Prion protein impairs kinesin-driven transport

Our previous studies have demonstrated that prion protein (PrP) leads to disassembly of microtubular cytoskeleton through binding to tubulin and its oligomerization. Here we found that PrP-treated cells exhibited improper morphology of mitotic spindles. Formation of aberrant spindles may result not only from altered microtubule dynamics - as expected from PrP-induced tubulin oligomerization - but also from impairing the function of molecular motors. Therefore we checked whether binding of PrP to microtubules affected movement generated by Ncd - a kinesin responsible for the proper organization of division spindles. We found that PrP inhibited Ncd-driven transport of microtubules. Most probably, the inhibition of the microtubule movement resulted from PrP-induced changes in the microtubule structure since Ncd-microtubule binding was reduced already at low PrP to tubulin molar ratios. This study suggests another plausible mechanism of PrP cytotoxicity related to the interaction with tubulin, namely impeding microtubule-dependent transport.     

Skeletal muscle-derived fibroblast-like cells fail to enable HeLa cells to induce bone in the murine kidney

HeLa cells fail to induce bone in murine kidneys, despite being highly chondro/osteogenic when implanted into thigh muscles. Bone induction in the kidneys failed also when HeLa cells were grafted together with skeletal-muscle-derived cell cultures. It is postulated that kidney parenchyma releases unidentified factcor(s) which prevent activation by inducer of cells termed Friedenstein's (1976) "inducible osteoprogenitor cells", while this hypothetical factor does not prevent further differentiation of"determined osteoprogenitor cells", thus allowing bone to form in the renal parenchyma.     

Importance of N- and C-terminal regions of IbpA, Escherichia coli small heat shock protein, for chaperone function and oligomerization

Small heat shock proteins are ubiquitous molecular chaperones that, during cellular stress, bind to misfolded proteins and maintain them in a refolding competent state. Two members of the small heat shock protein family, IbpA and IbpB, are present in Escherichia coli. Despite 48% sequence identity, the proteins have distinct activities in promoting protein disaggregation. Cooperation between IbpA and IbpB is crucial for prevention of the irreversible aggregation of proteins. In this study, we investigated the importance of the N- and C-terminal regions of IbpA for self-oligomerization and chaperone functions. Deletion of either the N- or C-terminal region of IbpA resulted in a defect in the IbpA fibril formation process. The deletions also impaired IbpA chaperone function, defined as the ability to stabilize, in cooperation with IbpB, protein aggregates in a disaggregation-competent state. Our results show that the defect in chaperone function, observed in truncated versions of IbpA, is due to the inability of these proteins to interact with substrate proteins and consequently to change the properties of aggregates. At the same time, these versions of IbpA interact with IbpB similarly to the wild type protein. Competition experiments performed with the pC peptide, which corresponds to the IbpA C terminus, suggested the importance of IbpA intermolecular interactions in the stabilization of aggregates in a state competent for disaggregation. Our results suggest that these interactions are not only dependent on the universally conserved IEI motif but also on arginine 133 neighboring the IEI motif. IbpA mutated at arginine 133 to alanine lacked chaperone activity.     

Comparison of UTCI to selected thermal indices

Over the past century more than 100 indices have been developed and used to assess bioclimatic conditions for human beings. The majority of these indices are used sporadically or for specific purposes. Some are based on generalized results of measurements (wind chill, cooling power, wet bulb temperature) and some on the empirically observed reactions of the human body to thermal stress (physiological strain, effective temperature). Those indices that are based on human heat balance considerations are referred to as "rational indices". Several simple human heat balance models are known and are used in research and practice. This paper presents a comparative analysis of the newly developed Universal Thermal Climate Index (UTCI), and some of the more prevalent thermal indices. The analysis is based on three groups of data: global data-set, synoptic datasets from Europe, and local scale data from special measurement campaigns of COST Action 730. We found the present indices to express bioclimatic conditions reasonably only under specific meteorological situations, while the UTCI represents specific climates, weather, and locations much better. Furthermore, similar to the human body, the UTCI is very sensitive to changes in ambient stimuli: temperature, solar radiation, wind and humidity. UTCI depicts temporal variability of thermal conditions better than other indices. The UTCI scale is able to express even slight differences in the intensity of meteorological stimuli.     

