophagy is perfectly positioned to link environmental conditions with synaptic growth and plasticity. As such, it is intriguing to speculate on a role for autophagy in learning and memory (Shen, 2009).

Search PubMed for articles about Drosophila atg1

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date revised: 20 December 2009

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