Mitochondrial Redox Sensor for Drosophila Female Germline Stem Cells

Abstract

Mitochondrial reactive oxygen species (mROS), a major source of ROS within cells, functions as an important signaling molecule and has the ability to damage cellular macromolecules including DNA and proteins. Monitoring mROS levels is therefore essential to understand cell-cell communication and programmed cell death in all types of cell including stem cells. Here, we describe generation and characterization of a redox sensor for mROS that is specifically expressed in the germline stem cells (GSCs) in Drosophila. This redox sensor can be used to monitor the production of mROS and mitophagy in the GSCs during oogenesis.

Autophagy slows the aging of Germline stem cells in Drosophila through modulation of E-cadherin

“…Importantly, as cells, tissues and organisms age there is increased production of mROS due to inefficient clearing of damaged mitochondria by mitophagy. To test if autophagy block leads to mROS accumulation in GSCs, we used a germline specific mitochondrial redox sensor, mito-roGFP2-Grx1, previously characterized by our lab (Albrecht et al, 2014; Nilangekar and Shravage, 2019). This reporter senses the redox state of glutaredoxin (Grx1) within the cells and the redox potential is determined by the 405/488 ratiometric(Pan et al, 2007; Zhao et al, 2008) shift in fluorescence of roGFP2.…”

“…OregonR, ywhsFLP1; mCherry-Atg8a/CyO; GFP-Ref(2)P/TM6b (Nilangekar et al, 2019) nanos-mito-roGFP2-Grx1 (Nilangekar and Shravage, 2019). The following flies were obtained from Bloomington Drosophila Stock Center, USA; yw; UASp.mCherry.Atg8a; Dr/ TM3, Ser (BL37750, RRID:BDSC_37750), w; +; nosGal4VP16 (BL4937, RRID:BDSC_4937), y sc v; +; Atg8a-RNAi (BL34340, RRID:BDSC_34340 ),…”

“…The image acquisition and analysis was performed as mentioned in (Nilangekar and Shravage, 2019; Nilangekar et al, 2019)…”

Generation and Characterization of Germline-Specific Autophagy and Mitochondrial Reactive Oxygen Species Reporters in Drosophila

“…We created a pCasper4 based plasmid that possesses 935 bp nanos promoter and a 1236 bp 3′ UTR of nanos that stabilizes transcripts in the germline to generate GFP-Ref(2)P (Doren et al, 1998; Rørth, 1998; Nilangekar and Shravage, 2018; Figure 1). Nanos is a maternally expressed and is required for the process of oogenesis and egg production.…”

“…The ovaries were mounted in 80% glycerol and imaged the same day. The redox treatment procedure is modified from Albrecht et al, 2011; (Nilangekar and Shravage, 2018).…”