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

Understanding how intracellular organelles are organized and dynamically remodeled is essential for elucidating fungal growth, differentiation, and pathogenicity. The human fungal pathogen Cryptococcus neoformans exhibits remarkable morphological diversity, yet the contribution of intracellular organelles to adaptation across environmental and host-relevant conditions has not been systematically examined. Here, we establish a comprehensive toolkit of fluorescent organelle marker plasmids and strains expressing mCherry or GFP fusions, enabling high-resolution live-cell imaging of ten major subcellular compartments: the nucleolus, endoplasmic reticulum (ER), Golgi apparatus, mitochondria, peroxisome, endosome, autophagosome, vacuole, the plasma membrane, and processing bodies (P-bodies). With this platform, we analyzed organelle organization under host-relevant conditions, including elevated temperature, 5% CO₂, and capsule- and melanin-inducing media, as well as throughout sexual development from zygote formation to basidiospore production. We further show that the toolkit supports colocalization analyses via diploid formation or dual-labeling strategies. Collectively, our results reveal condition- and stage-specific remodeling of organelle architecture during vegetative growth and mating. This imaging platform provides a robust framework for investigating how subcellular organization supports fungal adaptation, development, and virulence, and for inferring the functions of uncharacterized genes from their spatial localization.