Neurospora Crassa Polarisome Architecture Figure 12. BNI-1 is a constituent of the Spk, but also localized to an apical cap. (A) BNI-1-GFP colocalized with the Spk, but also was present in an apical cap (see inset in A). (B) Time sequence of BNI-1 dynamics during mature hyphal tip growth and lateral branch initiation. 0–2 min: in the straight growing hyphal tip small crescents (arrowheads) of BNI-1 were located on either side of the Spk. 4–6 min: shortly before branch initiation, tip extension transiently ceased – evident by rounding-off of the tip - and apical BNI-1 fluorescence disappeared. 8–10 min: extension of the main tip resumed with a brighter cluster of BNI-1 at the left hand side of the apex, followed by displacement of the Spk and left-orientation of the tip. A small crescent of formin fluorescence localized to the tip of the emerging branch (arrow). 12–14 min: a brighter cluster of the formin accumulated at the right hand side preceding reorientation of tip extension into this direction. Scale bars, 10 mm. doi:10.1371/journal.pone.0030372.g012 Finally, we related the live-cell imaging data to the phenotypic defects observed in the corresponding gene deletion mutants. The key findings of our analyses are summarized in Table 3 and the accompanying schematic diagram in Figure 15. Cell symmetry breaking in N. crassa is a polarisome- independent process We found no evidence for BUD-6 or SPA-2 being involved in the establishment of polarized growth in Neurospora crassa. Cortical recruitment during cell symmetry breaking or lateral branching was only observed for BNI-1.The earliest recruitment of BUD-6 and SPA- 2 was observed during germ tube extension (Figures 1 and 2) [59]. Together with BNI-1, all three proteins localized to an apical cap, suggesting that a functional polarisome complex became constituted for the maintenance of polarized tip growth. The same applied for CAT homing during germling fusion (Figures 2 and 11), and extension of vegetative fusion hyphae in mature colonies (Figures 5 and 11). During lateral branching apical BUD-6 recruitment coincided with but did not precede Spk appearance (Figure 3C). Together, this data suggests an exclusive role for BUD-6 in the maintenance of polarized tip growth in N. crassa, which contrasts with findings from budding yeast. Here punctate, cortical accumulations of Bud6-GFP have been observed prior to bud emergence [40]. However, the Aip3/Bud6 mutants of S. cerevisiae have not been found to be defective in the establishment of cytoskeletal polarity during budding. Loss over polarity control occurred later during bud enlargement, resulting in abnormally swollen cells. In fission yeast, Bud6 was found to localize to both cell tips and the cytokinetic ring [64]. Considering that Dbud6 cells of S. pombe had a specific defect in the efficient initiation of polarized growth indeed indicates an important role for Bud6 in the establishment of cell polarity, at least in fission yeast. In A. nidulans BudA-GFP has only been reported to accumulate at septation sites, but not at any other location associated with polarized growth [65]. Severe defects in polarity regulation in the A. nidulans DbudA mutant, however, do suggest an important function for BudA in hyphal tip growth. In C. albicans and A. gossypii Bud6 has been localized to apical crescents/caps during tip growth maintenance [18,66]; cortical recruitment prior to or during cell symmetry breaking has not been reported in these species. Considered together, it appears that the role of BUD-6 in filamentous fungi is focused on the maintenance of cell polarity, PLoS ONE | www.plosone.org 14 January 2012 | Volume 7 | Issue 1 | e30372