Estimation of Membrane Potential Δψ in Reconstituted Plasma Membrane Vesicles Using a Numerical Model of Oxonol VI Distribution

A model of membrane potential-dependent distribution of oxonol VI to estimate the electrical potential difference across Schizosaccharomyces pombe plasma membrane vesicles (PMV) has been developed. was generated by the H⁺-ATPase reconstituted in the PMV. The model treatment was necessary since the usual calibration of the dye fluorescence changes by diffusion potentials (K⁺ + valinomycin) failed. The model allows for fitting of fluorescence changes at different vesicle and dye concentrations, yielding in ATP-energized PMV of 80 mV. The described model treatment to estimate may be applicable for other reconstituted membrane systems.     

Transport of Cl– across the Plasma Membrane during Pollen Grain Germination in Tobacco

The role of Cl- transport across the plasma membrane was studied in an early step of pollen grain germination in tobacco Nicotiana tabacum L. The Cl- channel blockers, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid, completely suppress the germination with IC(50) approximately 8 micro M. At this concentration NPPB reduces the rate of Cl- efflux out of pollen grain by 1.8-fold in the interval 5-12 min, and niflumic acid reduces the rate 1.2-fold. 4,4;-Diisothiocyanatostilbene-2,2;-disulfonic acid, a known inhibitor of Cl- channels and antiporters, completely suppresses germination as well (IC(50) = 240 micro M), but has no effect on the rate of Cl- efflux. Inhibitors of chloride co-transporters, such as furosemide, bumetanide, and bis(1,3-dibutylbarbituric acid)pentamethine oxonol, suppress the germination by less than 50%. This set of data suggests that NPPB-sensitive anion channels are involved in the activation of pollen grains in the early stage of germination.     

Differential Distribution of Both IL-12Rβ Chains in the Plasma Membrane of Human T Cells

IL-12 is a cytokine that stimulates the expression of CD26, a T cell– and raft-associated ectopeptidase. IL-12 also enhances the interaction between CD26 and CD45RO, which removes the phosphatase CD45RO from raft microdomains. Since Janus kinases are known CD45 substrates, our hypothesis was that this relocation of CD45RO in nonraft areas of the membrane could be important to switch off the signaling via cytokine receptors, e.g., the IL-12 receptor (IL-12R). Accordingly, both IL-12R and CD45RO should be equally positioned in the cell membrane upon IL-12R ligation. However, there were no data available on the membrane distribution of IL-12R on human T cells. Working with phytohemagglutinin (PHA) lymphoblasts, we tried to fill that gap. The high-affinity IL-12R is made of two chains: IL-12Rβ1 and IL-12Rβ2. Using flow cytometry, Western blot and confocal microscopy, we obtained data suggesting that IL-12Rβ1 is mainly associated to phospholipid-rich membrane areas, a location even enhanced upon IL-12 incubation of PHA blasts. Instead, IL-12Rβ2 is found more segregated into membrane rafts, which could explain why two IL-12-triggered events, T-cell proliferation and ERK1/2 activation, are both methyl-β-cyclodextrin-sensitive events. Ligation of IL-12R with IL-12 seems to induce a partial enrichment of IL-12Rβ2 in phospholipid-rich areas, where according to our data IL-12Rβ1 is already present. Therefore, although new data will be required, the present results support the initial hypothesis.     

Regulation of Constitutive Cargo Transport from the trans-Golgi Network to Plasma Membrane by Golgi-localized G Protein βγ Subunits

Observations of Golgi fragmentation upon introduction of G protein βγ (Gβγ) subunits into cells have implicated Gβγ in a pathway controlling the fission at the trans-Golgi network (TGN) of plasma membrane (PM)-destined transport carriers. However, the subcellular location where Gβγ acts to provoke Golgi fragmentation is not known. Additionally, a role for Gβγ in regulating TGN-to-PM transport has not been demonstrated. Here we report that constitutive or inducible targeting of Gβγ to the Golgi, but not other subcellular locations, causes phospholipase C- and protein kinase D-dependent vesiculation of the Golgi in HeLa cells; Golgi-targeted β₁γ₂ also activates protein kinase D. Moreover, the novel Gβγ inhibitor, gallein, and the Gβγ-sequestering protein, GRK2ct, reveal that Gβγ is required for the constitutive PM transport of two model cargo proteins, VSV-G and ss-HRP. Importantly, Golgi-targeted GRK2ct, but not a PM-targeted GRK2ct, also blocks protein transport to the PM. To further support a role for Golgi-localized Gβγ, endogenous Gβ was detected at the Golgi in HeLa cells. These results are the first to establish a role for Golgi-localized Gβγ in regulating protein transport from the TGN to the cell surface.     

