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Organization and dynamics of the Aspergillus nidulans Golgi during apical extension and mitosis.

ABSTRACT: Aspergillus nidulans hyphae grow exclusively by apical extension. Golgi equivalents (GEs) labeled with mRFP-tagged PH(OSBP) domain form a markedly polarized, dynamic network of ring-shaped and fenestrated cisternae that remains intact during "closed" mitosis. mRFP-PH(OSBP) GEs advance associated with the growing apex where secretion predominates but do not undergo long-distance movement toward the tip that could account for their polarization. mRFP-PH(OSBP) GEs overlap with the trans-Golgi resident Sec7 but do not colocalize with also polarized accretions of the early Golgi marker GrhA(Grh1)-GFP, indicating that early and late Golgi membranes segregate spatially. AnSec23-GFP ER exit sites (ERES) are numerous, relatively static foci localizing across the entire cell. However, their density is greatest near the tip, correlating with predominance of early and trans-Golgi elements in this region. Whereas GrhA-GFP structures and ERES reach the apical dome, mRFP-PH(OSBP) GEs are excluded from this region, which contains the endosome dynein loading zone. After latrunculin-mediated F-actin disruption, mRFP-PH(OSBP) GEs fragment and, like AnSec23-GFP ERES, depolarize. Brefeldin A transiently collapses late and early GEs into distinct aggregates containing Sec7/mRFP-PH(OSBP) and GrhA-GFP, respectively, temporarily arresting apical extension. Rapid growth reinitiates after washout, correlating with reacquisition of the normal Golgi organization that, we conclude, is required for apical extension.

SUBMITTER: Pantazopoulou A

PROVIDER: S-EPMC2762137 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Organization and dynamics of the Aspergillus nidulans Golgi during apical extension and mitosis.

Pantazopoulou Areti A Peñalva Miguel A MA

Molecular biology of the cell 20090819 20

Aspergillus nidulans hyphae grow exclusively by apical extension. Golgi equivalents (GEs) labeled with mRFP-tagged PH(OSBP) domain form a markedly polarized, dynamic network of ring-shaped and fenestrated cisternae that remains intact during "closed" mitosis. mRFP-PH(OSBP) GEs advance associated with the growing apex where secretion predominates but do not undergo long-distance movement toward the tip that could account for their polarization. mRFP-PH(OSBP) GEs overlap with the trans-Golgi resid ...[more]

PMID: 19692566

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Project description: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.

Dataset Information

Organization and dynamics of the Aspergillus nidulans Golgi during apical extension and mitosis.

Publications

Organization and dynamics of the Aspergillus nidulans Golgi during apical extension and mitosis.

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