Comparative Live-Cell Imaging Analyses of SPA-2, BUD-6 and BNI-1 in Neurospora crassa Reveal Novel Features of the Filamentous Fungal Polarisome Alexander Lichius1,2., Mario E. Ya´ n˜ ez-Gutie´ rrez1., Nick D. Read2, Ernestina Castro-Longoria1* 1 Department of Microbiology, Center for Scientific Research and Higher Education of Ensenada (CICESE), Ensenada, Baja California, Mexico, 2 Fungal Cell Biology Group, Institute of Cell Biology, Rutherford Building, The University of Edinburgh, Edinburgh, United Kingdom Abstract A key multiprotein complex involved in regulating the actin cytoskeleton and secretory machinery required for polarized growth in fungi, is the polarisome. Recognized core constituents in budding yeast are the proteins Spa2, Pea2, Aip3/Bud6, and the key effector Bni1. Multicellular fungi display a more complex polarized morphogenesis than yeasts, suggesting that the filamentous fungal polarisome might fulfill additional functions. In this study, we compared the subcellular organization and dynamics of the putative polarisome components BUD-6 and BNI-1 with those of the bona fide polarisome marker SPA- 2 at various developmental stages of Neurospora crassa. All three proteins exhibited a yeast-like polarisome configuration during polarized germ tube growth, cell fusion, septal pore plugging and tip repolarization. However, the localization patterns of all three proteins showed spatiotemporally distinct characteristics during the establishment of new polar axes, septum formation and cytokinesis, and maintained hyphal tip growth. Most notably, in vegetative hyphal tips BUD-6 accumulated as a subapical cloud excluded from the Spitzenko¨ rper (Spk), whereas BNI-1 and SPA-2 partially colocalized with the Spk and the tip apex. Novel roles during septal plugging and cytokinesis, connected to the reinitiation of tip growth upon physical injury and conidial maturation, were identified for BUD-6 and BNI-1, respectively. Phenotypic analyses of gene deletion mutants revealed additional functions for BUD-6 and BNI-1 in cell fusion regulation, and the maintenance of Spk integrity. Considered together, our findings reveal novel polarisome-independent functions of BUD-6 and BNI-1 in Neurospora, but also suggest that all three proteins cooperate at plugged septal pores, and their complex arrangement within the apical dome of mature hypha might represent a novel aspect of filamentous fungal polarisome architecture. Citation: Lichius A, Ya´n˜ ez-Gutie´rrez ME, Read ND, Castro-Longoria E (2012) Comparative Live-Cell Imaging Analyses of SPA-2, BUD-6 and BNI-1 in Neurospora crassa Reveal Novel Features of the Filamentous Fungal Polarisome. PLoS ONE 7(1): e30372. doi:10.1371/journal.pone.0030372 Editor: Alexandra Carolyn Brand, University of Aberdeen, United Kingdom Received August 26, 2011; Accepted December 14, 2011; Published January 24, 2012 Copyright: 2012 Lichius et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding for this project was provided by a SEP-CONACyT (www.conacyt.mx) grant (CB-2006-1-61524) to EC-L and a Biotechnology and Biological Sciences Research Council (www.bbsrc.ac.uk) grant (# BB/E010741/1) to NDR. We thank CONACyT for a grant to MEY-G., and a postdoctoral fellowship to AL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ecastro@cicese.mx . These authors contributed equally to this work. Introduction Cell polarity regulation is a central process during cell morphogenesis. The establishment and maintenance of cell polarity occurs in response to diverse external and internal signals, which control processes such as cell symmetry breaking, polarized tip growth, and cellular compartmentalization, as well as intracellular transport of RNA, proteins and organelles. Many key aspects of the molecular basis of cell polarity regulation have been elucidated using yeast models, including Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans (reviewed for example in [1,2,3,4,5,6,7]), but certain facets of filamentous fungal morphogenesis are more complex and cannot be explained by the yeast paradigm [8,9,10]. These most notably include: (1) the ability to simultaneously establish several axes of polarized growth from the individual spore thereby giving rise to functionally distinct cell protrusions (e.g. germ tubes and conidial anastomosis tubes [CATs]) [11,12,13], (2) the ability to perma- nently maintain polarized tip growth and form tubular hyphae which can achieve much higher tip growth rates than can yeasts [14,15], and (3) to establish interconnected germling and hyphal networks by cell fusion [16]. Some of the molecular components conserved between yeasts and filamentous fungi appear to be used in different morphogenetic contexts during filamentous fungal development, and proteins no longer encoded in the yeast genome are additional key features responsible for the more complex, multicellular morphology of filamentous fungi. The tip growth apparatus of vegetative hyphae consists of three major components: the Spitzenko¨ rper (Spk), the polarisome and the exocyst [17,18,19]. Together, they contain more than 40 different proteins [20] which, in interaction with the three cytoskeletal polymers F-actin, microtubules and septins, regulate hyphal morphogenesis and tip growth [21,22]. Targeted secretion of plasma membrane and cell wall components through the exocyst drives tip extension, and is coupled to compensatory endocytosis within a subapical collar [23,24,25], rich in F-actin patches [26,27]. The newly emerging ‘Apical Recycling Model’ accounts for the need to balance exocytosis and endocytosis at the hyphal tip in order to control growth and cell shape, maintain high tip extension rates and recover key plasma membrane proteins PLoS ONE | www.plosone.org 1 January 2012 | Volume 7 | Issue 1 | e30372