Early Effects of Extracellular Vesicles Secreted by Adipose Tissue Mesenchymal Cells in Renal Ischemia Followed by Reperfusion: Mechanisms Rely on a Decrease in Mitochondrial Anion Superoxide Production.

Acute kidney injury (AKI) caused by ischemia followed by reperfusion (I/R) is characterized by intense anion superoxide (O2•-) production and oxidative damage. We investigated whether extracellular vesicles secreted by adipose tissue mesenchymal cells (EVs) administered during reperfusion can suppress the exacerbated mitochondrial O2•- formation after I/R. We used Wistar rats subjected to bilateral renal arterial clamping (30 min) followed by 24 h of reperfusion. The animals received EVs (I/R + EVs group) or saline (I/R group) in the kidney subcapsular space. The third group consisted of false-operated rats (SHAM). Mitochondria were isolated from proximal tubule cells and used immediately. Amplex Red™ was used to measure mitochondrial O2•- formation and MitoTracker™ Orange to evaluate inner mitochondrial membrane potential (Δψ). In vitro studies were carried out on human renal proximal tubular cells (HK-2) co-cultured or not with EVs under hypoxic conditions. Administration of EVs restored O2•- formation to SHAM levels in all mitochondrial functional conditions. The gene expression of catalase and superoxide dismutase-1 remained unmodified; transcription of heme oxygenase-1 (HO-1) was upregulated. The co-cultures of HK-2 cells with EVs revealed an intense decrease in apoptosis. We conclude that the mechanisms by which EVs favor long-term recovery of renal structures and functions after I/R rely on a decrease of mitochondrial O2•- formation with the aid of the upregulated antioxidant HO-1/Nuclear factor erythroid 2-related factor 2 system, thus opening new vistas for the treatment of AKI.

Phase separation of p53 precedes aggregation and is affected by oncogenic mutations and ligands.

Mutant p53 tends to form aggregates with amyloid properties, especially amyloid oligomers inside the nucleus, which are believed to cause oncogenic gain-of-function (GoF). The mechanism of the formation of the aggregates in the nucleus remains uncertain. The present study demonstrated that the DNA-binding domain of p53 (p53C) underwent phase separation (PS) on the pathway to aggregation under various conditions. p53C phase separated in the presence of the crowding agent polyethylene glycol (PEG). Similarly, mutant p53C (M237I and R249S) underwent PS; however, the process evolved to a solid-like phase transition faster than that in the case of wild-type p53C. The data obtained by microscopy of live cells indicated that transfection of mutant full-length p53 into the cells tended to result in PS and phase transition (PT) in the nuclear compartments, which are likely the cause of the GoF effects. Fluorescence recovery after photobleaching (FRAP) experiments revealed liquid characteristics of the condensates in the nucleus. Mutant p53 tended to undergo gel- and solid-like phase transitions in the nucleus and in nuclear bodies demonstrated by slow and incomplete recovery of fluorescence after photobleaching. Polyanions, such as heparin and RNA, were able to modulate PS and PT in vitro. Heparin apparently stabilized the condensates in a gel-like state, and RNA apparently induced a solid-like state of the protein even in the absence of PEG. Conditions that destabilize p53C into a molten globule conformation also produced liquid droplets in the absence of crowding. The disordered transactivation domain (TAD) modulated both phase separation and amyloid aggregation. In summary, our data provide mechanistic insight into the formation of p53 condensates and conditions that may result in the formation of aggregated structures, such as mutant amyloid oligomers, in cancer. The pathway of mutant p53 from liquid droplets to gel-like and solid-like (amyloid) species may be a suitable target for anticancer therapy.

Nanometer scale insight on the analysis of limpets mineralized teeth: Special focus on the silica-containing regions.

