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In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice
Published on: November 22, 2017
Quantification of mitophagy using mKeima-mito in cultured human primary retinal pigment epithelial cells
Cody R Fisher1, Mara C Ebeling2, Deborah A Ferrington1
1Department of Ophthalmology and Visual Neurosciences, University of Minnesota, Minneapolis, MN, 55455, USA; Graduate Program in Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, 55455, USA.
Abstract:
The retinal pigment epithelium is a pigmented monolayer of cells that help maintain a healthy retina. Loss of this essential cell layer is implicated in a number of visual disorders, including age-related macular degeneration (AMD). Utilizing primary RPE cultures to investigate disease is an important step in understanding disease mechanisms. However, the use of primary RPE cultures presents a number of challenges, including the limited number of cells available and the presence of auto-fluorescent pigment that interferes with quantifying fluorescent probes. Additionally, primary RPE are difficult to transfect with exogenous nucleic acids traditionally used for fluorescent imaging. To overcome these challenges, we used an adeno-associated viral (AAV) vector to express a pH sensitive fluorescent protein, mKeima, fused to the mitochondrial targeting sequence of cytochrome oxidase subunit 8A (mKeima-mito). mKeima-mito allows for quantification of mitochondrial autophagy (mitophagy) in live-cell time-lapse imaging experiments. We also developed an image analysis pipeline to selectively quantify mKeima-mito while removing the signal of auto-fluorescent pigment from the dataset by utilizing information from the mKeima fluorescent channels. These techniques are demonstrated in primary RPE cultures expressing mKeima-mito treated with 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile (FCCP), an uncoupler that depolarizes the mitochondrial membrane and leads to mitochondrial fragmentation and mitophagy. The techniques outlined provide a roadmap for investigating disease mechanisms or the effect of treatments utilizing fluorescent probes in an important cell culture model.
Insights
Researchers developed new methods to study retinal pigment epithelium (RPE) cells, overcoming challenges in imaging and analysis for diseases like age-related macular degeneration (AMD). This enables better investigation of RPE cell function and disease progression.
Area of Science:
- Ophthalmology
- Cell Biology
- Biotechnology
Background:
- The retinal pigment epithelium (RPE) is crucial for retinal health, and its loss is linked to visual disorders like age-related macular degeneration (AMD).
- Primary RPE cultures are valuable for disease research but present challenges, including limited cell availability, autofluorescent pigment interference, and difficulty in transfection for fluorescent imaging.
- Existing methods struggle to accurately quantify cellular processes in RPE due to inherent pigment autofluorescence and transfection difficulties.
Purpose of the Study:
- To develop novel techniques for overcoming challenges in studying primary RPE cell cultures.
- To enable accurate quantification of mitophagy in RPE cells using live-cell imaging.
- To establish a robust methodology for investigating disease mechanisms and treatment effects in RPE models.
Main Methods:
- Adeno-associated viral (AAV) vector used to express mKeima-mito, a pH-sensitive fluorescent protein targeted to mitochondria.
- Live-cell time-lapse imaging employed to monitor mitophagy.
- A specialized image analysis pipeline developed to isolate mKeima-mito signal and eliminate autofluorescent pigment interference.
Main Results:
- Successfully expressed and visualized mKeima-mito in primary RPE cultures.
- Developed and validated an image analysis pipeline capable of accurately quantifying mitophagy by distinguishing mKeima-mito signal from RPE autofluorescence.
- Demonstrated the technique's efficacy in tracking mitochondrial fragmentation and mitophagy induced by FCCP treatment.
Conclusions:
- The developed AAV-mediated expression of mKeima-mito and the specialized image analysis pipeline provide a powerful tool for studying mitophagy in RPE cells.
- These techniques overcome significant limitations of primary RPE cultures, offering a reliable roadmap for future research into retinal diseases and therapeutic interventions.
- This methodology enhances the study of mitochondrial dynamics and autophagy in RPE, crucial for understanding and treating vision disorders.
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