Reconstitution of clathrin-coated pit budding from plasma membranes

Receptor-mediated endocytosis begins with the binding of ligand to receptors in clathrin-coated pits followed by the budding of the pits away from the membrane. We have successfully reconstituted this sequence in vitro. Highly purified plasma membranes labeled with gold were obtained by incubating cells in the presence of anti-LDL receptor IgG-gold at 4 degrees C, attaching the labeled cells to a poly-L-lysine-coated substratum at 4 degrees C and then gently sonicating them to remove everything except the adherent membrane. Initially the gold label was clustered over flat, clathrin-coated pits. After these membranes were warmed to 37 degrees C for 5-10 min in the presence of buffer that contained cytosol extract, Ca2+, and ATP, the coated pits rounded up and budded from the membrane, leaving behind a membrane that was devoid of LDL gold. Simultaneous with the loss of the ligand, the clathrin triskelion and the AP-2 subunits of the coated pit were also lost. These results suggest that the budding of a coated pit to form a coated vesicle occurs in two steps: (a) the spontaneous rounding of the flat lattice into a highly invaginated coated pit at 37 degrees C; (b) the ATP, 150 microM Ca2+, and cytosolic factors(s) dependent fusion of the adjoining membrane segments at the neck of the invaginated pit.     

Inefficient signalase cleavage promotes efficient nucleocapsid incorporation into budding flavivirus membranes

The mechanism for efficient nucleocapsid (NC) uptake into flavivirus particles which form by budding through the membranes of the endoplasmic reticulum (ER) was investigated by using Murray Valley encephalitis virus as a model. Budding of flavivirus membranes is driven by the viral transmembrane proteins prM and E independently of NC interaction. We show that control of signalase cleavage of the multimembrane-spanning flavivirus polyprotein by the catalytic function of the viral protease is critical for efficient virus morphogenesis. In wild-type virus, signalase cleavage of prM remains inefficient until cleavage of capsid at the cytosolic side of the signal sequence separating the two proteins has occurred. This obligatory sequence of cleavages was uncoupled in a mutant virus with the consequence of greatly reduced incorporation of NC into budding membranes and augmented release of NC-free virus-like particles. Efficient signalase cleavage of prM in the mutant virus resulted in partial inhibition of cleavage of capsid by the viral NS2B-3 protease. Our results support a model for flavivirus morphogenesis involving temporal and spatial coordination of NC assembly and envelopment by regulated cleavages of an ER membrane-spanning capsid-prM intermediate.     

Entropy-driven instability and rupture of fluid membranes.

A computer simulation is used to investigate hole formation in a model membrane. The model parameters are the stress applied to the membrane, and the edge energy per unit length along the hole boundary (edge tension). Even at zero stress, the membrane has an entropically driven instability against hole formation. Within the model, the minimum edge tension required for the stability of a typical biological membrane is in the region of 1 x 10(-11) J/m, which is similar to the edge tension obtained in many measurements of biomembranes. At the zero-stress instability threshold, the hole shape is the same as a self-avoiding ring, but under compression, the hole shape assumes a branched polymer form. In the presence of large holes at zero stress, the membrane itself behaves like a branched polymer. The boundaries of the phase diagram for membrane stability are obtained, and general features of the rate of membrane rupture under stress are investigated. A model in which the entropy of hole formation is proportional to the hole perimeter is used to interpret the simulation results at small stress near the instability threshold.     

Coordination of Kinesin motors pulling on fluid membranes

Intracellular transport relies on the action of motor proteins, which work collectively to either carry small vesicles or pull membranes tubes along cytoskeletal filaments. Although the individual properties of kinesin-1 motors have been extensively studied, little is known on how several motors coordinate their action and spatially organize on the microtubule when pulling on fluid membranes. Here we address these questions by studying, both experimentally and numerically, the growth of membrane tubes pulled by molecular motors. Our in vitro setup allows us to simultaneously control the parameters monitoring tube growth and measure its characteristics. We perform numerical simulations of membrane tube growth, using the experimentally measured values of all parameters, and analyze the growth properties of the tube considering various motor cooperation schemes. The comparison of the numerical results and the experimental data shows that motors use simultaneously several protofilaments of a microtubule to pull a single tube, as motors moving along a single protofilament cannot generate the forces required for tube extraction. In our experimental conditions, we estimate the average number of motors pulling the tube to be approximately nine, distributed over three contiguous protofilaments. Our results also indicate that the motors pulling the tube do not step synchronously.     