We report the electron microscopy-based analysis of the major lateral tooth of the limpet Colisella subrugosa during early and intermediate stages of development. We aimed to analyze the structural relationship among the needle-like crystals of the iron oxide goethite, the amorphous silica phase that forms the tooth base and occupy inter-crystalline spaces in the cusp, and the chitin fibers of the matrix. Goethite crystals followed the three dimensional organization pattern of the chitin fibers in the cusp. In the tooth base, spherical individual silica granules were found in regions where the chitin fibers cross. The spherical granules near the interface between the tooth base and the cusp (junction zone) formed an almost continuous medium that could easily be ultrathin-sectioned for further analysis. By contrast, the nearby silica-rich region localized on the other side of the junction zone contained needle-like goethite crystals immersed in the matrix and presented a conchoidal fracture. The chitin fibers from the silica granules of the tooth base were dotted or undulating in projection with a periodicity of about 6 nm when observed by high magnification transmission electron microscopy. Very thin goethite crystals were present in the base of the cusp near the junction zone surrounded by silica. On several occasions, crystals presented internal thin straight white lines parallel to the major axis, indicating a possible growth around fibers. We propose that silica and iron oxide phases mineralization may occur simultaneously at least for some period and that silica moderates the dimensions of the iron oxide crystals.

Current Microscopy Strategies to Image Fungal Vesicles: From the Intracellular Trafficking and Secretion to the Inner Structure of Isolated Vesicles.

Extracellular vesicles (EVs) are nano-sized structures that play important roles in a variety of biological processes among members of the Eukaryota domain. They have been studied since the 1940s and a broader use of different microscopy techniques to image either isolated vesicles or vesicles within the intracellular milieu (trafficking) has been limited by their nanometric size, usually below the resolution limit of most standard light microscopes. The development of genetically encoded fluorescent proteins and fluorescent probes able to switch between "on" and "off" states, as well the improvement in computer-assisted microscopy, photon detector devices, illumination designs, and imaging strategies in the late Twentieth century, boosted the use of light microscopes to provide structural and functional information at the sub-diffraction resolution, taking advantage of a nondestructive analytical probe such light, and opening new possibilities in the study of life at the nanoscale. As well, traditional and novel electron microscopy techniques have been widely used in the characterization of subcellular compartments, either isolated or in situ, providing a comprehensive understanding of their functional role in many cellular processes. Here, we present basic aspects of some of these techniques that have already been applied and their potential application to the study of fungal vesicles.

Uptake and persistence of bacterial magnetite magnetosomes in a mammalian cell line: Implications for medical and biotechnological applications.

Magnetotactic bacteria biomineralize intracellular magnetic nanocrystals surrounded by a lipid bilayer called magnetosomes. Due to their unique characteristics, magnetite magnetosomes are promising tools in Biomedicine. However, the uptake, persistence, and accumulation of magnetosomes within mammalian cells have not been well studied. Here, the endocytic pathway of magnetite magnetosomes and their effects on human cervix epithelial (HeLa) cells were studied by electron microscopy and high spatial resolution nano-analysis techniques. Transmission electron microscopy of HeLa cells after incubation with purified magnetosomes showed the presence of magnetic nanoparticles inside or outside endosomes within the cell, which suggests different modes of internalization, and that these structures persisted beyond 120 h after internalization. High-resolution transmission electron microscopy and electron energy loss spectra of internalized magnetosome crystals showed no structural or chemical changes in these structures. Although crystal morphology was preserved, iron oxide crystalline particles of approximately 5 nm near internalized magnetosomes suggests that minor degradation of the original mineral structures might occur. Cytotoxicity and microscopy analysis showed that magnetosomes did not result in any apparent effect on HeLa cells viability or morphology. Based on our results, magnetosomes have significant biocompatibility with mammalian cells and thus have great potential in medical, biotechnological applications.

Effect of applied magnetic fields on motility and magnetotaxis in the uncultured magnetotactic multicellular prokaryote 'Candidatus Magnetoglobus multicellularis'.

Magnetotactic bacteria are found in the chemocline of aquatic environments worldwide. They produce nanoparticles of magnetic minerals arranged in chains in the cytoplasm, which enable these microorganisms to align to magnetic fields while swimming propelled by flagella. Magnetotactic bacteria are diverse phylogenetically and morphologically, including cocci, rods, vibria, spirilla and also multicellular forms, known as magnetotactic multicellular prokaryotes (MMPs). We used video-microscopy to study the motility of the uncultured MMP 'Candidatus Magnetoglobus multicellularis' under applied magnetic fields ranging from 0.9 to 32 Oersted (Oe). The bidimensional projections of the tridimensional trajectories where interpreted as plane projections of cylindrical helices and fitted as sinusoidal curves. The results showed that 'Ca. M. multicellularis' do not orient efficiently to low magnetic fields, reaching an efficiency of about 0.65 at 0.9-1.5 Oe, which are four to six times the local magnetic field. Good efficiency (0.95) is accomplished for magnetic fields ≥10 Oe. For comparison, unicellular magnetotactic microorganisms reach such efficiency at the local magnetic field. Considering that the magnetic moment of 'Ca. M. multicellularis' is sufficient for efficient alignment at the Earth's magnetic field, we suggest that misalignments are due to flagella movements, which could be driven by photo-, chemo- and/or other types of taxis.

