Neurospora Crassa Polarisome Architecture Colocalization{ BUD-6 & BNI-1 in random cortical clusters of unknown function - - SPA-2, BUD-6 & BNI-1: apical - cap prior to Spk appearance SPA-2, BUD-6 & BNI-1: apical - cap during CAT homing and transient ring during fusion pore formation SPA-2 & BNI-1: partial overlap in the Spk and potentially in the tip apex BUD-6 & BNI-1: at incipient septation site and part of contractile ring SPA-2, BUD-6 & BNI-1: transient during all stages of the fusion process BUD-6 & BNI-1: during cell separation Dbud-6 & Dbni-1 phenotypically very similar Unique roles BUD-6: persistent clusters at cell poles BNI-1: recruitment during polarity establishment BUD-6: subapical cloud, excluded from Spk BNI-1: cortical recruitment during branch initiation BUD-6: remains at septal pore/ Woronin body BUD-6: persistence at septal pore (.4 h) - - BUD-6: persists at completed cell separation sites/ secondary septa Conidium CSB CAT Germ tube Hyphal tip Branching Septal plugging & regeneration of tip growth Septum formation in mature hyphae VHF Conidiophore KO phenotype - -2 - diffuse Spk, i rregular hyphal growth2 apical cap at emerging and growing branch2 subapical accumulation cortical recruitment to coincides with Spk assembly incipient branch site recruitment to septal plug permanently at septal pore, prior to repolarization, concentrates at septal plug, apical cap at repolarizing tip apical cap at repolarizing tip recruitment to septal plug prior to repolarization, apical cap at repolarizing tip SPA-2, BUD-6 & BNI-1: at septal pore and as apical cap at repolarizing tip apical cap, transient ring at apical cap, transient ring at apical cap, transient ring opening fusion pore opening fusion pore at opening fusion pore SPA-2 cytoplasmic1 apical cap2 apical cap, transient ring at opening fusion pore at Spk and fan-shaped apical dome2 BUD-6 cytoplasmic, random cortical clusters, clusters at cell poles - apical cap, at septa apical cap, transient ring at opening fusion pore, random cortical clusters subapical cloud, excluded from Spk BNI-1 cytoplasmic, random cortical clusters recruitment to incipient polarisation sites apical cap apical cap, at opening fusion pore, random cortical clusters in Spk core, apical cap3 at incipient septation site, contractile ring, persistence at septal pore at incipient septation site, contractile ring3 cytokinetic ring, persistence cytokinetic ring, at secondary septa, random transient at separation cortical clusters sites, random cortical clusters hyperbraching, apical branching, no Spk, aconidiate, aseptate, derepressed VHF at colony edge hyperbraching, apical branching, no Spk, aconidiate, aseptate, derepressed VHF at colony edge Table 3. Subcellular localization patterns of polarisome components during key developmental stages in N. crassa. (-) no specific subcellular recruitment observed/no colocalization indicated/no unique role observed; {Colocalization inferred from identical subcellular localization patterns; CSB = cell symmetry breaking, CAT = conidial anastomosis tube, Spk = Spitzenko¨ rper, VHF = vegetative hyphal fusion (in the mature colony), KO = knock-out (gene deletion) mutant; References: 1Araujo-Palmorales, Master Thesis, CICESE, 2007; 2Araujo-Palmorales et al., 2009; 3Justa-Schuch et al., 2010. doi:10.1371/journal.pone.0030372.t003 structure (Figure 12). To our knowledge, BUD-6 of N. crassa is the first Bud6 homolog localized as part of the apical growth apparatus in a filamentous fungus. It will be interesting to see if similar observations can be made in other filamentous fungal species. All three polarisome proteins are required to build a functional tip growth apparatus including the Spitzenko¨ rper We could show that deletion of bud-6 or bni-1 results in the complete absence of a detectable Spk (Figures 8 and 9). Although Dspa-2 did possess a Spk, its structure, however, was found to be distorted and accompanied by tip growth defects [59]. This demonstrates that all three polarisome components are required to establish and maintain the Spk, and furthermore to organise a functional tip growth apparatus. Other N. crassa mutants that fail to establish a Spk are the conventional kinesin-1 (nkin-1) mutant [67,68], the exocyst component knock-out Dsec-5 [69], and the recently characterized GTPase module mutants Dcdc42, Drac-1 and Dcdc24 [46]. Similar links between Spk integrity and hyphal morphogenesis have been identified in C. albicans and A. gossypii. Deletion of either CaSPA2 or CaBUD6 resulted in germ tubes that were broader and less polarized than the wild type, and caused the regulatory light chain of Myosin 2 (Mlc1) to no longer localise to the Spk, but instead into a polarisome-like crescent [66]. A Spk-like accumu- lation of polarity components only existed in the hyphal growth form of this yeast. In all other cases, Mlc1 and Spa2, as well as FM4–64-stained vesicles, exclusively localized into an apical cap resembling the polarisome of pseudohyphae and yeast growth form of C. albicans. In A. gossypii, the accumulation of polarisome and exocyst components into an apical cap alone or an apical cap plus subtending Spk was associated with hyphal growth velocity, in that an increased extension rate of the tip coincided with the formation of a Spk [18]. Notably, very recently Spk-like vesicle PLoS ONE | www.plosone.org 16 January 2012 | Volume 7 | Issue 1 | e30372