Neurospora Crassa Polarisome Architecture Figure 10. Dbni-1 and Dbud-6 strains displayed derepressed hyphal fusion at the colony edge. (A) The lack of septa in Dbni-1 resulted in extensive accumulation of vacuoles at the leading edge of the colony. Scale bar, 50 mm. (B) Hyphal fusion at the leading edge of the colony, which is usually suppressed in the wild type through apical dominance, was observed in Dbni-1. Several fusion pores are indicated with arrowheads. Scale bar, 10 mm. See Movie S9 for time course sequence showing vacuolar passage through fusion connections. (C) After closer inspection, the same phenotype could be observed at the leading edge of Dbud-6 colonies. Scale bar 10 mm. (D–F) In the wild type, derepression of hyphal fusion at the leading edge occurred in the presence of conidial germlings. The establishment of cytoplasmic continuity – here visualized through the transfer of nuclei fluorescently labeled with histone H1-GFP (green) from conidial germlings into mature hyphae - involved either CAT-mediated cell fusion (D), or fusion pegs from the mature hypha (E and F) induced through the presence of conidial germlings. Arrowheads indicate fusion sites; C denotes the spore body; GT denotes the germ tube; H denotes mature hypha. Note that fluorescently labeled nuclei originating from the germling have only migrated into the upper part of the unlabeled wild type hypha. The arrowheads in (F) mark fusion pegs emerging from the mature hypha. Scale bar in D, 10 mm. doi:10.1371/journal.pone.0030372.g010 formin at specific locations inside the apical dome might underlie the regulation of directional tip growth, probably by actin- mediated interaction with the Spk. It furthermore confirmed the function of BNI-1 in polarity establishment as already indicated by the findings in conidial germlings (Figure 11). BNI-1 became transiently recruited to septal plugs and showed identical dynamics at repolarizing hyphal tips as SPA-2, but only partial spatiotemporal overlap with BUD-6 About 2 minutes after physical injury had been produced, BNI- 1-GFP fluorescence localized around the septal plug closest to the severed end of the hypha (Figure 13A). Shortly after, fluorescence focussed at an even smaller area around the Woronin body from which a new hyphal tip repolarized and extended into the emptied compartment. Different to BUD-6, the formin did not remain associated with the plug complex, but rather migrated with the repolarizing tip apex and later on, in addition, concentrated into a small subapical spot resembling a forming Spk (Figure 13A). It is likely that the formation of this spot coincided with the appearance of a FM4–64-stained vesicle cluster. This however cannot be concluded with certainty as the FM4–64 background fluorescence from cell debris in the damaged compartment hindered its detection (see Movie S11). The dynamics of BNI-1 during this PLoS ONE | www.plosone.org 12 January 2012 | Volume 7 | Issue 1 | e30372