Vesicle-associated membrane protein 2 mediates trafficking of α5β1 integrin to the plasma membrane

Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of α5β1 integrin. VAMP2 was present on vesicles containing endocytosed β1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cell surface α5β1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of α5β1, without altering cell surface expression of α2β1 integrin or α3β1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of α5β1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.     

Regulation of β-catenin trafficking to the membrane in living cells

β-catenin is a key mediator of the Wnt signaling process and accumulates in the nucleus and at the membrane in response to Wnt-mediated inhibition of GSK-3β. In this study we used live cell photobleaching experiments to determine the dynamics and rate of recruitment of β-catenin at membrane adherens junctions (cell adhesion) and membrane ruffles (cell migration). First, we confirmed the nuclear-cytoplasmic shuttling of GFP-tagged β-catenin, and found that a small mobile pool of β-catenin can move from the nucleus to membrane ruffles in NIH 3T3 fibroblasts with a t_0.5 of ~ 30 s. Thus, β-catenin can shuttle between the nucleus and plasma membrane. The localized recruitment of β-catenin-GFP to membrane ruffles was more rapid, and the strong recovery observed after bleaching (mobile fraction 53%, t_0.5 ~5 s) is indicative of high turnover and transient association. In contrast, β-catenin-GFP displayed poor recovery at adherens junctions in MDCK epithelial cells (mobile fraction 10%, t_0.5 ~8 s), indicating stable retention at these membrane structures. We previously identified IQGAP1 as an upstream regulator of β-catenin at the membrane, and this is supported by photobleaching assays which now reveal IQGAP1 to be more stably anchored at membrane ruffles than β-catenin. Further analysis showed that LiCl-mediated inactivation of the kinase GSK-3β increased β-catenin membrane ruffle staining; this correlated with a faster rate of recruitment and not increased membrane retention of β-catenin. In summary, β-catenin displays a high turnover rate at membrane ruffles consistent with its dynamic internalization and recycling at these sites by macropinocytosis.     

Increase of β2-integrin on adhesion of THP-1 cells to collagen vitrigel membrane

When human monocyte-derived leukemia (THP-1) cells, which are floating cells, are stimulated with lipid peroxides, or Streptococcus suis, these cells adhere to a plastic plate or endothelial cells. However, it is unclear whether or not non-stimulated THP-1 cells adhere to collagen vitrigel membrane (CVM). In this study, firstly, we investigated the rate of adhesion of THP-1 cells to CVM. When THP-1 cells were not stimulated, the rate of adhesion to CVM was high. Then, to identify adhesion molecules involved in adhesion of THP-1 cells to CVM, expressions of various cell adhesion molecules on the surface of THP-1 cells adhering to CVM were measured. β-actin, β-catenin, and β1-integrin expressions did not change in non-stimulated THP-1 cells cultured on CVM compared with those in cells cultured in a flask, but β2-integrin expression markedly increased.     

Initial steps of αand β-d-glucose binding to intact red cell membrane

Summary The kinetics of the initial phases of d-glucose binding to the glucose transport protein (GLUT1) of the human red cell can be followed by stopped-flow measurements of the time course of tryptophan (trp) fluorescence enhancement. A number of control experiments have shown that the trp fluorescence kinetics are the result of conformational changes in GLUT1. One shows that nontransportable l-glucose has no kinetic response, in contrast to d-glucose kinetics. Other controls show that d-glucose binding is inhibited by cytochalasin B and by extracellular d-maltose. A typical time course for a transportable sugar, such as d-glucose, consists of a zero-time displacement, too fast for us to measure, followed by three rapid reactions whose exponential time courses have rate constants of0.5–100 sec^+–1 at 20°C. It is suggested that the zero-time displacement represents the initial bimolecular ligand/GLUT1 association. Exponential 1 appears to be located at, or near, the external membrane face where it is involved in discriminating among the sugars. Exponential 3 is apparently controlled by events at the cytosolic face. Trp kinetics distinguish the K _d of the epimer, d-galactose, from the K _dfor d-glucose, with results in agreement with determinations by other methods. Trp kinetics distinguish between the binding of the - and -d-glucose anomers. The exponential 1 activation energy of the -anomer, 13.6 ± 1.4 kcal mol^+–1, is less than that of -d-glucose, 18.4 ± 0.8 kcal mol^+–1, and the two Arrhenius lines cross at 23.5°C. The temperature dependence of the kinetic response following -d-glucose binding illustrates the interplay among the exponentials and the increasing dominance of exponential 2 as the temperature increases from 22.3 to 36.6°C. The existence of these interrelations means that previously acceptable approximations in simplified reaction schemes for sugar transport will now have to be justified on a point-to-point basis.We should like to express our thanks to Michael R. Toon for his important contributions. This work was supported in part by a grant-in-aid from the American Heart Association, by the Squibb Institute for Medical Research and by The Council for Tobacco Research.     

