The GATOR2 complex maintains lysosomal-autophagic function by inhibiting the protein degradation of MiT/TFEs.

Lysosomes are central to metabolic homeostasis. The microphthalmia bHLH-LZ transcription factors (MiT/TFEs) family members MITF, TFEB, and TFE3 promote the transcription of lysosomal and autophagic genes and are often deregulated in cancer. Here, we show that the GATOR2 complex, an activator of the metabolic regulator TORC1, maintains lysosomal function by protecting MiT/TFEs from proteasomal degradation independent of TORC1, GATOR1, and the RAG GTPase. We determine that in GATOR2 knockout HeLa cells, members of the MiT/TFEs family are ubiquitylated by a trio of E3 ligases and are degraded, resulting in lysosome dysfunction. Additionally, we demonstrate that GATOR2 protects MiT/TFE proteins in pancreatic ductal adenocarcinoma and Xp11 translocation renal cell carcinoma, two cancers that are driven by MiT/TFE hyperactivation. In summary, we find that the GATOR2 complex has independent roles in TORC1 regulation and MiT/TFE protein protection and thus is central to coordinating cellular metabolism with control of the lysosomal-autophagic system.

Wdr59 promotes or inhibits TORC1 activity depending on cellular context.

Target of Rapamycin Complex I (TORC1) is a central regulator of metabolism in eukaryotes that responds to a wide array of negative and positive inputs. The GTPase-activating protein toward Rags (GATOR) signaling pathway acts upstream of TORC1 and is comprised of two subcomplexes. The trimeric GATOR1 complex inhibits TORC1 activity in response to amino acid limitation by serving as a GTPase-activating protein (GAP) for the TORC1 activator RagA/B, a component of the lysosomally located Rag GTPase. The multi-protein GATOR2 complex inhibits the activity of GATOR1 and thus promotes TORC1 activation. Here we report that Wdr59, originally assigned to the GATOR2 complex based on studies performed in tissue culture cells, unexpectedly has a dual function in TORC1 regulation in Drosophila. We find that in the ovary and the eye imaginal disc brain complex, Wdr59 inhibits TORC1 activity by opposing the GATOR2-dependent inhibition of GATOR1. Conversely, in the Drosophila fat body, Wdr59 promotes the accumulation of the GATOR2 component Mio and is required for TORC1 activation. Similarly, in mammalian HeLa cells, Wdr59 prevents the proteolytic destruction of GATOR2 proteins Mio and Wdr24. Consistent with the reduced levels of the TORC1-activating GATOR2 complex, Wdr59KOs HeLa cells have reduced TORC1 activity which is restored along with GATOR2 protein levels upon proteasome inhibition. Taken together, our data support the model that the Wdr59 component of the GATOR2 complex functions to promote or inhibit TORC1 activity depending on cellular context.

Wiring dendrites in layers and columns.

The most striking structure in the nervous system is the complex yet stereotyped morphology of the neuronal dendritic tree. Dendritic morphologies and the connections they make govern information flow and integration in the brain. The fundamental mechanisms that regulate dendritic outgrowth and branching are subjects of extensive study. In this review, we summarize recent advances in the molecular and cellular mechanisms for routing dendrites in layers and columns, prevalent organizational structures in the brain. We highlight how dendritic patterning influences the formation of synaptic circuits.

Multiple redundant medulla projection neurons mediate color vision in Drosophila.

The receptor mechanism for color vision has been extensively studied. In contrast, the circuit(s) that transform(s) photoreceptor signals into color percepts to guide behavior remain(s) poorly characterized. Using intersectional genetics to inactivate identified subsets of neurons, we have uncovered the first-order interneurons that are functionally required for hue discrimination in Drosophila. We developed a novel aversive operant conditioning assay for intensity-independent color discrimination (true color vision) in Drosophila. Single flying flies are magnetically tethered in an arena surrounded by blue and green LEDs (light-emitting diodes). The flies' optomotor response is used to determine the blue-green isoluminant intensity. Flies are then conditioned to discriminate between equiluminant blue or green stimuli. Wild-type flies are successfully trained in this paradigm when conditioned to avoid either blue or green. Functional color entrainment requires the function of the narrow-spectrum photoreceptors R8 and/or R7, and is within a limited range, intensity independent, suggesting that it is mediated by a color vision system. The medulla projection neurons, Tm5a/b/c and Tm20, receive direct inputs from R7 or R8 photoreceptors and indirect input from the broad-spectrum photoreceptors R1-R6 via the lamina neuron L3. Genetically inactivating these four classes of medulla projection neurons abolished color learning. However, inactivation of subsets of these neurons is insufficient to block color learning, suggesting that true color vision is mediated by multiple redundant pathways. We hypothesize that flies represent color along multiple axes at the first synapse in the fly visual system. The apparent redundancy in learned color discrimination sharply contrasts with innate ultraviolet (UV) spectral preference, which is dominated by a single pathway from the amacrine neuron Dm8 to the Tm5c projection neurons.

