Graphene as a subnanometre trans-electrode membrane.

Isolated, atomically thin conducting membranes of graphite, called graphene, have recently been the subject of intense research with the hope that practical applications in fields ranging from electronics to energy science will emerge. The atomic thinness, stability and electrical sensitivity of graphene motivated us to investigate the potential use of graphene membranes and graphene nanopores to characterize single molecules of DNA in ionic solution. Here we show that when immersed in an ionic solution, a layer of graphene becomes a new electrochemical structure that we call a trans-electrode. The trans-electrode's unique properties are the consequence of the atomic-scale proximity of its two opposing liquid-solid interfaces together with graphene's well known in-plane conductivity. We show that several trans-electrode properties are revealed by ionic conductance measurements on a graphene membrane that separates two aqueous ionic solutions. Although our membranes are only one to two atomic layers thick, we find they are remarkable ionic insulators with a very small stable conductance that depends on the ion species in solution. Electrical measurements on graphene membranes in which a single nanopore has been drilled show that the membrane's effective insulating thickness is less than one nanometre. This small effective thickness makes graphene an ideal substrate for very high resolution, high throughput nanopore-based single-molecule detectors. The sensitivity of graphene's in-plane electronic conductivity to its immediate surface environment and trans-membrane solution potentials will offer new insights into atomic surface processes and sensor development opportunities.

Spectrin: on the path from structure to function.

New structural analyses of the spectrin family of actin cross-linking proteins are providing molecular explanations for both the interchain binding between the alpha and beta chains of spectrin and the intermolecular associations between spectrin and other proteins. Additionally, the analyses bring into focus a conformation which may explain aspects of spectrin's interaction with lipids.

Partial purification and characterization of an actin-bundling protein, band 4.9, from human erythrocytes.

Band 4.9 (a 48,000-mol-wt polypeptide) has been partially purified from human erythrocyte membranes. In solution, band 4.9 polypeptides exist as trimers with an apparent molecular weight of 145,000 and a Stokes radius of 50 A. Electron microscopy shows that the protein is a three-lobed structure with a radius slightly greater than 50 A. When gel-filtered rabbit muscle actin is polymerized in the presence of band 4.9, actin bundles are generated that are similar in appearance to those induced by "vinculin" or fimbrin. The bundles appear brittle and when they are centrifuged small pieces of filaments break off and remain in the supernatant. At low band 4.9 to actin molar ratios (1:30), band 4.9 lowers the apparent steady-state low-shear falling ball viscosity by sequestering filaments into thin bundles; at higher ratios, the bundles become thicker and obstruct the ball's movement leading to an apparent increase in steady-state viscosity. Band 4.9 increases the length of the lag phase and decreases the rate of elongation during actin polymerization as measured by high-shear Ostwald viscometry or by the increase in the fluorescence of pyrene-labeled actin. Band 4.9 does not alter the critical actin monomer concentration. We hypothesize that band 4.9, together with actin, erythrocyte tropomyosin, and spectrin, forms structures in erythroid precursor cells analogous to those formed by fimbrin, actin, tropomyosin, and TW 260/240 in epithelial brush borders. During erythroid development and enucleation, the actin filaments may depolymerize up to the membrane, leaving a membrane skeleton with short stubs of actin bundled by band 4.9 and cross-linked by spectrin.

Intramembrane particle aggregation in erythrocyte ghosts. I. The effects of protein removal.

We have used freeze-etching and SDS-polyacrylamide gel electrophoresis to study the conditions under which the intramembrane particles of the human erythrocyte ghost may be aggregated. The fibrous membrane protein, spectrin, can be almost entirely removed from erythrocyte ghosts with little or no change in the distribution of the particles. However, after spectrin depletion, particle aggregation in the plane of the membrane may be induced by conditions which cause little aggregation in freshly prepared ghosts. This suggests that the spectrin molecules form a molecular meshwork which limits the translational mobility of the erythrocyte membrane particles.

Freeze-fracture autoradiography: feasibility.

