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Fig. 6. The polarisome components AgSpa2 and AgPea2 and the formin AgBni1 localize to the Spitzenkörper at high growth speeds. (A) Radial growth of polarisome deletion strains. Mycelium of wt, Agspa2
, Agpea2 and Agbud6 was inoculated on AFM agar, spores of a heterokaryonic Agbni1 strain were spotted on selective medium and incubated for 6 days at 30°C. Scale bar: 2 cm. (B) DIC and fluorescence images of GFP-AgBNI1, AgSPA2-GFP, AgPEA2-YFP and GFP-AgBUD6 hyphae. The white numbers indicate growth speed in µm/minute. (C) Correlation between localization and growth speed of the polarisome components. Growth speeds are plotted on the y-axis in µm/minute, the localization of the GFP- or YFP-tagged proteins are shown on the x-axis. (D) Overlay of fluorescently labeled polarisome components (green) and FM4-64 staining (red). (E) Stacks of GFP-AgBni1-expressing hypha that grew faster than 1.50 µm/minute were subjected to blind deconvolution. Maximum projections of central planes are shown. Seventy-one displayed a core-like localization of GFP-AgBni1 (black arrowhead, n=31). The gray arrowhead indicates a zone of reduced GFP-AgBni1 fluorescence. (F) Deconvoluted fluorescence images of AgSPA2-GFP hyphae. Fifty-two percent of fast hyphae displayed an enrichment of AgSpa2-GFP in a core region in the Spitzenkörper (top row, black arrowhead). Thirty-four percent of the hyphae displayed a uniform Spitzenkörper localization (bottom row, n=29). (G) GFP-AgBni1 in polarisome deletion strains. The hyphae grew with speeds of between 0.9 and 1.1 µm/minute. (H) Fluorescence ratios between the tip and the cytoplasm. Maximal GFP or YFP fluorescence was determined in two circular areas of 5 µm diameter, the centers of which were located in the tip and in the cytoplasm 10 µm away from the tip. The average ratios tip/cytoplasm were plotted. More than 15 hyphae with a growth speed between 0.75 and 1.3 µm/minute were assessed per strain. Error bars indicate s.e.m. Scale bars for B,D-G: 5 µm.
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