Protoplasma

, Volume 213, Issue 3–4, pp 203–217 | Cite as

Calcium and tip growth inNeurospora crassa

  • L. B. Silverman-Gavrila
  • R. R. Lew
Original Papers

Summary

We examined the ionic regulation of tip growth inNeurospora crassa by a combination of electrophysiology and confocal microscopy. To determine if transmembrane ionic fluxes are required for tip growth, we voltage clamped the membrane from −200 to +50 mV. In this voltage range, transmembrane ionic fluxes would either reverse (e.g., K+) or change dramatically (e.g., Ca2+ influx) but had no effect on hyphal growth rates. Therefore, ionic fluxes (including Ca2+ influx) may not be required for tip growth. However, intracellular Ca2+ may still play an obligatory role in tip growth. To assess this possibility, we first increased cytosolic Ca2+ directly by ionophoresis. Elevated Ca2+ induced subapical branch initiation, often multiple tips. At hyphal tips, fluorescence ratio imaging using fluo-3 and fura-red revealed a pronounced tip-high Ca2+ gradient within 10 μm of the tip in growing hyphae which was not observed in nongrowing hyphae. Injection of the Ca2+ chelator 1,2-bis(ortho-aminophenoxy)ethane-N,N,N′,N′-tetrapotassium acetate consistently inhibited growth concomitantly with a depletion of intracellular Ca2+ and dissipation of the tip-high gradient. We conclude that Ca2+ plays a regulatory 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 ascomyceteN. crassa.

Keywords

Calcium gradient Tip growth Ratio fluorescence imaging Ca2+ chelator Voltage clamping Neurospora crassa 

Abbreviations

BAPTA

1,2-bis(ortho-aminophenoxy)ethane-N,N,N′,N′-tetrapotassium acetate

[Ca2+]i

intracellular Ca2+ concentration

fluo-3

2,7-dichloro-6-hydroxy-3-oxo-9-xanthenyl-4′-methyl-2,2′-(ethylenedioxy)dianiline-N,N,N′,N′-tetraacetic acid

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Copyright information

© Springer-Verlag 2000

Authors and Affiliations

  • L. B. Silverman-Gavrila
    • 1
  • R. R. Lew
    • 1
  1. 1.Biology DepartmentYork UniversityTorontoCanada

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