We have shown that the combination of freeze-fracture with electron microscope autoradiography can be developed into a technique for correlating the molecular structure of the biological membrane with its chemical and functional characteristics. Within the limits of electron microscope autoradiographic resolution, FARG has the potential to detect the relative distribution of molecules in each half of the membrane and within the plane of the membrane. The use of radioisotopic labels in combination with freezing techniques requires minimal perturbation of the system being studied and may be suitable for the examination of substances which would be extracted or would diffuse during the normal fixation and embedding procedures used in standard electron microscope autoradiography.

Analytical characterization of beetroot vacuole membrane.

Vacuoles from beetroot (Beta vulgaris L. var. esculenta Gürke) isolated by a mechanical procedure were osmotically lysed to separate the membrane and sap components for analysis. Approximately 62% of the vacuole proteins, 70% of the nondialyzable carbohydrates and almost all of the phospholipids and sterols were recovered in the membrane fraction. The vacuole membrane had a phospholipid protein ratio of 0.68 and a sterol:phospholipid ratio of 0.21. 17 complex polar lipids including phosphatides and glycolipids have been tentatively identified. Phosphatidylcholine (54%) and phosphatidylethanolamine (24%) were the most prominent phosphoglycerides besides phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid (1, 4, 5, and 12%, respectively). A putative sulfoglycoside and two major ceramide glycoside-like lipids, resembling those of animal lysosomes, were identified by thin-layer chromatography. High-resolution SDS-acrylamide gel electrophoresis of the polypeptides from the vacuole revealed 15 major bands with apparent molecular weights ranging from 91,000 to 12,000. Selective elution experiments delineated those polypeptides that were peripheral membrane proteins or sap proteins adsorbed to the membrane, and those that exhibited hydrophobic interactions with the lipid core. Lectin labeling results indicated that most of the polypeptides from the membrane and from the sap were glycoproteins probably of the high-mannose type characteristic of lysosomal enzymes that have undergone several stages of posttranslational modification.

Identification of coated vesicles in Saccharomyces cerevisiae.

Clathrin-coated vesicles were found in yeast, Saccharomyces cerevisiae, and enriched from spheroplasts by a rapid procedure utilizing gel filtration on Sephacryl S-1000. The coated vesicles (62-nm diam) were visualized by negative stain electron microscopy and clathrin triskelions were observed by rotary shadowing. The contour length of a triskelion leg was 490 nm. Coated vesicle fractions contain a prominent band with molecular weight of approximately 185,000 when analyzed by SDS PAGE. The presence of coated vesicles in yeast cells suggests that this organism will be useful for studying the function of clathrin-coated vesicles.

Membrane splitting in freeze-ethching. Covalently bound ferritin as a membrane marker.

The freeze-etch technique was used to observe red blood cell ghosts labeled on both surfaces with covalently bound ferritin. Ferritin molecules were never observed on fracture faces, thus indicating that fracture does not show membrane-surface detail. Subliming away the surrounding ice did expose the ferritin on the membrane surface. These results were consistent with the concept that membranes split during the fracture process of freeze-etching.

Rotary replication for freeze-etching.

Rotary replication has been adapted to freeze-etching and evaluated using T4 polyheads, erythrocyte ghosts, and chloroplast membranes. Conventional electron microscopy, electron diffraction, and optical diffraction and filtering indicate that platinum-carbon rotary replication renders radially symmetrical contrast and 25 A resolution to freeze-etched specimens so as to clarify subunit structure not normally evident in unidirectional shadow replicas.

Solubilization of proteins from bovine brain coated vesicles by protein perturbants and Triton X-100.

To identify integral and peripheral membrane proteins, highly purified coated vesicles from bovine brain were exposed to solutions of various pH, ionic strength, and concentrations of the nonionic detergent Triton X-100. At pH 10.0 or above most major proteins were liberated, but four minor polypeptides sedimented with the vesicles. From quantitative analysis of phospholipids in the pellet and extract, we determined that at a pH of up to 12 all phospholipids could be recovered in the pellet. Electron microscopic examination of coated vesicles at pH 12.0 showed all vesicles devoid of coat structures. Treatment with high ionic strength solutions (0-1.0 M KCl) at pH 6.5-8.5 also liberated all major proteins, except tubulin, which remained sedimentable. The addition of Triton X-100 to coated vesicles or to stripped vesicles from which 90% of the clathrin had been removed resulted in the release of four distinct polypeptides of approximate Mr 38,000, 29,000, 24,000 and 10,000. The 38,000-D polypeptide (pK approximately 5.0), which represents approximately 50% of the protein liberated by Triton X-100, appears to be a glycoprotein on the basis of its reaction with periodic acid-Schiff reagent. Extraction of 90% of the clathrin followed by extraction of 90% of the phospholipids with Triton X-100 produced a protein residue that remained sedimentable and consisted of structures that appeared to be shrunken stripped vesicles. Together our data indicate that most of the major polypeptides of brain coated vesicles behave as peripheral membrane proteins and at least four polypeptides behave as integral membrane proteins. By use of a monoclonal antibody, we have identified one of these polypeptides (38,000 mol wt) as a marker for a subpopulation of calf brain coated vesicles.

