Freeze-Fracture Replica Immunolabelling Reveals Urothelial Plaques in Cultured Urothelial Cells

The primary function of the urothelium is to provide the tightest and most impermeable barrier in the body, i.e. the blood-urine barrier. Urothelial plaques are formed and inserted into the apical plasma membrane during advanced stages of urothelial cell differentiation. Currently, it is supposed that differentiation with the final formation of urothelial plaques is hindered in cultured urothelial cells. With the aid of the high-resolution imaging technique of freeze-fracture replica immunolabelling, we here provide evidence that urothelial cells in vitro form uroplakin-positive urothelial plaques, localized in fusiform-shaped vesicles and apical plasma membranes. With the establishment of such an in vitro model of urothelial cells with fully developed urothelial plaques and functional properties equivalent to normal bladder urothelium, new perspectives have emerged which challenge prevailing concepts of apical plasma membrane biogenesis and blood-urine barrier development. This may hopefully provide a timely impulse for many ongoing studies and open up new questions for future research

Urothelial Plaque Formation in Post-Golgi Compartments

Urothelial plaques are specialized membrane domains in urothelial superficial (umbrella) cells, composed of highly ordered uroplakin particles. We investigated membrane compartments involved in the formation of urothelial plaques in mouse umbrella cells. The Golgi apparatus did not contain uroplakins organized into plaques. In the post-Golgi region, three distinct membrane compartments containing uroplakins were characterized: i) Small rounded vesicles, located close to the Golgi apparatus, were labelled weakly with anti-uroplakin antibodies and they possessed no plaques; we termed them “uroplakin-positive transporting vesicles” (UPTVs). ii) Spherical-to-flattened vesicles, termed “immature fusiform vesicles” (iFVs), were uroplakin-positive in their central regions and contained small urothelial plaques. iii) Flattened “mature fusiform vesicles” (mFVs) contained large plaques, which were densely labelled with anti-uroplakin antibodies. Endoytotic marker horseradish peroxidase was not found in these post-Golgi compartments. We propose a detailed model of de novo urothelial plaque formation in post-Golgi compartments: UPTVs carrying individual 16-nm particles detach from the Golgi apparatus and subsequently fuse into iFV. Concentration of 16-nm particles into plaques and removal of uroplakin-negative membranes takes place in iFVs. With additional fusions and buddings, iFVs mature into mFVs, each carrying two urothelial plaques toward the apical surface of the umbrella cell

Effect of phosphorus on the attenuation of lead and chromium

This study analyses the adsorption of Pb(II) and Cr(III) in soils. These metals are commonly found together in nature in urban wastes or industrial spillages, and the theoretical approach of the work was to evaluate the response of the soil to continuous Cr and Pb spillages to soil in terms of several physicochemical parameters. The influence of an anthropogenic input of phosphorus was evaluated. Continuous flow experiments were run in duplicates in acrylic columns (25 cm × 3.2 cm). The influent Cr(III) and Pb(II) solutions of 10 mg l−1 and 25 mg l−1 at pH 5 were pumped upward through the bottom of the columns to ensure saturation flow conditions. Also, successive experiments were run with the above concentrations of Cr(III) and Pb(II) and NaH2PO4, keeping metal to phosphorus ratio of 1:0, 1:0.1 and 1:1. Modelling parameters included Freundlich and Langmuir equations, together with the Two-site adsorption model using CXTFIT code. Results obtained allowed concluding that Pb(II) adsorption presents a certain degree of irreversibility and the continued spillages over soil increment the fraction which is not easily desorbed. Cr(III) desorption was almost complete, evidencing its high mobility in nature. The presence of an anthropogenic input of phosphorus leads to a marked increase of both Pb(II) and Cr(III) adsorption in soils. Z-potential measurements allow to discard the electrostatic attraction of Cr(III) and Pb(II) with the surface charged soil as the dominant process of metal sorption. Instead, CheaqsPro simulation allows to identify PbH2PO4 +, PbHPO4 (aq) and CrHPO4 + as the dominant species which regulate Cr(III) and Pb(II) transport in soils.Fundação para a Ciência e a Tecnologi

TRY plant trait database - enhanced coverage and open access

Plant traits-the morphological, anatomical, physiological, biochemical and phenological characteristics of plants-determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait-based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits-almost complete coverage for 'plant growth form'. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait-environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives

Electron Tomography of Fusiform Vesicles and Their Organization in Urothelial Cells

The formation of fusiform vesicles (FVs) is one of the most distinctive features in the urothelium of the urinary bladder. FVs represent compartments for intracellular transport of urothelial plaques, which modulate the surface area of the superficial urothelial (umbrella) cells during the distension-contraction cycle. We have analysed the three-dimensional (3D) structure of FVs and their organization in umbrella cells of mouse urinary bladders. Compared to chemical fixation, high pressure freezing gave a new insight into the ultrastructure of urothelial cells. Electron tomography on serial sections revealed that mature FVs had a shape of flattened discs, with a diameter of up to 1.2 µm. The lumen between the two opposing asymmetrically thickened membranes was very narrow, ranging from 5 nm to 10 nm. Freeze-fracturing and immunolabelling confirmed that FVs contain two opposing urothelial plaques connected by a hinge region that made an omega shaped curvature. In the central cytoplasm, 4–15 FVs were often organized into stacks. In the subapical cytoplasm, FVs were mainly organized as individual vesicles. Distension-contraction cycles did not affect the shape of mature FVs; however, their orientation changed from parallel in distended to perpendicular in contracted bladder with respect to the apical plasma membrane. In the intermediate cells, shorter and more dilated immature FVs were present. The salient outcome from this research is the first comprehensive, high resolution 3D view of the ultrastructure of FVs and how they are organized differently depending on their location in the cytoplasm of umbrella cells. The shape of mature FVs and their organization into tightly packed stacks makes them a perfect storage compartment, which transports large amounts of urothelial plaques while occupying a small volume of umbrella cell cytoplasm

Voronoi Tessellation Captures Very Early Clustering of Single Primary Cells as Induced by Interactions in Nascent Biofilms

Biofilms dominate microbial life in numerous aquatic ecosystems, and in engineered and medical systems, as well. The formation of biofilms is initiated by single primary cells colonizing surfaces from the bulk liquid. The next steps from primary cells towards the first cell clusters as the initial step of biofilm formation remain relatively poorly studied. Clonal growth and random migration of primary cells are traditionally considered as the dominant processes leading to organized microcolonies in laboratory grown monocultures. Using Voronoi tessellation, we show that the spatial distribution of primary cells colonizing initially sterile surfaces from natural streamwater community deviates from uniform randomness already during the very early colonisation. The deviation from uniform randomness increased with colonisation — despite the absence of cell reproduction — and was even more pronounced when the flow of water above biofilms was multidirectional and shear stress elevated. We propose a simple mechanistic model that captures interactions, such as cell-to-cell signalling or chemical surface conditioning, to simulate the observed distribution patterns. Model predictions match empirical observations reasonably well, highlighting the role of biotic interactions even already during very early biofilm formation despite few and distant cells. The transition from single primary cells to clustering accelerated by biotic interactions rather than by reproduction may be particularly advantageous in harsh environments — the rule rather than the exception outside the laboratory