Date Published: April 8, 2019
Publisher: Public Library of Science
Author(s): Alessia Forgiarini, Zifei Wang, Claudio D’Amore, Morgan Jay-Smith, Freda Fan Li, Brian Hopkins, Margaret Anne Brimble, Andrea Pagetta, Sara Bersani, Sara De Martin, Barbara Napoli, Sergio Bova, David Rennison, Genny Orso, Christian Wegener.
In this study we investigated the performance of two norbormide (NRB)-derived fluorescent probes, NRBMC009 (green) and NRBZLW0047 (red), on dissected, living larvae of Drosophila, to verify their potential application in live cell imaging confocal microscopy. To this end, larval tissues were exposed to NRB probes alone or in combination with other commercial dyes or GFP-tagged protein markers. Both probes were rapidly internalized by most tissues (except the central nervous system) allowing each organ in the microscope field to be readily distinguished at low magnification. At the cellular level, the probes showed a very similar distribution (except for fat bodies), defined by loss of signal in the nucleus and plasma membrane, and a preferential localization to endoplasmic reticulum (ER) and mitochondria. They also recognized ER and mitochondrial phenotypes in the skeletal muscles of fruit fly models that had loss of function mutations in the atlastin and mitofusin genes, suggesting NRBMC009 and NRBZLW0047 as potentially useful screening tools for characterizing ER and mitochondria morphological alterations. Feeding of larvae and adult Drosophilae with the NRB-derived dyes led to staining of the gut and its epithelial cells, revealing a potential role in food intake assays. In addition, when flies were exposed to either dye over their entire life cycle no apparent functional or morphological abnormalities were detected. Rapid internalization, a bright signal, a compatibility with other available fluorescent probes and GFP-tagged protein markers, and a lack of toxicity make NRBZLW0047 and, particularly, NRBMC009 highly performing fluorescent probes for live cell microscopy studies and food intake assays in Drosophila.
Norbormide [5-(α-hydroxy-α-2-pyridylbenzyl)-7-(α-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide] (NRB) is a selective rat toxicant that exhibits little or no non-target effects , and was developed and commercialized as an ecologic pesticide in the 1980s. Evidence suggests that the rat-selective action of NRB is mediated by a generalized vasoconstrictor effect that has only been observed in the rat peripheral blood vessels, both in vivo and in vitro. In contrast, NRB displays a vasorelaxant action in arteries from non-rat species, as well as in rat aorta and extravascular smooth muscle, that has been proposed to be the result of a reduction of Ca2+ entry through L-type Ca2+ channels [2,3]. The molecular mechanism underlying NRB-induced vasoconstriction is not known, however, it has been proposed that the compound acts on rat vascular myocytes where it activates the PLC-IP3-PKC pathway , a signaling cascade stimulated by most receptor-coupled vasoconstrictor agents . In an attempt to identify the cellular targets of NRB, we previously developed fluorescent derivatives of the parent compound by linking it to either nitrobenzoxadiazole (NBD) or boron-dipyrromethene (BODIPY FL) fluorophores, and found that both were able to clearly label intracellular structures such as endoplasmic reticulum (ER), Golgi apparatus, mitochondria, and lipid droplets (LDs) in various cell lines, in the absence of cytotoxic effects. Based on these results, we proposed NRB as a scaffold for the development of new, high performing, non-toxic fluorescent probes for live cell imaging [5,6].