Neurospora Crassa Polarisome Architecture Figure 13. BNI-1 localization during septal plugging, tip repolarization and septum formation. (A) Two minutes after damaging leading hyphae BNI-1 became recruited to the septal plug (position of the Woronin body is indicated with an arrow, 0 min). Fluorescence focused into a smaller area from which a new hyphal tip repolarized, and shortly after condensed into an subapical spot (arrowhead, 16 min) with flanking BNI-1 crescents on either side (inset, 20 min). In parallel, a new septum was being formed about 25 mm behind the severed end, and an additional site of polarity was established (arrowhead, 20 min). Scale bar, 5 mm. See Movie S11 for full sequence. (B) Continuation of (A) but with an extended field of view including an old septum (asterisk). The part of the hypha shown in (A) is outlined with a dashed box. A selection of individual optical slices shows the formation of several septa. Upon septum completion, BNI-1 gradually disappeared from the septal pore. Barely visible remains are indicated with circles at the 68 min time point. BNI-1 fluorescence was usually not observed at ‘old’ septa (asterisk). Recruitment of BNI-1 to a vegetative hyphal fusion site is indicated by an arrowhead. doi:10.1371/journal.pone.0030372.g013 whereas in yeasts BUD-6 also seems to operate during its establishment. Novel localization pattern of BUD-6 identified in the mature hyphal tip Revealing was the finding that the apical localization pattern of BUD-6 in mature hyphal tips differed significantly from that of any other known polarisome component. This unexpected and so far unique localization pattern confirms the notion that the polarisome and Spk are distinct structures [66]. It furthermore suggests an entirely new function of the protein in the organization of the fungal tip growth apparatus. Absence from the Spk was surprising considering that the other two polarisome proteins partially (SPA-2, [59]) or fully (BNI-1) colocalized with this Figure 14. BNI-1 localization during conidiogenesis. (A) During cytological compartmentalization of conidiophores BNI-1 localized to forming septa (arrowheads). Apart from occasional cortical clusters no specialized localizations (e.g. to cell poles) of the formin could be observed at mature stages of conidial development. (B) In contrast, apart from weak cytoplasmic fluorescence, no specific accumulation of SPA-2 could be observed during conidiophore formation, cytokinesis or (C) upon physical separation of mature conidia. Scale bars, 5 mm. doi:10.1371/journal.pone.0030372.g014 PLoS ONE | www.plosone.org 15 January 2012 | Volume 7 | Issue 1 | e30372