In filamentous fungi, early endosomes are continuously trafficked to, and from, the growing hyphal tip by microtubule-based motor proteins, serving as platforms for the long-distance transport of diverse cargos including mRNA, signaling molecules, and other organelles which hitchhike on them. While the cellular machinery for early endosome motility in filamentous fungi is fairly well characterized, the broader physiological significance of this process remains less well understood. We set out to determine the importance of long-distance early endosome trafficking in Aspergillus fumigatus, an opportunistic human pathogenic fungus that can cause devastating pulmonary infections in immunocompromised individuals. We first characterized normal early endosome motile behavior in A. fumigatus, then generated a mutant in which early endosome motility is severely perturbed through targeted deletion of the gene encoding for FtsA, one of a complex of proteins that links early endosomes to their motor proteins. Using a microfluidics-based approach we show that contact-induced hyphal branching behaviors are impaired in ΔftsA mutants, but that FtsA-mediated early endosome motility is dispensable for virulence in an invertebrate infection model. Overall, our study provides new insight into early endosome motility in an important human pathogenic fungus.
The causal agent of rice blast disease, the ascomycete fungus Magnaporthe grisea, infects rice (Oryza sativa) plants by means of specialized infection structures called appressoria, which are formed on the leaf surface and mechanically rupture the cuticle. We have identified a gene, Magnaporthe metallothionein 1 (MMT1), which is highly expressed throughout growth and development by M. grisea and encodes an unusual 22-amino acid metallothionein-like protein containing only six Cys residues. The MMT1-encoded protein shows a very high affinity for zinc and can act as a powerful antioxidant. Targeted gene disruption of MMT1 produced mutants that show accelerated hyphal growth rates and poor sporulation but had no effect on metal tolerance. Mmt1 mutants are incapable of causing plant disease because of an inability to bring about appressorium-mediated cuticle penetration. Mmt1 appears to be distributed in the inner side of the cell wall of the fungus. These findings indicate that Mmt1-like metallothioneins may play a novel role in fungal cell wall biochemistry that is required for fungal virulence.
Atmospheric pressure chemical ionisation (APCI) and electrospray (ES) are compared as ion sources in the analysis of polyhydroxyalkaloids (PHAs) by liquid chromatography mass spectrometry (LC-MS) and collision induced dissociation (CID) product ion spectra, from tandem mass spectrometry (MS-MS) experiments in a quadrupole ion trap, are reported for 12 naturally occurring PHAs. APCI was found to be a more useful source than ES, as APCI could be used to generate deprotonated molecule ions in negative mode and for some isomeric PHAs the negative CID product ion spectra were more diagnostic than the positive product ion spectra. On-column detection limits were also approximately 32 times lower by positive APCI than ES. The work provides data that will facilitate screening and characterisation of this group of important natural products in plant and fungal extracts.
Eukaryotes have evolved multiple strategies for maintaining cellular protein homeostasis. One such mechanism involves neutralization of deleterious protein aggregates via their defined spatial segregation. Here, using the molecular disaggregase Hsp104 as a marker for protein aggregation, we describe the spatial and temporal dynamics of protein aggregates in the filamentous fungus Aspergillus nidulans. Filamentous fungi, such as A. nidulans, are a diverse group of species of major health and economic importance and also serve as model systems for studying highly polarized eukaryotic cells. We find that microtubules promote the formation of Hsp104-positive aggregates, which coalesce into discrete subcellular structures in a process dependent on the microtubule-based motor cytoplasmic dynein. Finally, we find that impaired clearance of these inclusions negatively impacts retrograde trafficking of endosomes, a conventional dynein cargo, indicating that microtubule-based transport can be overwhelmed by chronic cellular stress.
Eukaryotic cells use microtubule-based intracellular transport for the delivery of many subcellular cargos, including organelles. The canonical view of organelle transport is that organelles directly recruit molecular motors via cargo-specific adaptors. In contrast with this view, we show here that peroxisomes move by hitchhiking on early endosomes, an organelle that directly recruits the transport machinery. Using the filamentous fungus Aspergillus nidulans we found that hitchhiking is mediated by a novel endosome-associated linker protein, PxdA. PxdA is required for normal distribution and long-range movement of peroxisomes, but not early endosomes or nuclei. Using simultaneous time-lapse imaging, we find that early endosome-associated PxdA localizes to the leading edge of moving peroxisomes. We identify a coiled-coil region within PxdA that is necessary and sufficient for early endosome localization and peroxisome distribution and motility. These results present a new mechanism of microtubule-based organelle transport in which peroxisomes hitchhike on early endosomes and identify PxdA as the novel linker protein required for this coupling.
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