Live applications of norbormide-based fluorescent probes in Drosophila melanogaster

PLoS One. 2019 Apr 8;14(4):e0211169. doi: 10.1371/journal.pone.0211169. eCollection 2019.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Drosophila melanogaster / anatomy & histology
  • Drosophila melanogaster / physiology*
  • Eating
  • Female
  • Fluorescent Dyes / administration & dosage*
  • Fluorescent Dyes / chemistry
  • Fluorescent Dyes / toxicity
  • Gastrointestinal Absorption
  • Gastrointestinal Tract / diagnostic imaging
  • Green Fluorescent Proteins / chemistry
  • Intravital Microscopy / methods*
  • Larva / physiology
  • Male
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Models, Animal
  • Norbornanes / administration & dosage*
  • Norbornanes / chemistry
  • Norbornanes / toxicity
  • Toxicity Tests, Chronic

Substances

  • Fluorescent Dyes
  • Norbornanes
  • Green Fluorescent Proteins
  • norbormide

Grants and funding

This project was supported by the New Zealand Ministry of Business, Innovation and Employment’s Endeavour Fund C09X1710 (BH, SBo, MB, GO, DR): https://www.mbie.govt.nz/ and by the University of Padova, project n. 148125/14 (SBo) and SID18-01 (GO): https://www.unipd.it/en/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.