IMPAIRMENT OF CALCINEURIN FUNCTION IN NEUROSPORA-CRASSA REVEALS ITS ESSENTIAL ROLE IN HYPHAL GROWTH, MORPHOLOGY AND MAINTENANCE OF THE APICAL CA2+ GRADIENT

The function of Neurospora crassa calcineurin was investigated in N. c rassa strains transformed with a construct that provides for the induc ible expression of antisense RNA for the catalytic subunit of calcineu rin (cna-1). Induction of antisense RNA expression was associated with reduced levels of cna-1 mRNA and of immunodetectable CNA1 protein and decreased calcineurin enzyme activity, indicating that a conditional reduction of the target function had been achieved in antisense transf ormants with multiple construct integrations. Induction conditions cau sed growth arrest which indicated that the cna-1 gene is essential for growth of N. crassa. Growth arrest was preceded by an increase in hyp hal branching, changes in hyphal morphology and concomitant loss of th e distinctive tip-high Ca2+ gradient typical for growing wild-type hyp hae. This demonstrates a novel and specific role for calcineurin in th e precise regulation of apical growth, a common form of cellular proli feration. In vitro inhibition of N. crassa calcineurin by the complex of cyclosporin A (CsA) and cyclophilin20, and increased sensitivity of the induced transformants to the calcineurin-specific drugs CsA and F K506 imply that the drugs act in N. crassa, as in T-cells and Saccharo myces cerevisiae, by inactivating calcineurin. The finding that exposu re of growing wild-type mycelium to these drugs leads to a phenotype v ery similar to that of the cna-1 antisense mutants is consistent with this idea.