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.     

Polysialic acid can mediate membrane interactions by interacting with phospholipids

Polysialic acid (polySia) is expressed on the surface of neural cells, neuroinvasive bacterial cells and several tumor cells. PolySia chains attached to NCAM can influence both trans interactions between membranes of two cells and cis interactions. Here, we report on the involvement of phospholipids in regulation of membrane interactions by polySia. The pH at the surface of liposomes, specific molecular area of phosphatidylcholine molecules, phase transition of DPPC bilayers, cyclic voltammograms of BLMs, and electron micrographs of phosphatidylcholine vesicles were studied after addition of polysialic acid free in solution. The results indicate that polySia chains can associate with phosphatidylcholine bilayers, incorporate into the polar part of a phospholipid monolayer, modulate cis interactions between phosphatidylcholine molecules, and facilitate trans interactions between apposing phospholipid vesicles. These observations imply that polySia attached to NCAM or to lipids can behave similarly.     

Polychaete fauna associated with holdfasts of the large brown alga Himantothallus grandifolius in Admiralty Bay, King George Island, Antarctic

The polychaete community associated with holdfasts of the brown alga Himantothallus grandifolius in Admiralty Bay has been studied. It is the first study of its kind in this area and only the second in the Antarctic. Samples were collected in the summer season of 1979/1980 from a depth range of 10–75 m. Seventy-eight species were found on 19 holdfasts. The community was dominated by Brania rhopalophora and Neanthes kerguelensis. Analysis of similarity showed that polychaete fauna associated with this habitat did not show any partitioning related to depth. Regression analysis showed that densities of both species and individuals decreased with increased holdfast volume. A positive correlation was found between the number of individuals and holdfast volume. Polychaetes from 10 feeding guilds were found with dominance of macrophagous motile herbivores and sessile filter feeders. The complex habitat provided by holdfasts is a shelter for a rich polychaete fauna and may function as important protection from disturbance in the shallow areas of Admiralty Bay.     

Molecular characteristics of community-associated methicillin-resistant Staphylococcus aureus strains for clinical medicine

Infections caused by methicillin-resistant S. aureus strains are mainly associated with a hospital setting. However, nowadays, the MRSA infections of non-hospitalized patients are observed more frequently. In order to distinguish them from hospital-associated methicillin-resistant S. aureus (HA-MRSA) strains, given them the name of community-associated methicillin-resistant S. aureus (CA-MRSA). CA-MRSA strains most commonly cause skin infections, but may lead to more severe diseases, and consequently the patient’s death. The molecular markers of CA-MRSA strains are the presence of accessory gene regulator (agr) of group I or III, staphylococcal cassette chromosome mec (SCCmec) type IV, V or VII and genes encoding for Panton–Valentine leukocidin (PVL). In addition, CA-MRSA strains show resistance to β-lactam antibiotics. Studies on the genetic elements of CA-MRSA strains have a key role in the unambiguous identification of strains, monitoring of infections, improving the treatment, work on new antimicrobial agents and understanding the evolution of these pathogens.     

Neuroprotective action of FK-506 (tacrolimus) after seizures induced with pilocarpine: quantitative and topographic elemental analysis of brain tissue

In the present work, X-ray fluorescence microscopy with a synchrotron source for the exciting radiation was applied for topographic and quantitative elemental analysis of rat brain tissue in pilocarpine-induced epilepsy and neuroprotection with FK-506. The mass per unit area of the elements P, S, Cl, K, Ca, Fe, Cu, Zn, Se, Br, and Rb was determined in four fields of the hippocampal formation (sectors 1 and 3 of Ammon’s horn–CA1, CA3; dentate gyrus; hilus of dentate gyrus) and the parietal cortex. The results obtained for epileptic rats treated with FK-506 (SNF) were compared with data obtained previously for epileptic rats (SNS) and a control group. Many statistically significant differences in elemental composition were observed between the SNF and SNS groups. Higher mass per unit area of P was noticed in CA1 and CA3 regions of the hippocampus of SNF rats in comparison with SNS rats. A similar relation was observed for K in all five brain areas analyzed. Also, Fe in CA3 and dentate gyrus, Cu in the parietal cortex, and Zn in CA3 and in the cortex were present at a higher level in the SNF group in comparison with the SNS group. The findings obtained in the present study suggest that the neuroprotective action of FK-506 in epileptic rat brain may involve not only the inhibition of calcineurin but also blockade of the K⁺ channels.