Figure 7.

Modifications to arfA expression shift the actin ring toward the hyphal apex. In order to assess the location of the endocytic actin ring, strains MF58.8 and MK6.1 were utilized, which both express the actin binding protein AbpA tagged with cyan fluorescent protein (AbpA::CFP). Isolate MK6.1 was used as control to assess the actin ring position under native arfA expression. Strain MF58.5 has the native arfA deleted, and titratable arfA expression using the Tet-on system. Isolates were cultivated at 22°C for 2 days on MM supplemented with various concentrations of doxycycline and analyzed via confocal microscopy. Z-stacks of 15 individual hyphal tips were taken (A), and representative pictures of DIC (upper panels) and projected 3D images of Z-stacks (middle and bottom panels) are shown. These data indicated structural disruption of the actin ring following reduced/increased arfA expression, and a clear shift of AbpA::CFP toward the hyphal apex. Quantification of AbpA::CFP fluorescence along the cell membrane 20 μm from the tip apex (B) revealed a shift in actin ring position in ArfA loss- and gain-of-function mutants. Fifteen hyphae/strain/condition were assessed, with bars depicting standard error. Dotted lines denote the predicted position of the actin ring. Note two possible positions are shown for MF58.5 under 10 μg/ml dox due to deterioration of ring structure observed in (A).