Biophysics of α-synuclein membrane interactions

Membrane proteins participate in nearly all cellular processes; however, because of experimental limitations, their characterization lags far behind that of soluble proteins. Peripheral membrane proteins are particularly challenging to study because of their inherent propensity to adopt multiple and/or transient conformations in solution and upon membrane association. In this review, we summarize useful biophysical techniques for the study of peripheral membrane proteins and their application in the characterization of the membrane interactions of the natively unfolded and Parkinson's disease (PD) related protein, α-synuclein (α-syn). We give particular focus to studies that have led to the current understanding of membrane-bound α-syn structure and the elucidation of specific membrane properties that affect α-syn-membrane binding. Finally, we discuss biophysical evidence supporting a key role for membranes and α-syn in PD pathogenesis. This article is part of a Special Issue entitled: Membrane protein structure and function.     

How do Bax and Bak lead to permeabilization of the outer mitochondrial membrane?

Bcl-2 family members, like the structurally similar translocation domain of diphtheria toxin, can form ion-selective channels and larger-diameter pores in artificial lipid bilayers. Recent studies show how Bcl-2 family members change topology in membranes during apoptosis and that these different states may either promote or inhibit apoptosis. Binding of BH3-only proteins alters the subcellular localization and/or membrane topology and probably affects the channel formation of Bcl-2, Bcl-xL and Bcl-w. However, it remains unclear how the pore-forming activity functions in cells to regulate mitochondrial membrane permeabilization and cell death. Bcl-2 family members in flies and worms regulate apoptosis by mechanisms seemingly unrelated to membrane permeabilization, leaving a unifying model for the biochemical activity of this protein family unknown. Work linking Bcl-2 family members to mitochondrial morphogenesis in worms and mammals suggests some common functions of Bcl-2 family proteins may exist.     

Adsorption of phage λ to Salmonella typhimurium lamB + requires the presence of lipopolysaccharide in the outer membrane

Summary Two S. typhimurium strains TA1534 (rfa ⁺) and TA1538 (rfaE) were transformed with the lamB expression plasmid pAMH70. Transposition events with placMu55 hybrid phage were successful only with TA1534/pAMH70 strain. Using SDS-PAGE, the LamB protein was present in the total cell proteins but not in the outer membrane proteins of the TA1538/pAMH70 strain. The LamB protein must linked to the LPS of the outer membrane to allow adsorption of phage in S. typhimurium.     

The major basement membrane components localize to the chondrocyte pericellular matrix--a cartilage basement membrane equivalent?