Conserved alternative splicing and expression patterns of arthropod N-cadherin.

Metazoan development requires complex mechanisms to generate cells with diverse function. Alternative splicing of pre-mRNA not only expands proteomic diversity but also provides a means to regulate tissue-specific molecular expression. The N-Cadherin gene in Drosophila contains three pairs of mutually-exclusive alternatively-spliced exons (MEs). However, no significant differences among the resulting protein isoforms have been successfully demonstrated in vivo. Furthermore, while the N-Cadherin gene products exhibit a complex spatiotemporal expression pattern within embryos, its underlying mechanisms and significance remain unknown. Here, we present results that suggest a critical role for alternative splicing in producing a crucial and reproducible complexity in the expression pattern of arthropod N-Cadherin. We demonstrate that the arthropod N-Cadherin gene has maintained the three sets of MEs for over 400 million years using in silico and in vivo approaches. Expression of isoforms derived from these MEs receives precise spatiotemporal control critical during development. Both Drosophila and Tribolium use ME-13a and ME-13b in "neural" and "mesodermal" splice variants, respectively. As proteins, either ME-13a- or ME-13b-containing isoform can cell-autonomously rescue the embryonic lethality caused by genetic loss of N-Cadherin. Ectopic muscle expression of either isoform beyond the time it normally ceases leads to paralysis and lethality. Together, our results offer an example of well-conserved alternative splicing increasing cellular diversity in metazoans.

Dissection of the peripheral motion channel in the visual system of Drosophila melanogaster.

In the eye, visual information is segregated into modalities such as color and motion, these being transferred to the central brain through separate channels. Here, we genetically dissect the achromatic motion channel in the fly Drosophila melanogaster at the level of the first relay station in the brain, the lamina, where it is split into four parallel pathways (L1-L3, amc/T1). The functional relevance of this divergence is little understood. We now show that the two most prominent pathways, L1 and L2, together are necessary and largely sufficient for motion-dependent behavior. At high pattern contrast, the two pathways are redundant. At intermediate contrast, they mediate motion stimuli of opposite polarity, L2 front-to-back, L1 back-to-front motion. At low contrast, L1 and L2 depend upon each other for motion processing. Of the two minor pathways, amc/T1 specifically enhances the L1 pathway at intermediate contrast. L3 appears not to contribute to motion but to orientation behavior.

Tiling of r7 axons in the Drosophila visual system is mediated both by transduction of an activin signal to the nucleus and by mutual repulsion.

The organization of neuronal wiring into layers and columns is a common feature of both vertebrate and invertebrate brains. In the Drosophila visual system, each R7 photoreceptor axon projects within a single column to a specific layer of the optic lobe. We refer to the restriction of terminals to single columns as tiling. In a genetic screen based on an R7-dependent behavior, we identified the Activin receptor Baboon and the nuclear import adaptor Importin-alpha3 as being required to prevent R7 axon terminals from overlapping with the terminals of R7s in neighboring columns. This tiling function requires the Baboon ligand, dActivin, the transcription factor, dSmad2, and retrograde transport from the growth cone to the R7 nucleus. We propose that dActivin is an autocrine signal that restricts R7 growth cone motility, and we demonstrate that it acts in parallel with a paracrine signal that mediates repulsion between R7 terminals.

Neural mechanism of spatio-chromatic opponency in the Drosophila amacrine neurons.

