Drosophila Unfolded Protein Response (UPR) Assays In Vitro and In Vivo.

Wildtype or mutant proteins expressed beyond the capacity of a cell's protein folding system could be detrimental to general cellular function and survival. In response to misfolded protein overload in the endoplasmic reticulum (ER), eukaryotic cells activate the Unfolded Protein Response (UPR) that helps cells restore protein homeostasis in the endoplasmic reticulum (ER). As part of the UPR, cells attenuate general mRNA translation and activate transcription factors that induce stress-responsive gene expression.UPR signaling draws research interest in part because conditions that cause chronic protein misfolding in the ER or those that impair UPR signaling underlie several diseases including neurodegeneration, diabetes, and cancers. Model organisms are frequently employed in the field as the UPR pathways are generally well-conserved throughout phyla. Here, we introduce experimental procedures to detect UPR in Drosophila melanogaster.

A protein-trap allele reveals roles for Drosophila ATF4 in photoreceptor degeneration, oogenesis and wing development.

Metazoans have evolved various quality control mechanisms to cope with cellular stress inflicted by external and physiological conditions. ATF4 is a major effector of the integrated stress response, an evolutionarily conserved pathway that mediates adaptation to various cellular stressors. Loss of function of Drosophila ATF4, encoded by the gene cryptocephal (crc), results in lethality during pupal development. The roles of crc in Drosophila disease models and in adult tissue homeostasis thus remain poorly understood. Here, we report that a protein-trap Minos-mediated integration cassette insertion in the crc locus generates a Crc-GFP fusion protein that allows visualization of Crc activity in vivo. This allele also acts as a hypomorphic mutant that uncovers previously unknown roles for crc. Specifically, the crc protein-trap line shows Crc-GFP induction in a Drosophila model for retinitis pigmentosa. This crc allele renders flies more vulnerable to amino acid deprivation and age-dependent retinal degeneration. These mutants also show defects in wing veins and oocyte maturation. Together, our data reveal previously unknown roles for crc in development, cellular homeostasis and photoreceptor survival. This article has an associated First Person interview with the first author of the paper.

Neurochemical Organization of the Drosophila Brain Visualized by Endogenously Tagged Neurotransmitter Receptors.

Neurotransmitters often have multiple receptors that induce distinct responses in receiving cells. Expression and localization of neurotransmitter receptors in individual neurons are therefore critical for understanding the operation of neural circuits. Here we describe a comprehensive library of reporter strains in which a convertible T2A-GAL4 cassette is inserted into endogenous neurotransmitter receptor genes of Drosophila. Using this library, we profile the expression of 75 neurotransmitter receptors in the brain. Cluster analysis reveals neurochemical segmentation of the brain, distinguishing higher brain centers from the rest. By recombinase-mediated cassette exchange, we convert T2A-GAL4 into split-GFP and Tango to visualize subcellular localization and activation of dopamine receptors in specific cell types. This reveals striking differences in their subcellular localization, which may underlie the distinct cellular responses to dopamine in different behavioral contexts. Our resources thus provide a versatile toolkit for dissecting the cellular organization and function of neurotransmitter systems in the fly brain.

Tango knock-ins visualize endogenous activity of G protein-coupled receptors in Drosophila.

G protein-coupled receptors (GPCRs) represent a family of seven-pass transmembrane protein receptors whose ligands include neuropeptides and small-molecule neuromodulators such as dopamine and serotonin. These neurotransmitters act at long distances and are proposed to define the ground state of the nervous system. The Drosophila genome encodes approximately 50 neuropeptides and their functions in physiology and behavior are now under intensive studies. Key information currently lacking in the field is the spatiotemporal activation patterns of endogenous GPCRs. Here we report application of the Tango system, a reporter assay to detect GPCR activity, to endogenous GPCRs in the fly genome. We developed a method to integrate the sensor component of the Tango system to the C-terminus of endogenous genes by using genome editing techniques. We demonstrate that Tango sensors in the Sex-peptide receptor (SPR) locus allow sensitive detection of mating-dependent SPR activity in the female reproductive organ. The method is easily applicable to any GPCR and will provide a way to systematically characterize GPCRs in the fly brain.

Regulation of axon arborization pattern in the developing chick ciliary ganglion: Possible involvement of caspase 3.

During a certain critical period in the development of the central and peripheral nervous systems, axonal branches and synapses are massively reorganized to form mature connections. In this process, neurons search their appropriate targets, expanding and/or retracting their axons. Recent work suggested that the caspase superfamily regulates the axon morphology. Here, we tested the hypothesis that caspase 3, which is one of the major executioners in apoptotic cell death, is involved in regulating the axon arborization. The embryonic chicken ciliary ganglion was used as a model system of synapse reorganization. A dominant negative mutant of caspase-3 precursor (C3DN) was made and overexpressed in presynaptic neurons in the midbrain to interfere with the intrinsic caspase-3 activity using an in ovo electroporation method. The axon arborization pattern was 3-dimensionally and quantitatively analyzed in the ciliary ganglion. The overexpression of C3DN significantly reduced the number of branching points, the branch order and the complexity index, whereas it significantly elongated the terminal branches at E6. It also increased the internodal distance significantly at E8. But, these effects were negligible at E10 or later. During E6-8, there appeared to be a dynamic balance in the axon arborization pattern between the "targeting" mode, which is accompanied by elongation of terminal branches and the pruning of collateral branches, and the "pathfinding" mode, which is accompanied by the retraction of terminal branches and the sprouting of new collateral branches. The local and transient activation of caspase 3 could direct the balance towards the pathfinding mode.

Optogenetic probing and manipulation of the calyx-type presynaptic terminal in the embryonic chick ciliary ganglion.

The calyx-type synapse of chick ciliary ganglion (CG) has been intensively studied for decades as a model system for the synaptic development, morphology and physiology. Despite recent advances in optogenetics probing and/or manipulation of the elementary steps of the transmitter release such as membrane depolarization and Ca(2+) elevation, the current gene-manipulating methods are not suitable for targeting specifically the calyx-type presynaptic terminals. Here, we evaluated a method for manipulating the molecular and functional organization of the presynaptic terminals of this model synapse. We transfected progenitors of the Edinger-Westphal (EW) nucleus neurons with an EGFP expression vector by in ovo electroporation at embryonic day 2 (E2) and examined the CG at E8-14. We found that dozens of the calyx-type presynaptic terminals and axons were selectively labeled with EGFP fluorescence. When a Brainbow construct containing the membrane-tethered fluorescent proteins m-CFP, m-YFP and m-RFP, was introduced together with a Cre expression construct, the color coding of each presynaptic axon facilitated discrimination among inter-tangled projections, particularly during the developmental re-organization period of synaptic connections. With the simultaneous expression of one of the chimeric variants of channelrhodopsins, channelrhodopsin-fast receiver (ChRFR), and R-GECO1, a red-shifted fluorescent Ca(2+)-sensor, the Ca(2+) elevation was optically measured under direct photostimulation of the presynaptic terminal. Although this optically evoked Ca(2+) elevation was mostly dependent on the action potential, a significant component remained even in the absence of extracellular Ca(2+). It is suggested that the photo-activation of ChRFR facilitated the release of Ca(2+) from intracellular Ca(2+) stores directly or indirectly. The above system, by facilitating the molecular study of the calyx-type presynaptic terminal, would provide an experimental platform for unveiling the molecular mechanisms underlying the morphology, physiology and development of synapses.