Neurospora Crassa Polarisome Architecture Figure 1. BUD-6 localization in conidia, conidial germlings and sites of septum formation. (A) Intense and locally defined clusters of BUD- 6 were localized at the cell poles of dormant macroconidia. Scale bars, 5 mm. (B–C) Specific recruitment of BUD-6 during isotropic expansion, cell symmetry breaking and germ tube outgrowth was not observed. Scale bars, 2.5 mm. (D) Localized recruitment of BUD-6-GFP to apical caps of growing germ tubes (arrowhead), as well as to septa (arrow), occurred in germlings $35 mm in length. FM4–64 stained vesicle clusters were observed at the same positions. (E) Enlarged view of the germ tube tip highlighted in (D). The merged image shows that BUD-6-GFP fluorescence only partially colocalizes with the FM4–64 stained apical cap which extends over a larger crecent. (F) Staining with FM4–64 revealed that BUD-6 recruitment to the incipient septation site preceded plasma membrane invagination (arrowheads), and that it constantly remained associated with the leading edge of the closing septum. Scale bar, 5 mm. See Movie S1 for time course sequences. Note: the bright FM-4–64 stained spot appearing at time point 0 sec does not colocalize with the cortical BUD-6 accumulation indicated by the arrowhead. (G) Reconstruction of BUD-6-GFP localization at the inner perimeter of the closing septal pore. Scale bar, 2.5 mm. doi:10.1371/journal.pone.0030372.g001 35 mm (Figure 1D). In that case, BUD-6-GFP formed an apical cap with the highest concentration of fluorescence at the very tip, which colocalized with an accumulation of vesicles stained with FM4–64 (arrowhead in Figure 1D; enlarged view shown in Figure 1E). Notably, the apical area stained by FM4–64 localizes over a wider crescent than that labelled with BUD-6-GFP. Taken together, these findings are consistent with a role for BUD-6 in the maintenance of germ tube polarity rather than its establishment. BUD-6 was part of the contractile acto-myosin ring during septum formation BUD-6 participated in septum formation in germlings (arrow in Figure 1D) and mature hyphae (Figures 1F and G). Small clusters of BUD-6 fluorescence localized to the incipient site of septum formation shortly before FM4–64 staining indicated plasma membrane invagination (arrowheads Figure 1F, Movie S1). Subsequently, BUD-6-GFP remained associated with the leading edge of the progressively inward growing septum, and finally PLoS ONE | www.plosone.org 3 January 2012 | Volume 7 | Issue 1 | e30372