Biology subjects

Wappner, P.

Publications and source records attributed to Wappner, P..

6 recordsLinked to original sources

bioRxiv2024

Autophagy controls differentiation of Drosophila blood cells by regulating Notch levels in response to nutrient availability

Drosophila larval hematopoiesis takes place at the lymph gland, where blood cell progenitors differentiate into two possible cell types: plasmatocytes, analogous to mammalian macrophages, or crystal cells that share features with mammalian megakaryocytes; a third cell type, the lamellocytes, can develop only upon specific immune challenges. In this work, we investigate the role of autophagy in Drosophila hematopoiesis. We found that autophagy inhibition in blood cell progenitors results in augmented crystal cell differentiation due to accumulation of high levels of Notch protein. Notch activation during hematopoiesis depends on the endocytic pathway, which cross-talks with autophagy: While endocytosis and endosomal maturation are essential for Notch activation, autophagosomes are required for Notch lysosomal degradation. TOR signaling inhibits autophagosome biogenesis, which in turn prevents the formation of Notch-containing amphisomes, being the latter necessary for Notch lysosomal destruction. Reduction of Notch lysosomal degradation shifts the balance towards Notch activation at late endosomal membranes, thereby enhancing differentiation of crystal cells. Our work defines a novel mechanism of regulation of immune cell differentiation in response to the nutritional status of the organism: High nutrient availability induces TOR activation, thereby inhibiting autophagy, hindering lysosomal degradation of Notch, and promoting crystal cell differentiation.

developmental biology↗

bioRxiv2023

The exocyst complex controls multiple events in the pathway of regulated exocytosis.

Eukaryotic cells depend on exocytosis to direct intracellularly synthesized material towards the extracellular space or the plasma membrane, so exocytosis constitutes a basic function for cellular homeostasis and communication between cells. The secretory pathway includes biogenesis of secretory granules (SGs), their maturation and fusion with the plasma membrane (exocytosis), resulting in release of SG content to the extracellular space. The larval salivary gland of Drosophila melanogaster is an excellent model for studying exocytosis. This gland synthesizes mucins that are packaged in SGs that sprout from the trans-Golgi network and then undergo a maturation process that involves homotypic fusion, condensation and acidification. Finally, mature SGs are directed to the apical domain of the plasma membrane with which they fuse, releasing their content into the gland lumen. The exocyst is a hetero-octameric complex that participates in tethering of vesicles to the plasma membrane during constitutive exocytosis. By precise temperature-dependent gradual activation of the Gal4-UAS expression system, we have induced different levels of silencing of exocyst complex subunits, and identified three temporarily distinctive steps of the regulated exocytic pathway where the exocyst is critically required: SG biogenesis, SG maturation and SG exocytosis. Our results shed light on previously unidentified functions of the exocyst along the exocytic pathway. We propose that the exocyst acts as a general tethering factor in various steps of this cellular process. O_FIG O_LINKSMALLFIG WIDTH=124 HEIGHT=200 SRC="FIGDIR/small/555734v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@4057f5org.highwire.dtl.DTLVardef@4ed694org.highwire.dtl.DTLVardef@1e9a859org.highwire.dtl.DTLVardef@12b971f_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract.C_FLOATNO Proposed model of the action of the exocyst in maintenance of normal Golgi complex structure, maturation and exocytosis of secretory granules in Drosophila larval salivary gland cells. 1-Before secretory granule (SG) biogenesis (<96 hours AEL), the exocyst (pink dots) localizes at the Golgi complex, where it is required to maintain the normal Golgi structure. The mucine Sgs3 (brown dots) moves through the secretory pathway from the endoplasmic reticulum (ER) to the Golgi complex, from where immature SGs containing the mucine sprout out. 2-After sprouting, SGs undergo maturation (96-116 hours AEL). During maturation, the exocyst localizes in between immature SGs, where it is required for homotypic fusion. The exocyst is also required for incorporation of maturation factors to the membrane of SGs. These maturation factors include Syt-1 (purple line), DC63 (orange line), Rab11 (green oval) and Rab1 (light blue oval). At this stage, the exocyst no longer localizes at the Golgi complex. 3-When maturation has been completed SGs fuse with the apical plasma membrane and exocytosis takes place. During exocytosis (116-120 hours AEL), the exocyst localizes at mature SGs, in contact with the apical plasma membrane, where it is required for tethering and subsequent fusion, prior to release of the SG content to the salivary gland lumen. C_FIG

cell biology↗

bioRxiv2023

Metabolic FRET sensors in intact organs: Applying spectral unmixing to acquire reliable signals