Phosphorus-rich structures and capsular architecture in Cryptococcus neoformans.

AIM: In this study, we aimed to analyze the relationship of phosphorus-rich structures with surface architecture in Cryptococcus neoformans. METHODS: Phosphorus-rich structures in C. neoformans were analyzed by combining fluorescence microscopy, biochemical extraction, scanning electron microscopy, electron probe x-ray microanalysis and 3D reconstruction of high pressure frozen and freeze substituted cells by focused ion beam-scanning electron microscopy (FIB-SEM). RESULTS & CONCLUSION: Intracellular and surface phosphorus-enriched structures were identified. These molecules were required for capsule assembly, as demonstrated in experiments using polysaccharide incorporation by capsule-deficient cells and mutants with defects in polyphosphate synthesis. The demonstration of intracellular and cell wall-associated polyphosphates in C. neoformans may lead to future studies involving their participation in both physiologic and pathogenic events.

Membrane cholesterol removal changes mechanical properties of cells and induces secretion of a specific pool of lysosomes.

In a previous study we had shown that membrane cholesterol removal induced unregulated lysosomal exocytosis events leading to the depletion of lysosomes located at cell periphery. However, the mechanism by which cholesterol triggered these exocytic events had not been uncovered. In this study we investigated the importance of cholesterol in controlling mechanical properties of cells and its connection with lysosomal exocytosis. Tether extraction with optical tweezers and defocusing microscopy were used to assess cell dynamics in mouse fibroblasts. These assays showed that bending modulus and surface tension increased when cholesterol was extracted from fibroblasts plasma membrane upon incubation with MßCD, and that the membrane-cytoskeleton relaxation time increased at the beginning of MßCD treatment and decreased at the end. We also showed for the first time that the amplitude of membrane-cytoskeleton fluctuation decreased during cholesterol sequestration, showing that these cells become stiffer. These changes in membrane dynamics involved not only rearrangement of the actin cytoskeleton, but also de novo actin polymerization and stress fiber formation through Rho activation. We found that these mechanical changes observed after cholesterol sequestration were involved in triggering lysosomal exocytosis. Exocytosis occurred even in the absence of the lysosomal calcium sensor synaptotagmin VII, and was associated with actin polymerization induced by MßCD. Notably, exocytosis triggered by cholesterol removal led to the secretion of a unique population of lysosomes, different from the pool mobilized by actin depolymerizing drugs such as Latrunculin-A. These data support the existence of at least two different pools of lysosomes with different exocytosis dynamics, one of which is directly mobilized for plasma membrane fusion after cholesterol removal.

Swimming behaviour of the multicellular magnetotactic prokaryote 'Candidatus Magnetoglobus multicellularis' under applied magnetic fields and ultraviolet light.

Magnetotactic bacteria move by rotating their flagella and concomitantly are aligned to magnetic fields because they present magnetosomes, which are intracellular organelles composed by membrane-bound magnetic crystals. This results in magnetotaxis, which is swimming along magnetic field lines. Magnetotactic bacteria are morphologically diverse, including cocci, rods, spirilla and multicellular forms known as magnetotactic multicellular prokaryotes (MMPs). 'Candidatus Magnetoglobus multicellularis' is presently the best known MMP. Here we describe the helical trajectories performed by these microorganisms as they swim forward, as well as their response to UV light. We measured the radius of the trajectory, time period and translational velocity (velocity along the helix axis), which enabled the calculation of other trajectory parameters such as pitch, tangential velocity (velocity along the helix path), angular frequency, and theta angle (the angle between the helix path and the helix axis). The data revealed that 'Ca. M. multicellularis' swims along elongated helical trajectories with diameters approaching the diameter of the microorganism. In addition, we observed that 'Ca. M. multicellularis' responds to UV laser pulses by swimming backwards, returning to forward swimming several seconds after the UV laser pulse. UV light from a fluorescence microscope showed a similar effect. Thus, phototaxis is used in addition to magnetotaxis in this microorganism.

