How a RING finger protein and a steroid hormone control autophagy

Previous work in our laboratory has indicated that the steroid hormone ecdysone triggers programmed autophagy in the fat body of Drosophila larvae by downregulating the class I phosphoinositide 3-kinase (PI3K) pathway. We recently found evidence that Deep orange (Dor), a Drosophila RING finger protein implicated in late-endosomal trafficking, controls ecdysone signaling as well as autolysosome fusion, thus exerting a dual regulation of autophagy. We found that dor mutants are defective in programmed autophagy. The mutant larvae showed impaired upregulation of ecdysone signaling during development, accompanied by a failure to downregulate the PI3K pathway. Downregulation of the PI3K pathway could be restored by feeding the dor mutants with ecdysone. Even though ecdysone signaling and autophagy were impaired in the dor mutants, we detected an accumulation of autophagosomes in dor mutant fat bodies. This could probably be attributed to the failure of autophagosomes to fuse with lysosomes. In this Addendum we review these findings and provide some speculations about how Dor may control both ecdysone signalling and autolysosomal fusion.     

How a RING Finger Protein and a Steroid Hormone Control Autophagy?

Previous work in our laboratory has indicated that the steroid hormone ecdysone triggers programmed autophagy in the fat body of Drosophila larvae by downregulating the class I phosphoinositide 3-kinase (PI3K) pathway. We recently found evidence that Deep orange (Dor), a Drosophila RING finger protein implicated in late-endosomal trafficking, controls ecdysone signaling as well as autolysosome fusion, thus exerting a dual regulation of autophagy. We found that dor mutants are defective in programmed autophagy. The mutant larvae showed impaired upregulation of ecdysone signaling during development, accompanied by a failure to downregulate the PI3K pathway. Downregulation of the PI3K pathway could be restored by feeding the dor mutants with ecdysone. Even though ecdysone signaling and autophagy were impaired in the dor mutants, we detected an accumulation of autophagosomes in dor mutant fat bodies. This could probably be attributed to the failure of autophagosomes to fuse with lysosomes. In this Addendum we review these findings and provide some speculations about how Dor may control both ecdysone signalling and autolysosomal fusion.

Addendum to:

A Dual Function for Deep Orange in Programmed Autophagy in the Drosophila melanogaster Fat Body

K. Lindmo, A. Simonsen, A. Brech, K. Finley, T.E. Rusten and H. Stenmark

Exp Cell Res 2006; Epub ahead of print     

Programmed autophagy in the Drosophila fat body is induced by ecdysone through regulation of the PI3K pathway

Eukaryotic cells catabolize their own cytoplasm by autophagy in response to amino acid starvation and inductive signals during programmed tissue remodeling and cell death. The Tor and PI3K signaling pathways have been shown to negatively control autophagy in eukaryotes, but the mechanisms that link these effectors to overall animal development and nutritional status in multicellular organisms remain poorly understood. Here, we reveal a complex regulation of programmed and starvation-induced autophagy in the Drosophila fat body. Gain-of-function genetic analysis indicated that ecdysone receptor signaling induces programmed autophagy whereas PI3K signaling represses programmed autophagy. Genetic interaction studies showed that ecdysone signaling downregulates PI3K signaling and that this represents the effector mechanism for induction of programmed autophagy. Hence, these studies link hormonal induction of autophagy to the regulatory function of the PI3K signaling pathway in vivo.     

deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster

Endosomal degradation is severely impaired in primary hemocytes from larvae of eye color mutants of Drosophila. Using high resolution imaging and immunofluorescence microscopy in these cells, products of eye color genes, deep-orange (dor) and carnation (car), are localized to large multivesicular Rab7-positive late endosomes containing Golgi-derived enzymes. These structures mature into small sized Dor-negative, Car-positive structures, which subsequently fuse to form tubular lysosomes. Defective endosomal degradation in mutant alleles of dor results from a failure of Golgi-derived vesicles to fuse with morphologically arrested Rab7-positive large sized endosomes, which are, however, normally acidified and mature with wild-type kinetics. This locates the site of Dor function to fusion of Golgi-derived vesicles with the large Rab7-positive endocytic compartments. In contrast, endosomal degradation is not considerably affected in car1 mutant; fusion of Golgi-derived vesicles and maturation of large sized endosomes is normal. However, removal of Dor from small sized Car-positive endosomes is slowed, and subsequent fusion with tubular lysosomes is abolished. Overexpression of Dor in car1 mutant aggravates this defect, implicating Car in the removal of Dor from endosomes. This suggests that, in addition to an independent role in fusion with tubular lysosomes, the Sec1p homologue, Car, regulates Dor function.     

The class III PI(3)K Vps34 promotes autophagy and endocytosis but not TOR signaling in Drosophila

Degradation of cytoplasmic components by autophagy requires the class III phosphatidylinositol 3 (PI(3))-kinase Vps34, but the mechanisms by which this kinase and its lipid product PI(3) phosphate (PI(3)P) promote autophagy are unclear. In mammalian cells, Vps34, with the proautophagic tumor suppressors Beclin1/Atg6, Bif-1, and UVRAG, forms a multiprotein complex that initiates autophagosome formation. Distinct Vps34 complexes also regulate endocytic processes that are critical for late-stage autophagosome-lysosome fusion. In contrast, Vps34 may also transduce activating nutrient signals to mammalian target of rapamycin (TOR), a negative regulator of autophagy. To determine potential in vivo functions of Vps34, we generated mutations in the single Drosophila melanogaster Vps34 orthologue, causing cell-autonomous disruption of autophagosome/autolysosome formation in larval fat body cells. Endocytosis is also disrupted in Vps34(-/-) animals, but we demonstrate that this does not account for their autophagy defect. Unexpectedly, TOR signaling is unaffected in Vps34 mutants, indicating that Vps34 does not act upstream of TOR in this system. Instead, we show that TOR/Atg1 signaling regulates the starvation-induced recruitment of PI(3)P to nascent autophagosomes. Our results suggest that Vps34 is regulated by TOR-dependent nutrient signals directly at sites of autophagosome formation.     

Interaction of the HOPS complex with Syntaxin 17 mediates autophagosome clearance in Drosophila

Homotypic fusion and vacuole protein sorting (HOPS) is a tethering complex required for trafficking to the vacuole/lysosome in yeast. Specific interaction of HOPS with certain SNARE (soluble NSF attachment protein receptor) proteins ensures the fusion of appropriate vesicles. HOPS function is less well characterized in metazoans. We show that all six HOPS subunits (Vps11 [vacuolar protein sorting 11]/CG32350, Vps18/Dor, Vps16A, Vps33A/Car, Vps39/CG7146, and Vps41/Lt) are required for fusion of autophagosomes with lysosomes in Drosophila. Loss of these genes results in large-scale accumulation of autophagosomes and blocks autophagic degradation under basal, starvation-induced, and developmental conditions. We find that HOPS colocalizes and interacts with Syntaxin 17 (Syx17), the recently identified autophagosomal SNARE required for fusion in Drosophila and mammals, suggesting their association is critical during tethering and fusion of autophagosomes with lysosomes. HOPS, but not Syx17, is also required for endocytic down-regulation of Notch and Boss in developing eyes and for proper trafficking to lysosomes and eye pigment granules. We also show that the formation of autophagosomes and their fusion with lysosomes is largely unaffected in null mutants of Vps38/UVRAG (UV radiation resistance associated), a suggested binding partner of HOPS in mammals, while endocytic breakdown and lysosome biogenesis is perturbed. Our results establish the role of HOPS and its likely mechanism of action during autophagy in metazoans.     

