Immuno-isolation of a plasma membrane fraction from the Fao cell.

A plasma membrane was immuno-isolated from a post-nuclear supernatant of a cultured rat hepatocyte, the Fao cell, using a cellulose immuno-adsorbent and antibodies raised against a variety of endogenous antigens of hepatocytes: 5'-nucleotidase, a plasma membrane fraction and the whole Fao cell. The antibodies which recognize antigens on the cell surface were selected from the total serum by first binding the antiserum to suspension cells. Alternatively, the plasma membrane and Fao antisera were affinity purified on a column prepared from a Triton X-114 extract of a plasma membrane fraction. The immuno-isolation was most efficient when carried out with either the plasma membrane or the Fao anti-serum. When alkaline phosphodiesterase I or 5'-nucleotidase was used as the plasma membrane marker, 40-60% of the plasma membrane of the post-nuclear supernatant was isolated representing a maximum 34-fold increase in the specific activity of the enzymes in the bound material. Using the NaB-[3H]4-labelled glycoproteins of the plasma membrane or the IgG bound to the plasma membrane as alternative markers, an 80% isolate of the plasma membrane of the post-nuclear supernatant was achieved, resulting in an estimated 40-fold purification. The non-specific binding was low despite the use of a post-nuclear supernatant as the input fraction. The characterization of the bound materials suggested that the whole plasma membrane was immuno-isolated and not a particular domain.     

Lipid Characterization of an Enriched Plasma Membrane Fraction of Dunaliella salina Grown in Media of Varying Salinity

We have developed a rapid procedure for isolating a fraction enriched in plasma membrane from Dunaliella salina using an aqueous two-phase system (dextran/polyethylene glycol, 6.7%/6.7%). An enriched plasma membrane fraction, free of chloroplast and mitochondrial contamination, could be obtained in 2.5 hours. Plasma membrane proteins, which accounted for approximately 1% of the total membrane protein, contained a number of unique proteins compared with the other cell fractions, as shown by gel electrophoresis. The lipids of the plasma membrane fraction from 1.7 molar NaCl-grown cells were extracted and characterized. Phosphatidylethanolamine and phosphatidylcholine were the two most prevalent phospholipids, at 20.6% and 6.0% of the total lipid, respectively. In addition, inositol phospholipids were a significant component of the D. salina plasma membrane fraction. Phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate accounted for 5.2% and 1.5% of the plasma membrane phospholipid, respectively. Diacylglyceryltrimethylhomoserine accounted for 7.9% of the plasma membrane total lipid. Free sterols were the major component of the plasma membrane fraction, at 55% of the total lipid, and consisted of ergosterol and 7-dehydroporiferasterol. Sterol peroxides were not present in the plasma membrane fraction. The lipid composition of enriched plasma membrane fractions from cells grown at 0.85 molar NaCl and 3.4 molar NaCl were compared with those grown at 1.7 molar NaCl. The concentration of diacylglyceryltrimethylhomoserine and the degree of plasma membrane fatty acid saturation increased in 3.4 molar plasma membranes. The relative concentration of sterols in the plasma membrane fraction was similar in all three NaCl concentrations tested.     

The spermatid plasma membrane comes of age

Spermiogenesis affords a unique opportunity to examine the formation of plasma membrane domains. Recent attempts to chart the life cycles of well-characterized integral plasma membrane proteins during spermiogenesis have suggested that spermatids are at least as adept as epithelial cells or neurons at establishing their plasma membrane domains. They appear to expand upon the standard recipe involving concurrent domain-specific protein targeting and diffusion barriers by using a combination of intracellular storage within the secretory pathway, developmentally-regulated delivery to provisional plasma membrane domains, large-scale redistributions of diffusion barriers and integral plasma membrane proteins, and the shedding of an entire plasma membrane domain.     

Phosphatidylinositol 4,5-bisphosphate functions as a second messenger that regulates cytoskeleton-plasma membrane adhesion

Binding interactions between the plasma membrane and the cytoskeleton define cell functions such as cell shape, formation of cell processes, cell movement, and endocytosis. Here we use optical tweezers tether force measurements and show that plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) acts as a second messenger that regulates the adhesion energy between the cytoskeleton and the plasma membrane. Receptor stimuli that hydrolyze PIP2 lowered adhesion energy, a process that could be mimicked by expressing PH domains that sequester PIP2 or by targeting a 5'-PIP2-phosphatase to the plasma membrane to selectively lower plasma membrane PIP2 concentration. Our study suggests that plasma membrane PIP2 controls dynamic membrane functions and cell shape by locally increasing and decreasing the adhesion between the actin-based cortical cytoskeleton and the plasma membrane.     