Visual animals detect spatial variations of light intensity and wavelength composition. Opponent coding is a common strategy for reducing information redundancy. Neurons equipped with both spatial and spectral opponency have been identified in vertebrates but not yet in insects. The Drosophila amacrine neuron Dm8 was recently reported to show color opponency. Here, we demonstrate Dm8 exhibits spatio-chromatic opponency. Antagonistic convergence of the direct input from the UV-sensing R7s and indirect input from the broadband receptors R1-R6 through Tm3 and Mi1 is sufficient to confer Dm8's UV/Vis (ultraviolet/visible light) opponency. Using high resolution monochromatic stimuli, we show the pale and yellow subtypes of Dm8s, inheriting retinal mosaic characteristics, have distinct spectral tuning properties. Using 2D white-noise stimulus and reverse correlation analysis, we found that the UV receptive field (RF) of Dm8 has a center-inhibition/surround-excitation structure. In the absence of UV-sensing R7 inputs, the polarity of the RF is inverted owing to the excitatory input from the broadband photoreceptors R1-R6. Using a new synGRASP method based on endogenous neurotransmitter receptors, we show that neighboring Dm8s form mutual inhibitory connections mediated by the glutamate-gated chloride channel GluClα, which is essential for both Dm8's spatial opponency and animals' phototactic behavior. Our study shows spatio-chromatic opponency could arise in the early visual stage, suggesting a common information processing strategy in both invertebrates and vertebrates.

Antagonistic regulation by insulin-like peptide and activin ensures the elaboration of appropriate dendritic field sizes of amacrine neurons.

Establishing appropriate sizes and shapes of dendritic arbors is critical for proper wiring of the central nervous system. Here we report that Insulin-like Peptide 2 (DILP2) locally activates transiently expressed insulin receptors in the central dendrites of Drosophila Dm8 amacrine neurons to positively regulate dendritic field elaboration. We found DILP2 was expressed in L5 lamina neurons, which have axonal terminals abutting Dm8 dendrites. Proper Dm8 dendrite morphogenesis and synapse formation required insulin signaling through TOR (target of rapamycin) and SREBP (sterol regulatory element-binding protein), acting in parallel with previously identified negative regulation by Activin signaling to provide robust control of Dm8 dendrite elaboration. A simulation of dendritic growth revealed trade-offs between dendritic field size and robustness when branching and terminating kinetic parameters were constant, but dynamic modulation of the parameters could mitigate these trade-offs. We suggest that antagonistic DILP2 and Activin signals from different afferents appropriately size Dm8 dendritic fields.

The Rag GTPase Regulates the Dynamic Behavior of TSC Downstream of Both Amino Acid and Growth Factor Restriction.

The dysregulation of the metabolic regulator TOR complex I (TORC1) contributes to a wide array of human pathologies. Tuberous sclerosis complex (TSC) is a potent inhibitor of TORC1. Here, we demonstrate that the Rag GTPase acts in both the amino-acid-sensing and growth factor signaling pathways to control TORC1 activity through the regulation of TSC dynamics in HeLa cells and Drosophila. We find that TSC lysosomal-cytosolic exchange increases in response to both amino acid and growth factor restriction. Moreover, the rate of exchange mirrors TSC function, with depletions of the Rag GTPase blocking TSC lysosomal mobility and rescuing TORC1 activity. Finally, we show that the GATOR2 complex controls the phosphorylation of TSC2, which is essential for TSC exchange. Our data support the model that the amino acid and growth factor signaling pathways converge on the Rag GTPase to inhibit TORC1 activity through the regulation of TSC dynamics.

Visual circuit development in Drosophila.

Fly visual circuits are organized into lattice-like arrays and layers. Recent genetic studies have provided insights into how these reiterated structures are assembled through stepwise processes and how precise connections are established during development. Afferent-derived morphogens, such as Hedgehog, play a key role in organizing the overall structure by inducing and recruiting target neurons and glia. In turn, the target-derived ligand DWnt4 guides Frizzled2-expressing photoreceptor afferents to their proper destination. Photoreceptor afferents select specific synaptic targets by forming adhesive interactions and regulating actin cytoskeleton in growth cones. Target specificity is probably achieved by restricting the expression of adhesive molecules, such as Capricious, to appropriate presynaptic and postsynaptic partners, and by differentially regulating the function of broadly expressed adhesive molecules such as N-cadherin.

From form to function: the ways to know a neuron.

The shape of a neuron, its morphological signature, dictates the neuron's function by establishing its synaptic partnerships. Here, we review various anatomical methods used to reveal neuron shape and the contributions these have made to our current understanding of neural function in the Drosophila brain, especially the optic lobe. These methods, including Golgi impregnation, genetic reporters, and electron microscopy (EM), necessarily incorporate biases of various sorts that are easy to overlook, but that filter the morphological signatures we see. Nonetheless, the application of these methods to the optic lobe has led to reassuringly congruent findings on the number and shapes of neurons and their connection patterns, indicating that morphological classes are actually genetic classes. Genetic methods using, especially, GAL4 drivers and associated reporters have largely superceded classical Golgi methods for cellular analyses and, moreover, allow the manipulation of neuronal activity, thus enabling us to establish a bridge between morphological studies and functional ones. While serial-EM reconstruction remains the only reliable, albeit labor-intensive, method to determine actual synaptic connections, genetic approaches in combination with EM or high-resolution light microscopic techniques are promising methods for the rapid determination of synaptic circuit function.

