Fluorescent organelle markers in Cryptococcus neoformans: a versatile toolkit for live-cell subcellular localization

Area of Science:

• Mycology
• Cell Biology
• Microbiology

Background:

• Intracellular organelle dynamics are crucial for fungal growth, differentiation, and pathogenicity.
• The role of organelles in adaptation for the human fungal pathogen *Cryptococcus neoformans* remains understudied.
• Understanding organelle remodeling is key to addressing fungal virulence.

Purpose of the Study:

• To develop and validate a comprehensive toolkit for live-cell imaging of ten major subcellular compartments in *Cryptococcus neoformans*.
• To investigate the dynamic remodeling of organelle architecture under various host-relevant conditions and during sexual development.
• To provide a platform for colocalization analyses and functional inference of uncharacterized genes.

Main Methods:

• Creation of a toolkit with fluorescent organelle marker plasmids and strains (mCherry or GFP fusions).
• High-resolution live-cell imaging of ten subcellular compartments: nucleolus, ER, Golgi, mitochondria, peroxisome, endosome, autophagosome, vacuole, plasma membrane, and P-bodies.
• Analysis of organelle organization under conditions like elevated temperature, 5% CO2, capsule/melanin induction, and during sexual reproduction.

Main Results:

• Established a robust imaging platform for visualizing multiple organelles simultaneously in *Cryptococcus neoformans*.
• Revealed condition- and stage-specific remodeling of organelle architecture during vegetative growth and sexual development.
• Demonstrated the utility of the toolkit for colocalization studies and inferring gene function based on spatial localization.

Conclusions:

• The developed imaging toolkit provides a powerful framework for studying fungal organelle dynamics.
• Subcellular organization plays a significant role in fungal adaptation, development, and virulence.
• This platform facilitates future research into fungal pathogenesis and the function of novel genes.