Spatiotemporal distribution of the magnetotactic multicellular prokaryote Candidatus Magnetoglobus multicellularis in a Brazilian hypersaline lagoon and in microcosms

Candidatus Magnetoglobus multicellularis is an unusual morphotype of magnetotactic prokaryotes. These microorganisms are composed of a spherical assemblage of gram-negative prokaryotic cells capable of swimming as a unit aligned along a magnetic field. While they occur in many aquatic habitats around the world, high numbers of Ca. M. multicellularis have been detected in Araruama Lagoon, a large hypersaline lagoon near the city of Rio de Janeiro, in Brazil. Here, we report on the spatiotemporal distribution of one such population in sediments of Araruama Lagoon, including its annual distribution and its abundance compared with the total bacterial community. In microcosm experiments, Ca. M. multicellularis was unable to survive for more than 45 days: the population density gradually decreased coinciding with a shift to the upper layers of the sediment. Nonetheless, Ca. M. multicellularis was detected throughout the year in all sites studied. Changes in the population density seemed to be related to the input of organic matter as well as to salinity. The population density of Ca. M. multicellularis did not correlate with the total bacterial counts; instead, changes in the microbial community structure altered their counts in the environment (AU)

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Spatiotemporal distribution of the magnetotactic multicellular prokaryote Candidatus Magnetoglobus multicellularis in a Brazilian hypersaline lagoon and in microcosms.

Candidatus Magnetoglobus multicellularis is an unusual morphotype of magnetotactic prokaryotes. These microorganisms are composed of a spherical assemblage of gram-negative prokaryotic cells capable of swimming as a unit aligned along a magnetic field. While they occur in many aquatic habitats around the world, high numbers of Ca. M. multicellularis have been detected in Araruama Lagoon, a large hypersaline lagoon near the city of Rio de Janeiro, in Brazil. Here, we report on the spatiotemporal distribution of one such population in sediments of Araruama Lagoon, including its annual distribution and its abundance compared with the total bacterial community. In microcosm experiments, Ca. M. multicellularis was unable to survive for more than 45 days: the population density gradually decreased coinciding with a shift to the upper layers of the sediment. Nonetheless, Ca. M. multicellularis was detected throughout the year in all sites studied. Changes in the population density seemed to be related to the input of organic matter as well as to salinity. The population density of Ca. M. multicellularis did not correlate with the total bacterial counts; instead, changes in the microbial community structure altered their counts in the environment.

Flagellar apparatus of south-seeking many-celled magnetotactic prokaryotes.

Magnetotactic bacteria orient and migrate along geomagnetic field lines. Each cell contains membrane-enclosed, nano-scale, iron-mineral particles called magnetosomes that cause alignment of the cell in the geomagnetic field as the bacteria swim propelled by flagella. In this work we studied the ultrastructure of the flagellar apparatus in many-celled magnetotactic prokaryotes (MMP) that consist of several Gram-negative cells arranged radially around an acellular compartment. Flagella covered the organism surface, and were observed exclusively at the portion of each cell that faced the environment. The flagella were helical tubes never as long as a complete turn of the helix. Flagellar filaments varied in length from 0.9 to 3.8 micro m (average 2.4 +/- 0.5 micro m, n = 150) and in width from 12.0 to 19.5 nm (average 15.9 +/- 1.4 nm, n = 52), which is different from previous reports for similar microorganisms. At the base of the flagella, a curved hook structure slightly thicker than the flagellar filaments was observed. In freeze-fractured samples, macromolecular complexes about 50 nm in diameter, which possibly corresponded to part of the flagella basal body, were observed in both the P-face of the cytoplasmic membrane and the E-face of the outer membrane. Transmission electron microscopy showed that magnetosomes occurred in planar groups in the cytoplasm close and parallel to the organism surface. A striated structure, which could be involved in maintaining magnetosomes fixed in the cell, was usually observed running along magnetosome chains. The coordinated movement of the MMP depends on the interaction between the flagella of each cell with the flagella of adjacent cells of the microorganism.