Aspergillus nidulans ArfB Plays a Role in Endocytosis and Polarized Growth

Abstract

Filamentous fungi undergo polarized growth throughout most of their life cycles. The Spitzenkörper is an apical organelle composed primarily of vesicles that is unique to filamentous fungi and is likely to act as a vesicle supply center for tip growth. Vesicle assembly and trafficking are therefore important for hyphal growth. ADP ribosylation factors (Arfs), a group of small GTPase proteins, play an important role in nucleating vesicle assembly. Little is known about the role of Arfs in filamentous hyphal growth. We found that Aspergillus nidulans is predicted to encode six Arf family proteins. Analysis of protein sequence alignments suggests that A. nidulans ArfB shares similarity with ARF6 of Homo sapiens and Arf3p of Saccharomyces cerevisiae. An arfB null allele (arfB disrupted by a transposon [arfB::Tn]) was characterized by extended isotropic growth of germinating conidia followed by cell lysis or multiple, random germ tube emergence, consistent with a failure to establish polarity. The mutant germ tubes and hyphae that do form initially meander abnormally off of the axis of polarity and frequently exhibit dichotomous branching at cell apices, consistent with a defect in polarity maintenance. FM4-64 staining of the arfB::Tn strain revealed that another phenotypic characteristic seen for arfB::Tn is a reduction and delay in endocytosis. ArfB is myristoylated at its N terminus. Green fluorescent protein-tagged ArfB (ArfB::GFP) localizes to the plasma membrane and endomembranes and mutation (ArfB G2A ::GFP) of the N-terminal myristoylation motif disperses the protein to the cytoplasm rather than to the membranes. These results demonstrate that ArfB functions in endocytosis to play important roles in polarity establishment during isotropic growth and polarity maintenance during hyphal extension.

Aspergillus nidulans Ambient pH Signaling Does Not Require Endocytosis

“…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.…”

“…ArfB and FimA are the A. nidulans orthologues of yeast Arf3 (36) and Sac6 (37), respectively. As judged by the severe defects in morphogenesis (19,20) and colony growth (Fig. 2B) displayed by the corresponding null mutants, these proteins were expected to be crucial for endocytosis.…”

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

“…For A. nidulans and Ustilago maydis, evidence strongly suggests that endocytosis is intimately associated with hyphal tip growth (2,30,45,46,49,51). However, the actual contribution of endocytosis to apical extension remains to be clarified.…”

“…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.…”

“…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.…”