Environmental suppression of Neurospora crassa cot-1 hyperbranching: a link between COT1 kinase and stress sensing

Abstract

cot-1 mutants belong to a class of Neurospora crassa colonial temperature-sensitive (cot) mutants that exhibit abnormal polar extension and branching patterns when grown at restrictive temperatures. cot-1 encodes a Ser/Thr protein kinase that is structurally related to the human myotonic dystrophy kinase which, when impaired, confers a disease that involves changes in cytoarchitecture and ion homeostasis. When grown under restrictive conditions, cot-1 cultures exhibited enhanced medium acidification rates, increased relative abundance of sodium, and increased intracellular glycerol content, indicating an ion homeostasis defect in a hyperbranching mutant. The application of ion transport blockers led to only mild suppression of the cot-1 phenotype. The presence of increased medium NaCl or sorbitol, H(2)O(2), or ethanol levels significantly suppressed the cot-1 phenotype, restored ion homeostasis, and was accompanied by reduced levels of cyclic AMP-dependent protein kinase (PKA) activity. The cot-1 phenotype could also be partially suppressed by direct inhibition of PKA with KT-5720. A reduced availability of fermentable carbon sources also had a suppressive effect on the cot-1 phenotype. In contrast to the effect of extragenic ropy suppressors of cot-1, environmental stress-related suppression of cot-1 did not change COT1 polypeptide expression patterns in the mutant. We suggest that COT1 function is linked to environmental stress response signaling and that altering PKA activity bypasses the requirement for fully functional COT1.

FIG. 1.

Growth of N. crassa strains under different conditions. (A) Medium acidification and biomass accumulation during the growth of N. crassa wild-type (wt) and cot-1 strains in Vogel's minimal salts medium. Error bars indicate standard deviations. (B) Medium pH of wt or cot-1 cultures 6 h after a shift to 37°C in the presence of DES, amiloride, or ouabain. (C) Effect of DES, amiloride, or ouabain on cot-1 culture morphology. Pictures were taken 8 h after the cultures were shifted from permissive (25°C) to restrictive (37°C) growth conditions (inhibitor concentrations were as shown in panel B).

FIG. 2.

Relative growth of N. crassa strains. (A and B) Relative growth, as a percentage of that of the untreated control, was determined on the basis of radial growth of N. crassa strains in the presence of different concentrations of NaCl or sorbitol, respectively. Full and empty circles indicate culture incubation temperatures of 37 and 25°C, respectively. Error bars indicate standard deviations. (C) Effect of NaCl or sorbitol on cot-1 culture morphology. Pictures were taken 8 h after the cultures were shifted from permissive (25°C) to restrictive (37°C) growth conditions.

FIG. 3.

Effect of medium amendments on the morphology of cot-1 cultures grown at the restrictive temperature (37°C). Pictures were taken 6 h after the amendments were added and after the cultures were shifted from permissive (25°C) to restrictive (37°C) growth conditions. (A) Nonamended medium. (B) H₂O₂ (7 mM). (C) Ethanol (7.5%). (D) Sorbitol (1.0 M) plus 8-Br-cAMP (1 mM). (E) Glucose (3%). (F) Sorbitol (1.0 M) plus glucose(3%).

FIG. 4.

Relative in vitro PKA activities measured in protein extracts prepared from N. crassa wild-type and cot-1 cultures 5 h after a shift from permissive (25°C) to restrictive (37°C) growth conditions. Cultures were grown in the presence of NaCl (1.0 M), sorbitol (1.25 M), ethanol (EtOH) (7.5%), or H₂O₂ (10 mM). NT, not treated. The nonphosphorylated and phosphorylated (indicating activity) fluorescent Kemptide substrates migrated to the anode and cathode of the agarose gel, respectively.

FIG. 5.

Suppression of the cot-1 phenotype by KT-5720. (A) Effect of KT-5720 on cot-1 culture morphology at the restrictive temperature. (B) Relative in vitro PKA activities in proteins extracted from cot-1 cultures grown in the presence of KT-5720 or a PKA activator (8-Br-cAMP) 5 min after introduction of the PKA effector. NT, not treated.

FIG. 6.

Immunodetection of COT1 in extracts prepared from the cot-1 strain. The effects of phenotypically suppressive concentrations of ion pump inhibitors (A), NaCl or sorbitol (B), H₂O₂ ethanol (EtOH) or NaCl (C), or ro-1 or ro-3 extragenic suppressors (D) are shown. Temp., temperature; NT, not treated.

FIG. 6.

Immunodetection of COT1 in extracts prepared from the cot-1 strain. The effects of phenotypically suppressive concentrations of ion pump inhibitors (A), NaCl or sorbitol (B), H₂O₂ ethanol (EtOH) or NaCl (C), or ro-1 or ro-3 extragenic suppressors (D) are shown. Temp., temperature; NT, not treated.