(e.g. receptors) back to the growing apex [28]. Therefore, recycling endocytosis can be considered a fourth key component of the hyphal tip growth apparatus. The polarisome is involved in the establishment, maintenance and termination of polarized cell growth. Proteins known to constitute this complex in budding yeast include the three core components Spa2, Pea2 and Aip3/Bud6, as well as the formin Bni1 [29,30,31]. Localization and activation of Bni1 at the cell cortex requires the presence of all three core proteins [31,32,33,34], which together localize in an apical cap driving the directed extension of the bud, mating projection or pseudohypha. All are equally required to delocalize apical actin and terminate mating projection growth in budding yeast [35]. Spa2 is considered to be the central polarisome scaffolding protein that physically interacts with all other components through specific binding domains [29,36,37]. Pea2 contains a predicted coiled-coil domain suggesting a possible function in targeted vesicle delivery; its precise molecular role, however, remains obscure. Nevertheless, it has been shown to display interdependent localization with Spa2, and to be required for bipolarization and mating cell fusion [38,39]. The actin-interacting protein Aip3/ Bud6 was initially identified as a protein that besides its association with actin also contains domains which suggested binding to Spa2, Pea2 and Bni1 [29,40,41]. The formin Bni1 is stimulated by Bud6 in a positive feedback loop and together they reinforce the assembly of robust actin cables from the cell cortex during budding and mating projection formation, and contractile actomyosin ring formation during cytokinesis [32,40,41]. More recent data suggested an additional function of Bud6 in microtubule plus- end capture at the cell cortex, with contributions of formins [42]. Localized assembly of these polarity regulators in the polarisome is maintained through a positive feedback loop from the Cdc42/ Cdc24/Bem1 module whose components shuttle between the cytoplasm and plasma membrane [43,44]. Due to its vital importance in cell polarity regulation, this Cdc42 GTPase module can be considered a fifth core component of the tip growth apparatus. Homologs of Spa2, Aip3/Bud6 and Bni1 have been identified in a number of other filamentous growing fungi, and the majority already successfully localized in at least one of those species (Table 1). A homolog of Pea2 has so far only been identified in the filamentous yeast, Ashbya gossypii [45]. Recent work in Neurospora crassa has demonstrated how deletion or loss-of-function of CDC42, RAC-1 or CDC24 lead to severe defects in apical polarity and consequently hyphal morphologies [46], thereby demonstrating the vital role of this GTPase module in filamentous fungal cell polarity regulation. Although many new insights into the inner workings of the polarisome have been gained over the past decades, it is very likely that additional components localizing to this structure will be identified in the near future, revealing further details of its functional differences between yeasts and filamentous fungi. In this study we set out to analyze the subcellular organization and dynamics of SPA-2, BUD-6 and BNI-1 in a wide range of developmental stages of Neurospora crassa, in order to characterize the filamentous fungal polarisome more comprehensively, and identify potential differences to other fungal species. Our analysis showed that during early, unicellular developmental stages the filamentous fungal polarisome closely resembles the apical cap configuration known from yeasts, but during later, multicellular developmental stages the three polarisome components SPA-2, BUD-6 and BNI-1 become spatiotemporally separated within the apical dome, and thus adopt a so far unknown polari- some architecture. Furthermore, novel polarisome-independent Neurospora Crassa Polarisome Architecture Table 1. Polarisome components in yeast and filamentous fungi. S. cerecisiae Spa2 Pea2* Bni1 Aip3/Bud6 Filamentous fungi AgSpa2p Spa2 SpaA Spa2 SpaA SPA-2 (AGR135Cp) AgBni1 Bni1 SepA SepA BNI-1 AgBud6 Bud6 BudA Apex Apex Apex Apex Apex ? Apex Apex Apex Apex Apex Apex Septa Localization Apex and Septa A. gosypii [45] Species (Reference) Apex and Septa A. nidulans [74] C. albicans [66] A. nidulans [65] U. maydis [93] A. niger [94] N. crassa [59] A. gosypii [18] A. gossypii [87] C. albicans [66,86] A. niger [95] N. crassa [62] A. gossypii [18] C. albicans [66] A. nidulans [65] *apart from A. gosypii, Pea2 homologues have so far not been identified in the genomes of any other filamentous fungal species. doi:10.1371/journal.pone.0030372.t001 functions of BUD-6 and BNI-1 have been identified, including the maintenance of Spitzenko¨ rper integrity, cell fusion, septum formation and cytokinesis. Results Heterologous expression of fluorescently labeled BUD-6 Expression of fluorescently labeled BUD-6 occurred under control of the glucose-repressible ccg-1 promoter from an ectopically integrated plasmid. However, we did not find evidence that the lack of native expression levels interfered with normal cell biology and colony development in Neurospora crassa (Figure S2), suggesting that the constructs were functional and the observed localization patterns reflect the dynamics of the native protein. Due to the absence of conidia, transformation of the Dbud-6 gene deletion mutant with bud-6-gfp in order to demonstrate phenotypic rescue, was not possible. Therefore, functionality of the fusion protein has not yet been formally proven. BUD-6 functioned during polarized tip growth maintenance rather than its establishment In dormant conidia weak BUD-6 fluorescence could be observed in the cytoplasm and as strongly fluorescent, discrete clusters at one or both cell poles (Figure 1A). Upon hydration and isotropic cell expansion, BUD-6 fluorescence became dispersed throughout the cytoplasm (Figure 1B), but discrete cortical accumulations at incipient site of germ tube emergence could not be observed. Apart from occasional concentration in what appeared to be membranous compartments, and obvious exclusion from nuclei, the overall fluorescence pattern in the cytoplasm did not significantly change upon cell symmetry breaking and germ tube protrusion (Figure 1C). A more focused and specific recruitment to the growing germ tube tip occurred when the germling reached a length of about PLoS ONE | www.plosone.org 2 January 2012 | Volume 7 | Issue 1 | e30372