Our work focusses on proteins that localize to hyphal tips and septation sites. In Aspergillus nidulans, Protein kinase C (PkcA) is one such protein. Our previous work identified a mutation in the pkcA gene (calC2) which results in reduced cell wall integrity. We also showed that wild type PkcA localizes to growing hyphal apices and septation sites, and we identified amino acid sequences within PkcA that are required for PkcA to localize to these sites of cell wall synthesis. Our work, as well as work done in other labs, has also brought to light many other proteins that play roles in polarized growth and that also localize to septation sites and growing tips. Among these are the formin SepA which has been observed at septation sites and at the hyphal tip in both a spot and crescent pattern. SepA is associated with contractile actomyosin ring (CAR) formation, and a mutation in the sepA gene (sepA1) renders A. nidulans aseptate at elevated temperature. Using time lapse fluorescence microscopy we have observed overlap of PkcA::RFP and SepA::GFP signals in some tips in the crescent pattern, while other tips displayed SepA spot and PkcA crescent localization within the same tip or separately. At septation sites PkcA::RFP and SepA::GFP consistently appear concurrently; both initially appear at the outer edges of the septation site and contract in a lockstep fashion until both signals dissipate. A functional interaction between the two proteins is shown by the ability of the sepA1 mutation to block PkcA::GFP localization to septation sites, but not to hyphal tips. Additionally, we have demonstrated reciprocal complementation of the septation defect in the sepA1 mutant by pkcA overexpression and of hypersensitivity to cell wall perturbing agents calcofluor white, Congo red, and sodium dodecyl sulfate by sepA overexpression in the calC2 mutant. Using a bimolecular fluorescence complementation strategy we have found evidence that SepA and PkcA physically interact at both hyphal tips and septation sites. Interaction of these two proteins was confirmed using a GAL4-based yeast two-hybrid assay.
Full conference title:
- Asperfest 14 (2017)