Comparative Live-Cell Imaging Analyses of SPA-2, BUD-6 and BNI-1 in Neurospora crassa Reveal Novel Features of the Filamentous Fungal Polarisome

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 Spitzenkörper (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.

F-actin localization dynamics during appressorium formation inColletotrichum graminicola

“…S. cerevisiae formin Bni1 also is required for the de novo assembly of actin filaments and of actin contractile rings in budding yeast , Sagot et al 2002, Tolliday et al 2002, Pring et al 2003. Formin orthologs in A. nidulans (SepA ) and N. crassa (Bni1) also were required for septum formation and localized to septation sites (Sharpless and Harris 2002, Lichius et al 2012, Delgado-Alvarez et al 2014. These studies suggest that the formation of actin contractile rings required de novo-assembled actin filaments in animals and fungi.…”

CDC-42 and RAC-1 regulate opposite chemotropisms inNeurospora crassa

“…The fact that addition of NSC23766 does not remove the CRIB reporter from septa, furthermore, suggests that CDC-42 takes the primary role in septation. In a previous study, we have shown that the actin-binding protein BUD-6 becomes recruited to the incipient septation site and, together with the formin BNI-1, remains associated with the leading edge of the constricting actomyosin ring (CAR) (Lichius et al, 2012b). It has been suggested that the CDC-42-RAC-1-CDC-24 module acts as a negative regulator of the septation process, counteracting RHO-1 and RHO-4 which have been positively attributed to septation (Rasmussen and Glass, 2005;Rasmussen and Glass, 2007;JustaSchuch et al, 2010; see also reviews by Seiler and Justa-Schuch, 2010;Mouriño-Pérez and Riquelme, 2013).…”

Signal exchange and integration during self-fusion in filamentous fungi

“…A similar but so far unknown mechanism might steer tip growth in fusing germlings and hyphae. The Bni1 homolog of N.crassa localizes to sites of cell fusion, is, however, not essential for this process, indicating that other cytoskeleton organizing factors contribute to MAK-2-mediated self-tropism [47].…”