OBSERVATIONS ON SPERM PENETRATION IN THE RAT

The structural aspects of sperm penetration in the rat egg were investigated by electron microscopy. Eggs were recovered at intervals between 8 and 10:30 A.M. from females which had mated during the previous night. The oviducts were flushed with hyaluronidase and the eggs transferred into a 2 per cent osmium tetroxide solution, buffered at pH 7.8. After fixation, the eggs were mounted individually in agar, dehydrated in ethyl alcohol, and embedded in butyl-methyl methacrylate (3:1). The sperm penetrating the egg is covered by a plasma membrane which is present only on the side facing toward the zona pellucida; no membrane is visible on the side facing toward the vitellus. The sperm plasma membrane becomes continuous with the egg plasma membrane and forms a deep fold around the entering sperm. Cross-sections through the sperm midpiece in the perivitelline space show an intact plasma membrane. At the place of entrance, the plasma membrane of the sperm appears to fuse with the egg plasma membrane. After the sperm has penetrated the vitellus, it has no plasma membrane at all. The nuclear membrane is also absent. These observations suggest a new hypothesis for sperm penetration. After the sperm has come to lie on the plasma membrane of the egg, the egg and sperm plasma membranes rupture and then fuse with one another to form a continuous cell membrane over the egg and the outer surface of the sperm. As a result the sperm comes to lie inside the vitellus, leaving its own plasma membrane incorporated into the egg membrane at the surface of the egg.     

Modeling the effect of stretch and plasma membrane tension on Na+-K+-ATPase activity in alveolar epithelial cells

While a number of whole cell mechanical models have been proposed, few, if any, have focused on the relationship among plasma membrane tension, plasma membrane unfolding, and plasma membrane expansion and relaxation via lipid insertion. The goal of this communication is to develop such a model to better understand how plasma membrane tension, which we propose stimulates Na(+)-K(+)-ATPase activity but possibly also causes cell injury, may be generated in alveolar epithelial cells during mechanical ventilation. Assuming basic relationships between plasma membrane unfolding and tension and lipid insertion as the result of tension, we have captured plasma membrane mechanical responses observed in alveolar epithelial cells: fast deformation during fast cyclic stretch, slower, time-dependent deformation via lipid insertion during tonic stretch, and cell recovery after release from stretch. The model estimates plasma membrane tension and predicts Na(+)-K(+)-ATPase activation for a specified cell deformation time course. Model parameters were fit to plasma membrane tension, whole cell capacitance, and plasma membrane area data collected from the literature for osmotically swollen and shrunken cells. Predictions of membrane tension and stretch-stimulated Na(+)-K(+)-ATPase activity were validated with measurements from previous studies. As a proof of concept, we demonstrate experimentally that tonic stretch and consequent plasma membrane recruitment can be exploited to condition cells against subsequent cyclic stretch and hence mitigate stretch-induced responses, including stretch-induced cell death and stretch-induced modulation of Na(+)-K(+)-ATPase activity. Finally, the model was exercised to evaluate plasma membrane tension and potential Na(+)-K(+)-ATPase stimulation for an assortment of traditional and novel ventilation techniques.     

Plasma membrane stress induces relocalization of Slm proteins and activation of TORC2 to promote sphingolipid synthesis

The plasma membrane delimits the cell, and its integrity is essential for cell survival. Lipids and proteins form domains of distinct composition within the plasma membrane. How changes in plasma membrane composition are perceived, and how the abundance of lipids in the plasma membrane is regulated to balance changing needs remains largely unknown. Here, we show that the Slm1/2 paralogues and the target of rapamycin kinase complex 2 (TORC2) play a central role in this regulation. Membrane stress, induced by either inhibition of sphingolipid metabolism or by mechanically stretching the plasma membrane, redistributes Slm proteins between distinct plasma membrane domains. This increases Slm protein association with and activation of TORC2, which is restricted to the domain known as the membrane compartment containing TORC2 (MCT; ref.?). As TORC2 regulates sphingolipid metabolism, our discoveries reveal a homeostasis mechanism in which TORC2 responds to plasma membrane stress to mediate compensatory changes in cellular lipid synthesis and hence modulates the composition of the plasma membrane. The components of this pathway and their involvement in signalling after membrane stretch are evolutionarily conserved.     

