machinery, nor components of the CAR driving septum constriction [59,65]. Furthermore, we found that Dspa-2 strains of N. crassa were able to form septa and showed no defects in cytokinesis/conidiation (data not shown), clearly demonstrating that both processes occur independently of the central polarisome scaffolding protein. BUD-6 and BNI-1, on the other hand, were recruited to the incipient septation site before membrane invagination became visible by FM4–64 staining. During the actual septum formation process both proteins showed identical dynamics by remaining associated to the CAR. Whereas BNI-1 disappeared upon septum completion (Figure 13, and previously shown [62]), BUD-6 remained associated to the inner perimeter of the septal pore (Figure 1) for periods of about 4–5 hours and within a peripheral zone of about 1 mm behind the leading colony edge. This behavior indicates additional functions of the protein at this location. Key Rho signaling components, including RHO-1, RHO-2, RHO-3 and RHO-4, the associated GEFs BUD-3 and RGF-3, as well as the landmarking protein BUD-4, have been shown to exclusively function during septation, but not polarized tip growth [62,72,73]. Interestingly, most of these components also persist at septal pores, similar to BUD-6. Thus, it seems likely that these proteins interact at the septal pore, to allow rapid sealing in case physical injury occurs (see further discussion below). Interestingly, although a very similar phenotype for the A. nidulans DbudA mutant has been reported [65,74], in contrast to N. crassa Dbud-6, DbudA was able to form septa, indicating that BudA has only a minor role in septum formation in Aspergillus. Another difference seems to be that A. nidulans BudA did not remain at the pore for prolonged periods of time after septum completion. A second polarisome-independent process is cytokinesis result- ing in cell separation. In fungi this involves the formation of a primary cross wall, the inward growth of which is led by the CAR and results in two adjacent, cytologically separated cell compart- ments. Subsequently, additional layers of cell wall material are laid down at either side of the primary septum to form two secondary septa. Finally, the primary septum is dissolved by hydrolytic enzymes to allow physical cell separation, followed by removal of the septation machinery. In N. crassa, this sequence of events occurs during conidophore development and leads to the differentiation of asexual macroconidia [49]. During this process, BUD-6 and BNI-1 (but not SPA-2) are present in the CAR ultimately forming the primary septum, and contribute to the formation of secondary septa. In analogy to the formation of porous septa, BUD-6 remained associated with the cell poles upon completion of the secondary cross wall, whereas BNI-1 was only transiently present and did not last beyond the point when physical cell separation was achieved. A potential function of BUD-6 could involve recruitment of secretory vesicles containing cell wall material in order to strengthen the initially weak secondary septa. With progressing age of the colony, the number of conidia showing these accumulations decreased, suggesting that with maturation of the conidium, BUD-6 gradually dispersed from secondary septa and cell poles, respectively. Septal plugging, consolidation and repolarization Hyphal wounding assays showed that immediately after the physical injury has been produced all three polarisome proteins rapidly localized to the septal plug, initiated by Woronin body recruitment [75]. Interestingly the three polarisome proteins exhibited slightly different dynamics: BUD-6, which was already present around the pore, became focused to the plug (Figure 4A); BNI-1 became newly recruited to an area around the pore, then focused to the plug (Figure 13A); and SPA-2 directly appeared at the plug (Figure 4D). This seemingly sequential occurrence might Neurospora Crassa Polarisome Architecture reflect a certain order of events during pore sealing. As neither Woronin bodies nor their key protein components are conserved in the Saccharomycotina [76], this probably polarisome-depen- dent function of BUD-6, SPA-2 and BNI-1 must be an ancient role removed during the evolution of the yeast forms from the filamentous fungal ancestor [77]. Complete pore sealing requires consolidation [78,79], i.e. the establishment of a permanent seal through the accumulation of secretory vesicles depositing new cell wall material over the cytoplasmic side of the pore-occluding Woronin body, as well as plasma membrane resealing. Consolidation is usually followed by the reinitiation of polarized hyphal tip growth [80,81]. The polarisome proteins are likely to be involved in the coordination of these processes, as all three components – the Woronin body, the polarisome proteins BUD-6, SPA-2 and BNI-1, and secretory vesicles – colocalized during this step (Figure 4). The notion that the Woronin body acts as an assembly platform for the polarity machinery, is supported by the fact that N. crassa mutants lacking the Woronin body core protein HEX-1 displayed a defect in the reinitiation of polarized growth [82]. The association between Woronin body and septum becomes established very early on during synthesis of the cross-wall [83], thus it would be interesting to investigate the spatiotemporal relationship between fluores- cently labelled HEX-1 and polarisome proteins in greater detail. Astonishing in these wounding assays was the speed at which a new hyphal tip regenerated from the sealed septum. Five to ten minutes after cutting the colony edge new growing tips were readily established (Figure 4), and sometimes several hyphal tips emerged from one sealed septum. Even more revealing were the quick changes in the apical localization patterns of the three polarisome proteins. Their spatial rearrangements occurred within the 10 minutes following septal plugging, and represent a ‘time- lapse’ of the morphogenetic transition from germling to mature hypha, which normally takes several hours. Thus, hyphal wounding assays provide an excellent model system to study the rearrangement of the tip growth apparatus in a time window of about 20 minutes, and can be studied with even greater spatiotemporal precision and less overall mycelial damage when applying laser dissection to selectively cut individual hyphae. This approach has provided the first evidence that components of the cell fusion machinery are involved in septal pore plugging and consolidation [84]. Is the spatial location of cell fusion in the mature colony regulated by polarisome components? All three polarisome proteins showed identical dynamics at the tips of CATs and fusion hyphae, terminating with their disappearance from the established fusion pore (Figures 2, 5 and 11). Interestingly, the fusion phenotype of the corresponding gene deletion mutants revealed some differences. Conidia of Dspa-2 only showed a slightly delayed onset of CAT-mediated cell fusion. However, in the end this mutant successfully established germling networks, and VHF in the mature colony was indistinguishable from the wild type [89]. Both, Dbni-1 and Dbud-6 mutants showed an intriguing phenotype in that VHF was established right at the leading edge of the colony (Figure 10); an unusual feature not seen in the wild type [53]. Due to the lack of conidia, germling fusion assays could not be performed in these two mutants. However, as evidence for VHF in the mature colonies of BUD-6 and BNI-1 deletion mutants has been found, and furthermore the presence of SPA-2 also was not essential for cell fusion, these data strongly suggest that a functional polarisome is dispensable for hyphal fusion but might assist in its regulation. PLoS ONE | www.plosone.org 18 January 2012 | Volume 7 | Issue 1 | e30372