Selected papers from the international conference on reconfigurable computing and FPGAs (ReConFig'10)

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Condition-Dependent Functional Shift of Two Drosophila Mtmr Lipid Phosphatases in Autophagy Control

Myotubularin (MTM) and myotubularin-related (MTMR) lipid phosphatases catalyze the removal of a phosphate group from certain phosphatidylinositol derivatives. Because some of these substrates are required for macroautophagy/autophagy, during which unwanted cytoplasmic constituents are delivered into lysosomes for degradation, MTM and MTMRs function as important regulators of the autophagic process. Despite its physiological and medical significance, the specific role of individual MTMR paralogs in autophagy control remains largely unexplored. Here we examined two Drosophila MTMRs, EDTP and Mtmr6, the fly orthologs of mammalian MTMR14 and MTMR6 to MTMR8, respectively, and found that these enzymes affect the autophagic process in a complex, condition-dependent way. EDTP inhibited basal autophagy, but did not influence stress-induced autophagy. In contrast, Mtmr6 promoted the process under nutrient-rich settings, but effectively blocked its hyperactivation in response to stress. Thus, Mtmr6 is the first identified MTMR phosphatase with dual, antagonistic roles in the regulation of autophagy, and shows conditional antagonism/synergism with EDTP in modulating autophagic breakdown. These results provide a deeper insight into the adjustment of autophagy. Abbreviations: Atg, autophagy-related; BDSC, Bloomington Drosophila Stock Center; DGRC, Drosophila Genetic Resource Center; EDTP, Egg-derived tyrosine phosphatase; FYVE, zinc finger domain from Fab1 (yeast ortholog of PIKfyve), YOTB, Vac1 (vesicle transport protein) and EEA1 cysteine-rich proteins; LTR, LysoTracker Red; MTM, myotubularin; MTMR, myotubularin-related; PI, phosphatidylinositol; Pi3K59F, Phosphotidylinositol 3 kinase 59F; PtdIns3P, phosphatidylinositol-3-phosphate; PtdIns(3,5)P(2), phosphatidylinositol-3,5-bisphosphate; PtdIns5P, phosphatidylinositol-5-phosphate; ref(2)P, refractory to sigma P; Syx17, Syntaxin 17; TEM, transmission electron microscopy; UAS, upstream activating sequence; Uvrag, UV-resistance associated gene; VDRC, Vienna Drosophila RNAi Center; Vps34, Vacuolar protein sorting 34

Developmentally regulated autophagy is required for eye formation in Drosophila

The compound eye of the fruit fly Drosophila melanogaster is one of the most intensively studied and best understood model organs in the field of developmental genetics. Herein we demonstrate that autophagy, an evolutionarily conserved selfdegradation process of eukaryotic cells, is essential for eye development in this organism. Autophagic structures accumulate in a specific pattern in the developing eye disc, predominantly in the morphogenetic furrow (MF) and differentiation zone. Silencing of several autophagy genes (Atg) in the eye primordium severely affects the morphology of the adult eye through triggering ectopic cell death. In Atg mutant genetic backgrounds however genetic compensatory mechanisms largely rescue autophagic activity in, and thereby normal morphogenesis of, this organ. We also show that in the eye disc the expression of a key autophagy gene, Atg8a, is controlled in a complex manner by the anterior Hox paralog lab (labial), a master regulator of early development. Atg8a transcription is repressed in front of, while activated along, the MF by lab. The amount of autophagic structures then remains elevated behind the moving MF. These results indicate that eye development in Drosophila depends on the cell death-suppressing and differentiating effects of the autophagic process. This novel, developmentally regulated function of autophagy in the morphogenesis of the compound eye may shed light on a more fundamental role for cellular self-digestion in differentiation and organ formation than previously thought

Specialized Cortex Glial Cells Accumulate Lipid Droplets in Drosophila melanogaster.

Lipid droplets (LDs) are common organelles of the majority of eukaryotic cell types. Their biological significance has been extensively studied in mammalian liver cells and white adipose tissue. Although the central nervous system contains the highest relative amount and the largest number of different lipid species, neither the spatial nor the temporal distribution of LDs has been described. In this study, we used the brain of the fruitfly, Drosophila melanogaster, to investigate the neuroanatomy of LDs. We demonstrated that LDs are exclusively localised in glial cells but not in neurons in the larval nervous system. We showed that the brain's LD pool, rather than being constant, changes dynamically during development and reaches its highest value at the beginning of metamorphosis. LDs are particularly enriched in cortex glial cells located close to the brain surface. These specialized superficial cortex glial cells contain the highest amount of LDs among glial cell types and encapsulate neuroblasts and their daughter cells. Superficial cortex glial cells, combined with subperineurial glial cells, express the Drosophila fatty acid binding protein (Dfabp), as we have demonstrated through light- and electron microscopic immunocytochemistry. To the best of our best knowledge this is the first study that describes LD neuroanatomy in the Drosophila larval brain

Atg6/UVRAG/Vps34-Containing Lipid Kinase Complex Is Required for Receptor Downregulation through Endolysosomal Degradation and Epithelial Polarity during Drosophila Wing Development