The variable transmembrane domain of Drosophila N-cadherin regulates adhesive activity.

Drosophila N-cadherin (CadN) is an evolutionarily conserved classic cadherin which has a large, complex extracellular domain and a catenin-binding cytoplasmic domain. The CadN locus contains three modules of alternative exons (7a/b, 13a/b, and 18a/b) and undergoes alternative splicing to generate multiple isoforms. Using quantitative transcript analyses and green fluorescent protein-based cell sorting, we found that during development CadN alternative splicing is regulated in a temporal but not cell-type-specific fashion. In particular, exon 18b is predominantly expressed during early developmental stages, while exon 18a is prevalent at the late developmental and adult stages. All CadN isoforms share the same molecular architecture but have different sequences in their extracellular and transmembrane domains, suggesting functional diversity. In vitro quantitative cell aggregation assays revealed that all CadN isoforms mediate homophilic interactions, but the isoforms encoded by exon 18b have a higher adhesive activity than those by its alternative, 18a. Domain-swapping experiments further revealed that the different sequences in the transmembrane domains of isoforms are responsible for their differential adhesive activities. CadN alternative splicing might provide a novel mechanism to fine-tune its adhesive activity at different developmental stages or to restrict the use of high-affinity 18b-type isoforms at the adult stage.

The GATOR complex regulates an essential response to meiotic double-stranded breaks in Drosophila.

The TORC1 regulator GATOR1/SEACIT controls meiotic entry and early meiotic events in yeast. However, how metabolic pathways influence meiotic progression in metazoans remains poorly understood. Here we examine the role of the TORC1 regulators GATOR1 and GATOR2 in the response to meiotic double-stranded breaks (DSB) during Drosophila oogenesis. We find that in mutants of the GATOR2 component mio, meiotic DSBs trigger the constitutive downregulation of TORC1 activity and a permanent arrest in oocyte growth. Conversely, in GATOR1 mutants, high TORC1 activity results in the delayed repair of meiotic DSBs and the hyperactivation of p53. Unexpectedly, we found that GATOR1 inhibits retrotransposon expression in the presence of meiotic DSBs in a pathway that functions in parallel to p53. Thus, our studies have revealed a link between oocyte metabolism, the repair of meiotic DSBs and retrotransposon expression.

Sumoylation of p45/NF-E2: nuclear positioning and transcriptional activation of the mammalian beta-like globin gene locus.

NF-E2 is a transcription activator for the regulation of a number of erythroid- and megakaryocytic lineage-specific genes. Here we present evidence that the large subunit of mammalian NF-E2, p45, is sumoylated in vivo in human erythroid K562 cells and in mouse fetal liver. By in vitro sumoylation reaction and DNA transfection experiments, we show that the sumoylation occurs at lysine 368 (K368) of human p45/NF-E2. Furthermore, p45 sumoylation enhances the transactivation capability of NF-E2, and this is accompanied by an increase of the NF-E2 DNA binding affinity. More interestingly, we have found that in K562 cells, the beta-globin gene loci in the euchromatin regions are predominantly colocalized with the nuclear bodies promyelocytic leukemia protein (PML) oncogenic domains that are enriched with the PML, SUMO-1, RNA polymerase II, and sumoylatable p45/NF-E2. Chromatin immunoprecipitation assays further showed that the intact sumoylation site of p45/NF-E2 is required for its binding to the DNase I-hypersensitive sites of the beta-globin locus control region. Finally, we demonstrated by stable transfection assay that only the wild-type p45, but not its mutant form p45 (K368R), could efficiently rescue beta-globin gene expression in the p45-null, erythroid cell line CB3. These data together point to a model of mammalian beta-like globin gene activation by sumoylated p45/NF-E2 in erythroid cells.

Analyzing Dendritic Morphology in Columns and Layers.

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Mapping chromatic pathways in the Drosophila visual system.

