Areti Pantazopoulou

Long-distance movement of Aspergillus nidulans early endosomes on microtubule tracks.

In fungal hyphal cells, intracellular membrane trafficking is constrained by the relatively long intracellular distances and the mode of growth, exclusively by apical extension. Endocytosis plays a key role in hyphal tip growth, which involves the coupling of secretory membrane delivery to the apical region with subapical compensatory endocytosis. However, the identity, dynamics and function of filamentous fungal endosomal compartments remain largely unexplored. Aspergillus nidulans RabARab5 localizes to a population of endosomes that show long range bidirectional movement on microtubule (MT) tracks and are labelled with FM4‐64 shortly after dye internalization. RabARab5 membranes do not overlap with largely static mature endosomes/vacuoles. Impaired delivery of dynein to the MT plus ends or downregulation of cytoplasmic dynein using the dynein heavy chain nudA1tsmutation results in accumulation of RabARab5 endosomal membranes in an abnormal NudA1 compartment at the tip, strongly supporting the existence in A. nidulans hyphal tips of a dynein loading region. We show that the SynA synaptobrevin endocytic recycling cargo traffics through this region, which strongly supports the contention that polarized hyphal growth involves the association of endocytic recycling with the plus ends of MTs located at the tip, near the endocytic internalization collar.

Aspergillus RabBRab5 Integrates Acquisition of Degradative Identity with the Long Distance Movement of Early Endosomes

Of the two Aspergillus early endosomal Rab5 paralogues, RabB recruits, in its GTP conformation, Vps19, Vps45, and Vps34, and the CORVET complex and couples acquisition of PI(3)P degradative identity with the long-distance movement of early endosomes. RabA also recruits CORVET, albeit less efficiently. The simultaneous loss of RabA and RabB is lethal.

Maturation of late Golgi cisternae into RabERAB11 exocytic post-Golgi carriers visualized in vivo

Maturation of Golgi cisternae into post-Golgi carriers is directly visualized using the Aspergillus nidulans Ypt31/RAB11 homologue RabE as a reporter of post-Golgi identity. A microtubule-based conveyor belt fuels carriers to actin microfilaments radiating from the apex, which carry out the membrane-proximal transport step of exocytosis.

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

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.

Characterization of Aspergillus nidulans RabC/Rab6

The Aspergillus nidulans Golgi is not stacked. Early and late Golgi equivalents (GEs) are intermingled but can be resolved by epifluorescence microscopy. RabC, the Aspergillus ortholog of mammalian Rab6, is present across the Golgi, preferentially associated with early GEs near the tip and with late GEs in tip‐distal regions. rabCΔ mutants, showing markedly impaired apical extension, have conspicuously fragmented, brefeldin A‐insensitive early and late GEs, indicating that the Golgi network organization requires RabC. rabCΔ Golgi fragmentation is paralleled by an increase in early endosome abundance. rabCΔ reduces extracellular levels of the major secretable protease, suggesting that it impairs secretion. Notably, the Spitzenkörper, an apical intracellular structure in which secretory carriers accumulate awaiting fusion with the adjacent plasma membrane (PM), contains RabC. rabCΔ leads to abnormally increased accumulation of carriers, detectable with secretory v‐SNARE GFP‐SynA and FM4‐64, in this structure. VpsTVps10, present across the Golgi, recycles between endosomes and Golgi and is mislocalized to a cytosolic haze by rabCΔ that, in contrast, does not affect SynA recycling between endosomes and the PM, indicating that SynA follows a RabC‐independent pathway. tlg2Δ mutants grow normally but are synthetically lethal with rabCΔ, indicating that RabC plays Tlg2‐independent roles.

Endosomal maturation by Rab conversion in Aspergillus nidulans is coupled to dynein- mediated basipetal movement

Highly motile fungal early endosomes can be easily distinguished from more static late endosomes and vacuoles, a feature that is exploited to study endosomal maturation. RabA/RabB early endosomes mature into RabSRab7 late endosomes as they move away from the tip where endocytosis predominates, augmenting their size, with concomitant loss of motility.

Acute inactivation of the Aspergillus nidulans Golgi membrane fusion machinery: correlation of apical extension arrest and tip swelling with cisternal disorganization

The mechanisms governing traffic across the Golgi are incompletely understood. We studied, by live‐cell microscopy, the consequences of disorganizing the Aspergillus nidulans Golgi, using an extended set of fluorescent protein markers to resolve early from late cisternae. The early Golgi syntaxin SedVSed5 and the RabORab1 regulatory GTPase play essential roles in secretion, cooperating in the ER–Golgi interface. Following a temperature shift‐up ‘on‐the‐stage’, hyphae carrying engineered sedVR258G and rabOA136D ts mutations arrest polarized growth. This arrest correlates with overall Golgi disorganization and characteristic hyphal tip swelling. Using v‐SNARE SynA as reporter, we show that the sedVR258G phenotypes correlate with arrested secretion. Both the morphogenetic defect and the secretory deficit are reversible. Thus downregulation of secretion, like that of endocytosis, has morphogenetic consequences, implying that mechanisms tuning the secretory pathway might be involved in developmental processes. According to the cisternal maturation model, acute impairment of traffic in the ER–Golgi interface should lead to disorganization of both the early and the late Golgi cisternae. Thus, the relatively rapid late Golgi disorganization observed upon shifting ER–Golgi interface mutants to the restrictive temperature seems incompatible with an A. nidulans Golgi network organized on the basis of stable early and late compartments, supporting instead cisternal maturation.