Paramyxovirus budding

Our knowledge about envelope virus budding has been dramatically increased, since L-domain motifs were identified within their matrix and retroviral Gag proteins which drive virus budding. These viral proteins have been shown to interact with host cellular proteins involved in endocytosis and/or multi-vesicular body (MVB) sorting via their L-domains. Since budding of many enveloped viruses have been reported to be dependent on the activity of cellular Vps4, which catalyzes the disassembly of ESCRT machinery in the final step of protein sorting, this cellular function is believed to be utilized for efficient virus budding. However, for many enveloped viruses, L-domain motifs have not yet been identified, and the involvement of MVB sorting machinery in virus budding is still unknown. In this review, we will focus on paramyxoviruses among such viruses, and discuss their budding with the latest information.     

Aspirin inhibits formation of cholesterol rafts in fluid lipid membranes

Aspirin and other non-steroidal anti-inflammatory drugs have a high affinity for phospholipid membranes, altering their structure and biophysical properties. Aspirin has been shown to partition into the lipid head groups, thereby increasing membrane fluidity. Cholesterol is another well known mediator of membrane fluidity, in turn increasing membrane stiffness. As well, cholesterol is believed to distribute unevenly within lipid membranes leading to the formation of lipid rafts or plaques. In many studies, aspirin has increased positive outcomes for patients with high cholesterol. We are interested if these effects may be, at least partially, the result of a non-specific interaction between aspirin and cholesterol in lipid membranes.We have studied the effect of aspirin on the organization of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) membranes containing cholesterol. Through Langmuir–Blodgett experiments we show that aspirin increases the area per lipid and decreases compressibility at 32.5 mol% cholesterol, leading to a significant increase of fluidity of the membranes. Differential scanning calorimetry provides evidence for the formation of meta-stable structures in the presence of aspirin. The molecular organization of lipids, cholesterol and aspirin was studied using neutron diffraction. While the formation of rafts has been reported in binary DPPC/cholesterol membranes, aspirin was found to locally disrupt membrane organization and lead to the frustration of raft formation. Our results suggest that aspirin is able to directly oppose the formation of cholesterol structures through non-specific interactions with lipid membranes.     

Rotary movements and fluid membranes in termite flagellates.

We previously described a remarkable type of cell motility that provided direct, visual evidence for the fluid nature of cell membranes. The movement involved continual, unidirectional rotation of one part of a protozoan, including the plasma membrane and cytoplasmic organelles, in relation to a neighbouring part. The cell membrane in the 'shear zone' appeared continuous with that of the rest of the cell. The rotary motor consisted, at least in part, of a non-contractile, microtubular axostyle which extended centrally through the cell. The protozoan was a devescovinid flagellate found in the hindgut of a Florida termite. In this paper, we have confirmed earlier reports of this type of motility in other kinds of devescovinids from Australian termites. By demonstrating continuity of the plasma membrane in the shear zone of the Australian devescovinids as well, we have obtained additional examples that provide direct, visual evidence for fluid membranes. A comparative analysis of rotational motility in various devescovinids revealed 2 different kinds of rotary mechanisms. Hyperdevescovina probably have an internal motor, in which one part of the cell exerts forces against another part, as in the Florida termite devescovinid. Devescovina species, on the other hand, have an external motor, in which flagellar and/or papillar movements exert forces against the surrounding medium. The structure of the axostyle in different devescovinids was compared, and its role in rotational motility discussed with respect to the behavioural data.     

Impact of a model synovial fluid on supported lipid membranes

The interaction of a model synovial fluid, here a solution of 3mg/mL hyaluronic acid (HA) in heavy water (D(2)O), with an oligolamellar stack of lipid (DMPC) membranes on silicon support has been studied by neutron reflectometry and infrared spectroscopy on the molecular scale at non-physiological and physiological conditions. The system under investigation represents a simple model for lipid-coated mammalian joints and other artificial implant surfaces. When exposed to pure D(2)O at 21°C, i.e. below the main phase transition of the system, the lipid membranes show a lamellar spacing of 65?. Heating to 26°C results in detachment of all lipid bilayers except for the innermost lipid lamella directly adsorbed to the surface of the silicon support. On the contrary, when incubated in the solution of HA in D(2)O the oligolamellar lipid system starts swelling. In addition, heating to 39°C does not result in loss of the lipid membranes into the liquid phase. The interfacial lipid coating adopts a new stable lamellar state with an increase in d-spacing by 380% to 247? measured after 43 days of incubation with the model synovial fluid. Potential consequences for joint lubrication and protective wear functionality are considered.     