In multicellular organisms, metabolic coordination across multiple tissues and cell types is essential to satisfy regionalized energetic requirements and respond coherently to changing environmental conditions. However, most metabolic assays require the destruction of the biological sample, with a concomitant loss of spatial information. Fluorescent metabolic sensors and probes are among the most user-friendly techniques for collecting metabolic information with spatial resolution. In a previous work, we have adapted to an animal system, Drosophila melanogaster, genetically encoded metabolic FRET-based sensors that had been previously developed in single-cell systems. These sensors provide semi-quantitative data on the stationary concentrations of key metabolites of the bioenergetic metabolism: lactate, pyruvate, and 2-oxoglutarate. The use of these sensors in intact organs required the development of an image processing method that minimizes the contribution of spatially complex autofluorescence patterns, that would obscure the FRET signals. In this article, we describe the fundamentals of intramolecular hetero-FRET, the technology on which these sensors are based. Finally, using data from the lactate sensor expressed in the larval brain as a case study, we show step by step how to process the fluorescence signal to obtain reliable FRET values.

developmental biology↗

bioRxiv2020

The immunophilin Zonda controls regulated exocytosis in endocrine and exocrine tissues

Exocytosis is a fundamental process in physiology, communication between cells, organs and even organisms. Hormones, neuropeptides and antibodies, among other cargoes are packed in exocytic vesicles that need to reach and fuse with the plasma membrane to release their content to the extracellular milieu. Hundreds of proteins participate in this process and several others in its regulation. We report here a novel component of the exocytic machinery, the Drosophila transmembrane immunophilin Zonda (Zda), previously found to participate in autophagy. Zda is highly expressed in secretory tissues, and regulates exocytosis in at least three of them: the ring gland, insulin-producing cells and the salivary gland. Using the salivary gland as a model system, we found that Zda is required at final steps of the exocytic process for fusion of secretory granules to the plasma membrane. In a genetic screen we identified the small GTPase RalA as a crucial regulator of secretory granule exocytosis that operates upstream of Zda in the process.

cell biology↗

bioRxiv2019

A genetic toolkit for the analysis of metabolic changes in Drosophila provides new insights into metabolic responses to stress and malignant transformation

Regulation of the energetic metabolism occurs fundamentally at the cellular level, so analytical strategies must aim to attain single cell resolution to fully embrace its inherent complexity. We have developed methods to utilize a toolset of metabolic FRET sensors for assessing lactate, pyruvate and 2-oxoglutarate levels of Drosophila tissues in vivo by imaging techniques. We show here how the energetic metabolism is altered by hypoxia: While larval tissues that contribute directly to adult organs respond to low oxygen levels by executing a metabolic switch towards lactic fermentation, polytene tissues that are degraded during metamorphosis do not alter their energetic metabolism. Analysis of tumor metabolism revealed that depending on the genetic background, some tumors undergo a lactogenic switch typical of the Warburg effect, while other tumors dont. This toolset allows for developmental and physiologic studies in genetically manipulated Drosophila individuals in vivo.

cell biology↗

bioRxiv2019

Context-specific functions of Notch in Drosophila blood cell progenitors

Drosophila Larval hematopoiesis takes place at the lymph gland, where myeloid-like progenitors differentiate into Plasmatocytes and Crystal Cells, under regulation of conserved signaling pathways. It has been established that the Notch pathway plays a specific role in Crystal Cell differentiation and maintenance. In mammalian hematopoiesis, the Notch pathway has been proposed to fulfill broader functions, including Hematopoietic Stem Cell maintenance and cell fate decision in progenitors. In this work we describe different roles that Notch plays in the lymph gland. We show that Notch, activated by its ligand Serrate, expressed at the Posterior Signaling Center, is required to restrain Core Progenitor differentiation. We define a novel population of blood cell progenitors that we name Distal Progenitors, where Notch, activated by Serrate expressed in Lineage Specifying Cells at the Medullary Zone/Cortical Zone boundary, regulates a binary decision between Plasmatocyte and Crystal Cell fates. Thus, Notch plays context-specific functions in different blood cell progenitor populations of the Drosophila lymph gland. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/682658v2_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@8f3c25org.highwire.dtl.DTLVardef@1b5d8d1org.highwire.dtl.DTLVardef@1ad99cforg.highwire.dtl.DTLVardef@4d0bd9_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