The PI 3-kinase regulator Vps15 is required for autophagic clearance of protein aggregates

Autophagy is involved in cellular clearance of aggregate-prone proteins, thereby having a cytoprotective function. Studies in yeast have shown that the PI 3-kinase Vps34 and its regulatory protein kinase Vps15 are important for autophagy, but the possible involvement of these proteins in autophagy in a multicellular animal has not been addressed genetically. Here, we have created a Drosophila deletion mutant of vps15 and studied its role in autophagy and aggregate clearance. Homozygous Deltavps15 Drosophila died at the early L3 larval stage. Using GFP-Atg8a as an autophagic marker, we employed fluorescence microscopy to demonstrate that fat bodies of wild type Drosophila larvae accumulated autophagic structures upon starvation whereas vps15 fat bodies showed no such response. Likewise, electron microscopy revealed starvation-induced autophagy in gut cells from wild type but not Deltavps15 larvae. Fluorescence microscopy showed that Deltavps15 mutant tissues accumulated profiles that were positive for ubiquitin and Ref(2)P, the Drosophila homolog of the sequestosome marker SQSTM1/p62. Biochemical fractionation and Western blotting showed that these structures were partially detergent insoluble, and immuno-electron microscopy further demonstrated the presence of Ref(2)P positive membrane free protein aggregates. These results provide the first genetic evidence for a function of Vps15 in autophagy in multicellular organisms and suggest that the Vps15-containing PI 3-kinase complex may play an important role in clearance of protein aggregates.     

Lysosomal membrane protein composition, acidic pH and sterol content are regulated via a light-dependent pathway in metazoan cells

In metazoans, lysosomes are characterized by a unique tubular morphology, acidic pH, and specific membrane protein (LAMP) and lipid (cholesterol) composition as well as a soluble protein (hydrolases) composition. Here we show that perturbation to the eye-color gene, light, results in impaired lysosomal acidification, sterol accumulation, altered endosomal morphology as well as compromised lysosomal degradation. We find that Drosophila homologue of Vps41, Light, regulates the fusion of a specific subset of biosynthetic carriers containing characteristic endolysosomal membrane proteins, LAMP1, V0-ATPase and the cholesterol transport protein, NPC1, with the endolysosomal system, and is then required for the morphological progression of the multivesicular endosome. Inhibition of Light results in accumulation of biosynthetic transport intermediates that contain these membrane cargoes, whereas under similar conditions, endosomal delivery of soluble hydrolases, previously shown to be mediated by Dor, the Drosophila homologue of Vps18, is not affected. Unlike Dor, Light is recruited to endosomes in a PI3P-sensitive fashion wherein it facilitates fusion of these biosynthetic cargoes with the endosomes. Depletion of the mammalian counterpart of Light, hVps41, in a human cell line also inhibits delivery of hLAMP to endosomes, suggesting an evolutionarily conserved pathway in metazoa.     

The PI 3-kinase regulator Vps15 is required for autophagic clearance of protein aggregates

Autophagy is involved in cellular clearance of aggregate-prone proteins, thereby having a cytoprotective function. Studies in yeast have shown that the PI 3-kinase Vps34 and its regulatory protein kinase Vps15 are important for autophagy, but the possible involvement of these proteins in autophagy in a multicellular animal has not been addressed genetically. Here, we have created a Drosophila deletion mutant of vps15 and studied its role in autophagy and aggregate clearance. Homozygous Δvps15 Drosophila died at the early L3 larval stage. Using GFP-Atg8a as an autophagic marker, we employed fluorescence microscopy to demonstrate that fat bodies of wild type Drosophila larvae accumulated autophagic structures upon starvation whereas vps15 fat bodies showed no such response. Likewise, electron microscopy revealed starvation-induced autophagy in gut cells from wild type but not Δvps15 larvae. Fluorescence microscopy showed that Δvps15 mutant tissues accumulated profiles that were positive for ubiquitin and Ref(2)P, the Drosophila homolog of the sequestosome marker SQSTM1/p62. Biochemical fractionation and Western blotting showed that these structures were partially detergent insoluble, and immuno-electron microscopy further demonstrated the presence of Ref(2)P positive membrane free protein aggregates.. These results provide the first genetic evidence for a function of Vps15 in autophagy in multicellular organisms and suggest that the Vps15-containing PI 3-kinase complex may play an important role in clearance of protein aggregates.     

