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

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-yeast-like 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 (∼0.5 μm/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 recycling (i.e., efficient endocytosis of membrane proteins followed by their redelivery to the plasma membrane) can generate and maintain polarity, as shown with the v-SNARE and secretory-vesicle-resident SynA, which is a substrate of the subapical endocytic ring (1, 49, 52). It is plausible that such a mechanism could drive the polarization of one or more proteins acting as positional cues to mediate polarity maintenance.Genetic evidence strongly supported the conclusion that endocytosis is required for apical extension. Mutational inactivation of the A. nidulans fimbrin FimA or of the small GTPase ArfB^Arf6 (a regulator of endocytosis from fungi to mammals), led to delayed polarity establishment and morphologically aberrant tips indicative of polarity maintenance defects (30, 51). These mutations, although very severely debilitating, are not lethal. In contrast, heterokaryon rescue showed that SlaB, a key F-actin regulator of the endocytic internalization machinery (26), is essential in A. nidulans (2). slaBΔ cells are able to establish polarity, but they arrest in apical extension during the very early steps of polarity maintenance with a bud-like germ tube (2). However, work with Aspergillus oryzae using a thiamine-repressible promoter to drive A. oryzae End4 (AoEnd4) (SlaB) expression showed that although endocytosis was prevented and hyphal morphology altered under repressing conditions, hyphal tip extension and polarity maintenance were not completely abolished (20), perhaps suggesting that the phenotype of the thiamine-repressed conditional allele might not fully resemble the null phenotype.F-actin strongly predominates in the hyphal tips (2, 14, 17, 49, 51). Because endocytic internalization is powered by F-actin (27), predominance of endocytic “patches” (i.e., sites of endocytic internalization) in the tip accounts, at least in part, for F-actin polarization. However, F-actin plays fundamental nonendocytic roles in the tip; for example, actin cables nucleated by the SepA formin localizing to the apical crescent are thought to play a major role in secretion, whereas a network of F-actin microfilaments appears to be a major component of the Spitzenkörper (4, 21, 43, 49). Notably, all genes used to address the role of endocytosis in apical extension share in common their involvement in regulating F-actin. Thus, the Saccharomyces cerevisiae ArfB orthologue Arf3p regulates endocytosis but also appears to regulate F-actin at multiple levels (12, 28, 44). Like their respective S. cerevisiae orthologues Sla2p and Sac6p, SlaB and FimA are key components of endocytic patches, but in budding yeast their orthologues appear to regulate F-actin dynamics beyond endocytosis (27, 35, 56).To gain insight into the essential role of SlaB in A. nidulans, we designed a conditional expression allele. Time-lapse microscopy under restrictive conditions demonstrated that polarity establishment is essentially normal but that these mutant germ tubes arrested in apical extension subsequently undergo swelling, acquiring the characteristic bud-like shape of abortive slaBΔ germlings. Medium shift experiments allowed us to address the role of SlaB in apical extension beyond these early stages of polarity maintenance. We demonstrate the key role that SlaB plays in endocytosis and polarity maintenance, but we also show that deficiency of SlaB affects the actin cytoskeleton.