Loss of growth polarity and mislocalization of septa in a Neurospora mutant altered in the regulatory subunit of cAMP-dependent protein kinase.

Abstract

In filamentous fungi, growth polarity (i.e. hyphal extension) and formation of septa require polarized deposition of new cell wall material. To explore this process, we analyzed a conditional Neurospora crassa mutant, mcb, which showed a complete loss of growth polarity when incubated at the restrictive temperature. Cloning and DNA sequence analysis of the mcb gene revealed that it encodes a regulatory subunit of cAMP‐dependent protein kinase (PKA). Unexpectedly, the mcb mutant still formed septa when grown at the restrictive temperature, indicating that polarized deposition of wall material during septation is a process that is, at least in part, independent of polarized deposition during hyphal tip extension. However, septa formed in the mcb mutant growing at the restrictive temperature are mislocalized. Both polarized growth and septation are actin‐dependent processes, and a concentration of actin patches is observed at growing hyphal tips and sites where septa are being formed. In the mcb mutant growing at the restrictive temperature, actin patches are uniformly distributed over the cell cortex; however, actin patches are still concentrated at sites of septation. Our results suggest that the PKA pathway regulates hyphal growth polarity, possibly through organizing actin patches at the cell cortex.

A catalytic subunit of cyclic AMP-dependent protein kinase, PKAC-1, regulates asexual differentiation in Neurospora crassa

“…The mcb (microcyclic conidiation) mutant of N. crassa, which is known to be a mutant of the PKA regulatory subunit gene, exhibits loss of growth polarity at restrictive temperatures, possibly due to altered organization of actin patches at the cell cortex (Bruno et al 1996). The morphology of the double mutant pkac-1 mcb resembles that of the pkac-1 mutants (Fig.…”

“…Suppression of the abnormal growth polarity of the mcb strain by the pkac-1 gene mutation indicates that PKAC-1 activity is essential for the mcb phenotype. The mcb mutant has increased expression of two overlapping transcriptional units, and loss of polarity is suppressed by cr-1 (Bruno et al 1996), suggesting that the mcb mutant is not a null mutant of the regulatory subunit gene. Further studies will be necessary to clarify the function of the regulatory subunit in cell polarity in the mcb mutant of N. crassa.…”

“…The mcb (microcyclic conidiation) mutant, a morphological mutant known to have a defect in the regulatory subunit of PKA, forms defined hyphae at 25 ° C, but it shows a complete loss of growth polarity when hyphal filaments are transferred to 37 ° C. The phenotype of the mcb mutant is restored by transformation with the wild-type mcb gene. The cr-1 mutation is epistatic to the mcb mutation, because a cr-1 mcb double mutant shows normal conidial germination at 37 ° C (Bruno et al 1996). The cot-1 gene encodes a Ser/ Thr protein kinase (Yarden et al 1992), and the colonial temperature-sensitive ( cot ) mutants exhibit abnormal polar extension and branching patterns when grown at the restrictive temperature.…”

The Nuclear Dbf2-Related Kinase COT1 and the Mitogen-Activated Protein Kinases MAK1 and MAK2 Genetically Interact to Regulate Filamentous Growth, Hyphal Fusion and Sexual Development in Neurospora crassa

“…As environmental suppression of both kinases was correlated with reduced PKA activity levels, we analyzed PKA activity in the Dmak-2 strain and found several lines of evidence for reduced PKA activity. mcb is a temperaturesensitive mutant defective in the regulatory subunit of PKA, which displays elevated PKA activity levels at restrictive temperature, resulting in apolar growth and irregular chains of spherical cells (Bruno et al 1996a;Seiler et al 2006;Ziv et al 2008). Genetic analysis of a Dmak-2;mcb(14-4) double mutant demonstrated that Dmak-2;mcb(14-4) grew slower than the parental strains at permissive temperature, suggesting a genetic interaction between MAK2 and PKA signaling.…”

Systematic Epistasis Analysis of the Contributions of Protein Kinase A- and Mitogen-Activated Protein Kinase-Dependent Signaling to Nutrient Limitation-Evoked Responses in the Yeast Saccharomyces cerevisiae

“…Taken together with our results, it is clear that, despite the similarities in cellular behaviors that accompany FG in diploids and haploids, and despite the fact that the same kinase pathways are involved, cell type-specific differences impose different regulatory dependencies and outcomes in the face of similar inputs. It will be interesting to compare the detailed regulatory arrangements in other yeasts and pathogenic fungi, whose filamentous behaviors generally require components orthologous to the MAPK and PKA signaling pathways (and the downstream transcription factors) first described in S. cerevisiae (Bruno et al 1996;Banuett 1998;Martinez-Espinoza et al 2004;Verstrepen and Klis 2006;Klosterman et al 2007;Rispail et al 2009;Hoi and Dumas 2010).…”