Neurospora Crassa Polarisome Architecture Figure 3. BUD-6 recruitment to polarized growing mature hyphal tips and branches. (A) BUD-6-GFP accumulated as a subapical cloud with a distinct exclusion zone at the very tip apex (arrowhead). FM4–64 staining revealed that this exclusion zone was occupied by the Spk. Scale bars, 10 mm; in inset, 5 mm. (B) Recording BUD-6-GFP dynamics during hyphal tip growth showed that the BUD-6-GFP exclusion zone (arrowheads and inset for magnified view) consistently remained at the extending tip apex. Scale bars, 10 mm. See Movie S4 for time course sequences. (C) Recruitment of BUD-6 to incipient lateral branch points was not observed. Detectable BUD-6 accumulation occurred after the Spk became stained with FM4–64 in newly established branches (arrowheads). doi:10.1371/journal.pone.0030372.g003 the localization of these brighter, more fluorescent regions changed with time indicating fast dynamics of the protein (Figure 3B). The region at the hyphal apex which is usually occupied by the Spk was completely free of BUD-6 fluorescence (Figure 3A), but clearly visible with FM4–64 staining. Figure 3B shows the dynamic behaviour of BUD-6 in the apical dome during polarized tip growth (see Movie S4). Cortical accumulation of BUD-6 at incipient branch points was never observed, and detection of apical BUD-6-GFP signal in branch tips coincided with but did not precede the appearance of a FM4–64-stained vesicle cluster (Figure 3C). This observation is in line with the finding from conidial germlings that BUD-6’s major role is in the maintenance of polarity but not its establishment. Re-establishment of tip growth after physical injury involved recruitment of BUD-6 and SPA-2 to the septal plug and regenerating hyphal tip To test potential functions of the persisting ring of BUD-6 at completed septal pores (Figure 1H) we performed wounding assays that trigger immediate pore occlusion with Woronin bodies, subsequent consolidation of the sealed pore, and the rapid reinitiation of tip polarity from the severed end (for a recent review see Jedd, 2010). Within two minutes of a hypha being physically injured, a Woronin body occluded the septal pore, and BUD-6-GFP that was originally localized around the septal pore (Figures 1F–H) disappeared within 4 min once a new hyphal tip emerged from this septum (Figure 4A). Often further recruitment of BUD-6 into a cortical ring preceding and accompanying the formation of a new septum 20–30 mm behind the sealed septum could be observed (Figure 4A). Interestingly, coinciding with the onset of reinitiation of polarized tip growth from the septum closest to the severed end of the hypha, BUD-6 became focussed at the septal plug. This occurred in parallel with the recruitment of FM4–64-stained lipophilic, possibly membranous, material in the vicinity of the Woronin body (Figure 4B). This plug complex, consisting of Woronin body, BUD-6 and FM-64 stained material, either became pushed aside by the emerging tip (Figure 4B, 25 min), or remained in place (Figure 4C). In any case, while one population of BUD-6 remained at the plug complex, another population became recruited to an apical cap of the regenerating PLoS ONE | www.plosone.org 5 January 2012 | Volume 7 | Issue 1 | e30372