Identification of homologous binding proteins in porcine and bovine gametes

The purpose of this study was to determine if sperm and oocyte proteins that mediate plasma membrane interaction during mammalian fertilization are conserved among porcine and bovine gametes. We examined homologous and heterologous sperm and zona-free oocyte interactions to determine the extent of cross-reactivity between the gametes of these two ungulate species. First, the numbers of ejaculated porcine and bovine sperm bound to the oocyte plasma membrane of intact porcine and bovine oocytes were determined in vitro. There was no significant difference between the number of porcine or bovine sperm that bound to porcine or bovine oocytes (P > 0.25). Second, individual porcine and bovine sperm plasma membrane proteins were identified by binding of homologous or heterologous oocyte plasma membrane to whole sperm plasma membrane on Western ligand blots. The relative amount of labeled oocyte plasma membrane bound to individual sperm plasma membrane proteins was analyzed by laser densitometry. Eight porcine sperm plasma membrane proteins and seven bovine sperm plasma membrane proteins were bound by both porcine and bovine oocyte plasma membrane. A significantly greater relative amount of porcine oocyte plasma membrane than bovine oocyte plasma membrane was bound to the 14- and 10-kD porcine sperm plasma membrane proteins (P < 0.001 and P < 0.01, respectively). A 27-kD bovine sperm plasma membrane protein bound proportionally more bovine oocyte plasma membrane probe than porcine oocyte plasma membrane probe (P < 0.04). These results are consistent with conservation of similar receptor ligand interactions at the gamete plasma membrane among porcine and bovine gametes.     

VDAC1 is a transplasma membrane NADH-ferricyanide reductase

Porin isoform 1 or VDAC (voltage-dependent anion-selective channel) 1 is the predominant protein in the outer mitochondrial membrane. We demonstrated previously that a plasma membrane NADH-ferricyanide reductase activity becomes up-regulated upon mitochondrial perturbation, and therefore suggested that it functions as a cellular redox sensor. VDAC1 is known to be expressed in the plasma membrane; however, its function there remained a mystery. Here we show that VDAC1, when expressed in the plasma membrane, functions as a NADH-ferricyanide reductase. VDAC1 preparations purified from both plasma membrane and mitochondria fractions exhibit NADH-ferricyanide reductase activity, which can be immunoprecipitated with poly- and monoclonal antibodies directed against VDAC(1). Transfecting cells with pl-VDAC1-GFP, which carries an N-terminal signal peptide, directs VDAC1 to the plasma membrane, as shown by confocal microscopy and FACS analysis, and significantly increases the plasma membrane NADH-ferricyanide reductase activity of the transfected cells. This novel enzymatic activity of the well known VDAC1 molecule may provide an explanation for its role in the plasma membrane. Our data suggest that a major function of VDAC1 in the plasma membrane is that of a NADH(-ferricyanide) reductase that may be involved in the maintenance of cellular redox homeostasis.     

Isolation of Plasma Membrane from Porcine Spermatozoa

A procedure is reported for isolating plasma membrane from porcine spermatozoa. Sperm were separated from gel particles and seminal plasma and subjected to low-intensity sonication that fragmented the plasma membrane, but minimized damage to the acrosome, nucleus and mitochondria. After cytoplasmic droplets and plasma membrane-depleted sperm were removed by differential centrifugation, the plasma membrane fragments were purified by flotation through a discontinuous sucrose gradient. During isolation, the effectiveness of each step was evaluated by light or electron microscopy. Plasma membrane fragments were identified using a stain specific for that membrane. Preparations were shown, by morphometry, to contain at least 70 per cent plasma membrane.     

Purification of plasma membrane from Acanthamoeba castellanii

A simple method for isolation of plasma membrane from Acanthamoeba using self-generating gradients of Percoll is described. To obtain a membrane marker, intact amoebae were radioiodinated and the distribution of the radiolabel was followed through the plasma membrane isolation procedure. The purity of isolated plasma membrane was assessed by enrichment of radiolabel, by electron microscopy, and by enzymatic assays for contaminating membranes. As judged from enrichment of radiolabel, a 37-fold purification of plasma membrane was obtained. We estimate that 80% of the total protein was from plasma membrane and 10% from membrane-associated actin.     

Integration of a two-phase partition method into proteomics research on rat liver plasma membrane proteins

To comprehensively identify proteins of the rat liver plasma membrane (PM), we have adopted a proteomics strategy that utilizes sucrose density centrifugation in conjunction with aqueous two-phase partition for plasma membrane isolation, followed by SDS-PAGE, mass spectrometry and bioinformatics. Western blot analysis showed that this method results in highly purified plasma membrane fractions, which is a key to successful plasma membrane proteomics. The PM proteins were separated by SDS-PAGE and digested with trypsin. Through nano-ESI-LC MS/MS analysis we identified 428 rat liver membrane proteins, of which 304 had a gene ontology (GO) annotation indicating a cellular component, and 204 (67%) of the latter were known integral membrane proteins or membrane-associated proteins. In addition to proteins known to be associated with the plasma membrane, several hypothetical proteins have also been identified. This study not only provides a tool to study plasma membrane proteins with low levels of contamination, but also provides a data set for proteins of high to moderate abundance in rat liver plasma membranes, thus allowing for more comprehensive characterization of membrane proteins and a better understanding of membrane dynamics.     

