Neurospora Crassa Polarisome Architecture Figure 4. BUD-6 and SPA-2 concentrated at the septal plug prior to reinitiation of polarized tip growth. (A) Upon physical injury a Woronin body occluded the septal pore which was surrounded by pre-existing BUD-6 fluorescence (arrow). At the same time, a new septum was being initiated 25 mm further back (arrowheads), again leaving BUD-6 at the septal pore after its completion. (B) At the onset of repolarization from the septum at the severed end of the hypha, BUD-6-GFP fluorescence became concentrated at the sealed pore, in the vicinity of the Woronin body and within an accumulation of lipophilic, possibly membranous, material stained by FM4–64 (arrows). See Movie S5 for full sequence. (C) The BUD-6 cluster often remained in place while the new tip emerged (arrowhead). Subsequently and coinciding with the condensation of a recognizable Spk, apical BUD-6 fluorescence became increasingly diffuse (arrowheads). See Movie S6 for full sequence. (D) SPA-2 rapidly became recruited to the occluded septal pore forming an intense spot associated with the Woronin body (arrow). The protein did not reside at consolidated septal pores, but rather relocated into an apical cap of the emerging tip (arrowhead) which accompanied extension of the new hyphae (E). See Movie S7 for full sequences of D. Scale bars A to D, 5 mm. Scale bar E, 10 mm. doi:10.1371/journal.pone.0030372.g004 hyphal tip (Figure 4C). Subsequently and coinciding with the appearance of a FM4–64 stained Spk, apical BUD-6 fluorescence became increasingly diffuse (arrowheads in Figure 4C, 8 min), suggesting rearrangement of the protein into a subapical cloud within a time window of about 10 minutes. Interestingly, SPA-2 which generally does not reside at completed septal pores [59] also became recruited to the plug, and colocalized as intense spot in the vicinity of the Woronin body (Figure 4D). SPA-2, however, did not remain associated with this site, but rather relocated to an apical cap at the emerging and extending hyphal tip (Figure 4D, 2 and 4 min; Movies S5, S6, S7). Together, these observations indicate a role for BUD-6 and SPA-2 in the completion of the septal plug, and the rapid reestablishment of polarized tip growth. Recruitment of BUD-6 and SPA-2 during vegetative hyphal fusion in the mature colony followed identical dynamics as observed during CAT-mediated cell fusion Apical clusters of BUD-6-GFP became recruited to homing tips of fusion hyphae in the mature colony, suggesting the participation of BUD-6 function in vegetative hyphal fusion (VHF). As seen during germling fusion (Figure 2), BUD-6 fluorescence peaked at the incipient fusion site upon cell wall attachment (Figure 5, Movie S8). A ring of fluorescence that increased in diameter could be observed during fusion pore formation, and then gradually disappeared after cytoplasmic continuity was established (Figure 5, 32 min onwards). Thus, BUD-6 did not persist at the completed fusion site as observed upon septum formation (Figure 5B). SPA-2-GFP was also found to accumulate at VHF sites (Figure 5C), and exhibited the same dynamics as BUD-6. Taken together, the BUD-6 VHF localization pattern is distinct from its dynamics during septum formation, and together with the fact that SPA-2 participates in VHF – but not septum formation – provides further evidence that the two processes are distinct from each other. BUD-6 participated in cytokinesis during conidiogenesis, and persisted for several days at secondary septa after cell separation In line with the findings of BUD-6 behaviour during septum formation (Figures 1F and G, and 5B), persistent BUD-6-GFP fluorescence was also localized to sites of cytokinesis in developing conidiophores (Figure 6). In addition, in cytologically separated but physically still attached conidia, BUD-6 fluorescence persisted at the cell poles; either at both or only at one pole in case of the Figure 5. BUD-6 dynamics during vegetative hyphal fusion. (A) BUD-6 became recruited to the tips of homing fusion hyphae, then concentrated at the attachment point and surrounded the opening fusion pore. Shortly after the pore was fully established BUD-6 fluorescence disappeared from this site. Scale bar, 5 mm. See Movie S8 for time course sequence. (B) Transient BUD-6 fluorescence accumulated at incipient fusion sites in the mature colony (arrowhead) and persistent BUD-6 signal at septal pores (all other fluorescently marked sites). Scale bar, 10 mm. (C) SPA-2- GFP became recruited to vegetative hyphal fusion sites (arrow). As it was never seen at completed fusion connections (arrowheads) it must follow the transient dynamics of BUD-6 in this context. Scale bar, 10 mm. doi:10.1371/journal.pone.0030372.g005 PLoS ONE | www.plosone.org 7 January 2012 | Volume 7 | Issue 1 | e30372