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. crassa strains transformed with a construct that provides for the inducible expression of antisense RNA for the catalytic subunit of calcineurin (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 transformants with multiple construct integrations. Induction conditions caused growth arrest which indicated that the cna-1 gene is essential for growth of N. crassa. Growth arrest was preceded by an increase in hyphal branching, changes in hyphal morphology and concomitant loss of the distinctive tip-high Ca2+ gradient typical for growing wild-type hyphae. This demonstrates a novel and specific role for calcineurin in the precise regulation of apical growth, a common form of cellular proliferation. 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 FK506 imply that the drugs act in N. crassa, as in T-cells and Saccharomyces cerevisiae, by inactivating calcineurin. The finding that exposure of growing wild-type mycelium to these drugs leads to a phenotype very similar to that of the cna-1 antisense mutants is consistent with this idea.
Prokisch
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H
Institut für Angewandte Genetik, Universität Hannover, Germany. Holger.Prokisch@mbox.genetik.uni-hannover.de
Yarden
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O
Dieminger
M
M
Tropschug
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M
Barthelmess
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IB
D019703:Calcineurin / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D016572:Cyclosporine / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D007166:Immunosuppressive Agents / Q000494:pharmacology; D009492:Neurospora crassa / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development*; D019696:Peptidylprolyl Isomerase / Q000494:pharmacology; D010957:Plasmids; D016372:RNA, Antisense / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D011995:Recombination, Genetic; D016559:Tacrolimus / Q000494:pharmacology; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D007166:Immunosuppressive Agents; D016372:RNA, Antisense; D012333:RNA, Messenger; D016572:Cyclosporine; D019703:Calcineurin; D019696:Peptidylprolyl Isomerase; D002118:Calcium; D016559:Tacrolimus