Localization and function of calmodulin in live-cells of Aspergillus nidulans

Abstract

Calmodulin (CaM) is a small, eukaryotic protein that reversibly binds Ca 2+. Study of CaM localization in genetically tractable organisms has yielded many insights into CaM function. Here, we described the dynamic localization of Aspergillus nidulans CaM (AnCaM) in live-cells by using recombination strains with homologous, single cross-over insertions at the target gene which placed the GFP fused copy under the inducible alcA promoter and the RFP–CaM integration under the native cam promoter. We found that the localization of CaM fusion was quite dynamic throughout the hypha and was concentrated to the active growing sites during germination, hyphal growth, cytokinesis and conidiation. The depletion of CaM by alcA promoter repression induced the explicit abnormalities of germlings with the swollen germ tubes. In addition, the position of highly concentrated GFP–CaM in the extreme apex seemed to determine the hyphal orientation. These data collectively suggest that CaM is constantly required for new hyphal growth. In contrast to this constant accumulation at the apex, GFP–CaM was only transiently localized at septum sites during cytokinesis. Notably, depletion of CaM caused the defect of septation with a completely blocked septum formation indicating that the transient CaM accumulation at the septum site is essential for septation. Moreover, the normal localization of CaM at a hyphal tip required the presence of the functional actin cytoskeleton and the motor protein KipA, which is indispensable for positioning Spitzenkörper. This is the first report of CaM localization and function in live-cells by the site-specific homologous integration in filamentous fungi.