Atg6 (Beclin 1 in mammals) is a core component of the Vps34 PI3K (III) complex, which promotes multiple vesicle trafficking pathways. Atg6 and Vps34 form two distinct PI3K (III) complexes in yeast and mammalian cells, either with Atg14 or with UVRAG. The functions of these two complexes are not entirely clear, as both Atg14 and UVRAG have been suggested to regulate both endocytosis and autophagy. In this study, we performed a microscopic analysis of UVRAG, Atg14, or Atg6 loss-of-function cells in the developing Drosophila wing. Both autophagy and endocytosis are seriously impaired and defective endolysosomes accumulate upon loss of Atg6. We show that Atg6 is required for the downregulation of Notch and Wingless signaling pathways; thus it is essential for normal wing development. Moreover, the loss of Atg6 impairs cell polarity. Atg14 depletion results in autophagy defects with no effect on endocytosis or cell polarity, while the silencing of UVRAG phenocopies all but the autophagy defect of Atg6 depleted cells. Thus, our results indicate that the UVRAG-containing PI3K (III) complex is required for receptor downregulation through endolysosomal degradation and for the establishment of proper cell polarity in the developing wing, while the Atg14-containing complex is involved in autophagosome formation

Schematic illustration of the relative distribution of lipid droplets between glial cell types and the localization of Dfabp.

<p>LDs are concentrated in large clusters in the perinuclear region of glial cells but are not present at all in neurons (N). Specialized superficial cortex glial cells (CG) insulating neuroblasts (NB) and their daughter cells (asterisks) accumulating the highest amount of LDs. Neighboring subperineurial cells (SPG) establish septate junctions (SJ), while SPG and superficial cortex glial cells are connected to each other through adherens junctions (AJ). The Drosophila fatty acid binding protein (Dfabp) is expressed in LD accumulating superficial cortex glial cells and subperineurial (SPG) cells, and is localized in the cytosol and in the nucleus. NL: neural lamella, PG: perineural glia, NG: neuropil glia, ax: axon.</p

Cell type-specific distribution of LDs.

<p>(A) Single cell clones of different glial cell types. Glial cell membranes are higlighted by myr-RFP (red) and LDs are highlighted by Lsd2-GFP (green). (B) Higher magnification image of a superficial cortex glia shown in panel A. (C) A superficial cortex glial clone (myr-RFP, red) on a pan-cortex glial cell labeled background (Nrv2-GFP, green). Note the similar morphology of the labeled glia as shown in panel B. (D) Pattern of LDs in the larval brain, visualized by ectopically expressed Lsd2-GFP shows similar distribution as the cortex glial specific GFP signal. (E) Box plot representing LD per cell area ratios from particular glial cell types. Scalebar: (A,B,C) 20 μm (D) 100 μm</p

Lipid droplets in the <i>Drosophila</i> brain.

<p>(A-E) Pictures taken from third instar larval brains. (A) Oil Red O staining, note an intense staining in the dorso-medial part of the central brain. (B) Toluidine blue-stained semithin section from the brain cortex. LDs (green) are organized in large clusters between neurons. (C) Pseudocolored electron micrograph taken from the brain cortex. LDs (green) are found in the cytoplasm of glial cells (G, red) but not in neurons (N). (D) High power electron micrograph from the perinuclear region of a cortex glia (G) LDs are rounded electron-opaque structures, delineated by a phospholipid monolayer (inset, arrow). N: neuron. (E) LDs (brown) are generally found in the closest vicinity of neuroblasts (asterisks). Unstained semithin section. (F) Diagram representing the distribution of LDs between neurons and glial cells. (G) Number of LDs in order of their distance from neuroblasts. (H) Time-course changes in the amount of accumulated LDs per brain tissue area ratio. Standard deviations are indicated. Scalebars: (A) 100 μm (B) 10 μm (C) 1μm, (D) 500nm, inset: 200nm, (E) 10 μm.</p

Ultrastructural localisation of Dfabp.

<p>Post-embedding silver-intensified immunogold labeling for Dfabp on freeze-substituted LR White-embedded material. An intense labeling can be seen over subperineurial (SPG) and superficial cortex (CG) glial cells. Note the absence of labeling over perineurial glia (PG), neurons (N), or neuroblasts (NB). Arrows: lipid droplets, arrowheads: mitochondria. Scalebar: 1 μm</p

Representative electron micrographs of particular glial cell types.

<p>LDs are marked by arrows. (A) Perineurial (PG) and subperineurial cells (SPG) located on the surface of the brain. (B) A superficial glial cell located in the outer layer of the brain cortex, containing high amount of LDs. (C) A neuropil glia (NP) located at the cortex-neuropil boundary ensheating axons. (D) A deeper cortex glia (CG) found close to the cortex-neuropil boundary encapsulating a large peptiderg neuron (PN) and several other neurons (N) with its processes. No LDs seen in the cytoplasm of such a cortex glia. Note the presence of large clusters of neurosecretory vesicles (arrowheads) in the cytoplasm of PN. Scalebar: 2μm.</p