Live applications of norbormide-based fluorescent probes in Drosophila melanogaster

Abstract

20 In this study we investigated the performance of two norbormide (NRB)-derived fluorescent probes, 21 NRB MC009 (green) and NRB ZLW0047 (red), on dissected, living larvae of Drosophila, to verify their 22 potential application in confocal microscopy imaging in vivo. To this end, larval tissues were exposed 23 to NRB probes alone or in combination with other commercial dyes or GFP-tagged protein markers.24 Both probes were rapidly internalized by most tissues (except the central nervous system) allowing 25 each organ in the microscope field to be readily distinguished at low magnification. At the cellular 26 level, the probes showed a very similar distribution (except for fat bodies), defined by loss of signal 27 in the nucleus and plasma membrane, and a preferential localization to endoplasmic reticulum (ER) 28 and mitochondria. They also recognized ER and mitochondrial phenotypes in the skeletal muscles of 29 fruit fly models that had loss of function mutations in the atlastin and mitofusin genes, suggesting 30 NRB MC009 and NRB ZLW0047 as potentially useful in vivo screening tools for characterizing ER and 31 mitochondria morphological alterations. Feeding of larvae and adult Drosophilae with the NRB-32 derived dyes led to staining of the gut and its epithelial cells, revealing a potential role in food intake 33 assays. In addition, when flies were exposed to either dye over their entire life cycle no apparent 34 functional or morphological abnormalities were detected. Rapid internalization, a bright signal, a 35 compatibility with other available fluorescent probes and GFP-tagged protein markers, and a lack of 36 toxicity make NRB ZLW0047 and, particularly, NRB MC009 one of the most highly performing fluorescent 37 probes available for in vivo microscopy studies and food intake assay in Drosophila. 38 39 40 3 41 Introduction 42 Norbormide[5-(α-hydroxy-α-2-pyridylbenzyl)-7-(α-2-pyridylbenzylidene)-5-norbornene-2,3-43 dicarboximide] (NRB) is a selective rat toxicant that exhibits little or no non-target effects (1), and 44 was developed and commercialized as an ecologic pesticide in the 1980s (Roszkowski, 1965).45 Evidence suggests that the rat-selective action of NRB is mediated by a generalized vasoconstrictor 46 effect that has only been observed in the rat peripheral blood vessels, both in vivo and in vitro. In 47 contrast, NRB displays a vasorelaxant action in arteries from non-rat species, as well as in rat aorta 48 and extravascular smooth muscle, that has been proposed to be the result of a reduction of Ca 2+ entry 49 through L-type Ca 2+ channels (2,3). The molecular mechanism underlying NRB-induced 50 vasoconstriction is not known, however, it has been proposed that the compound acts on rat vascular 51 myocytes where it activates the PLC-IP3-PKC pathway (2), a signaling cascade stimulated by most 52 receptor-coupled vasoconstrictor agents (4). In an attempt to identify the cellular targets of NRB, we 53 previously developed fluorescent derivatives of the parent compound by linking it to either 54 nit...