Endocytic Machinery Protein SlaB Is Dispensable for Polarity Establishment but Necessary for Polarity Maintenance in Hyphal Tip Cells of Aspergillus nidulans

Abstract

The Aspergillus nidulans endocytic internalization protein SlaB is essential, in agreement with the key role in apical extension attributed to endocytosis. We constructed, by gene replacement, a nitrate-inducible, ammonium-repressible slaB1 allele for conditional SlaB expression. Video microscopy showed that repressed slaB1 cells are able to establish but unable to maintain a stable polarity axis, arresting growth with budding-yeastlike morphology shortly after initially normal germ tube emergence. Using green fluorescent protein (GFP)-tagged secretory v-SNARE SynA, which continuously recycles to the plasma membrane after being efficiently endocytosed, we establish that SlaB is crucial for endocytosis, although it is dispensable for the anterograde traffic of SynA and of the t-SNARE Pep12 to the plasma and vacuolar membrane, respectively. By confocal microscopy, repressed slaB1 germlings show deep plasma membrane invaginations. Ammonium-to-nitrate medium shift experiments demonstrated reversibility of the null polarity maintenance phenotype and correlation of normal apical extension with resumption of SynA endocytosis. In contrast, SlaB downregulation in hyphae that had progressed far beyond germ tube emergence led to marked polarity maintenance defects correlating with deficient SynA endocytosis. Thus, the strict correlation between abolishment of endocytosis and disability of polarity maintenance that we report here supports the view that hyphal growth requires coupling of secretion and endocytosis. However, downregulated slaB1 cells form F-actin clumps containing the actin-binding protein AbpA, and thus F-actin misregulation cannot be completely disregarded as a possible contributor to defective apical extension. Latrunculin B treatment of SlaB-downregulated tips reduced the formation of AbpA clumps without promoting growth and revealed the formation of cortical "comets" of AbpA.Germinating asexual spores (conidiospores) of Aspergillus nidulans transiently undergo isotropic growth ("swelling") before establishing a polarity axis that grows by apical extension, leading to the characteristic tubular morphology of the fungal cell (15,16,33). Stable maintenance of a polarity axis at the high apical extension rates of A. nidulans (ϳ0.5 m/min at 25°C) (23) can be attributable, at least in part, to the polarization of the secretory apparatus and the predominant and highly efficient delivery of secretory vesicles to the apex (8,18,40,49). In addition, work from several laboratories strongly indicated that hyphal tip growth also involves endocytosis. A key observation supporting this involvement was that despite the fact that endocytosis can occur elsewhere, the endocytic internalization machinery predominates in the hyphal tip, forming a subapical collar. The spatial association of this collar with the apical region where secretory materials are delivered would allow removal of excess lipids/proteins reaching the plasma membrane with secretory vesicles (1,2,30,49,51,57), but, most importantly, rapid endocytic recycli...

Aspergillus nidulans Ambient pH Signaling Does Not Require Endocytosis

“…We have previously demonstrated, using heterokaryon rescue, that slaB deletion is lethal (21), a conclusion that was further buttressed using slaB1, a conditional expression allele having the niiA (encoding nitrite reductase) promoter, which is repressible by ammonium and inducible by nitrate (22) (Fig. 2C).…”

“…On ammonium, slaB1 spores germinate but do not maintain polarity beyond yeast-like cells and do not internalize SynA, forming characteristic pits in the plasma membrane (Fig. 2C) (22). Contrary to the heterogeneous populations observed with arfB⌬ and fimA⌬ strains, slaB1 yeast-like cells represented the sole cellular type found under ammonium conditions.…”

“…However, endocytosis is essential for A. nidulans, because it plays a key role in hyphal growth (19)(20)(21)(22); thus, the degree to which mutations preventing endocytosis permit hyphal growth is expected to correlate inversely with the extent to which they impair endocytosis, with a complete endocytic block being lethal (21)(22)(23). Here, we demonstrate that PalB also is recruited to cortical structures.…”

“…Protein extraction and Western blots. The protein extraction procedure was a modification of a reported protocol (22). Lyophilized mycelia were homogenized using an FP120 Fast Prep cell disruptor and a 0.5-mm ceramic bead with a 10-s pulse at a setting of 4.0.…”

Fungal Morphogenesis

“…Formins assist nucleating microfilament formation at polarization sites (Evangelista et al 2003;Pearson et al 2004;Schmitz et al 2006), but the formation of a stable axis of hyphal polarity can occur in their absence, as shown for A. nidulans (Sharpless and Harris 2002). On the other hand, regulators of actin patch formation and dynamics appear to be essential for the formation of a stable axis Upadhyay and Shaw 2008;Hervas-Aguilar and Penalva 2010). These observations underscore the importance of endocytosis in polarized growth, but also imply that the localized delivery of cell wall material to polarization sites can occur in the absence of actin filaments.…”