Effect of cholesterol on the lateral nanoscale dynamics of fluid membranes

Inelastic neutron scattering was used to study the effect of 5 and 40?mol% cholesterol on the lateral nanoscale dynamics of phospholipid membranes. By measuring the excitation spectrum at several lateral q _|| values (up to q _||?=?3 ?^?1), complete dispersion curves were determined of gel, fluid and liquid-ordered phase bilayers. The inclusion of cholesterol had a distinct effect on the collective dynamics of the bilayer’s hydrocarbon chains; specifically, we observed a pronounced stiffening of the membranes on the nanometer length scale in both gel and fluid bilayers, even though they were experiencing a higher degree of molecular disorder. Also, for the first time we determined the nanoscale dynamics in the high-cholesterol liquid-ordered phase of bilayers containing cholesterol. Namely, this phase appears to be “softer” than fluid bilayers, but better ordered than bilayers in the gel phase.     

Oxygen distribution in the fluid/gel phases of lipid membranes

The interactions between oxygen and lipid membranes play fundamental roles in basic biological processes (e.g., cellular respiration). Obviously, membrane oxidation is expected to be critically dependent on the distribution and concentration of oxygen in the membrane. Here, we combined theoretical and experimental methods to investigate oxygen partition and distribution in lipid membranes of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in a temperature range between 298 and 323 K, specifically focusing on the changes caused by the lipid phase and phase transition. Even though oxygen is known to be more concentrated in the center of fluid phase membranes than on the headgroup regions, the distribution profile of oxygen inside gel-phase bilayers remained to be determined. Molecular dynamics simulations now show that the distribution of oxygen inside DPPC bilayers dramatically changes upon crossing the main transition temperature, with oxygen being nearly depleted halfway from the headgroups to the membrane center below the transition temperature. In a parallel approach, singlet oxygen luminescence emission measurements employing the photosensitizer Pheophorbide-a (Pheo) confirmed the differences in oxygen distribution and concentration profiles between gel- and fluid-phase membranes, revealing changes in the microenvironment of the embedded photosensitizer. Our results also reveal that excited triplet state lifetime, as it can be determined from the singlet oxygen luminescence kinetics, is a useful probe to assess oxygen distribution in lipid membranes with distinct lipid compositions.     

Proteolytic enzymes in human fetal membranes and amniotic fluid. A comparison of normal and premature ruptured membranes

The amniotic and chorionic membranes obtained at term and term amniotic fluid contain a soluble protease activity which cleaves [14C]-labeled globin at acid pH. In contrast, a salt extract of the pellet fraction obtained from the fetal membranes displays only negligible protease activities at the pH range of 4-8. Specific activities of the proteases in the soluble and salt-extractable fractions of fetal membranes which were intact before onset of labor were not significantly different from the respective activities in cases of premature rupture of fetal membranes (PROM). However, the protease activity of the amniotic fluid was found to increase with advancing gestational age and to reach maximal activity at term. A heat-sensitive and nondializable protease inhibitory activity was found in term amniotic fluid. This inhibitory activity acted on the cytosolic protease of amniotic membranes from control and PROM cases, but not on the soluble protease of chorionic membranes, and had a similar potency in fluids from PROM cases or fluids collected at term. These results do not support a role for fetal membrane proteases, amniotic fluid proteases, or amniotic fluid protease inhibitory activities in the etiology of PROM. However, the observed changes in amniotic fluid protease activity with fetal age suggest a physiological role for the enzyme in normal fetal development.     

Purification and characterization of lutropin receptor from membranes of pig follicular fluid