BIOCHEMICAL AND MORPHOLOGICAL COMPARISON OF PLASMA MEMBRANE AND MILK FAT GLOBULE MEMBRANE FROM BOVINE MAMMARY GLAND

Purified plasma membrane fractions from lactating bovine mammary glands and membranes of milk fat globules from the same source were similar in distribution and fatty acid composition of phospholipids. The sphingomyelin content of the phospholipid fraction of both membranes was higher than in these fractions from other cell components, β-carotene, a constituent characteristic of milk fat, was present in the lipid fraction of the plasma membrane. Cholesterol esters of plasma membrane were similar in fatty acid composition to those of milk fat globule membranes. Disc electrophoresis of either membrane preparation on polyacrylamide gels revealed a single major protein component characteristic of plasma membrane from other sources. Distinct morphological differences between plasma membrane and milk fat globule membranes were observed in both thin sections and in negatively stained material. Plasma membrane was vesicular in appearance while milk fat globule membranes had a platelike aspect. These observations are consistent with derivation of fat globule membrane from plasma membrane accompanied by structural rearrangement of membrane constituents.     

Hierarchical organization of the plasma membrane: Investigations by single-molecule tracking vs. fluorescence correlation spectroscopy

Single-molecule tracking and fluorescence correlation spectroscopy (FCS) applied to the plasma membrane in living cells have allowed a number of unprecedented observations, thus fostering a new basic understanding of molecular diffusion, interaction, and signal transduction in the plasma membrane. It is becoming clear that the plasma membrane is a heterogeneous entity, containing diverse structures on nano-meso-scales (2-200 nm) with a variety of lifetimes, where certain membrane molecules stay together for limited durations. Molecular interactions occur in the time-dependent inhomogeneous two-dimensional liquid of the plasma membrane, which might be a key for plasma membrane functions.     

Phenothiazines alter plasma membrane properties and sensitize cancer cells to injury by inhibiting annexin-mediated repair

Repair of damaged plasma membrane in eukaryotic cells is largely dependent on the binding of annexin repair proteins to phospholipids. Changing the biophysical properties of the plasma membrane may provide means to compromise annexin-mediated repair and sensitize cells to injury. Since, cancer cells experience heightened membrane stress and are more dependent on efficient plasma membrane repair, inhibiting repair may provide approaches to sensitize cancer cells to plasma membrane damage and cell death. Here, we show that derivatives of phenothiazines, which have widespread use in the fields of psychiatry and allergy treatment, strongly sensitize cancer cells to mechanical-, chemical-, and heat-induced injury by inhibiting annexin-mediated plasma membrane repair. Using a combination of cell biology, biophysics, and computer simulations, we show that trifluoperazine acts by thinning the membrane bilayer, making it more fragile and prone to ruptures. Secondly, it decreases annexin binding by compromising the lateral diffusion of phosphatidylserine, inhibiting the ability of annexins to curve and shape membranes, which is essential for their function in plasma membrane repair. Our results reveal a novel avenue to target cancer cells by compromising plasma membrane repair in combination with noninvasive approaches that induce membrane injuries.     

Isolation of the dorsal, ventral and intracellular domains of HeLa cell plasma membranes following adhesion to a gelatin substrate

The plasma membrane is a complex organelle responsible for many cellular functions. In addition to mediating the exchange of components with the extracellular fluid, the plasma membrane is involved in cell adhesion to matrix proteins in vivo and in vitro. In vitro, adherent cells have three distinct plasma membrane domains to carry out these functions: one attached to the substrate (ventral); another exposed to the media (dorsal); and an intracellular domain involved in endocytosis and secretion. A technique has been developed for the rapid isolation of these specific domains from HeLa cells immediately following adhesion to a gelatin substrate. The isolation procedure utilizes the tight binding of cationic colloidal silica to the dorsal plasma membrane domain of attached cells. Following silica binding and cell lysis, the silica-coated dorsal plasma membrane domain is readily separated from intracellular plasma membrane components by virtue of the high density of the silica pellicle, and the intact ventral plasma membrane domain remains attached to the gelatin substrate. Fluorescence and electron microscopy and biochemical studies using 125I-lactoperoxidase labeling, 125I-labeled wheat germ agglutinin binding, and [3H]-fucose incorporation into plasma membrane glycoproteins confirmed the separation of these three topologically distinct plasma membrane domains. The fractions isolated by the technique contained essentially all of the plasma membrane components present in intact cells. This unique membrane-isolation procedure is now being used to analyze membrane flow during plasma membrane domain formation accompanying cell adhesion to an extracellular matrix.     

