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

Abstract

Oogenesis is a fundamental process that forms the egg and, is crucial for the transmission of genetic information to the next generation. Drosophila oogenesis has been used extensively as a genetically tractable model to study organogenesis, niche-germline stem cell communication, and more recently reproductive aging including germline stem cell (GSC) aging. Autophagy, a lysosome-mediated degradation process, is implicated in gametogenesis and aging. However, there is a lack of genetic tools to study autophagy in the context of gametogenesis and GSC aging. Here we describe the generation of three transgenic lines mcherry-Atg8a, GFP-Ref(2)P and mito-roGFP2-Orp1 that are specifically expressed in the germline compartment including GSCs during Drosophila oogenesis. These transgenes are expressed from the nanos promoter and present a better alternative to UASp mediated overexpression of transgenes. These fluorescent reporters can be used to monitor and quantify autophagy, and the production of reactive oxygen species during oogenesis. These reporters provide a valuable tool that can be utilized in designing genetic screens to identify novel regulators of autophagy and redox homeostasis during oogenesis.

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

“…To monitor autophagy we followed the expression of two candidates Atg8a fused to mCherry (LC3/GABARAP in mammals) and Ref(2)P fused to GFP (p62 in mammals) for our study. Atg8a is a ubiquitin-like protein that labels the inner and outer membranes of all autophagic structures: phagophore, autophagosomes, and autolysosomes, and has been widely used to monitor autophagy (Bali and Shravage, 2017; Klionsky et al, 2021; Nilangekar et al, 2019; Scott et al, 2004). Similarly, Ref(2) P (p62 in mammals), is a selective autophagy cargo receptor that binds polyubiquitinated substrates via ubiquitin-binding domain and interacts with Atg8a with the help of an Atg8-interacting motif (AIM/LIR) to recruit substrates to autophagosomes (Klionsky et al, 2021; Nezis et al, 2008).…”

“…The number and distribution of GSCs was assayed. We have previously shown that KD of Atg8a within the germline cells leads to disruption of autophagy (Nilangekar et al, 2019). In contrast to Atg8a OE , Atg8aRNAi in the germline led to decrease in the average GSCs within the germarium at 1 week and 4 weeks of age as compared to the controls ( Figure 4A, B ).…”

“…Flies were dissected as described in (Nilangekar et al, 2019). For RNA extraction, ovaries were dissected in Grace’s medium and transferred to a 1.5 ml microfuge tube containing TriZol (Invitrogen, USA) and frozen at −80 C till further processing.…”

“…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 ),…”

ROCK inhibitor fasudil reduces the expression of inflammatory factors in LPS-induced rat pulmonary microvascular endothelial cells via ROS/NF-κB pathway

“…It is necessary to further study how fasudil achieved these beneficial effects. Reactive oxygen species (ROS), particularly the hydrogen peroxide and superoxide anion, are considered as the vital signal molecules in a lot of biological events [ 37 , 38 ]. It is well known that various pro-inflammatory substances can activate neutrophils of lungs in patients with ALI/ARDS [ 39 ], and the activated neutrophils are the main source of ROS.…”