A bird's-eye view of the endoplasmic reticulum in filamentous fungi.

SUMMARYThe endoplasmic reticulum (ER) is one of the most extensive organelles in eukaryotic cells. It performs crucial roles in protein and lipid synthesis and Ca2+ homeostasis. Most information on ER types, functions, organization, and domains comes from studies in uninucleate animal, plant, and yeast cells. In contrast, there is limited information on the multinucleate cells of filamentous fungi, i.e., hyphae. We provide an analytical review of existing literature to categorize different types of ER described in filamentous fungi while emphasizing the research techniques and markers used. Additionally, we identify the knowledge gaps that need to be resolved better to understand the structure-function correlation of ER in filamentous fungi. Finally, advanced technologies that can provide breakthroughs in understanding the ER in filamentous fungi are discussed.

The small Ras-like GTPase BUD-1 modulates conidial germination and hyphal growth guidance in the filamentous fungus Neurospora crassa.

In filamentous fungi, the hypha orientation is essential for polarized growth and morphogenesis. The ability to re-orient tip growth in response to environmental cues is critical for the colony survival. Therefore, hyphal tip orientation and tip extension are distinct mechanisms that operate in parallel during filamentous growth. In yeast, the axial growth orientation requires a pathway regulated by Rsr1p/Bud1p, a Ras-like GTPase protein, which determines the axial budding pattern. However, in filamentous fungi the function of the Rsr1/Bud1p gene (krev-1 homolog) has not been completely characterized. In this work, we characterized the phenotype of a homokaryon mutant Bud1p orthologous in Neurospora crassa (△bud-1) and tagged BUD-1 with the green fluorescent protein (GFP) to determine its localization and cell dynamics under confocal microscopy. During spore germination BUD-1 was localized at specific points along the plasma membrane and during germ tube emergence it was located at the tip of the germ tubes. In mature hyphae BUD-1 continued to be located at the cell tip and was also present at sites of branch emergence and at the time of septum formation. The △bud-1 mutant showed a delayed germination, and the orientation of hyphae was somewhat disrupted. Also, the hypha diameter was reduced approximately 37 % with respect to the wild type. The lack of BUD-1 affected the Spitzenkörper (Spk) formation, trajectory, the localization of polarisome components BNI-1 and SPA-2, and the actin cytoskeleton polarization. The results presented here suggest that BUD-1 participates in the establishment of a new polarity axis. It may also mediate the delivery of secretory vesicles for the efficient construction of new plasma membrane and cell wall.

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 culture's 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.

Dual function of EDTA with silver nanoparticles for root canal treatment-A novel modification.

The chelating and antimicrobial capacity of a novel modification of 17% EDTA with silver nanoparticles (AgNPs) (EDTA-AgNPs) was evaluated in-vitro for root canal treatment (RCT). The EDTA-AgNPs solution was characterized by UV-Vis spectroscopy, ζ-potential and high-resolution transmission electron microscopy (HRTEM). Antimicrobial capacity was evaluated against Candida albicans and Staphylococcus aureus in planktonic and biofilm cells by broth macrodilution (24 h) and XTT assays, (1, 10 and 30 min) respectively. The chelating capacity of EDTA-AgNPs was assessed indirectly (smear layer removal) and directly (demineralizing effect) in bovine dentin at two silver concentrations, 16 and 512 µg/ml at 1 and 10 minutes of exposure time. Smear layer removal was evaluated by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The demineralizing effect was determined by atomic absorption spectroscopy (AAS), microhardness test (MH) and X-ray diffractometer (XRD). Synthesized AgNPs were quasi-spherical in shape with an average size of 13.09 ± 8.05 nm. 17% EDTA-AgNPs was effective to inhibit C. albicans and S. aureus in planktonic and biofilm cultures. The smear layer removal and demineralizing effect were similar between 17% EDTA-AgNPs and 17% EDTA treatments. The 17% EDTA-AgNPs solution proved to be an effective antimicrobial agent, and has a similar chelating capacity to 17% EDTA alone. These in-vitro studies strongly suggest that EDTA-AgNPs could be used for effective smear layer removal, having an antimicrobial effect at the same time during RCT.