Membranes derived from free floating granulosa cells in porcine ovarian follicular fluid were used as a starting material for structural characterization of both LH/hCG and FSH receptors. The receptors were highly hormone-specific and showed single classes of high-affinity binding sites (Kd = 19-74 pM). Their molecular weights as determined by affinity cross-linking with their respective 125I-ligands were similarly 70,000. The membrane-localized receptors could be solubilized with reduced Triton X-100 in the presence of 20% glycerol with good retention of hormone binding activity. The Triton extracts of membranes also showed hormone specificity and equilibrium binding constants similar to the membrane receptors (Kd = 32-48 pM). Affinity chromatography on divinylsulfonyl-Sepharose-oLH columns was utilized to purify the solubilized LH/hCG receptor to a specific activity of 2000 pmol/mg of protein. The purified receptor exhibited a high specificity for hCG and hLH but not for hFSH nor bTSH. The purified receptor was iodinated and visualized to be composed of a major protein of Mr approximately 70,000 and other minor proteins of molecular weights ranging from 14,000 to 40,000. Except for the Mr 14,000 protein, all other protein species bound to the concanavalin A-Sepharose column. The data suggest that the ovarian LH/hCG and FSH receptors are structurally similar and consist of a single polypeptide chain, as recently documented for the LH/hCG receptor (Loosefelt et al., 1989; McFarland et al., 1989).     

HIV Pol inhibits HIV budding and mediates the severe budding defect of Gag-Pol

The prevailing hypothesis of HIV budding posits that the viral Gag protein drives budding, and that the Gag p6 peptide plays an essential role by recruiting host-cell budding factors to sites of HIV assembly. HIV also expresses a second Gag protein, p160 Gag-Pol, which lacks p6 and fails to bud from cells, consistent with the prevailing hypothesis of HIV budding. However, we show here that the severe budding defect of Gag-Pol is not caused by the absence of p6, but rather, by the presence of Pol. Specifically, we show that (i) the budding defect of Gag-Pol is unaffected by loss of HIV protease activity and is therefore an intrinsic property of the Gag-Pol polyprotein, (ii) the N-terminal 433 amino acids of Gag and Gag-Pol are sufficient to drive virus budding even though they lack p6, (iii) the severe budding defect of Gag-Pol is caused by a dominant, cis-acting inhibitor of budding in the HIV Pol domain, and (iv) Gag-Pol inhibits Gag and virus budding in trans, even at normal levels of Gag and Gag-Pol expression. These and other data support an alternative hypothesis of HIV budding as a process that is mediated by the normal, non-viral pathway of exosome/microvesicle biogenesis.     

Function curve of the membranes that regulate amniotic fluid volume in sheep

Seven singleton 120-day fetal lambs were prepared with a shunt from the lung to the gastric end of the esophagus, a bladder catheter, and multiple amniotic fluid and vascular catheters. The urachus was ligated. Beginning 7 days later, amniotic fluid volumes were determined by drainage, followed by replacement with 1 liter of lactated Ringer (LR) solution. Urine flow into the amnion was measured continuously. In 14 of 27 experiments, amniotic fluid volumes were determined again 2 days after the inflow into the amnion had consisted of urine only and in 13 experiments after the inflow of urine had been supplemented by an intraamniotic infusion of LR solution. Intramembranous absorption was calculated from the inflows and the changes in volume between the beginning and end of each experiment. The relations between absorption rate and amniotic fluid volume, the "function curves," were highly individual. Urine production during the infusion of LR solution did not decrease, fetal plasma renin activity decreased (P < 0.001), and amniotic fluid volume increased by 140% [SE (27%), P < 0.005], but the increase in the amniochorionic absorption rate of 411% [SE (48%), P < 0.001] was greater (P < 0.005) than the increase in volume. Each of the seven fetuses was proven capable of an average intramembranous absorption rate that exceeded 4.5 liters of amniotic fluid per day. During the infusion of LR solution, the increase in the rate of absorption matched the rate of infusion (both in ml/h), with a regression coefficient of 0.75 (P < 0.001). Thus, even for large amniotic fluid volumes, volume is not limited by the absorptive capacity of the amniochorion, and, at least in these preparations, the position of the function curve and not the natural rate of inflow was the major determinant of resting amniotic fluid volume.     

Lipid arrangement in fluid model membranes: analysis by cross-linking of phosphatidylethanolamines

Binary mixtures of fluid phase phosphatidylethanolamines at pH 10 were treated with the bifunctional cross-linking reagent dimethylsuberimidate. Analysis of the dimeric species formed demonstrated that the phospholipid species in dimyristoylphosphatidylethanolamine/dielaidoylphosphatidylethanolamine mixtures at 52 degrees C and dielaidoylphosphatidylethanolamine/dilauroylphosphatidylethanolamine mixtures at 41 degrees C were randomly arranged. Analysis of the dimeric species formed in dipalmitoylphosphatidylethanolamine/dioleoylphosphatidylethanolamine mixtures at 68 degrees C showed that this mixture was very close to being randomly arranged, with just a slight propensity of like phospholipid species to cluster.