Fig. 1. Mlc1 and FM4-64 colocalise to a Spitzenkörper-like structure at hyphal tips. (A) Colocalisation of Mlc1-YFP (green) and FM4-64 (red). Images shown are projections of the Z-stack of deconvolved images. In the merged panel, there is extensive overlap between Mlc1-YFP and FM4-64. (B) Detail of Mlc1 localisation at hyphal tips. Top left, An Mlc1 spot and crescent are visible. Right panels, the Mlc1-spot is present either at the tip (top) or just behind the tip (bottom). (C) Optical sections at 200 nm intervals through an individual germ tube tip. The crescent comes into focus at a different optical plane compared to the spot (arrow). Hyphal tips are outlined in panels B and C. (D) Hyphal germ tubes of an Mlc1-YFP-expressing strain were stained with filipin to reveal the plasma membrane. A 3D model was generated from the deconvolved Z-stack using Volocity^® software. Mlc1-YFP (green) forms a 3D ball located behind the cell membrane (blue). (E) Localisation of Mlc1 (red) and the septin, Cdc11 (green), revealed by immunocytofluorescence using polyclonal antisera to the respective S. cerevisiae proteins. Specificity of the Cdc11 antibody has been described elsewhere (Sudbery, 2001). The anti-Mlc1 antibody recognised a single band in western blots that was the same size as S. cerevisiae Mlc1 (data not shown). (F) The shape of a hypha can be predicted by the VSC model according to the distance of the spot from the hyphal tip. An Mlc1-YFP expressing strain was induced to form hyphae and cell walls were stained with Concanavalin A-Texas Red. Measurements were made of external width at various distances from the tip. These were compared with the predicted widths at these points made by the Fungus Simulator program version 4 according to the distance of the centre of the Mlc1-YFP spot to the outside of the hyphal tip. Bar, 5 µm (A); 1 µm (B,C,F); 0.5 µm (D); 2 µm (E).