Appearance and distribution of surface proteins of the human erythrocyte membrane. An electron microscope and immunochemical labeling study.

We have used freeze-etching, before and after immunoferritin labeling, to visualize spectrin molecules and other surface proteins of the human erythrocyte membrane. After intramembrane particle aggregation was induced, spectrin molecules, identified by labeling with ferritin-conjugated antispectrin, were clustered on the cytoplasmic surface of the membrane in patches directly underlying the particle clusters. This labeling pattern confirms the involvement of spectrin in such particle aggregates, as previously inferred from indirect evidence. Ferritin-conjugated antihapten molecules, directed against external and cytoplasmic surface proteins of the erythrocyte membrane which had been covalently labeled nonspecifically with the hapten p-diazoniumphenyl-beta-D-lactoside, were similarly found in direct association with such intramembrane particle aggregates. This indicates that when spectrin and the intramembrane particles are aggregated, all the major proteins of the erythrocyte membrane are constrained to coaggregate with them. Although giving no direct information concerning the freedom of translational movement of proteins in the unperturbed erythrocyte membrane, these experiments suggest that a close dynamic association may exist between the integral and peripheral protein components of the membrane, such that immobilization of one component can restrict the lateral mobility of others.

Structural comparison of several actin-binding macromolecules.

The cytoskeletal components, macrophage actin-binding protein and filamin, were dried from glycerol and examined by low-angle rotary shadowing electron microscopy. Both are elongate, flexible molecules whose general morphologi is similar to that of erythrocyte spectrin. Neither actin-binding protein nor filamin binds to spectrin-depleted erythrocyte membranes.

Two independent domains of hDlg are sufficient for subcellular targeting: the PDZ1-2 conformational unit and an alternatively spliced domain.

hDlg, a human homologue of the Drosophila Dig tumor suppressor, contains two binding sites for protein 4.1, one within a domain containing three PSD-95/Dlg/ZO-1 (PDZ) repeats and another within the alternatively spliced I3 domain. Here, we further define the PDZ-protein 4.1 interaction in vitro and show the functional role of both 4.1 binding sites in situ. A single protease-resistant structure formed by the entirety of both PDZ repeats 1 and 2 (PDZ1-2) contains the protein 4.1-binding site. Both this PDZ1-2 site and the I3 domain associate with a 30-kD NH2-terminal domain of protein 4.1 that is conserved in ezrin/radixin/moesin (ERM) proteins. We show that both protein 4.1 and the ezrin ERM protein interact with the murine form of hDlg in a coprecipitating immune complex. In permeabilized cells and tissues, either the PDZ1-2 domain or the I3 domain alone are sufficient for proper subcellular targeting of exogenous hDlg. In situ, PDZ1-2-mediated targeting involves interactions with both 4.1/ERM proteins and proteins containing the COOH-terminal T/SXV motif. I3-mediated targeting depends exclusively on interactions with 4.1/ERM proteins. Our data elucidates the multivalent nature of membrane-associated guanylate kinase homologue (MAGUK) targeting, thus beginning to define those protein interactions that are critical in MAGUK function.

Drosophila development requires spectrin network formation.