Endosymbiotic Origin of Chloroplasts in Plant Cells’ Evolution

Russian Journal of Plant Physiology - The theory of endosymbiotic origin of chloroplasts has become basal in present-day biology. In this regard, the emergence of eukaryotic photosynthesis has been...     

Direct Observation of α-Synuclein Amyloid Aggregates in Endocytic Vesicles of Neuroblastoma Cells

Aggregation of α-synuclein has been linked to both familial and sporadic Parkinson’s disease. Recent studies suggest that α-synuclein aggregates may spread from cell to cell and raise questions about the propagation of neurodegeneration. While continuous progress has been made characterizing α-synuclein aggregates in vitro, there is a lack of information regarding the structure of these species inside the cells. Here, we use confocal fluorescence microscopy in combination with direct stochastic optical reconstruction microscopy, dSTORM, to investigate α-synuclein uptake when added exogenously to SH-SY5Y neuroblastoma cells, and to probe in situ morphological features of α-synuclein aggregates with near nanometer resolution. We demonstrate that using dSTORM, it is possible to follow noninvasively the uptake of extracellularly added α-synuclein aggregates by the cells. Once the aggregates are internalized, they move through the endosomal pathway and accumulate in lysosomes to be degraded. Our dSTORM data show that α-synuclein aggregates remain assembled after internalization and they are shortened as they move through the endosomal pathway. No further aggregation was observed inside the lysosomes as speculated in the literature, nor in the cytoplasm of the cells. Our study thus highlights the super-resolution capability of dSTORM to follow directly the endocytotic uptake of extracellularly added amyloid aggregates and to probe the morphology of in situ protein aggregates even when they accumulate in small vesicular compartments.     

Transport of α-Ketoisocaproate in Neuroblastoma NB-2a Cells

Transport of α-ketoisocaproate (KIC), a ketoacid originating from leucine and proposed to be involved in the buffering of glutamate in neurones, was studied in neuroblastoma NB-2a cells. The accumulated KIC was mostly transaminated to leucine, while free ketoacid was detectable either only after prolonged times or after inhibiting transaminase with aminooxyacetate. Accumulation of KIC was found to be inhibited by other branched-chain ketoacids, while lactate and β-hydroxybutyrate were ineffective. The transport of KIC, resembling a facilitated diffusion, was decreased by phloretin, α-cyano-4-hydroxycinnamate, 4,4′-diisothiocyano-2,2′-stilbenedisulphonate, and p-chlorimercuribenzoate. The process of accumulation did not resemble a symport with protons; therefore an involvement of the known proton-coupled monocarboxylate transporters (MCT) was excluded. Distribution of KIC suggests a mechanism involving a cotransport with 2 [Na⁺].     

Secretion and Uptake of α-Synuclein Via Extracellular Vesicles in Cultured Cells

In Parkinson’s disease and other Lewy body disorders, the propagation of pathology has been accredited to the spreading of extracellular α-synuclein (α-syn). Although the pathogenic mechanisms are not fully understood, cell-to-cell transfer of α-syn via exosomes and other extracellular vesicles (EVs) has been reported. Here, we investigated whether altered molecular properties of α-syn can influence the distribution and secretion of α-syn in human neuroblastoma cells. Different α-syn variants, including α-syn:hemi-Venus and disease-causing mutants, were overexpressed and EVs were isolated from the conditioned medium. Of the secreted α-syn, 0.1–2% was associated with vesicles. The major part of EV α-syn was attached to the outer membrane of vesicles, whereas a smaller fraction was found in their lumen. For α-syn expressed with N-terminal hemi-Venus, the relative levels associated with EVs were higher than for WT α-syn. Moreover, such EV-associated α-syn:hemi-Venus species were internalized in recipient cells to a higher degree than the corresponding free-floating forms. Among the disease-causing mutants, A53T α-syn displayed an increased association with EVs. Taken together, our data suggest that α-syn species with presumably lost physiological functions or altered aggregation properties may shift the cellular processing towards vesicular secretion. Our findings thus lend further support to the tenet that EVs can mediate spreading of harmful α-syn species and thereby contribute to the pathology in α-synucleinopathies.     

Structure and Function of ε-Subunit of ATP Synthase in Higher Plant Chloroplast

Theε-subunit is the smallest subunit of chloroplast ATP synthase, and is known to inhibit ATPase activity in isolated CF1-ATPase. As a result ε is sometimes called an inhibitory subunit. In addition, and perhaps more importantly, theε-subunit is essential for the coupling of proton translocation to ATP synthesis (as proton gate). The relation between the structure and function ofε-subunit of ATP synthase in higher plant chloroplast has been studied by molecular biological methods such as site-directed mu-tagenesis and truncations for C- or N-terminus ofε-subunit. The results showed that: (1) Thr42 ofε-subunit is important for its structure and function; (2) compared with theε-subunit in E.. coli, theε-subunit of chloroplast ATP synthase is more sensitive to C- or N-terminus truncations.     

