Molecular and confocal microscopy comparisons of wild type and temperature sensitive alleles of the aspergillus nidulans morphogenetic locus HypA

Aspergillus nidulans is a filamentous fungus whose cells have highly polarized growth. This requires many genes including hypA, which affects hyphal morphogenesis by promoting tip cell growth and suppressing growth in basal regions. The hypA locus was cloned previously by complementing the hypA1 phenotype. The hypA orthologues in Saccharomyces cerevisiae and Schizosaccharomyces pombe are essential, whereas hypA deletion strains in Aspergillus nidulans are viable but morphologically abnormal. The A. nidulans hypA locus has two temperature sensitive alleles, hypA1 and hypA6. These alleles were generated independently, but using the same mutagen, and were identified and characterized by different groups. Although the hypA1 strain has been shown to sporulate at restrictive temperature, the hypA6 strain was described as having a restrictive arrest phenotype, which suggested it was a lethal defect and was at odds with the report that hypA was dispensable for growth. To resolve this discrepancy, my study compared the hypA1 and hypA6 strains using morphometry, and compared their genetic lesions. I show that the hypA1 and hypA6 phenotypes are indistinguishable and mutation events in their coding regions are identical. Both hypA1 and hypA6 strains were able to sporulate at restrictive temperature as shown by scanning electron microscopy. Their cell morphologies were indistinguishable after growth under restrictive conditions, as were their repolarization kinetics when restrictive-grown cells were shifted to permissive temperature. Sequencing the hypA locus from a hypA6 strain, and comparing it to a hypA1 strain showed that they had identical lesions (G329R), which is consistent with the morphometric analysis. Unexpectedly, the hypA1 and hypA6 strains shared additional non-conservative amino acid substitutions, K885F and E932K, with their wildtype parent A28 compared to the strain used for complementing hypA1. The repair of these two amino acid changes can not rescue hypA1 or hypA6 phenotype at 42°C. The S. cerevisiae homologue of hypA protein is TRS120p, which is involved in endomembrane trafficking. FM4-64 endomembrane staining of hypA1 and hypA6 strains showed they had aberrant endomembrane arrays at restrictive versus permissive temperature, which recovered the wildtype pattern after a return to permissive conditions. This is consistent with the similarities between the hypA and TRS120 sequences. The disparity between the published descriptions of the hypA1 and hypA6 restrictive phenotypes has been resolved, and the allele renamed hypA1/A6