The head-end associations of spectrin give rise to tetramers and make it possible for the molecule to form networks. We analyzed the head-end associations of Drosophila spectrin in vitro and in vivo. Immunoprecipitation assays using protein fragments synthesized in vitro from recombinant DNA showed that interchain binding at the head end was mediated by segment 0-1 of alpha-spectrin and segment 18 of beta-spectrin. Point mutations equivalent to erythroid spectrin mutations that are responsible for human hemolytic anemias diminished Drosophila spectrin head-end interchain binding in vitro. To test the in vivo consequence of deficient head-end interchain binding, we introduced constructs expressing head-end interchain binding mutant alpha-spectrin into the Drosophila genome and tested for rescue of an alpha-spectrin null mutation. An alpha-spectrin minigene lacking the codons for head-end interchain binding failed to rescue the lethality of the null mutant, whereas a minigene with a point mutation in these codons overcame the lethality of the null mutant in a temperature-dependent manner. The rescued flies were viable and fertile at 25 degrees C, but they became sterile because of defects in oogenesis when shifted to 29 degrees C. At 29 degrees C, egg chamber tissue disruption and cell shape changes were evident, even though the mutant spectrin remained stably associated with cell membranes. Our results show that spectrin's capacity to form a network is a crucial aspect of its function in nonerythroid cells.

Membrane isolation on polylysine-coated beads. Plasma membrane from HeLa cells.

HeLa cell plasma membranes have been purified after binding cells to polylysine-coated polyacrylamide beads. Cell attachment to beads and membrane recovery were maximal in a sucrose-acetate buffer, pH 5.0, at 25 degrees C. Measurements of ouabain-sensitive NaK-adenosine triphosphatase, membrane-bound 125I-wheat germ agglutinin, and chemical analyses showed that membranes on beads were of comparable or greater purity than membranes isolated by conventional methods. Because the isolation procedure is rapid (approximately 2.5 h), and produces membranes whose protoplasmic surfaces are fully exposed, it should be a useful supplement to standard isolation techniques.

Drosophilia spectrin. I. Characterization of the purified protein.

We purified a protein from Drosophila S3 tissue culture cells that has many of the diagnostic features of spectrin from vertebrate organisms: (a) The protein consists of two equimolar subunits (Mr = 234 and 226 kD) that can be reversibly cross-linked into a complex composed of equal amounts of the two subunits. (b) Electron microscopy of the native molecule reveals two intertwined, elongated strands with a contour length of 180 nm. (c) Antibodies directed against vertebrate spectrin react with the Drosophila protein and, similarly, antibodies to the Drosophila protein react with vertebrate spectrins. One monoclonal antibody has been found to react with both of the Drosophila subunits and with both subunits of vertebrate brain spectrin. (d) The Drosophila protein exhibits both actin-binding and calcium-dependent calmodulin-binding activities. Based on the above criteria, this protein appears to be a bona fide member of the spectrin family of proteins.

Cell shape and interaction defects in alpha-spectrin mutants of Drosophila melanogaster.

We show that the alpha-spectrin gene is essential for larval survival and development by characterizing several alpha-spectrin mutations in Drosophila. P-element minigene rescue and sequence analysis were used to identify the alpha-spectrin gene as the l(3)dre3 complementation group of the Dras-Roughened-ecdysoneless region of chromosome 3 (Sliter et al., 1988). Germ line transformants carrying an alpha-spectrin cDNA, whose expression is driven by the ubiquitin promoter, fully rescued the first to second instar lethality characteristic of the l(3)dre3 alleles. The molecular defects in two gamma-ray-induced alleles were identified. One of these mutations, which resulted in second instar lethality, contained a 73-bp deletion in alpha-spectrin segment 22 (starting at amino acid residue 2312), producing a premature stop codon between the two EF hands found in this segment. The second mutation, which resulted in first instar lethality, contained a 20 base pair deletion in the middle of segment 1 (at amino acid residue 92), resulting in a premature stop codon. Examination of the spectrin-deficient larvae revealed a loss of contact between epithelial cells of the gut and disruption of cell-substratum interactions. The most pronounced morphological change was seen in tissues of complex cellular architecture such as the middle midgut where a loss of cell contact between cup-shaped cuprophilic cells and neighboring interstitial cells was accompanied by disorganization of the cuprophilic cell brush borders. Our examination of spectrin deficient larvae suggests that an important role of non-erythroid spectrin is to stabilize cell to cell interactions that are critical for the maintenance of cell shape and subcellular organization within tissues.

Drosophila spectrin. II. Conserved features of the alpha-subunit are revealed by analysis of cDNA clones and fusion proteins.