Relationship between growth arrest and autophagy in midgut programmed cell death in Drosophila

Autophagy has been implicated in both cell survival and programmed cell death (PCD), and this may explain the apparently complex role of this catabolic process in tumourigenesis. Our previous studies have shown that caspases have little influence on Drosophila larval midgut PCD, whereas inhibition of autophagy severely delays midgut removal. To assess upstream signals that regulate autophagy and larval midgut degradation, we have examined the requirement of growth signalling pathways. Inhibition of the class I phosphoinositide-3-kinase (PI3K) pathway prevents midgut growth, whereas ectopic PI3K and Ras signalling results in larger cells with decreased autophagy and delayed midgut degradation. Furthermore, premature induction of autophagy is sufficient to induce early midgut degradation. These data indicate that autophagy and the growth regulatory pathways have an important relationship during midgut PCD. Despite the roles of autophagy in both survival and death, our findings suggest that autophagy induction occurs in response to similar signals in both scenarios.     

Retromer Ensures the Degradation of Autophagic Cargo by Maintaining Lysosome Function in Drosophila

The retromer is an evolutionarily conserved coat complex that consists of Vps26, Vps29, Vps35 and a heterodimer of sorting nexin (Snx) proteins in yeast. Retromer mediates the recycling of transmembrane proteins from endosomes to the trans‐Golgi network, including receptors that are essential for the delivery of hydrolytic enzymes to lysosomes. Besides its function in lysosomal enzyme receptor recycling, involvement of retromer has also been proposed in a variety of vesicular trafficking events, including early steps of autophagy and endocytosis. Here we show that the late stages of autophagy and endocytosis are impaired in Vps26 and Vps35 deficient Drosophila larval fat body cells, but formation of autophagosomes and endosomes is not compromised. Accumulation of aberrant autolysosomes and amphisomes in the absence of retromer function appears to be the consequence of decreased degradative capacity, as they contain undigested cytoplasmic material. Accordingly, we show that retromer is required for proper cathepsin L trafficking mainly independent of LERP, the Drosophila homolog of the cation‐independent mannose 6‐phosphate receptor. Finally, we find that Snx3 and Snx6 are also required for proper autolysosomal degradation in Drosophila larval fat body cells.      

Vps15 is required for stress induced and developmentally triggered autophagy and salivary gland protein secretion in Drosophila