In this study, we demonstrate that articular cartilage chondrocytes are surrounded by the defining basement membrane proteins laminin, collagen type IV, nidogen and perlecan, and suggest that these form the functional equivalent of a basement membrane. We found by real-time PCR that mouse chondrocytes express these four cardinal components of basement membranes and demonstrated by immunohistochemistry that the proteins are present in bovine and mouse cartilage tissues and are deposited in a thin pericellular structure. Immunoelectron microscopy confirmed high laminin concentration in the pericellular matrix. In cartilage from newborn mice, basement membrane components are widespread in the territorial and interterritorial matrix, while in mature cartilage of adult mice the basement membrane components are localized mainly to a narrow pericellular zone. With progression into old age, this layer becomes less distinct, especially in areas of obvious mechanical attrition. Interestingly, individual laminin subunits were located in different zones of the cartilage, with laminin alpha1 showing preferential localization around a select population of superficial layer chondrocytes. We propose that the chondrocyte, like several other cell types of mesenchymal origin, is surrounded by the functional equivalent of a basement membrane. This structure is presumably involved in maintaining chondrocyte phenotype and viability and may well allow a new understanding of cartilage development and provide clues to the progression of degenerative joint disorders.     

Biogenesis of β-barrel integral proteins of bacterial outer membrane

Gram-negative bacteria are enveloped by two membranes, the inner (cytoplasmic) (CM) and the outer (OM). The majority of integral outer membrane proteins are arranged in β-barrels of cylindrical shape composed of amphipathic antiparallel β-strands. In bacteria, β-barrel proteins function as water-filled pores, active transporters, enzymes, receptors, and structural proteins. Proteins of bacterial OM are synthesized in the cytoplasm as unfolded polypeptides with an N-terminal sequence that marks them for transport across the CM. Precursors of membrane proteins move through the aqueous medium of the cytosol and periplasm under the protection of chaperones (SecB, Skp, SurA, and DegP), then cross the CM via the Sec system composed of a polypeptide-conducting channel (SecYEG) and ATPase (SecA), the latter providing the energy for the translocation of the pre-protein. Pre-protein folding and incorporation in the OM require the participation of the Bam-complex, probably without the use of energy. This review summarizes current data on the biogenesis of the β-barrel proteins of bacterial OM. Data on the structure of the proteins included in the multicomponent system for delivery of the OM proteins to their destination in the cell and on their complexes with partners, including pre-proteins, are pre-sented. Molecular models constructed on the basis of structural, genetic, and biochemical studies that describe the mechanisms of β-barrel protein assembly by this molecular transport machinery are also considered.     

Peritrophic membrane contribution to Bt Cry δ-endotoxin susceptibility in Lepidoptera and the effect of Calcofluor

In an effort to determine a reason for differing susceptibilities to Bacillus thuringiensis (Bt) Cry δ-endotoxins amongst lepidopteran species, the peritrophic membrane (PM), and a number of agents that target the PM, were investigated to determine their effect on the efficacy of Bt toxins. In particular Calcofluor is able to disrupt the PM that acts as a barrier to microorganisms. Although Bt toxins have been shown to traverse the PM in some lepidopteran species, new data shows that toxins can bind the PM. Lepidopteran larval PMs also vary in thickness and composition that may determine the passage of Bt toxins. In non-susceptible insects the toxin associates with PM proteins and frass and is thought to be retained by the PM and then excreted before binding to the exposed target midgut membrane. However, the addition of Calcofluor to Bt toxins at an LC₅₀ for the recipient species did not result in an increase in the efficacy of the toxin. It is evident that Calcofluor does disrupt the PM but the toxin preferentially binds PM fragments and is excreted instead of binding the exposed target midgut membrane, therefore having little toxic effect. This study therefore concludes that Calcofluor is not as suitable as other toxin enhancing agents such as chitinase.     

The membrane binding kinetics of full-length PKCα is determined by membrane lipid composition

Protein kinase Cα (PKCα) is activated by its translocation to the membrane. Activity assays show the importance of PIP(2) in determining the specific activity of this enzyme. A FRET stopped flow fluorescence study was carried out to monitor the rapid kinetics of protein binding to model membranes containing POPC/POPS/DOG and eventually PIP(2). The results best fitted a binding mechanism in which protein bound to the membrane following a two-phase mechanism with a first bimolecular reaction followed by a slow unimolecular reaction. In the absence of PIP(2), the rapid protein binding rate was especially dependent on POPS concentration. Formation of the slow high affinity complex during the second phase seems to involve specific interactions with POPS and DOG since it is only sensitive to changes within relatively low concentration ranges of these lipids. Both the association and dissociation rate constants fell in the presence of PIP(2). We propose a model in which PKCα binds to the membranes via a two-step mechanism consisting of the rapid membrane initial recruitment of PKCα driven by interactions with POPS and/or PIP(2) although interactions with DOG are involved too. PKCα searches on the lipid bilayer in two dimensions to establish interactions with its specific ligands.     

