Cellular membrane processes, from signal transduction to membrane fusion and fission, depend on acute membrane deformations produced by small and short-lived protein complexes working in conditions ...far from equilibrium. Real-time monitoring and quantitative assessment of such deformations are challenging; hence, mechanistic analyses of the protein action are commonly based on ensemble averaging, which masks important mechanistic details of the action. In this protocol, we describe how to reconstruct and quantify membrane remodeling by individual proteins and small protein complexes in vitro, using an ultra-short (80- to 400-nm) lipid nanotube (usNT) template. We use the luminal conductance of the usNT as the real-time reporter of the protein interaction(s) with the usNT. We explain how to make and calibrate the usNT template to achieve subnanometer precision in the geometrical assessment of the molecular footprints on the nanotube membrane. We next demonstrate how membrane deformations driven by purified proteins implicated in cellular membrane remodeling can be analyzed at a single-molecule level. The preparation of one usNT takes ~1 h, and the shortest procedure yielding the basic geometrical parameters of a small protein complex takes 10 h.
Secretory proteins are sorted at the
-Golgi network (TGN) for export into specific transport carriers. However, the molecular players involved in this fundamental process remain largely elusive. ...Here, we identified the human transmembrane protein TGN46 as a receptor for the export of secretory cargo protein PAUF in CARTS - a class of protein kinase D-dependent TGN-to-plasma membrane carriers. We show that TGN46 is necessary for cargo sorting and loading into nascent carriers at the TGN. By combining quantitative fluorescence microscopy and mutagenesis approaches, we further discovered that the lumenal domain of TGN46 encodes for its cargo sorting function. In summary, our results define a cellular function of TGN46 in sorting secretory proteins for export from the TGN.
In vitro reconstitution and microscopic visualization of membrane processes is an indispensable source of information about a cellular function. Here we describe a conceptionally novel free-standing ...membrane template that facilitates such quantitative reconstitution of membrane remodelling at different scales. The Giant Suspended Bilayers (GSBs) spontaneously swell from lipid lamella reservoir deposited on microspheres. GSBs attached to the reservoir can be prepared from virtually any lipid composition following a fast procedure. Giant unilamellar vesicles can be further obtained by GSB detachment from the microspheres. The reservoir stabilizes GSB during deformations, mechanical micromanipulations, and fluorescence microscopy observations, while GSB-reservoir boundary enables the exchange of small solutes with GSB interior. These unique properties allow studying macro- and nano-scale membrane deformations, adding membrane-active compounds to both sides of GSB membrane and applying patch-clamp based approaches, thus making GSB a versatile tool for reconstitution and quantification of cellular membrane trafficking events.
This study presents the results of the geoenvironmental characterization of La Matildes riverbed, affected by mine tailings in the Cartagena–La Unión district, Murcia (southeast Spain). Soils and ...riverbeds in this area are highly polluted. Two Electrical Resistivity Imaging (ERI) profiles were carried out to obtain information about the thickness of the deposits and their internal structure. For the mine tailings deposits of La Murla, a tributary of the El Miedo riverbed, the geophysical method imaged two different units: the upper one characterized by low resistivity values and 5–8 m thickness, correlated with the mine tailings deposits; and the lower more resistive unit corresponding to the Paleozoic metasediments bedrock. The ERI profile transverse to the Las Matildes dry riverbed revealed the existence of three different units. The uppermost one has the lowest resistivity values and corresponds to the tailings deposits discharged to the riverbeds. An intermediate unit, with intermediate resistivity values, corresponds to the riverbed sediments before the mining operations. The lower unit is more resistive and corresponds to the bedrock. Significant amounts of pyrite, sphalerite, and galena were found both in tailings and riverbed sediments. The geochemical composition of borehole samples from the riverbed materials shows significantly high contents of As, Cd, Cu, Fe, Pb, and Zn being released to the environment. Mining works have modified the natural landscape near La Unión town. Surface extraction in three open-pit mines have changed the summits of Sierra de Cartagena–La Unión. Rock and metallurgical wastes have altered the drainage pattern and buried the headwaters of ephemeral channels. The environmental hazards require remediation to minimize the environmental impact on the Mar Menor coastal lagoon, one of the most touristic areas in SE Spain.