Drosophila alpha-spectrin cDNA sequences were isolated from a lambda gt11 expression library. These cDNA clones encode fusion proteins that include portions of the Drosophila alpha-spectrin polypeptide as shown by a number of structural and functional criteria. The fusion proteins elicited antibodies that reacted strongly with Drosophila and vertebrate alpha-spectrins and a comparison of cyanogen bromide peptide maps demonstrated a clear structural correspondence between one fusion protein and purified Drosophila alpha-spectrin. Alpha-spectrin fusion protein also displayed calcium-dependent calmodulin-binding activity in blot overlay experiments and one fusion protein bound specifically to both Drosophila and bovine brain beta-spectrin subunits on protein blots. A region of the Drosophila cDNA cross-hybridized at lowered stringency with an avian alpha-spectrin cDNA. Together these data show that the composition, structure, and binding properties of the spectrin family of proteins have been remarkably well conserved between arthropods and vertebrates. Drosophila cDNA hybridized to an mRNA of greater than or equal to 9 kb on blots of total Drosophila poly A+ RNA; and hybridized in situ to a single site in polytene region 62B, 1-7. This result and Southern blot analysis of genomic DNA indicate that the sequences are likely to be single copy in the Drosophila genome.

Drosophila spectrin: the membrane skeleton during embryogenesis.

The distribution of alpha-spectrin in Drosophila embryos was determined by immunofluorescence using affinity-purified polyclonal or monoclonal antibodies. During early development, spectrin is concentrated near the inner surface of the plasma membrane, in cytoplasmic islands around the syncytial nuclei, and, at lower concentrations, throughout the remainder of the cytoplasm of preblastoderm embryos. As embryogenesis proceeds, the distribution of spectrin shifts with the migrating nuclei toward the embryo surface so that, by nuclear cycle 9, a larger proportion of the spectrin is concentrated near the plasma membrane. During nuclear cycles 9 and 10, as the nuclei reach the cell surface, the plasma membrane-associated spectrin becomes concentrated into caps above the somatic nuclei. Concurrent with the mitotic events of the syncytial blastoderm period, the spectrin caps elongate at interphase and prophase, and divide as metaphase and anaphase progress. During cellularization, the regions of spectrin concentration appear to shift: spectrin increases near the growing furrow canal and concomitantly increases at the embryo surface. In the final phase of furrow growth, the shift in spectrin concentration is reversed: spectrin decreases near the furrow canal and concomitantly increases at the embryo surface. In gastrulae, spectrin accumulates near the embryo surface, especially at the forming amnioproctodeal invagination and cephalic furrow. During the germband elongation stage, the total amount of spectrin in the embryo increases significantly and becomes uniformly distributed at the plasma membrane of almost all cell types. The highest levels of spectrin are in the respiratory tract cells; the lowest levels are in parts of the forming gut. The spatial and temporal changes in spectrin localization suggest that this protein plays a role in stabilizing rather than initiating changes in structural organization in the embryo.

Transformation by Rous sarcoma virus induces clathrin heavy chain phosphorylation.

We have shown that the heavy chain of clathrin is phosphorylated in chicken embryo fibroblast cells transformed by Rous sarcoma virus, but not in normal cells. Approximately 1 mol of phosphate is bound for every 5 mol of heavy chain in the maximally phosphorylated transformed cells. Two-thirds of the phosphate is on serine and one-third on tyrosine residues. Clathrin heavy chain is a substrate for pp60v-src in vitro. Cleveland analysis of the in vivo and in vitro clathrin heavy chain phosphopeptides, generated by protease V8 digestion, show labeled proteolytic fragments of similar molecular weight, suggesting that pp60v-src could be directly responsible for the in vivo phosphorylation of clathrin. Phosphate is equally incorporated into clathrin in both the unassembled and the assembled clathrin pools, whereas [35S]methionine is preferentially incorporated into the assembled pool. In normal cells, clathrin visualized by immunofluorescent staining appears in a punctate pattern along the membrane surface and concentrated around the nucleus; in transformed cells the perinuclear staining is completely absent. The phosphorylation of clathrin heavy chain in transformed cells may be linked to previously observed transformation-dependent alterations in receptor-mediated endocytosis of ligands such as EGF and thrombin.