Live-cell imaging of Aspergillus nidulans autophagy: RAB1 dependence, Golgi independence and ER involvement.

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 RabSRAB7)-HOPS-(homotypic fusion and vacuole protein sorting complex)-dependent manner. However, by removing RabSRAB7 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 RabARAB5/RabBRAB5 (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 FYVE2-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 RabORAB1. TRAPPIII-specific factor AN7311 (yeast Trs85) localizes to the phagophore assembly site (PAS) and RabORAB1 localizes to phagophores and autophagosomes. The Golgi and autophagy roles of RabORAB1 are dissociable by mutation: rabOA136D 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.

TRAPPII regulates exocytic Golgi exit by mediating nucleotide exchange on the Ypt31 ortholog RabERAB11

Significance Ypt1 and Ypt31/32 RAB GTPases regulate traffic across the Golgi. Both are activated by TRAPP, an oligomeric GEF. Three TRAPP versions share the same core subunits. TRAPPI and TRAPPIII activate Ypt1. The third, TRAPPII, composed of TRAPPI plus specific subunits, appears to act specifically on Ypt31, but this role has been disputed. By combining the resolving power of fungal genetics with biochemical assays, we establish that the physiological target of TRAPPII is RabE, the Aspergillus Ypt31 ortholog. However, our data suggest that TRAPPII contains independent binding sites for RabE and RabO (Ypt1), possibly explaining its relative lack of discrimination in vitro. TRAPPII arrives at Golgi cisternae preceding their dissipation into carriers, determining the Golgi–to–post-Golgi transition through RabE recruitment. The oligomeric complex transport protein particle I (TRAPPI) mediates nucleotide exchange on the RAB GTPase RAB1/Ypt1. TRAPPII is composed of TRAPPI plus three additional subunits, Trs120, Trs130, and Trs65. Unclear is whether TRAPPII mediates nucleotide exchange on RAB1/Ypt1, RAB11/Ypt31, or both. In Aspergillus nidulans, RabORAB1 resides in the Golgi, RabERAB11 localizes to exocytic post-Golgi carriers undergoing transport to the apex, and hypA encodes Trs120. RabERAB11, but not RabORAB1, immunoprecipitates contain Trs120/Trs130/Trs65, demonstrating specific association of TRAPPII with RabERAB11 in vivo. hypA1ts rapidly shifts RabERAB11, but not RabORAB1, to the cytosol, consistent with HypATrs120 being specifically required for RabERAB11 activation. Missense mutations rescuing hypA1ts at 42 °C mapped to rabE, affecting seven residues. Substitutions in six, of which four resulted in 7- to 36-fold accelerated GDP release, rescued lethality associated to TRAPPII deficiency, whereas equivalent substitutions in RabORAB1 did not, establishing that the essential role of TRAPPII is facilitating RabERAB11 nucleotide exchange. In vitro, TRAPPII purified with HypATrs120-S-tag accelerates nucleotide exchange on RabERAB11 and, paradoxically, to a lesser yet substantial extent, on RabORAB1. Evidence obtained by exploiting hypA1-mediated destabilization of HypATrs120/HypCTrs130/Trs65 assembly onto the TRAPPI core indicates that these subunits sculpt a second RAB binding site on TRAPP apparently independent from that for RabORAB1, which would explain TRAPPII in vitro activity on two RABs. Using A. nidulans in vivo microscopy, we show that HypATrs120 colocalizes with RabERAB11, arriving at late Golgi cisternae as they dissipate into exocytic carriers. Thus, TRAPPII marks, and possibly determines, the Golgi–to–post-Golgi transition.

New Interfacial Microtubule Inhibitors of Marine Origin, PM050489/PM060184, with Potent Antitumor Activity and a Distinct Mechanism

We have investigated the target and mechanism of action of a new family of cytotoxic small molecules of marine origin. PM050489 and its dechlorinated analogue PM060184 inhibit the growth of relevant cancer cell lines at subnanomolar concentrations. We found that they are highly potent microtubule inhibitors that impair mitosis with a distinct molecular mechanism. They bind with nanomolar affinity to unassembled αβ-tubulin dimers, and PM050489 binding is inhibited by known Vinca domain ligands. NMR TR-NOESY data indicated that a hydroxyl-containing analogue, PM060327, binds in an extended conformation, and STD results define its binding epitopes. Distinctly from vinblastine, these ligands only weakly induce tubulin self-association, in a manner more reminiscent of isohomohalichondrin B than of eribulin. PM050489, possibly acting like a hinge at the association interface between tubulin heterodimers, reshapes Mg(2+)-induced 42 S tubulin double rings into smaller 19 S single rings made of 7 ± 1 αβ-tubulin dimers. PM060184-resistant mutants of Aspergillus nidulans map to β-tubulin Asn100, suggesting a new binding site different from that of vinblastine at the associating β-tubulin end. Inhibition of assembly dynamics by a few ligand molecules at the microtubule plus end would explain the antitumor activity of these compounds, of which PM060184 is undergoing clinical trials.