The hyporheic zone (HZ) is a critical area of all river ecosystems. It is the area beneath the stream and adjacent to the stream, where the surface water and groundwater are mixed. The HZ extends ...both vertically and laterally depending on the sediment configuration, namely their porosity and permeability. This influences the hyporheic communities’ structural pattern and their active dispersal among distinct rivers compartments and alluvial aquifers. It is still difficult to assess the spatial extent of the HZ and the distribution of the mixing zones. This study applies time-lapse images obtained using electrical resistivity tomography (ERT) of 20 m wide and 5 m deep alluvial streams, with regards to the structural pattern of hyporheic communities represented by cyclopoids and ostracods, in order to assess the extent of the HZ in the riverbed and the parafluvial sediment configurations. The ERT images obtained at the hyporheic Site 1 are characterized by alluvial deposits dominated by coarse and very coarse sands with resistivity values ranging from ~20 to 80 Ohm.m, indicating a permeable zone up to ~0.5 m thick and extending laterally for ca. 5 m from the channel and associated with the hyporheic zone. The sediment configurations, texture, and structure indicate an active surface–hyporheic water exchange and low water retention into the sediments. This is also indicated by the hyporheic copepods and ostracods communities’ structure formed by a mixture of non-stygobites (five species) and stygobites (two species). A low-resistivity (<70 Ohm.m) permeable zone located 2.3 m below the streambed and unconnected with the river channel was also detected and associated with the associated alluvial aquifer. In contrast, the resistivity image at Site 2 dominated by coarse, medium, and very fine sands, shows a low-permeability zone in the upper ~0.5 m of the profile, with a resistivity value ranging from ~45 to 80 Ohm.m, indicating a reduced HZ extension in both vertical and lateral dimensions. Here the sediment configurations indicate that the water retention and interaction with the sediment is higher, reflected by more diverse hyporheic communities and with highly abundant stygobite species. The two examples show that non-invasive ERT images and biological assessments provide complementary and valuable information about the characterization of the sub-channel architecture and its potential hydraulic connection to the floodplain aquifer.
Variations in the pore system of sandstones from the so-called Utrillas Formation (Lower Cretaceous, Iberian Peninsula) after CO2 injection have been investigated in a laboratory on a micro scale. In ...this study, we present results regarding variations in the pore spaces of sandstones caused by the injection of CO2 and its permanence in supercritical conditions in contact with a rock sample for two months. The modifications produced in the porosity and pore size distribution have been evaluated on two geological samples, using a 3D modelling of the results obtained by Hg intrusion porosimetry. Reconstructions of the pore structure of the rock before and after CO2 injection from mercury intrusion-extrusion curves, generating virtual models of pores that reproduce the experimental porosity. By analysing the results, a drastic modification in the mesoporosity of the rock is confirmed, which may have a paramount influence not only on the total storage capacity but also on the percolation of fluid through the rock.
The most used methods to evaluate the vulnerability to contamination of aquifers are based on overlay index maps, such us DRASTIC, GOD and AVI. These methods assign weighting and rating values to ...hydrogeological characteristics, introducing subjectivity in the evaluation. In this research, a new methodology is proposed to eliminate some of that subjectivity. The methodology evaluates the vulnerability to contamination of a detrital aquifer using K-means cluster analysis with a new set of parameters. The set is composed of some parameters extracted from these methods, as well as other new ones that have a significant influence on the movement of contaminants. Application of the Principal Components Analysis (PCA) technique before using K-means cluster allowed the selection of the most relevant parameters. To validate the methodology, this was applied to a detrital aquifer located at central Spain (the so-called “Aluviales Jarama-Tajuña” aquifer) with a significant agricultural development. To compare the traditional methods of vulnerability assessment with the K-means cluster, nitrate concentration was used as a pollution indicator. Thus, 23 groundwater quality samples were used to correlate (Spearman´s correlation coefficient) the vulnerability values with nitrate concentration to validate the most suitable method. The results showed that GOD and AVI were not appropriate methods to evaluate the vulnerability of the aquifer, because they have negative or very low correlation with nitrate concentration (− 0.5 and 0.01, respectively). This is due to the use of very few variables that do not represent relevant features for the vulnerability assessment. Alternatively, DRASTIC and K-means cluster analysis obtained higher Spearman´s correlation coefficients (0.34 and 0.48, respectively). The relevant features selected by PCA analysis to use in the K-means low-dimensional analysis were depth of groundwater (
D
), net recharge (
R
), and land use (
L
). The new proposed method grouped data in three clusters that represent low vulnerability (35.9% of the study area), moderate (41.4%) and high vulnerability (22.7%). K-means increases the Spearman’s correlation by 14% with respect to the most approximate conventional method (DRASTIC). Therefore, the results obtained confirm the advantage of joint application of PCA and K-Means analysis, which represents a novel approach for the assessment of groundwater vulnerability in detrital aquifers.
