Martinez-Andrade, J. M. | Search bioRxiv

bioRxiv2022

Nanosized extracellular vesicles released by Neurospora crassa hyphae

Extracellular vesicles (EVs) are nanosized structures containing proteins, lipids, and nucleic acids, released by living cells to the surrounding medium. EVs participate in diverse processes, such as intercellular communication, virulence, and disease. In pathogenic fungi, EVs carry enzymes that allow them to invade the host or undergo environmental adaptation successfully. In Neurospora crassa, a non-pathogenic filamentous fungus widely used as a model organism, the vesicle-dependent secretory mechanisms that lead to polarized growth are well studied. In contrast, biosynthesis of EVs in this fungus has been practically unexplored. In the present work, we analyzed N. crassa cultures supernatant for the presence of EVs by dynamic light scattering (DLS), transmission electron microscopy (TEM) and proteomic analysis. We identified spherical membranous structures, with a predominant subpopulation averaging a hydrodynamic diameter (dh) of 68 nm and a particle diameter (dp) of 38 nm. EV samples stained with osmium tetroxide vapors were better resolved than those stained with uranyl acetate. Mass spectrometry analysis identified 252 proteins, including enzymes involved in carbohydrate metabolic processes, oxidative stress response, cell wall organization/remodeling, and circadian clock-regulated proteins. Some of these proteins have been previously reported in exosomes from human cells or in EVs of other fungi. In view of the results, it is suggested a putative role for EVs in cell wall biosynthesis and vegetative development in N. crassa.

microbiology↗

bioRxiv2022

The C-terminal domain of SEC-10 is fundamental for exocyst function, Spitzenkorper organization and cell morphogenesis in Neurospora crassa.

The exocyst is a conserved multimeric complex that participates in the final steps of the secretion of vesicles. In the filamentous fungus Neurospora crassa, the exocyst is crucial for polar growth, morphology, and the organization of the Spitzenkorper (Spk), the apical body where secretory vesicles accumulate before being delivered to the plasma membrane. In the highly polarized cells of N. crassa, the exocyst subunits SEC-3, SEC-5, SEC-6, SEC-8, and SEC-15 were previously found localized at the plasma membrane of the cells apices, while EXO-70 and EXO-84 occupied the frontal outer layer of the Spk, occupied by vesicles. The localization of SEC-10 had remained so far elusive. In this work, SEC-10 was tagged with the green fluorescent protein (GFP) either at its N- or C-terminus and found localized at the plasma membrane of growing hyphal tips, similar to what was previously observed for some exocyst subunits. While expression of an N-terminally tagged version of SEC-10 at its native locus was fully viable, expression of a C-terminally tagged version at its native locus resulted in severe hyphal growth and polarity defects. Additionally, a sec-10 knockout mutant in a heterokaryotic state (with genetically different nuclei) was viable but showed a strongly aberrant phenotype, confirming that this subunit is essential to maintain hyphal morphogenesis. Transmission electron microscopy analysis revealed the lack of a Spk in the SEC-10-GFP strain, suggesting a critical role of the exocyst in the vesicular organization at the Spk. Mass spectrometry analysis revealed fewer peptides of exocyst subunits interacting with SEC-10-GFP than with GFP-SEC-10, suggesting an essential role of the C-terminus of SEC-10 in exocyst assembly and/or stability. Altogether, our data suggest that an unobstructed C-terminus of SEC-10 is indispensable for the exocyst complex function and that a GFP tag could be blocking important subunit-subunit interactions.

microbiology↗