concentrated as a ring surrounding the septal pore (Figure 1G). This data suggests, that BUD-6 might be part of the landmarking machinery that determines the site of septation, and clearly is part of the contractile actomyosin ring (CAR) driving septum constriction [22,26,27]. Interestingly, upon the completion of a septum a ring of BUD-6 persisted for more than four hours, suggesting additional roles of the protein at the inner perimeter of the septal pore. BUD-6 and SPA-2 participated in CAT-mediated cell fusion A marked increase in localized recruitment of BUD-6 occurred during CAT-mediated cell fusion between conidial germlings (Figure 2A, Movie S2). During CAT homing cortical clusters of BUD-6 formed at the tip apex and coalesced at the incipient fusion point as soon as both CATs made contact. BUD-6 fluorescence concentrated at the site of fusion pore formation, was present as a ring during pore opening and disappeared shortly after cytoplas- mic continuity had been established. Changes in the BUD-6 fluorescence pattern in germling networks marked the different Neurospora Crassa Polarisome Architecture stages of the cell-cell fusion process (Figure 2B). The polarisome scaffolding protein SPA-2 showed very similar dynamics during CAT-mediated cell fusion (Figure 2C), including the dispersal of the protein. In the example shown, GT extension transiently arrested during cell fusion, but as soon as cytoplasmic continuity has been established, a new cluster of SPA-2 assembled at the germ tube tip (arrowhead in Figure 2C), and polarized tip extension resumed (Figure 2D and Movie S3). Taken together, BUD-6 and SPA-2 are both part of the polarity machinery organized in an apical cap that drives CAT homing, and both are localized at the site of CAT fusion. BUD-6 showed a unique localization pattern forming a subapical cloud around the Spitzenko¨ rper In mature hyphae a very distinct localization pattern was observed for BUD-6. Instead of localizing to an apical cap or to the Spk, as seen for many other components of the polarity machinery, BUD-6 fluorescence exclusively appeared in a heterogeneous, subapical cloud around the Spk (Figure 3). BUD- 6 was more concentrated in some regions of the BUD-6 cloud and Figure 2. BUD-6 and SPA-2 recruitment during CAT-mediated cell fusion. (A) Clusters of BUD-6-GFP became recruited to CAT tips, concentrated at the incipient fusion site upon contact, and formed an opening ring of fluorescence during fusion pore formation (arrowhead). See Movie S2 for time course sequences. Scale bar, 5 mm. (B) Typical changes in the BUD-6 localization pattern accompanied distinct stages of the cell-cell fusion process in germling networks: (1) pronounced accumulation shortly after CAT attachment, (2) ring formation during fusion pore opening, and (3) BUD-6 disappearance shortly after cytoplasmic continuity was established. Scale bar, 5 mm. (C) Z-projections of confocal optical sections through conidial germlings expressing SPA-2-GFP during CAT homing and fusion. Shortly after cytoplasmic continuity was successfully established and SPA-2 disappeared from the fusion site, a new cluster of SPA-2 became recruited to the germ tube tip (arrowhead) that resumed polarized growth (transient arrest of germ tube growth during cell fusion, and resumed tip extension post-fusion is indicated with dotted line). Scale bars, 5 mm. See Movie S3 for full time sequence. (D) Shows an enlarged view of BUD-6 recruitment during the continuation of germ tube tip growth as shown in (C). doi:10.1371/journal.pone.0030372.g002 PLoS ONE | www.plosone.org 4 January 2012 | Volume 7 | Issue 1 | e30372