Mercury is transported globally in the atmosphere mostly in gaseous elemental form (GEM, Formula: see text), but still few worldwide studies taking into account different and contrasted environmental ...settings are available in a single publication. This work presents and discusses data from Argentina, Bolivia, Bosnia and Herzegovina, Brazil, Chile, China, Croatia, Finland, Italy, Russia, South Africa, Spain, Slovenia and Venezuela. We classified the information in four groups: (1) mining districts where this contaminant poses or has posed a risk for human populations and/or ecosystems; (2) cities, where the concentration of atmospheric mercury could be higher than normal due to the burning of fossil fuels and industrial activities; (3) areas with natural emissions from volcanoes; and (4) pristine areas where no anthropogenic influence was apparent. All the surveys were performed using portable LUMEX RA-915 series atomic absorption spectrometers. The results for cities fall within a low GEM concentration range that rarely exceeds 30 ng m⁻³, that is, 6.6 times lower than the restrictive ATSDR threshold (200 ng m⁻³) for chronic exposure to this pollutant. We also observed this behavior in the former mercury mining districts, where few data were above 200 ng m⁻³. We noted that high concentrations of GEM are localized phenomena that fade away in short distances. However, this does not imply that they do not pose a risk for those working in close proximity to the source. This is the case of the artisanal gold miners that heat the Au–Hg amalgam to vaporize mercury. In this respect, while GEM can be truly regarded as a hazard, because of possible physical–chemical transformations into other species, it is only under these localized conditions, implying exposure to high GEM concentrations, which it becomes a direct risk for humans.
It is possible to assess the harm that society suffers as a consequence of groundwater contamination in aquifers. Indexing methodologies are commonly applied to assess the social vulnerability to ...polluted aquifers. However, they assign weighting and rating values to the different factors involved, which makes them very subjective. This research aims to assess the social vulnerability to groundwater pollution taking into account three factors: the uses of groundwater resources, the exposed population, and the socio-economic losses. In order to eliminate the subjectivity of current indexing methodologies, this work uses a K-means cluster analysis for the assessment of social vulnerability. With this method, a social vulnerability map can be produced with greater objectivity. The proposed methodology is applied to an aquifer located in central Spain, an area with significant agricultural development. Low population density and unproductive zones result in low social vulnerability in most of the area. However, high social vulnerability is observed in the southern sector due to agricultural development, which leads to higher socio-economic variables and demand for groundwater resources. Similarly, high social vulnerability is observed in the northeast, mainly influenced by the groundwater use and the exposed population. These results show that social vulnerability in most of the study area is not very significant for assessing the risk of groundwater contamination, because the damage to the social, environmental, or economic sector is low. However, in the south and northeast of the study area, pesticides and fertilizers should be used with caution, as they significantly increase the risk of groundwater contamination. The K-means clustering method proved to be an objective and reliable option for assessing social vulnerability to groundwater pollution in aquifers.
The emergence of phase separation phenomena among macromolecules has identified biomolecular condensates as fundamental cellular organizers. These condensates concentrate specific components and ...accelerate biochemical reactions without relying on membrane boundaries. Although extensive studies have revealed a large variety of nuclear and cytosolic membraneless organelles, we are witnessing a surge in the exploration of protein condensates associated with the membranes of the secretory pathway, such as the endoplasmic reticulum and the Golgi apparatus. This review focuses on protein condensates in the secretory pathway and discusses their impact on the organization and functions of this cellular process. Moreover, we explore the modes of condensate-membrane association and the biophysical and cellular consequences of protein condensate interactions with secretory pathway membranes.
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