Fluorescent organelle markers in <i>Cryptococcus neoformans</i> : 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 <i>Cryptococcus neoformans</i> 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% CO ₂ , 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.