The Drosophila membrane receptor Toll can function to promote cellular adhesion.

The product of the Toll gene is a membrane protein required for the formation of dorso-ventral polarity during early embryogenesis in Drosophila melanogaster. It acts together with the other dorsal group gene products to specify a nuclear gradient of dorsal morphogen in the syncytial blastoderm stage embryo. Here we report the presence in Toll protein of additional sequences held in common with the human membrane receptor platelet glycoprotein 1b (Gp1b). We propose that these sequences in Toll form disulphide linked extracellular domains that are important for the binding of ligands in the perivitelline space of the embryo. In addition, we show that expression of Toll protein induced in a non-adhesive cell line promotes cellular adhesion, a property held in common with the related Drosophila glycoprotein chaoptin. Toll protein in such aggregates accumulates at sites of cell-cell interaction, a characteristic displayed by other cellular adhesion molecules. Taken together these findings suggest that the biochemical function of Toll protein is more closely analogous to that of Gp1b than previously thought.     

Drosophila chaoptin, a member of the leucine-rich repeat family, is a photoreceptor cell-specific adhesion molecule

Drosophila chaoptin, required for photoreceptor cell morphogenesis, is a member of the leucine-rich repeat family of proteins. On the basis of biochemical and genetic analyses we previously proposed that chaoptin might function as a cell adhesion molecule. To test this hypothesis, chaoptin cDNA driven by the hsp 70 promoter was transfected into non-self-adherent Drosophila Schneider line 2 (S2) cells. Following heat shock induction of chaoptin expression, the transfected S2 cells formed multicellular aggregates. Mixing experiments of chaoptin expressing and non-expressing cells suggest that chaoptin expressing cells adhere homotypically. Previously it was shown that chaoptin is exclusively localized to photoreceptor cells. Thus, chaoptin is a cell-type-specific adhesion molecule. Biochemical analyses presented in this paper demonstrate that chaoptin is linked to the extracellular surface of the plasma membrane by covalent attachment to glycosyl-phosphatidylinositol. We propose that chaoptin and several other members of the leucine-rich repeat family of proteins define a new class of cell adhesion molecules.     

Shiverer and Normal Peripheral Myelin Compared: Basic Protein Localization, Membrane Interactions, and Lipid Composition

We have correlated membrane structure and interactions in shiverer sciatic nerve myelin with its biochemical composition. Analysis of x-ray diffraction data from shiverer myelin swollen in water substantiates our previous localization of an electron density deficit in the cytoplasmic half of the membrane. The density loss correlates with the absence of the major myelin basic proteins and indicates that in normal myelin, the basic protein is localized to the cytoplasmic apposition. As in normal peripheral myelin, hypotonic swelling in the shiverer membrane arrays occurs in the extracellular space between membranes; the cytoplasmic surfaces remain closely apposed notwithstanding the absence of basic protein from this region. Surprisingly, we found that the interaction at the extracellular apposition of shiverer membranes is altered. The extracellular space swells to a greater extent than normal when nerves are incubated in distilled water, treated at a reduced ionic strength of 0.06 in the range of pH 4-9, or treated at constant pH (4 or 7) in the range of ionic strengths 0.02-0.20. To examine the biochemical basis of this difference in swelling, we compared the lipid composition of shiverer and normal myelin. We find that sulfatides, hydroxycerebroside, and phosphatidylcholine are 20-30% higher than normal; nonhydroxycerebroside and sphingomyelin are 15-20% lower than normal; and ethanolamine phosphatides, phosphatidylserine, and cholesterol show little or no change. A higher concentration of negatively charged sulfatides at the extracellular surface likely contributes to an increased electrostatic repulsion and greater swelling in shiverer. The cytoplasmic surfaces of the apposed membranes of normal and shiverer myelins did not swell apart appreciably in the pH and ionic strength ranges expected to produce electrostatic repulsion. This stability, then, clearly does not depend on basic protein. We propose that P0 glycoprotein molecules form the stable link between apposed cytoplasmic membrane surfaces in peripheral myelin.     

A secretion granule membrane protein (GRAMP 92) is found in non-granule membranes including those of the endocytic pathway.