Autophagy is a catabolic process used to deliver cellular material to the lysosome for degradation. The core Vps34/class III phosphatidylinositol 3-kinase (PI3K) complex, consisting of Atg6, Vps15, and Vps34, is highly conserved throughout evolution, critical for recruiting autophagy-related proteins to the preautophagosomal structure and for other vesicular trafficking processes, including vacuolar protein sorting. Atg6 and Vps34 have been well characterized, but the Vps15 kinase remains poorly characterized with most studies focusing on nutrient deprivation-induced autophagy. Here, we investigate the function of Vps15 in different cellular contexts and find that it is necessary for both stress-induced and developmentally programmed autophagy in various tissues in Drosophila melanogaster. Vps15 is required for autophagy that is induced by multiple forms of stress, including nutrient deprivation, hypoxia, and oxidative stress. Furthermore, autophagy that is triggered by physiological stimuli during development in the fat body, intestine, and salivary gland also require the function of Vps15. In addition, we show that Vps15 is necessary for efficient salivary gland protein secretion. These data illustrate the broad importance of Vps15 in multiple forms of autophagy in different animal cells, and also highlight the pleiotropic function of this kinase in multiple vesicle-trafficking pathways.Autophagy is an evolutionarily conserved process in which cytoplasmic proteins or organelles are packaged into lysosomes for degradation. This process can be initiated by a variety of stimuli, such as high levels of starvation or stress, to provide nutrients to the cells or to clear the cell of damaged organelles or protein aggregates.¹ In some circumstances, autophagy can promote an alternative form of cell death, such as in the clearance of larval tissues in Drosophila melanogaster.² As defects in autophagy have been implicated in several physiological and pathological conditions, such as cancer, neurodegenerative diseases, and aging,^3,4 it is important to obtain a complete understanding of the molecular mechanisms controlling autophagy.The induction of autophagy is regulated by the Atg1/Ulk1 complex, and this complex is regulated by mechanistic target of rapamycin (mTOR).⁵ Vesicle nucleation is controlled by the class III phosphoinositide 3-kinase (PI3K) complex that generates phosphatidylinositol 3-phosphate (PI3P).⁶ This conserved complex consists of vacuolar protein sorting 34 (Vps34; also known as Pik3c3), Atg6/Becn1 (also known as Vps30 in yeast), and the serine-threonine kinase Vps15/ird1 (p150 in mammals; also known as Pik3r4).^7,8 Localized production of PI3P by Vps34 can act to recruit proteins containing PX or FYVE domains to membrane compartments, such as the autophagosome isolation membrane.⁹ Vps34 is also required more broadly for several vesicular trafficking processes such as the sorting of hydrolytic enzymes to the yeast vacuole and mammalian lysosome, and endocytic trafficking.^{10, 11, 12} There is mounting evidence demonstrating the pleiotropic function of the PI3K/Vps34 complex, but this has not been well studied in the context of autophagy under different physiological and cell contexts in animals.Of the three core PI3K complex proteins, Vps15 remains an understudied kinase, and its function has not been rigorously investigated in multicellular organisms in vivo. Most of the focus on the role of this complex in autophagy regulation has been on nutrient deprivation-initiated autophagy. Indeed, previous studies determined Vps15 to be necessary for starvation-induced autophagy in the Drosophila larval fat body.^13,14 However, its role in hormone-regulated autophagy, a process that occurs in the intestine,¹⁵ salivary glands,¹⁶ and fat body¹⁷ of developing Drosophila, as well as its role in other stress-induced conditions have not yet been examined. In order to address the role of Vps15 in these and other processes regulated by autophagy, we utilized Vps15 knockdown as well as a previously described null mutant¹⁴ to examine its role in a multicellular organism in vivo. We found that Vps15 is required not only for stress-induced autophagy in multiple tissues, but it is also a broad regulator of developmentally programmed autophagy in Drosophila. In addition, Vps15 is necessary for efficient protein secretion, as indicated by its role in the secretion of glue proteins from the Drosophila salivary gland. Together, these results highlight the importance of Vps15 in multiple processes in vivo.     

Regulation of Mammalian Autophagy by Class II and III PI 3-Kinases through PI3P Synthesis

Synthesis of phosphatidylinositol-3-phosphate (PI3P) by Vps34, a class III phosphatidylinositol 3-kinase (PI3K), is critical for the initial steps of autophagosome (AP) biogenesis. Although Vps34 is the sole source of PI3P in budding yeast, mammalian cells can produce PI3P through alternate pathways, including direct synthesis by the class II PI3Ks; however, the physiological relevance of these alternate pathways in the context of autophagy is unknown. Here we generated Vps34 knockout mouse embryonic fibroblasts (MEFs) and using a higher affinity 4x-FYVE finger PI3P-binding probe found a Vps34-independent pool of PI3P accounting for ^~35% of the total amount of this lipid species by biochemical analysis. Importantly, WIPI-1, an autophagy-relevant PI3P probe, still formed some puncta upon starvation-induced autophagy in Vps34 knockout MEFs. Additional characterization of autophagy by electron microscopy as well as protein degradation assays showed that while Vps34 is important for starvation-induced autophagy there is a significant component of functional autophagy occurring in the absence of Vps34. Given these findings, class II PI3Ks (α and β isoforms) were examined as potential positive regulators of autophagy. Depletion of class II PI3Ks reduced recruitment of WIPI-1 and LC3 to AP nucleation sites and caused an accumulation of the autophagy substrate, p62, which was exacerbated upon the concomitant ablation of Vps34. Our studies indicate that while Vps34 is the main PI3P source during autophagy, class II PI3Ks also significantly contribute to PI3P generation and regulate AP biogenesis.     