The UTCI-clothing model

The Universal Thermal Climate Index (UTCI) was conceived as a thermal index covering the whole climate range from heat to cold. This would be impossible without considering clothing as the interface between the person (here, the physiological model of thermoregulation) and the environment. It was decided to develop a clothing model for this application in which the following three factors were considered: (1) typical dressing behaviour in different temperatures, as observed in the field, resulting in a model of the distribution of clothing over the different body segments in relation to the ambient temperature, (2) the changes in clothing insulation and vapour resistance caused by wind and body movement, and (3) the change in wind speed in relation to the height above ground. The outcome was a clothing model that defines in detail the effective clothing insulation and vapour resistance for each of the thermo-physiological model’s body segments over a wide range of climatic conditions. This paper details this model’s conception and documents its definitions.     

Characterization of Caulobacter crescentus FtsZ protein using dynamic light scattering

The self-assembly of the tubulin homologue FtsZ at the mid-cell is a critical step in bacterial cell division. We introduce dynamic light scattering (DLS) spectroscopy as a new method to study the polymerization kinetics of FtsZ in solution. Analysis of the DLS data indicates that the FtsZ polymers are remarkably monodisperse in length, independent of the concentrations of GTP, GDP, and FtsZ monomers. Measurements of the diffusion coefficient of the polymers demonstrate that their length is remarkably stable until the free GTP is consumed. We estimated the mean size of the FtsZ polymers within this interval of stable length to be between 9 and 18 monomers. The rates of FtsZ polymerization and depolymerization are likely influenced by the concentration of GDP, as the repeated addition of GTP to FtsZ increased the rate of polymerization and slowed down depolymerization. Increasing the FtsZ concentration did not change the size of FtsZ polymers; however, it increased the rate of the depolymerization reaction by depleting free GTP. Using transmission electron microscopy we observed that FtsZ forms linear polymers in solutions which rapidly convert to large bundles upon contact with surfaces at time scales as short as several seconds. Finally, the best studied small molecule that binds to FtsZ, PC190723, had no stabilizing effect on Caulobacter crescentus FtsZ filaments in vitro, which complements previous studies with Escherichia coli FtsZ and confirms that this class of small molecules binds Gram-negative FtsZ weakly.     

Activation of the plasma membrane H+-ATPase by acid stress: Antibodies as a tool to follow the phosphorylation status of the penultimate activating Thr

