Calcium and tip growth in Neurospora crassa

We examined the ionic regulation of tip growth in Neurospora crassa by a co mbination of electrophysiology and confocal microscopy. To determine if tra nsmembrane ionic fluxes are required for tip growth, we voltage clamped the membrane from -200 to +50 mV. In this voltage range, transmembrane ionic d uxes would either reverse (e.g., K+) or change dramatically (e.g., Ca2+ inf lux) but had no effect on hyphal growth rates. Therefore, ionic fluxes (inc luding Ca2+ influx) may not be required for tip growth. However, intracellu lar Ca2+ may still play an obligatory role in tip growth. To assess this po ssibility, we first increased cytosolic Ca2+ directly by ionophoresis. Elev ated Ca2+ induced subapical branch initiation, often multiple tips. At hyph al tips, fluorescence ratio imaging using fluo-3 and fura-red revealed a pr onounced tip-high Ca2+ gradient within 10 mu m of the tip in growing hyphae which was not observed in nongrowing hyphae. Injection of the Ca2+ chelato r 1,2-bis(ortho-aminophenoxy)ethane-N,N,N',N'-tetrapotassium acetate consis tently inhibited growth concomitantly with a depletion of intracellular Ca2 + and dissipation of the tip-high gradient. We conclude that Ca2+ plays a r egulatory role in tip initiation and the maintenance of tip growth. Because plasma membrane ionic fluxes do not play a role in tip growth, we suggest that the tip-high Ca2+ gradient is generated from intracellular Ca2+ stores in the ascomycete N. crassa.