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Methods to detect apoptotic-like cell death in filamentous fungi
Camile P Semighini1, Steven D Harris
1Department of Plant Pathology and Center for Plant Science Innovation, University of Nebraska, Lincoln, NE, USA.
Abstract:
Fungi are capable of undergoing apoptotic-like cell death, and display many of the characteristic features of apoptosis observed in multicellular organisms. These features include nuclear condensation, DNA fragmentation, translocation of phosphatidylserine from the cytoplasmic to the extracellular side of the plasma membrane, and increased levels of reactive oxygen species (ROS). Several assays can be used to detect apoptotic cells, and here we describe adaptations of assays such as TUNEL, Annexin V, and Evan's Blue for the investigation of apoptotic-like cell death in fungal hyphae. We also present approaches for monitoring nuclear condensation and production of ROS.
Insights
Fungi exhibit apoptotic-like cell death, a process involving nuclear changes and membrane alterations. This study adapts assays to detect these fungal cell death features, including reactive oxygen species (ROS) production.
Area of Science:
- Mycology
- Cell Biology
- Biochemistry
Background:
- Fungi can undergo programmed cell death, sharing features with apoptosis in multicellular organisms.
- Key apoptotic markers include nuclear condensation, DNA fragmentation, and phosphatidylserine translocation.
- Reactive oxygen species (ROS) are also implicated in fungal cell death pathways.
Purpose of the Study:
- To describe adaptations of established assays for detecting apoptotic-like cell death in fungi.
- To provide methods for monitoring specific fungal apoptosis markers.
- To investigate the role of ROS in fungal cell death.
Main Methods:
- Adaptation of TUNEL assay for DNA fragmentation detection in fungal hyphae.
- Utilization of Annexin V staining to identify phosphatidylserine externalization.
- Application of Evan's Blue dye exclusion assay for cell viability assessment.
- Methods for visualizing nuclear condensation and quantifying ROS production.
Main Results:
- Demonstrated successful adaptation of TUNEL, Annexin V, and Evan's Blue assays for fungal hyphae.
- Established protocols for monitoring nuclear condensation and ROS levels in fungi.
- Provided a framework for studying apoptotic-like cell death in fungal systems.
Conclusions:
- The described assay adaptations enable robust detection of fungal apoptotic-like cell death.
- These methods facilitate detailed investigation into the mechanisms of programmed cell death in fungi.
- This research contributes to understanding fungal biology and potential therapeutic targets.
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