In Drosophila, color vision and wavelength-selective behaviors are mediated by the compound eye's narrow-spectrum photoreceptors R7 and R8 and their downstream medulla projection (Tm) neurons Tm5a, Tm5b, Tm5c, and Tm20 in the second optic neuropil or medulla. These chromatic Tm neurons project axons to a deeper optic neuropil, the lobula, which in insects has been implicated in processing and relaying color information to the central brain. The synaptic targets of the chromatic Tm neurons in the lobula are not known, however. Using a modified GFP reconstitution across synaptic partners (GRASP) method to probe connections between the chromatic Tm neurons and 28 known and novel types of lobula neurons, we identify anatomically the visual projection neurons LT11 and LC14 and the lobula intrinsic neurons Li3 and Li4 as synaptic targets of the chromatic Tm neurons. Single-cell GRASP analyses reveal that Li4 receives synaptic contacts from over 90% of all four types of chromatic Tm neurons, whereas LT11 is postsynaptic to the chromatic Tm neurons, with only modest selectivity and at a lower frequency and density. To visualize synaptic contacts at the ultrastructural level, we develop and apply a "two-tag" double-labeling method to label LT11's dendrites and the mitochondria in Tm5c's presynaptic terminals. Serial electron microscopic reconstruction confirms that LT11 receives direct contacts from Tm5c. This method would be generally applicable to map the connections of large complex neurons in Drosophila and other animals.

Isodiospyrin as a novel human DNA topoisomerase I inhibitor.

Isodiospyrin is a natural product from the plant Diospyros morrisiana, which consists of an asymmetrical 1,2-binaphthoquinone chromophore. Isodiospyrin exhibits cytotoxic activity to tumor cell lines but very little is known about its cellular target and mechanism of action. Unlike the prototypic human topoisomerase I (htopo I) poison camptothecin, isodiospyrin does not induce htopo I-DNA covalent complexes. However, isodiospyrin antagonizes camptothecin-induced, htopo I-mediated DNA cleavage. Binding analysis indicated that isodiospyrin binds htopo I but not DNA. These results suggest that isodiospyrin inhibits htopo I by direct binding to htopo I, which limits htopo I access to the DNA substrate. Furthermore, isodiospyrin exhibits strong inhibitory effect on the kinase activity of htopo I toward splicing factor 2/alternate splicing factor in the absence of DNA. Thus, these findings have important implications on naphthoquinone and its derivatives' cellular mode of actions, i.e. these novel DNA topoisomerase I inhibitors can prevent both DNA relaxation and kinase activities of htopo I.

Photoreceptor-derived activin promotes dendritic termination and restricts the receptive fields of first-order interneurons in Drosophila.

How neurons form appropriately sized dendritic fields to encounter their presynaptic partners is poorly understood. The Drosophila medulla is organized in layers and columns and innervated by medulla neuron dendrites and photoreceptor axons. Here, we show that three types of medulla projection (Tm) neurons extend their dendrites in stereotyped directions and to distinct layers within a single column for processing retinotopic information. In contrast, the Dm8 amacrine neurons form a wide dendritic field to receive ∼16 R7 photoreceptor inputs. R7- and R8-derived Activin selectively restricts the dendritic fields of their respective postsynaptic partners, Dm8 and Tm20, to the size appropriate for their functions. Canonical Activin signaling promotes dendritic termination without affecting dendritic routing direction or layer. Tm20 neurons lacking Activin signaling expanded their dendritic fields and aberrantly synapsed with neighboring photoreceptors. We suggest that afferent-derived Activin regulates the dendritic field size of their postsynaptic partners to ensure appropriate synaptic partnership.

A hard-wired glutamatergic circuit pools and relays UV signals to mediate spectral preference in Drosophila.

Many visual animals have innate preferences for particular wavelengths of light, which can be modified by learning. Drosophila's preference for UV over visible light requires UV-sensing R7 photoreceptors and specific wide-field amacrine neurons called Dm8. Here we identify three types of medulla projection neurons downstream of R7 and Dm8 and show that selectively inactivating one of them (Tm5c) abolishes UV preference. Using a modified GRASP method to probe synaptic connections at the single-cell level, we reveal that each Dm8 neuron forms multiple synaptic contacts with Tm5c in the center of Dm8's dendritic field but sparse connections in the periphery. By single-cell transcript profiling and RNAi-mediated knockdown, we determine that Tm5c uses the kainate receptor Clumsy to receive excitatory glutamate input from Dm8. We conclude that R7s→Dm8→Tm5c form a hard-wired glutamatergic circuit that mediates UV preference by pooling ∼16 R7 signals for transfer to the lobula, a higher visual center.