An improved staining method of cell cycle analysis with Sybr Green I for fungi: Cryptococcus neoformans and Saccharomyces cerevisiae
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Updated: Nov 20, 2025
Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi
Published on: November 28, 2019
1Institutes of Biomedical Sciences, Fudan University , Shanghai, People's Republic of China.
Abstract:
Cryptococcus neoformans is a pathogenic fungus which causes millions of deaths and infections, especially threatening immunocompromised individuals. During the development of new drugs, the ubiquitination has been found to play an important role in the regulation of the virulence and cell cycle of this fungus. Based on this mechanism, ubiquitination-related mutant strains exhibiting cell cycle arrest have been established for drug development for the fungus. However, flow cytometry detection of the cell cycle in fungi is generally difficult because the thick cell wall and capsule of fungi generally contribute to a nonspecific signal of cytometry. In this study, an improved method, derived from Saccharomyces cerevisiae assays, is developed to specifically stain C. neoformans, in whose cell cycle the G1 and G2 peaks are separated enough to be allowed for cell cycle analysis. As a result, the improved method facilitates the detection of the alterations in the cell cycle of C. neoformans with a mutation that results in cell cycle arrest, which distinctly delays the cell division of C. neoformans. Thus, the improved method reported here provides detailed technical information regarding assays on C. neoformans and, more importantly, offers a solution for assessing the cell cycle in other fungi in the future. Abbreviation: PI: propidium iodide.
Insights
Researchers developed an improved flow cytometry method to analyze the cell cycle of Cryptococcus neoformans. This technique aids in developing new antifungal drugs by detecting cell cycle arrest in fungal pathogens.
Area of Science:
- Mycology
- Cell Biology
- Drug Development
Background:
- Cryptococcus neoformans is a major fungal pathogen, particularly dangerous for immunocompromised individuals.
- Ubiquitination regulates virulence and cell cycle in C. neoformans, making it a target for drug development.
- Existing flow cytometry methods struggle with fungal cell walls, hindering cell cycle analysis.
Purpose of the Study:
- To develop an improved, specific flow cytometry method for C. neoformans cell cycle analysis.
- To enable the study of ubiquitination-related cell cycle arrest in C. neoformans drug development.
- To provide a versatile method applicable to other fungal species.
Main Methods:
- Adapted a flow cytometry assay from Saccharomyces cerevisiae.
- Developed a specific staining protocol for Cryptococcus neoformans.
- Utilized propidium iodide (PI) for DNA staining.
Main Results:
- Achieved distinct G1 and G2 phase peaks in C. neoformans cell cycle analysis.
- Successfully detected cell cycle arrest in a C. neoformans mutant strain.
- Demonstrated delayed cell division in the mutant strain.
Conclusions:
- The improved method facilitates accurate cell cycle assessment in C. neoformans.
- This technique supports the development of novel antifungal therapies targeting fungal cell cycle regulation.
- The method offers a potential solution for cell cycle analysis in other challenging fungal species.
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