Nonuniform lateral distribution of transmembrane ΔpH in a coupling membrane as related to its curvature

The possibility is analyzed that the pH of the water space localized inside the invagination of a membrane can differ from the pH of the external bulk buffer outside the invagination. The proton flow responsible for decreased pH values inside mitochondrial cristae and membrane invaginations of cyanobacteria has been calculated. If deltapsi (electric potential difference) inside and outside the invaginations is the same, there may exist a lateral microheterogeneity of transmembrane deltapH, and hence deltamu-H+. It seems that the invagination is a kind of buffer for accumulating deltamu-H+ in membrane systems. In eutrified waters (pH > 9) and also under the conditions of a sudden decrease or increase of light, or of a respiratory substrate of O2, ATP synthesis should proceed in the invaginated rather than in the flat regions of a membrane.     

Does a lateral gradient of membrane potential on the plasma membrane of growing pollen tube of germinating pollen grain exist?

The data presented in the article by Breigina et al. (2009) "Changes in the membrane potential during pollen grain germination and pollen tube growth" (Tsitologiya. 51 (10): 815-823) and concerning the measurement of electric membrane potential (Delta Psi) on the plasma membrane of growing pollen tube of germinating pollen grain with the use of fluorescent potential-sensitive dye, di-4-ANEPPS, were critically analyzed in order to clarify whether a lateral gradient of Delta Psi on this membrane indeed exists. This analysis showed that the main conclusion of the authors of the above article on the existence of polar distribution of Delta Psi along the pollen tube plasma membrane is not in accordance with a number of known peculiarities of di-4-ANEPPS behavior in biological membranes and requires a significant revision. The findings in question reported by the authors, in my opinion, might be interpreted as evidence for the presence on the plasma membrane of growing pollen tube not only the membrane potential Delta Psi but also lateral gradient of so called intra-membrane dipole potential. Based on the comments made, another interpretation of the experimental results described by Breigina et al. has been offered. In addition, some drawbacks in the methodology used by the authors for measurement of Delta Psi with other fluorescent potential-sensitive dye, DiBAC3(3), are also shortly considered.     

l-Carnitine is essential to β-oxidation of quarried fatty acid from mitochondrial membrane by PLA2

Mitochondrial β-oxidation is an important system involved in the energy production of various cells. In this system, the function of l-carnitine is essential for the uptake of fatty acids to mitochondria. However, it is unclear whether or not endogenous respiration, ADP-induced O₂ consumption without substrates, is caused by l-carnitine treatment. In this study, we investigated whether l-carnitine is essential to the β-oxidation of quarried fatty acids from the mitochondrial membrane by phospholipase A₂ (PLA₂) using isolated mitochondria from the liver of rats. Intact mitochondria were incubated in a medium containing Pi, CoA and l-carnitine. The effect of l-carnitine treatment on ADP-induced mitochondrial respiration was observed without exogenous respiratory substrate. Increase in mitochondrial respiration was induced by treatment with l-carnitine in a concentration-dependent manner. Treatment with rotenone, a complex I blocker, completely inhibited ADP-induced oxygen consumption even in the presence of l-carnitine. Moreover, the l-carnitine dependent ADP-induced mitochondrial oxygen consumption did not increase when PLA₂ inhibitors were treated before ADP treatment. The l-carnitine-dependent ADP-induced oxygen consumption did contribute to ATP productions but not heat generation via an uncoupling system. These results suggest that l-carnitine might be essential to the β-oxidation of quarried fatty acids from the mitochondrial membrane by PLA₂.     