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, but the mechanisms of autophagy regulation and its role in TOR-mediated cell growth are unclear. Here, we show that 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.     

Distinct autophagosomal-lysosomal fusion mechanism revealed by thapsigargin-induced autophagy arrest

Autophagy, a catabolic pathway that delivers cellular components to lysosomes for degradation, can be activated by stressful conditions such as nutrient starvation and endoplasmic reticulum (ER) stress. We report that thapsigargin, an ER stressor widely used to induce autophagy, in fact blocks autophagy. Thapsigargin does not affect autophagosome formation but leads to accumulation of mature autophagosomes by blocking autophagosome fusion with the endocytic system. Strikingly, thapsigargin has no effect on endocytosis-mediated degradation of epidermal growth factor receptor. Molecularly, while both Rab7 and Vps16 are essential regulatory components for endocytic fusion with lysosomes, we found that Rab7 but not Vps16 is required for complete autophagy flux, and that thapsigargin blocks recruitment of Rab7 to autophagosomes. Therefore, autophagosomal-lysosomal fusion must be governed by a distinct molecular mechanism compared to general endocytic fusion.     

Vps34 and PLD1 take center stage in nutrient signaling: their dual roles in regulating autophagy

Autophagy is a critical pathway leading to lysosomal degradation of cellular components in response to changes in nutrient availability. Autophagy includes the biogenesis of autophagosomes and their sequential maturation through fusion with endo-lysosomes. The class III PI3 kinase Vps34 and its product phosphatidylinositol-3-phosphate (PI(3)P) play a critical role in this process, and enable the amino acid-mediated activation of mammalian target of rapamycin (mTOR), a suppressor of autophagy. Recent studies have shown that phospholipase PLD1, a downstream regulator of Vps34, is also closely involved in both mTOR activation and autophagy. This mini review summarizes recent findings in the regulation of Vps34 and PLD1 and highlights the role of these lipid-metabolizing enzymes in both mTOR activation and autophagy.     

The evolutionarily conserved domain of Beclin 1 is required for Vps34 binding, autophagy and tumor suppressor function

Atg6/Beclin 1 is an evolutionarily conserved protein family that has been shown to function in vacuolar protein sorting (VPS) in yeast; in autophagy in yeast, Drosophila, Dictyostelium, C.elegans, and mammals; and in tumor suppression in mice. Atg6/Beclin 1 is thought to function as a VPS and autophagy protein as part of a complex with Class III phosphatidylinositol 3'-kinase (PI3K)/Vps34. However, nothing is known about which domains of Atg6/Beclin 1 are required for its functional activity and binding to Vps34. We hypothesized that the most highly conserved region of human Beclin 1 spanning from amino acids 244-337 is essential for Vps34 binding, autophagy, and tumor suppressor function. To investigate this hypothesis, we evaluated the effects of wild-type and mutant beclin 1 gene transfer in autophagy-deficient MCF7 human breast carcinoma cells. We found that, unlike wild-type Beclin 1, a Beclin 1 mutant lacking aa 244-337 (Beclin 1DeltaECD), is unable to enhance starvation-induced autophagy in low Beclin 1-expressing MCF7 human breast carcinoma cells. In contrast to wild-type Beclin 1, mutant Beclin 1DeltaECD is unable to immunoprecipitate Vps34, has no Beclin 1-associated Vps34 kinase activity, and lacks tumor suppressor function in an MCF7 scid mouse xenograft tumor model. The maturation of cathepsin D, which requires intact Vps34-dependent VPS function, is comparable in autophagy-deficient low-Beclin 1 expressing MCF7 cells, autophagy-deficient MCF7 cells transfected with Beclin 1DeltaECD, and autophagy-competent MCF7 cells transfected with wild-type Beclin 1. These findings identify an evolutionarily conserved domain of Beclin 1 that is essential for Vps34 interaction, autophagy function, and tumor suppressor function. Furthermore, they suggest a connection between Beclin 1-associated Class III PI3K/Vps34-dependent autophagy, but not VPS, function and the mechanism of Beclin 1 tumor suppressor action in human breast cancer cells.     

