Aquaporin-1 (AQP1) facilitates the osmotic transport of water across the capillary endothelium, among other cell types, and thereby has a substantial role in ultrafiltration during peritoneal ...dialysis. At present, pharmacologic agents that enhance AQP1-mediated water transport, which would be expected to increase the efficiency of peritoneal dialysis, are not available. Here, we describe AqF026, an aquaporin agonist that is a chemical derivative of the arylsulfonamide compound furosemide. In the Xenopus laevis oocyte system, extracellular AqF026 potentiated the channel activity of human AQP1 by >20% but had no effect on channel activity of AQP4. We found that the intracellular binding site for AQP1 involves loop D, a region associated with channel gating. In a mouse model of peritoneal dialysis, AqF026 enhanced the osmotic transport of water across the peritoneal membrane but did not affect the osmotic gradient, the transport of small solutes, or the localization and expression of AQP1 on the plasma membrane. Furthermore, AqF026 did not potentiate water transport in Aqp1-null mice, suggesting that indirect mechanisms involving other channels or transporters were unlikely. Last, in a mouse gastric antrum preparation, AqF026 did not affect the Na-K-Cl cotransporter NKCC1. In summary, AqF026 directly and specifically potentiates AQP1-mediated water transport, suggesting that it deserves additional investigation for applications such as peritoneal dialysis or clinical situations associated with defective water handling.
Genetic stability requires coordination of a network of pathways including DNA repair/recombination and apoptosis. In addition to its canonical anti-apoptotic role, Bcl-2 negatively impacts genome ...stability. In this study, we identified the breast cancer tumor suppressor BRCA1, which plays an essential role in homologous recombination (HR), as a target for Bcl-2 in the repression of HR. Indeed, ionizing radiation-induced BRCA1 foci assembly was repressed when Bcl-2 was expressed ectopically, in human SV40 fibroblasts, or spontaneously, in lymphoma t(14:18) cells and in HeLa and H460 cancer cell lines. Moreover, we showed that the transmembrane (TM) domain of Bcl-2 was required for both inhibition of BRCA1 foci assembly and the inhibition of HR induced by a double-strand break targeted into an intrachromosomal HR substrate by the meganuclease I-SceI. Fluorescence confocal microscopy, proximity ligation assay, and electron microscopy analyses as well as Western blot analysis of subcellular fractions showed that Bcl-2 and BRCA1 colocalized to mitochondria and endoplasmic reticulum in a process requiring the TM domain of Bcl-2. Targeting BRCA1 to the endomembranes depletes BRCA1 from the nucleus and, thus, accounts for the inhibition of HR. Furthermore, our findings support an apoptosis-stimulatory role for the cytosolic form of BRCA1, suggesting a new tumor suppressor function of BRCA1. Together, our results reveal a new mode of BRCA1 regulation and for HR in the maintenance of genome stability.
The purpose of this study was to examine the presence of aquaporin water channels in human skin and to assess their functional role. On western blots of human epidermis obtained from plastic surgery, ...a strong signal was obtained with polyclonal anti-aquaporin-3 antibodies. By indirect immunofluorescence on 5 µm cryosections, anti-aquaporin-3 antibodies strongly stained keratinocyte plasma membranes in human epidermis, whereas no staining was observed in the dermis or the stratum corneum or when anti-aquaporin-3 antibodies were preabsorbed with the peptide used for immunization. Similarly, a strong signal with anti-aquaporin-3 antibodies was observed in keratinocyte plasma membranes of reconstructed human epidermis in culture at the air–liquid interface for up to 3 wk. The keratinocyte plasma membrane localization of aquaporin-3 was confirmed at the electron microscope level in prickle cells. In addition an intracellular localization of aquaporin-3 was also detected in epidermis basal cells. Osmotically induced transepidermal water permeability was measured on stripped human skin and on reconstructed epidermis. Water transport across both stripped human skin and 2–3 wk reconstructed epidermis was comparable, inhibited by > 50% by 1 mM HgCl2 and fully inhibited by acid pH. By stopped-flow light scattering, keratinocyte plasma membranes, where aquaporin-3 is localized, exhibited a high, pH-sensitive, water permeability. Although human skin is highly impermeable to water, this is primarily accounted for by the stratum corneum, where a steep water content gradient was demonstrated. In contrast, the water content of viable strata of the epidermis is remarkably constant. Our results suggest that the human epidermis, below the stratum corneum, exhibits a high, aquaporin-3-mediated, water permeability. We propose that the role of aquaporin-3 is to water-clamp viable layers of the epidermis in order to improve the hydration of the epidermis below the stratum corneum.
The development of automatic tools for the three-dimensional reconstruction of the microtubule cytoskeleton is crucial for large-scale analysis of mitotic spindles. Recently, we have published a ...method for the semiautomatic tracing of microtubules based on 3D template matching (Weber et al., J Struct Biol 178:129-138, 2012). Here, we give step-by-step instructions for the automatic tracing of microtubules emanating from centrosomes in the early mitotic Caenorhabditis elegans embryo. This approach, integrated in the visualization and data analysis software Amira, is applicable to tomographic data sets from other model systems.