Physical Determinants of β-Barrel Membrane Protein Folding in Lipid Vesicles

The spontaneous folding of two Neisseria outer membrane proteins, opacity-associated (Opa)₆₀ and Opa₅₀ into lipid vesicles was investigated by systematically varying bulk and membrane properties. Centrifugal fractionation coupled with sodium dodecyl sulfate polyacrylamide gel electrophoresis mobility assays enabled the discrimination of aggregate, unfolded membrane-associated, and folded membrane-inserted protein states as well as the influence of pH, ionic strength, membrane surface potential, lipid saturation, and urea on each. Protein aggregation was reduced with increasing lipid chain length, basic pH, low salt, the incorporation of negatively charged guest lipids, or by the addition of urea to the folding reaction. Insertion from the membrane-associated form was improved in shorter chain lipids, with more basic pH and low ionic strength; it is hindered by unsaturated or ether-linked lipids. The isolation of the physical determinants of insertion suggests that the membrane surface and dipole potentials are driving forces for outer membrane protein insertion and folding into lipid bilayers.     

Involvement of Rab28 in NF-κB Nuclear Transport in Endothelial Cells

Our previous proteomic analysis revealed the expression of Rab28 in arteries of rats. However, the function of Rab28 in mammalian cells, and its role in vessels are still unknown. Coarctation of abdominal aorta above left kidney artery in rat was used as hypertensive animal model. FX-4000 cyclic strain loading system was used to mimic the mechanical condition on vascular cells during hypertension in vitro. Immunofluorescence and co-immunoprecipitation (Co-IP) were used to identify distribution and interaction of Rab28 and nuclear factor kappa B (NF-κB). Rab28 expression was significantly increased in carotid arteries of hypertensive rats. High cyclic strain induced Rab28 expression of endothelial cells (ECs) through a paracrine control of vascular smooth muscles cells (VSMCs), which at least partly via angiotensin II (Ang II). Rab28 knockdown decreased proliferation of ECs, while increased apoptosis and migration. Immunofluorescence revealed that Ang II stimulated the co-translocation of Rab28 and NF-κB from cytoplasm into nucleus. Knockdown of Rab28 attenuated NF-κB activation. Co-IP of NF-κB p65 and Rab28 indicated their interaction. Our results revealed that Rab28, as a novel regulator of NF-κB nuclear transport, might participate in the disturbance of EC homeostasis.     

Evidence from ITIR-FCS Diffusion Studies that the Amyloid-Beta (Aβ) Peptide Does Not Perturb Plasma Membrane Fluidity in Neuronal Cells

The amyloid-beta (Aβ) peptide, commonly found in elevated levels in the brains of patients with Alzheimer's disease (AD) and in the cerebrospinal fluid of individuals presenting mild cognitive impairment, is thought to be one of the major factors resulting in the onset of AD. Although observed and studied at the molecular level for several decades, the exact disease pathology of AD is still not totally clear. One way in which Aβ is thought to affect neurons is by influencing cell membrane fluidity, which could result in abnormal synaptic or signaling function. The effects of Aβ on the fluidity of biological membranes have been studied using numerous membrane models such as artificial lipid bilayers and vesicles, living cells and membranes extracted from animal models of AD, yet there is still no consensus as to what effects Aβ has, if any, on membrane fluidity. As one of the most precise and accurate means of assaying membrane dynamics, we have thus chosen fluorescence correlation spectroscopy to investigate the issue, using fluorescent membrane-targeted probes on living cells treated with Aβ(1–42) oligomers and observing possible changes in membrane diffusion. Effects of Aβ on viability in different cell types varied from no detectable effect to extensive cell death by 72?h post-exposure. However, there was no change in the fluidity of either ordered membrane domains or the bulk membrane in any of these cells within this period. Our conclusion from these results is that perturbation of membrane fluidity is not likely to be a factor in acute Aβ-induced cytotoxicity.     

Influence of K＋ on the Coupling Between ATP Hydrolysis and Proton Transport by the Plasma Membrane H＋-ATPase from Soybean Hypocotyls

The plasma membrane vesicles were purified from soybean ( Glycine max L. ) hypocotyls by two-phase partitioning methods. The stimulatory effects of K+ on the coupling between ATP hydrolysis and proton transport by the plasma membrane H+-ATPase were studied. The results showed that the proton transport activity was increased by 850% in the presence of 100 mmol/L KC1, while ATP hydrolytic activity was only increased by 28.2%. Kinetic studies showed that Km of ATP hydrolysis decreased from 1.14 to 0.7 mmol/L, while Vmax of ATP hydrolysis increased from 285.7 to 344.8 nmol Pi·mg- l protein·min-1 in the presence of KC1. Experiments showed that the optimum pH was 6.5 and 6.0 in the presence and absence of KC1, respectively. Further studies revealed that K+ could promote the inhibitory effects of hydroxylamines and vanadates on the ATP hydrolytic activity. The above results suggested that K+ could regulate the coupling between ATP hydrolysis and proton transport of the plasma membrane H+ -ATPase through modulating the structure and function of the kinase and phosphatase domains of the plasma membrane H + -ATPase.     

