Searching for gold beyond mitosis. Mining intracellular membrane traffic in Aspergillus nidulans

Cellular Logistics 01/2012;


The genetically tractable filamentous ascomycete fungus
Aspergillus nidulans has been successfully exploited to gain
major insight into the eukaryotic cell cycle. More recently, its
amenability to in vivo multidimensional microscopy has fueled
a potentially gilded second age of A. nidulans cell biology
studies. This review specifically deals with studies on intracellular
membrane traffic in A. nidulans. The cellular logistics
are subordinated to the needs imposed by the polarized mode
of growth of the multinucleated hyphal tip cells, whereas
membrane traffic is adapted to the large intracellular distances.
Recent work illustrates the usefulness of this fungus for
morphological and biochemical studies on endosome and
Golgi maturation, and on the role of microtubule-dependent
motors in the long-distance movement of endosomes. The
fungus is ideally suited for genetic studies on the secretory
pathway, as mutations impairing secretion reduce apical
extension rates, resulting in phenotypes detectable by visual
inspection of colonies.

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    ABSTRACT: We exploited the amenability of the fungus Aspergillus nidulans to genetics and live-cell microscopy to investigate autophagy. Upon nitrogen starvation, GFP-Atg8-containing pre-autophagosomal puncta give rise to cup-shaped phagophores and circular (0.9-μm diameter) autophagosomes that disappear in the vicinity of the vacuoles after their shape becomes irregular and their GFP-Atg8 fluorescence decays. This 'autophagosome cycle' gives rise to characteristic cone-shaped traces in kymographs. Autophagy does not require endosome maturation or ESCRTs, as autophagosomes fuse with vacuoles directly in a RabS (homolog of Saccharomyces cerevisiae Ypt7 and mammalian RAB7; written hereafter as RabS (RAB7) )-HOPS-(homotypic fusion and vacuole protein sorting complex)-dependent manner. However, by removing RabS (RAB7) or Vps41 (a component of the HOPS complex), we show that autophagosomes may still fuse, albeit inefficiently, with the endovacuolar system in a process almost certainly mediated by RabA (RAB5) /RabB (RAB5) (yeast Vps21 homologs)-CORVET (class C core vacuole/endosome tethering complex), because acute inactivation of HbrA/Vps33, a key component of HOPS and CORVET, completely precludes access of GFP-Atg8 to vacuoles without affecting autophagosome biogenesis. Using a FYVE 2-GFP probe and endosomal PtdIns3P-depleted cells, we imaged PtdIns3P on autophagic membranes. PtdIns3P present on autophagosomes decays at late stages of the cycle, preceding fusion with the vacuole. Autophagy does not require Golgi traffic, but it is crucially dependent on RabO (RAB1) . TRAPPIII-specific factor AN7311 (yeast Trs85) localizes to the phagophore assembly site (PAS) and RabO (RAB1) localizes to phagophores and autophagosomes. The Golgi and autophagy roles of RabO (RAB1) are dissociable by mutation: rabO (A136D) hyphae show relatively normal secretion at 28°C but are completely blocked in autophagy. This finding and the lack of Golgi traffic involvement pointed to the ER as one potential source of membranes for autophagy. In agreement, autophagosomes form in close association with ring-shaped omegasome-like ER structures resembling those described in mammalian cells.
    No preview · Article · Apr 2013 · Autophagy