Neurospora Crassa Polarisome Architecture Figure 11. BNI-1 recruitment to sites of polarized growth, septum formation and cell fusion. (A) In conidial germlings recruitment of fluorescently labeled BNI-1 was observed during three different cellular processes. (1) During cell symmetry breaking BNI-1 appeared at the cell cortex. (2) During CAT-mediated cell fusion, very bright accumulations of BNI-1 could be seen at the tips of interacting CATs. Due to a spore torque response upon cell-cell attachment, the left cell moved out of the focal plane. This movement is commonly observed when imaging germling fusion in liquid medium. (3) During septum formation BNI-1 was part of contractile actomyosin rings. Scale bar, 5 mm. (B) BNI-1 also accumulated in apical crescents at growing germ tube tips (arrow). Recruitment to septal pores and a new sites of cell symmetry breaking (arrowhead) showed up as well. Also note that BNI-1 is associated with septum formation (asterisk) at the base of the germ tube. (C) Consistent with its participation in CAT-mediated cell fusion, BNI-1 also became recruited to sites of vegetative hyphal fusion, and disappeared shortly after cytoplasmic continuity was established. The arrow marks the opening fusion pore. Scale bar, 5 mm. doi:10.1371/journal.pone.0030372.g011 process were identical to those of SPA-2 (Figure 4D), and overlapped with those of BUD-6, which was already present at the septal pore before injury, and thus did not require de novo recruitment (Figure 4A and B). Apical recruitment of BNI-1 prior to the outgrowth of a second tip was also observed (Figure 13A), again confirming the presence of BNI-1 during polarity establishment. In parallel, the formation of a new septum was being initiated about 25 mm distal from the severed end (Figure 13A). Notably, at the 20 min time point the segregation of BNI-1 into an apical crescent and subtending spot is indicated in the lower tip (enlarged view in inset). This again fits into the 10 min time window spanning from the reinitiation of tip growth until the architecture of the apical tip growth apparatus adapts for a faster rate of tip extension. Several other septa formed in the vicinity, and in all cases BNI-1 did not reside at septal pores for much longer than an hour. The barely visible residual BNI-1 fluorescence at the two septal pores in the image centre is indicated by circles in Figure 13B. Notably, no BNI-1 fluorescence could be seen at older septa (Figure 13B). This data confirms the transient role of BNI-1 during septum formation as already reported by Justa- Schuch et al. (2010), and additionally revealed the rapid recruitment of the formin to sites of polarity re-establishment from the plugged septal pore, as well as the quick (within approx. 10 min) transformation of the tip growth apparatus from an apical cap configuration into an apical crescent with subtending Spk. BNI-1 became only transiently recruited to sites of cytokinesis Similar to BUD-6 (Figure 6), BNI-1 fluorescence became recruited to the septation sites in differentiating conidiophores (Figure 14A), indicating that both proteins participate in cytokinesis. However, BNI-1 did not reside at the cell poles/ secondary septa as observed for BUD-6 (Figure 6). In line with its transient role during septum formation in mature hyphae, upon physical separation of conidia BNI-1 fluorescence disappeared from secondary septa. Fluorescent accumulations at the cell poles of mature conidia were not observed. Small clusters of BNI-1 randomly distributed at the cell cortex, however, could be observed. In contrast to BNI-1 and BUD-6, but consistent with its absence during septum formation, SPA-2 has so far not been observed to localise to sites of cytokinesis (Figure 14B), nor accumulate elsewhere in the cell upon physical separation and maturation of conidia (Figure 14C). Taken together, this data suggests that BNI-1 only transiently participates in cytokinesis. It thus has no continued function in cell wall fortification in maturing conidia as suggested for BUD-6, and both BNI-1 and BUD-6 function in the absence of SPA-2 during conidiogenesis. Discussion In order to determine during which morphogenetic processes a polarisome complex is constituted, and whether its spatiotemporal dynamics differ from those observed in other fungal species, we generated a comprehensive localization map of the three polarisome components identified in Neurospora crassa, We analyzed the subcellular dynamics of the actin-interaction protein BUD-6 and the formin BNI-1 during various morphogenetic transitions that involve changes in the establishment, maintenance and termination of polarized growth, and compared their dynamics to the localization patterns of the bona fide polarisome marker SPA-2. PLoS ONE | www.plosone.org 13 January 2012 | Volume 7 | Issue 1 | e30372