Drosophila endosomal proteins hook and deep orange regulate synapse size but not synaptic vesicle recycling

To study the function of endosomes at synapses we analyzed the localization and function of two Drosophila endosomal proteins, Hook and Deep orange (Dor), at the larval neuromuscular junction. Hook, a negative regulator of endocytic trafficking, and Dor, a positive regulator of endocytic trafficking, are highly enriched at synapses, especially close to postsynaptic membranes. Mutations in hook (hk) and dor do not affect synaptic vesicle recycling, as assessed by electrophysiological analysis of synaptic transmission and behavioral studies of double mutants with shi(ts) mutations that alter vesicle recycling. However, hk and dor mutations alter the number of presynaptic varicosities (synapse size) in opposing ways. Synapse size is increased in hk(11) mutants and is decreased in dor(4) mutants. Double mutants for dor and hk show a dor-like phenotype. These effects on synapse size parallel known functions of Hook and Dor in endocytosis and strongly indicate a role for endocytic trafficking in the regulation of synapse size in vivo. Our observations suggest a model in which Hook and Dor function in later stages of endocytosis is essential for regulating synaptic plasma membrane composition but not synaptic vesicle recycling.     

Action of NF-kappaB on the delta opioid receptor gene promoter

The G protein-coupled delta opioid receptor gene (dor) is temporally and spatially expressed during development. The DOR receptor plays important roles in diverse biological processes, including pain control, immune functions, and cell survival. We previously found that PI3K/Akt/NF-kappaB signaling is important in the regulation of dor gene expression during nerve growth factor (NGF)-induced differentiation of PC12h cells, which prompted us to examine whether NF-kappaB p65 is directly or indirectly involved in the regulation of dor promoter activity. In this study, deletional and functional analysis of the dor promoter revealed a 94-bp NGF-responsive fragment upstream of the dor promoter region and involvement of NF-kappaB in regulating the promoter activity. Chromatin immunoprecipitation assays demonstrated that NF-kappaB p65 is directly bound to the dor promoter and such binding is related to NGF/PI3K signaling. Together, the results show that direct association of p65 with the promoter is important in NGF-induced dor promoter activity.     

CUP-5, the C. elegans ortholog of the mammalian lysosomal channel protein MLN1/TRPML1, is required for proteolytic degradation in autolysosomes

The process of macroautophagy (herein referred to as autophagy) involves the formation of a closed double-membrane structure, called the autophagosome, and its subsequent fusion with lysosomes to form an autolysosome. Lysosomes are regenerated from autolysosomes after degradation of the sequestrated materials. In this study, we showed that mutations in cup-5, encoding the C. elegans Mucolipin 1 homolog, cause defects in the autophagy pathway. In cup-5 mutants, a variety of autophagy substrates accumulate in enlarged vacuoles that display characteristics of late endosomes and lysosomes, indicating defective proteolytic degradation in autolysosomes. We further revealed that lysosomes in coelomocytes (scavenger cells located in the body cavity) are smaller in size and more numerous in mutants with loss of autophagy activity. Furthermore, the enlarged vacuole accumulation abnormality and embryonic lethality of cup-5 mutants are partially suppressed by reduced autophagy activity. Our results indicate that the basal constitutive level of autophagy activity regulates the size and number of lysosomes and provides insights into the molecular mechanisms underlying mucolipidosis type IV disease.