Tight regulation of the plasma membrane proton pump ATPase (H⁺-ATPase) is necessary for controlling the membrane potential that energizes secondary transporters. This regulation relies on the phosphorylation of the H⁺-ATPase penultimate residue, a theonine, and the subsequent binding of regulatory 14-3-3 proteins, which results in enzyme activation. Using phospho-specific antibodies directed against the phosphorylable Thr of either PMA2 (Plasma membrane H⁺-ATPase from N. plumbaginifolia) or PMA4, we showed that the kinetics and extent of phosphorylation differ between both isoforms according to the growth or environmental conditions like cold stress.¹ Here, we used phospho-specific antibodies to follow PMA2 Thr phosphorylation upon acidification of the cytosol by incubating N. tabacum BY2 cells with four different weak organic acids. Increased PMA2 phosphorylation was observed for three of them, thus highlighting the role of the H+-ATPase in cell pH homeostasis.Key words: H⁺-ATPase, regulation, phosphorylation, phospho-specific antibodies, pH homeostasis, cold stressThe H⁺-ATPase is a major enzyme of the plant plasma membrane. This P-type ATPase couples ATP hydrolysis with proton transport out of the cell and establishes pH and potential gradients across the plasma membrane, thereby activating secondary transporters. At the physiological level, this enzyme is implicated in diverse roles, such as cytosolic pH regulation, cell elongation or stomata aperture.^2,3 H⁺-ATPase consists of ten membrane spanning regions and four cytosolic domains, among which the auto-inhibitory C-terminal region. The activation mechanism of the enzyme is well known and involves phosphorylation of its penultimate residue, a threonine, by an as yet unidentified protein kinase; phosphorylation in turn leads to the binding of regulatory 14-3-3 protein dimers and to the formation of an activated complex consisting of six H⁺-ATPases and six 14-3-3 proteins.^4–7Additional conserved phosphorylation sites in the enzyme C-terminal region have been shown to positively or negatively contribute to the enzyme regulation.^8–10 More sites have been discovered by large-scale phospho-proteomics, but have not been studied to date.^10,11 Most of these additional phosphorylated residues are located in the enzyme C-terminal autoinhibitory domain. This domain contains two to three inhibitory regions and a 14-3-3 binding region, partially super-imposed with an inhibitory region.^12,13 All these recent data suggest that the activity of H⁺-ATPase is finely tuned. However, the complexity of this regulation makes it difficult at the present stage to propose a comprehensive view.To follow and compare the activation status of two H⁺-ATPase isoforms belonging to different subfamilies, antibodies were designed for specifically recognizing the phosphorylated form of the penultimate Thr of either PMA2 (Plasma membrane H⁺-ATPase from N. plumbaginifolia) or PMA4, two broadly expressed isoforms belonging to subfamily I and II, respectively. This allowed us to find, for example, that PMA2, as opposed to PMA4, is strongly dephosphorylated upon cold stress. Both isoforms are strongly activated, upon subculturing N. tabacum BY2 suspension cells into a new media.¹ However, they underwent dephosphorylation at different rates as the cell culture proceeded. These data showed the usefulness of these antibodies for determining the regulation of specific H⁺-ATPase isoforms and better understanding their physiological roles.The primary function of the plasma membrane H⁺-ATPase is to transfer protons outside the cell. H⁺-ATPase is therefore considered as a possible regulator of the cytosolic pH homeostasis, for instance by preventing internal acidification. However, few data so far support this role for H⁺-ATPase. We addressed this point by adding to a N. tabacum BY2 cell culture weak organic acids, which are expected to permeate the membrane as a protonated form and dissociate once inside, resulting in cytosol acidification. This is expected to activate the plasma membrane H⁺-ATPase and so remove the proton excess out off the cell. A N. tabacum BY2 cell culture was treated with 5 mM of either citric acid, aminobenzoic acid, 2,2-dimethylglutaric acid, or propionic acid (Fig. 1). After several periods of time, a microsomal fraction was isolated and analyzed by western blotting. Among the four weak acids tested, propionic acid and citric acid induced strong and stable increase of PMA2 penultimate Thr phosphorylation. 2,2-dimethylglutaric acid induced a temporary increase of phosphorylation while aminobenzoic acid had no effect. The different responses might be explained either by different diffusion rates of the organic acids across the plasma membrane or by their possible toxicity. In addition, homeostasis of the intracellular pH results from the activity of several different enzymatic systems such as vacuolar H⁺-ATPase and H⁺-pyrophosphatase. Therefore it is also possible that, depending on the rate and/or extent of cytosol acidification, different responses are activated.Open in a separate windowFigure 1Effect of various weak acids on the phosphorylation of the PMA 2 penultimate Thr residue of N. tabacum suspension cells. A 3-day old N. tabacum BY2 cell culture was treated with 1/10^th volume of 50 mM of either aminobenzoic acid, 2,2-dimethylglutaric acid, citric acid or propionic acid, dissolved in the culture medium and brought beforehand to the same pH as the culture. After the indicated periods of time, cells were collected and a microsomal fraction was isolated and analyzed by western blotting using antibodies pThr⁹⁵⁵PMA 2 recognizing the PMA 2 penultimate activating Thr¹ (upper) and pan PMA 2 recognizing a short sequence specific for PMA2,¹⁴ (lower). C, untreated cells.This data supports the role H⁺-ATPase in pH homeostasis and highlights the strong potential of using phospho-specific antibodies to follow enzyme activation in the plant according to different environmental conditions. In addition, one should also take advantage of them as a tool for in vitro phosphorylation tests using different subcellular fractions of N. tabacum BY2 cells. This approach might lead to the isolation of the kinase and phosphatase involved in the modification of the penultimate Thr residue. Indeed, these enzymes are still undiscovered in spite of the fact that phosphorylation of this residue has been demonstrated more than a decade ago.     

Electrostatic Compensation Restores Trafficking of the Autosomal Recessive Retinitis Pigmentosa E150K Opsin Mutant to the Plasma Membrane

Rhodopsin is the rod photoreceptor G protein-coupled receptor responsible for capturing light. Mutations in the gene encoding this protein can lead to a blinding disease called retinitis pigmentosa, which is inherited frequently in an autosomal dominant manner. The E150K opsin mutant associated with rarely occurring autosomal recessive retinitis pigmentosa localizes to trans-Golgi network membranes rather than to plasma membranes of rod photoreceptor cells. We investigated the molecular mechanisms underlying opsin retention in the Golgi apparatus. Electrostatic calculations reveal that the E150K mutant features an overall accumulation of positive charges between helices H-IV and H-II. Human E150K and several other closely related opsin mutants were then expressed in HEK-293 cells. Spectral characteristics and functional biochemistry of each mutant were analyzed after reconstitution with the cis-retinoid chromophore. UV-visible spectra and rhodopsin/transducin activation assays revealed only minor differences between the purified wild type control and rhodopsin mutants. However, partial restoration of the surface electrostatic charge in the compensatory R69E/E150K double mutant rescues the plasma membrane localization of opsin. These findings emphasize the fundamental importance of electrostatic interactions for appropriate membrane trafficking of opsin and advance our understanding of the pathophysiology of autosomal recessive retinitis pigmentosa due to the E150K mutation.