The role of IP3-activated calcium ion channels in fungal growth

Abstract

During my previous research, I showed that cytoplasmic Ca ²⁺ is important for branch initiation and hyphal elongation in the fungal organism Neurospora crassa, and that the tip-high Ca²⁺ gradient must be maintained internally. Decoding the regulation of the Ca²⁺ gradient during tip growth in Neurospora crassa represented the main objective of my PhD studies.

I imaged the cytoplasmic Ca²⁺ in hyphae treated with regulators of Ca²⁺ transporters and I found that the tip-high gradient is maintained by the cooperation between endoplasmic reticulum which sequesters Ca²⁺ subapically and apical vesicles which release Ca ²⁺ at the tip through an IP₃ receptor, and that the localization of Ca²⁺ storing organelles is controlled by actin. The importance of Ca²⁺-storing organelles was demonstrated in the morphological mutant spray. I showed that the cytoplasmic Ca²⁺ and plasma membrane ion transport are similar to wild-type. In this mutant, organellar Ca²⁺ is absent, reappearing in high external Ca²⁺ which also rescues the mutant phenotype of aberrant tip growth.

Next, using the black lipid membrane technique, I demonstrated the presence of two IP₃-activated Ca²⁺ channels: a large conductance channel, and a small conductance channel which was implicated in the regulation of tip growth. Since IP₃ production must be catalyzed by tip-localized phospholipase C (PLC), I examined the role of PLC in fungal growth. I showed that PLC inhibitors inhibit hyphal growth. Both direct inhibition of the IP ₃ channel by 2-APB and indirect inhibition by inhibiting PLC and thus IP₃ production have similar effects on hyphal growth, morphology and organellar Ca²⁺ causing the subapical retreat of vesicular Ca²⁺ and a hyperbranching phenotype after wash out.

I showed that stretching of the plasma membrane stimulates the PLC which produces DAG and IP₃. Thus, the PLC senses hyphal expansion during hyphal growth.

In conclusion, my results support the hypothesis that the tip-high Ca ²⁺ gradient required for hyphal growth is generated and maintained internally by Ca²⁺ sequestration subapically in the endoplasmic reticulum by a Ca²⁺ ATPase and Ca²⁺ release from tip-localized vesicles through a small conductance vesicular Ca ²⁺ channel activated by IP₃ produced at the tip by PLC activated by stretching of the plasma membrane.