Reduced sensitivity of Niemann-Pick C1-deficient cells to θ-toxin (perfringolysin O): sequestration of toxin to raft-enriched membrane vesicles

-Toxin (perfringolysin O) binds to cell surface cholesterol and forms oligomeric pores that cause membrane damage. Both in cytotoxicity and cell survival assays, a mutant Chinese hamster ovary cell line NPC1(–) that lacked Niemann-Pick C1 showed reduced sensitivity to -toxin, compared with wild-type (wt) cells. BC is a derivative of -toxin that retains cholesterol-binding activity but lacks cytotoxicity. Confocal and electron microscopy revealed the presence of multiple vesicles which bound BC, both on the cell surface and in the extracellular space of these cells. BC binding to raft microdomains was verified by its resistance to 1% Triton X-100 at 4°C and recovery of bound BC in floating low-density fractions on sucrose density gradient fractionation. BC-labeled vesicles were abolished when NPC1(–) cells were depleted of lipoproteins and also when treated with a Rho-associated kinase inhibitor Y-27632. In addition, similar vesicles were observed in wt cells treated with progesterone. In parallel with these results, -toxin sensitivity of NPC1(–) cells was increased when cells were depleted of lipoproteins or treated with Y-27632, whereas that of wt cells was decreased by progesterone. Our findings suggest that sequestration of toxin to raft-enriched cell surface vesicles may underlie reduced sensitivity of NPC1-deficient cells to -toxin.     

Rates of chilling to 0°C: implications for the survival of microorganisms and relationship with membrane fluidity modifications

The effects of slow chilling (2°C min⁻¹) and rapid chilling (2,000°C min⁻¹) were investigated on the survival and membrane fluidity of Escherichia coli, of Bacillus subtilis, and of Saccharomyces cerevisiae. Cell death was found to be dependent on the physiological state of cell cultures and on the rate of temperature downshift. Slow temperature decrease allowed cell stabilization, whereas the rapid chilling induced an immediate loss of viability of up to more than 90 and 70% for the exponentially growing cells of E. coli and B. subtilis, respectively. To relate the results of viability with changes in membrane physical state, membrane anisotropy variation was monitored during thermal stress using the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene. No variation in the membrane fluidity of all the three microorganisms was found after the slow chilling. It is interesting to note that fluorescence measurements showed an irreversible rigidification of the membrane of exponentially growing cells of E. coli and B. subtilis after the instantaneous cold shock, which was not observed with S. cerevisiae. This irreversible effect of the rapid cold shock on the membrane correlated well with high rates of cell inactivation. Thus, membrane alteration seems to be the principal cause of the cold shock injury.     

A guideline to proteome-wide α-helical membrane protein topology predictions

For current state-of-the-art methods, the prediction of correct topology of membrane proteins has been reported to be above 80%. However, this performance has only been observed in small and possibly biased data sets obtained from protein structures or biochemical assays. Here, we test a number of topology predictors on an "unseen" set of proteins of known structure and also on four "genome-scale" data sets, including one recent large set of experimentally validated human membrane proteins with glycosylated sites. The set of glycosylated proteins is also used to examine the ability of prediction methods to separate membrane from nonmembrane proteins. The results show that methods utilizing multiple sequence alignments are overall superior to methods that do not. The best performance is obtained by TOPCONS, a consensus method that combines several of the other prediction methods. The best methods to distinguish membrane from nonmembrane proteins belong to the "Phobius" group of predictors. We further observe that the reported high accuracies in the smaller benchmark sets are not quite maintained in larger scale benchmarks. Instead, we estimate the performance of the best prediction methods for eukaryotic membrane proteins to be between 60% and 70%. The low agreement between predictions from different methods questions earlier estimates about the global properties of the membrane proteome. Finally, we suggest a pipeline to estimate these properties using a combination of the best predictors that could be applied in large-scale proteomics studies of membrane proteins.