The ejectisomes of the flagellate Chilomonas paramecium: visualization by freeze-fracture and isolation techniques

Freeze-fracture procedures were used to visualize ejectisomes and adjacent plasma membrane specializations in the flagellate protozoan Chilomonas paramecium. The ejectisomes are membrane-bounded, cylindrically rolled, extrusive organelles. Small ones occur in large number beneath the plasma membrane of the body and considerably larger ones are located around the gullet membrane. The intra-membrane particle distribution is different in each type. In small ejectisomes, the portion of the membrane in contact with the plasma membrane of the body has a P-face rosette of five particles while the plasma membrane has not been observed with a rosette. Small ejectisomes and plasma membrane both contain aggregations of particles a short distance from the contact or docking site. Slightly beneath the plasma membrane is the periplastic sheet with which we speculate the small ejectisomes interact during the docking phenomenon. No obvious rosettes have been observed in large ejectisomes. Some other ejectisomal structures are presented and discussed.     

Specificity of plasma membrane targeting by the rous sarcoma virus gag protein

Budding of C-type retroviruses begins when the viral Gag polyprotein is directed to the plasma membrane by an N-terminal membrane-binding (M) domain. While dispersed basic amino acids within the M domain are critical for stable membrane association and consequent particle assembly, additional residues or motifs may be required for specific plasma membrane targeting and binding. We have identified an assembly-defective Rous sarcoma virus (RSV) Gag mutant that retains significant membrane affinity despite having a deletion of the fourth alpha-helix of the M domain. Examination of the mutant protein's subcellular distribution revealed that it was not localized to the plasma membrane but instead was mistargeted to intracytoplasmic membranes. Specific plasma membrane targeting was restored by the addition of myristate plus a single basic residue, by multiple basic residues, or by the heterologous hydrophobic membrane-binding domain from the cellular Fyn protein. These results suggest that the fourth alpha-helix of the RSV M domain promotes specific targeting of Gag to the plasma membrane, either through a direct interaction with plasma membrane phospholipids or a membrane-associated cellular factor or by maintaining the conformation of Gag to expose specific plasma membrane targeting sequences.     

Purification of Plasma Membrane from Acanthamoeba castellanii1

A simple method for isolation of plasma membrane from Acanthamoeba using self-generating gradients of Percoll is described. To obtain a membrane marker, intact amoebae were radioiodinated and the distribution of the radiolabel was followed through the plasma membrane isolation procedure. The purity of isolated plasma membrane was assessed by enrichment of radiolabel, by electron microscopy, and by enzymatic assays for contaminating membranes. As judged from enrichment of radiolabel, a 37-fold purification of plasma membrane was obtained. We estimate that 80% of the total protein was from plasma membrane and 10% from membrane-associated actin.     

Role of the Plasma Membrane H+-ATPase in K+ Transport

The role of the plant plasma membrane H+-ATPase in K+ uptake was examined using red beet (Beta vulgaris L.) plasma membrane vesicles and a partially purified preparation of the red beet plasma membrane H+-ATPase reconstituted in proteoliposomes and planar bilayers. For plasma membrane vesicles, ATP-dependent K+ efflux was only partially inhibited by 100 [mu]M vanadate or 10 [mu]M carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. However, full inhibition of ATP-dependent K+ efflux by these reagents occurred when the red beet plasma membrane H+-ATPase was partially purified and reconstituted in proteoliposomes. When reconstituted in a planar bilayer membrane, the current/voltage relationship for the plasma membrane H+-ATPase showed little effect of K+ gradients imposed across the bilayer membrane. When taken together, the results of this study demonstrate that the plant plasma membrane H+-ATPase does not mediate direct K+ transport chemically linked to ATP hydrolysis. Rather, this enzyme provides a driving force for cellular K+ uptake by secondary mechanisms, such as K+ channels or H+/K+ symporters. Although the presence of a small, protonophore-insensitive component of ATP-dependent K+ transport in a plasma membrane fraction might be mediated by an ATP-activated K+ channel, the possibility of direct K+ transport by other ATPases (i.e. K+-ATPases) associated with either the plasma membrane or other cellular membranes cannot be ruled out.