Regulation of the tip-high [Ca2+] gradient in growing hyphae of the fungusNeurospora crassa

Previous work has shown that hyphal elongation in the fungus Neurospora cra ssa requires a tip-high cytosolic Ca2+ gradient. The source of the Ca2+ app ears to be intracellular stores as there is no net transplasma membrane Ca2 + flux at the elongating hyphal tip and modification of ion fluxes across t he plasma membrane using voltage clamp is without effect on tip growth. To decode the internal mechanisms which generate and maintain the tip-high Ca2 + gradient we first identified calcium regulators which affect hyphal growt h and morphology, then determined how they modify cytosolic [Ca2+] and the actin cytoskeleton using fluorescent dyes and confocal microscopy. Cyclopia zonic acid (a known inhibitor of the endoplasmic reticulum calcium ATPase) inhibits growth and increases cytoplasmic [Ca2+] in the basal region 10-25 mum behind the hyphal tip. 2-APB (2-aminoethoxydiphenyl berate, an inhibito r of IP3-induced Ca2+ release) inhibits hyphal elongation and dissipates th e tip-high Ca2+ gradient 0-10 mum from the tip. Microinjections of the IP3 receptor agonists adenophostin A and IP3 (but not control microinjections o f the biologically inactive L-IP3) transiently inhibited growth and induced subapical branches. IP3 microinjections, but not L-IP3, lowered tip-locali zed [Ca2+] and increased basal [Ca2+]. Even though their effect on [Ca2+] g radients was different, both cyclopiazonic acid and 2-APB disrupted similar ly the normal actin pattern at the hyphal apex. Conversely, disruption of a ctin with latrunculin B dissipated tip-localized Ca2+. We conclude that the tip-high Ca2+ gradient is generated internally by Ca2 sequestration into endoplasmic reticulum behind the tip and Ca2+ release v ia an IP3 receptor from tip-localized vesicles whose location is maintained by the actin cytoskeleton.