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Calcium (Ca2+) is an important second messenger for activating stress response signalling and cell adaptation in eukaryotic cells yet intracellular Ca2+-dynamics in fungi is poorly understood due to lack of effective real-time Ca2+ reporters. We engineered the GCaMP6f construct for use in the fungal pathogen, Candida albicans, and used live-cell imaging to observe dynamic Ca2+ spiking as well as slower changes in ambient Ca2+-GCaMP levels elicited by stress or gene deletion. Short-term exposure to membrane, osmotic or oxidative stress generated immediate stress-specific responses and repeated exposure revealed differential recovery signatures. Osmotic stress caused yeast cell shrinkage and no adaptation response, where Ca2+-GCaMP spiking was inhibited by 1 M NaCl but not by 0.66 M CaCl2. Treatment with SDS caused a spike-burst, raised ambient Ca2+-GCaMP levels and significant cell death, but surviving cells adapted over subsequent exposures. Treatment with 5 mM H2O2 abolished spiking and caused transient autofluorescence but cells adapted such that spiking returned and autofluorescence diminished on repeated exposure. Adaptation to H2O2 was dependent on Cap1, extracellular Ca2+ and calcineurin, but not on its downstream target, Crz1. Ca2+-dynamics were not affected by H2O2 in the hog1Δ or yvc1Δ mutants, suggesting a pre-adapted, resistant state, possibly due to changes in membrane permeability. Live-cell imaging of Ca2+-GCaMP responses in individual cells has therefore revealed the dynamics of Ca2+-influx, signalling and homeostasis and their role in the temporal stress response signatures of C. albicans.

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