Neurospora Crassa Polarisome Architecture Figure 15. Schematic representation of the subcellular localization and dynamics of the three polarisome components SPA-2, BUD- 6 and BNI-1 during key developmental stages of N. crassa. (A) Prior to the constitution of the entire polarisome complex during germ tube growth and CAT-mediated cell fusion, BNI-1 accumulates at the incipient sites of cell symmetry breaking. (B) Equivalent to its dynamics during CAT fusion, the polarisome complex is present during homing and fusion of fusion hyphae, and removed once cytoplasmic continuity is achieved. (C) BNI- 1 accumulates at the incipient site of branch formation, and together with BUD-6 and SPA-2, subsequently constitutes the complete polarisome complex as an apical crescent at the tip of the emerging branch. Finally, the polarisome adopts the mature hyphal tip configuration (D). (D) Configuration of the polarized tip growth apparatus in mature hyphae, including an apical cap and Spitzenk o¨rper core of BNI-1, a fan-shaped distribution of SPA-2 inside the apical dome, and the subapical BUD-6 cloud. (E) Septal plugging and consolidation involves the Woronin body and all three polarisome proteins. During repolarization, a polarisome crescent is constituted at the emerging hyphal tip, and ultimately rearranged into its mature form (D). (F) Septum formation requires BNI-1 and BUD-6 – but not SPA-2 - as components of the CAR. Upon septum completion, only BUD-6 remains associated with the inner perimeter of the septal pore. (G) During cytokinesis BNI-1 and BUD-6 become recruited to the CAR and to forming secondary septa. Upon physical separation of macroconidia BNI-1 disappears, whereas BUD-6 remains at the cell poles. As during septum formation, SPA-2 has no role in this process. doi:10.1371/journal.pone.0030372.g015 accumulations have been identified at the tips of growing mating projections of S. cerevisiae and C. albicans, suggesting that more characteristics of hyphal tip growth have been conserved in yeasts than previously thought [70]. Our observation that during tip reorientation the apical BNI-1 clusters relocate before the Spk changes its position and the tip turns (Figure 12B), suggests that the formin exerts distinct functions in both structures to coordinate tip directionality. A possible scenario might include one population of BNI-1 that first initiates the nucleation of F-actin cables from the new tip location, before a second population of BNI-1 in the Spk core regulates the lateral displacement of the vesicle cluster towards the endpoint of the new cable track. Strains coexpressing BNI-1-GFP and the F- actin marker Lifeact-TagRFP-T [71] now provide excellent tools to study these dynamics in detail. Taken together, these results show that polarisome components are essential to establish a functional Spk, and that their absence provokes changes in tip growth apparatus architecture and function which can significantly restrict morphogenesis. These findings also confirm that the presence of a vesicle supply centre is not per se required for polarized growth, but that its formation is essential for increased and targeted vesicle flow in order to support the hyphal tip shape and achieve fast extension speeds, and to coordinate tip directionality probably in interaction with the polarisome. BUD-6 and BNI-1 have polarisome-independent functions during septum formation and cytokinesis It has been previously demonstrated that Spa2 homologs of N. crassa and A. nidulans are neither part of the landmarking PLoS ONE | www.plosone.org 17 January 2012 | Volume 7 | Issue 1 | e30372