The integral membrane light-harvesting (LH) proteins from purple photosynthetic bacteria form circular oligomers of an elementary unit that is composed of two very hydrophobic polypeptides, termed α ...and β. These apoprotein dimers are known to associate into closed circular arrays of 8, 9 and 16 α/β-mers. We report the existence of peripheral LH proteins purified from
Allochromatium vinosum with two intermediate ring sizes and postulate that one is a 13 α/β-mer. This shows that LH proteins are able to form membrane rings of continuously increasing diameter from 68 to 115
Å. The presence of these new ring sizes warrants further study, as it will help to further validate the structure–function models of LH proteins currently found in the literature.
Nanometer-sized gold nanoparticles have been prepared and surface-modified in order to stabilize alkane-in-water emulsions. A mixed hexane−undecanol ligand layer at the surface of the nanoparticles ...allowed us to tune their wettability and thus the adsorption at the oil−water interface. Oil droplets of the stable emulsions have been evidenced by confocal fluorescence microscopy, freeze-fracture transmission electron microscopy, and dynamic light scattering. Prepared emulsions were stable during performed cooling−heating cycles, in which the temperature stability of the emulsions has been studied by means of dynamic light scattering. The interfacial structure of the oil droplets was investigated by small-angle X-ray scattering. The obtained area per nanoparticle at the oil droplet interface was 30 nm2. The investigation of the nanoparticle adsorption at the curved interface of the emulsion droplets is in agreement with our previous study at a planar oil−water interface, in which the nanoparticles started to interact with each other at about the same area per particle.
Correlative light and electron microscopy (CLEM) encompasses a growing number of imaging techniques aiming to combine the benefits of light microscopy, which allows routine labeling of molecules and ...live-cell imaging of fluorescently tagged proteins with the resolution and ultrastructural detail provided by electron microscopy (EM). Here we review three different strategies that are commonly used in CLEM and we illustrate each approach with one detailed example of their application. The focus is on different options for sample preparation with their respective benefits as well as on the imaging workflows that can be used. The three strategies cover: (1) the combination of live-cell imaging with the high resolution of EM (time-resolved CLEM), (2) the need to identify a fluorescent cell of interest for further exploration by EM (cell sorting), and (3) the subcellular correlation of a fluorescent feature in a cell with its associated ultrastructural features (spatial CLEM). Finally, we discuss future directions for CLEM exploring the possibilities for combining super-resolution microscopy with EM.
The thermophilic cyanobacterium, Thermosynechococcus elongatus, has been grown in the presence of Sr2+ instead of Ca2+ with the aim of biosynthetically replacing the Ca2+ of the oxygen-evolving ...enzyme with Sr2+. Not only were the cells able to grow normally with Sr2+, they actively accumulated the ion to levels higher than those of Ca2+ in the normal cultures. A protocol was developed to purify a fully active Sr2+-containing photosystem II (PSII). The modified enzyme contained a normal polypeptide profile and 1 strontium/4 manganese, indicating that the normal enzyme contains 1 calcium/4 manganese. The Sr2+- and Ca2+-containing enzymes were compared using EPR spectroscopy, UV-visible absorption spectroscopy, and O2 polarography. The Ca2+/Sr2+ exchange resulted in the modification of the EPR spectrum of the manganese cluster and a slower turnover of the redox cycle (the so-called S-state cycle), resulting in diminished O2 evolution activity under continuous saturating light: all features reported previously by biochemical Ca2+/Sr2+ exchange in plant PSII. This allays doubts that these changes could be because of secondary effects induced by the biochemical treatments themselves. In addition, the Sr2+-containing PSII has other kinetics modifications: 1) it has an increased stability of the S3 redox state; 2) it shows an increase in the rate of electron donation from TyrD, the redox-active tyrosine of the D2 protein, to the oxygen-evolving complex in the S3-state forming S2; 3) the rate of oxidation of the S0-state to the S1-state by TyrD. is increased; and 4) the release of O2 is slowed down to an extent similar to that seen for the slowdown of the S3TyrZ. to S0TyrZ transition, consistent with the latter constituting the limiting step of the water oxidation mechanism in Sr2+-substituted enzyme as well as in the normal enzyme. The replacement of Ca2+ by Sr2+ appears to have multiple effects on kinetics properties of the enzyme that may be explained by S-state-dependent shifts in the redox properties of both the manganese complex and TyrZ as well as structural effects.
Mixtures of cationic and anionic surfactants crystallized at various ratios in the absence of added salt form micrometer-sized colloids. Here, we propose and test a general mechanism explaining how ...this ratio controls the shape of the resulting colloidal structure, which can vary from nanodiscs to punctured planes; during cocrystallization, excess (nonstoichiometric) surfactant accumulates on edges or pores rather than being incorporated into crystalline bilayers. Molecular segregation then produces a sequence of shapes controlled by the initial mole ratio only. Using freeze-fracture electron microscopy, we identified three of these states and their corresponding coexistence regimes. Fluorescence confocal microscopy directly showed the segregation of anionic and cationic components within the aggregate. The observed shapes are consistently reproduced upon thermal cycling, demonstrating that the icosahedral shape corresponds to the existence of a local minimum of bending energy for facetted icosahedra when the optimal amount of excess segregated material is present.
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