Floristics of Higher Plants in China——Report from Catalogue of Life: Higher Plants in China Database

Biodiversity research increasingly relies on distribution networks for dealing with large-scale primary data. Up-to-date information on biodiversity is critical for the proper management and conservation of any area. The first step towards conservation should be to compile an authoritative species inventory or checklist. Catalogue of Life: Higher Plants in China (CNPC) is an ongoing biodiversity informatics project with the aim to integrate existing higher plants inventory data, and provide access via an internet based web service to public user and the scientific community. The CNPC, for the first time, provides integrated and authoritative taxonomic information on higher plant species found in China, and this database will be permanent, free and continously updated. Presently, a total of 34377 species have been included in the database. Among of them, 16620 species are only found in China. Taxa are classified into 432 families and 3941 genera. The CNPC will be an important source for scientists working on Chinese flora, and will play an important role in helping to achieve the targets set under the Global Strategy for Plant Conservation in the future.     

Responses of Chrysanthemum Cells to Mechanical Stimulation Require Intact Microtubules and Plasma Membrane–Cell Wall Adhesion

Plant cells are highly susceptible and receptive to physical factors, both in nature and under experimental conditions. Exposure to mechanical forces dramatically results in morphological and microstructural alterations in their growth. In the present study, cells from chrysanthemum (Dendranthema morifolium) were subjected to constant pressure from an agarose matrix, which surrounded and immobilized the cells to form a cell-gel block. Cells in the mechanically loaded blocks elongated and divided, with an axis preferentially perpendicular to the direction of principal stress vectors. After a sucrose-induced plasmolysis, application of peptides containing an RGD motif, which interferes with plasma membrane-cell wall adhesion, reduced the oriented growth under stress conditions. Moreover, colchicines, but not cytochalasin B, abolished the effects of mechanical stress on cell morphology. Cellulose staining revealed that mechanical force reinforces the architecture of cell walls and application of mechanical force, and RGD peptides caused aggregative staining on the surface of plasmolyzed protoplasts. These results provide evidence that the oriented cell growth in response to compressive stress requires the maintenance of plasmalemma-cell wall adhesion and intact microtubules. Stress-triggered wall development in individual plant cells was also demonstrated.     

Comparison of Plasma Membrane H^＋-ATPase Activity in Two Ecotypes of Reed （Phragmites communis） Leaves from Different Habitats

Plasma membrane (PM) vesicles of the leaves of two ecotypes of reed (Phragrnites communis Trin.), swamp reed (SR) and heavy salt meadow reed (HSMR) growing in the desert region of Northwest China, were purified by two-phase partitioning and the properties of their PM H^ -ATPases (EC 3.6.1.35) were compared. The specific activity of this enzyme was greater in HSMR than in SR and the Km lower (1.27mmol/L in SR and 0.30mmol/L in HSMR), and the Vmax of ATP hydrolysis activity showed no significant difference between the two ecotypes. The PM H^ -ATPase was more sensitive to denaturing temperatures in HSMR than in SR, and the pH profile also showed a slight difference, suggesting that the catalytic mechanism of this enzyme was different in HSMR compared with that in SR. The p-nitrophenyl phosphate (PNPP) hydrolysis activity of H^ -ATPase was higher in HSMR than in SR at low concentrations of PNPP, but showed no difference at high PNPP concentration. The Km for PNPP hydrolysis was 3.61mmol/L and 1.92mmol/L in SR and HSMR, respectively. And the Vmax of PNPP hydrolysis showed no significant difference in the two reed ecotypes. An experiment with the inhibitor vanadate showed that the catalytic mechanisms of the phosphatase domain of the ATPase were different in the two ecotypes. The data obtained following trypsin treatment showed a difference in the enzyme activity pattern, suggesting that there existed a possible change in the C-terminus of the ATPase, either in the structure or in the property or both of them. In addition, compared with SR, the ATP-dependent H^ pumping activity of ATPase and the coupling between proton transport and ATP hydrolysis in HSMR were increased. These results indicated that the properties of PM H^ -ATPase were changed in HSMR with compared to those in SR, which might include enzyme modifications and different isoforms expressed. The alterations of the properties of this enzyme might be an adaptive response to the habitat.