GRAMP 92, a secretion granule-associated membrane protein, has been identified in exocrine and endocrine storage granule membranes using a monoclonal antibody against rat parotid secretion granule membranes. This integral membrane glycoprotein has a M(r) of 92,000 in pancreatic zymogen granule membranes, and is slightly smaller in endocrine granule membranes. In both cases, deglycosylation produces core proteins of M(r) 52,000, that have identical peptide fingerprints. Unlike the slightly smaller zymogen granule membrane glycoprotein GP-2, GRAMP 92 does not appear to be bound to the membrane by a glycophosphatidyl inositol anchor, is not found on the plasma membrane and is not released into the secretion. Within acinar cells, low levels of antigen are observed immunocytochemically over the membranes of most granules. Antigen is highly concentrated on small vesicles that are closely apposed to (and possibly interact with) granules. As well, antigen is localized to organelles in the Golgi and basolateral regions that are part of the endocytic pathway. In hepatocytes a glycoprotein similar if not identical to GRAMP 92 marks the endocytic pathway including lysosomes. These findings indicate that GRAMP 92 is a widely distributed endocytic component and suggest that cells specialized for regulated secretion may adapt such components for storage granule function. Granule-associated GRAMP 92-rich membranes may link the exocytotic and endocytic pathways.     

The Caenorhabditis elegans Behavioral Gene unc-24 Encodes a Novel Bipartite Protein Similar to Both Erythrocyte Band 7.2 (Stomatin) and Nonspecific Lipid Transfer Protein

Abstract: We report here the positional cloning and molecular characterization of the unc-24 gene of Caenorhabditis elegans . This gene is required for normal locomotion and interacts with genes that affect the worm's response to volatile anesthetics. The predicted gene product contains a domain similar to part of two ion channel regulators (the erythrocyte integral membrane protein stomatin and the C. elegans neuronal protein MEC-2) juxtaposed to a domain similar to nonspecific lipid transfer protein (nsLTP; also called sterol carrier protein 2). Sequence analysis suggests that the nsLTP-like domain of UNC-24 provides lipid carrier function and is tethered to the plasma membrane by the stomatin-like domain, which may be regulatory. We postulate that UNC-24 may be involved in lipid transfer between closely apposed membranes.     

Fast lipid disorientation at the onset of membrane fusion revealed by molecular dynamics simulations.

Membrane fusion is a key event in vesicular trafficking in every cell, and many fusion-related proteins have been identified. However, how the actual fusion event occurs has not been elucidated. By using molecular dynamics simulations we found that when even a small region of two membranes is closely apposed such that only a limited number of water molecules remain in the apposed area (e.g., by a fusogenic protein and thermal membrane fluctuations), dramatic lipid disorientation results within 100 ps-2 ns, which might initiate membrane fusion. Up to 12% of phospholipid molecules in the apposing layers had their alkyl chains outside the hydrophobic region, lying almost parallel to the membrane surface or protruding out of the bilayer by 2 ns after two membranes were closely apposed.     

Nuclear congression and membrane fusion: two distinct events in the yeast karyogamy pathway

Karyogamy is the process where haploid nuclei fuse to form a diploid nucleus during yeast mating. We devised a novel genetic screen that identified five new karyogamy (KAR) genes and three new cell fusion (FUS) genes. The kar mutants fell into two classes that represent distinct events in the yeast karyogamy pathway. Class I mutations blocked congression of the nuclei due to cytoplasmic microtubule defects. In Class II mutants, nuclear congression proceeded and the membranes of apposed nuclei were closely aligned but unfused. In vitro, Class II mutant membranes were defective in a homotypic ER/nuclear membrane fusion assay. We propose that Class II mutants define components of a novel membrane fusion complex which functions during vegetative growth and is recruited for karyogamy.     

Interaction of lipoproteins with isolated ovary plasma membranes

Plasma membranes of ovarian luteal and adrenal cortical cells from "microvillar channels," a unique extracellular compartment formed by the close apposition of flattened microvillar surfaces. Microvillar channels have unusual affinity for cholesterol-rich lipoproteins, and, in vivo, may provide an increased surface area for these particles. In this research, we have isolated a plasma membrane-enriched fraction from rat luteinized ovaries, in which closely apposed membrane (i.e. microvillar channels) comprise about 30% of the preparation. Following in vitro incubations (approximately 1 h) of this plasma membrane fraction with different plasma lipoproteins, the closely apposed plasma membrane surfaces widen and become filled with lipoprotein particles (up to about 30 nm), whereas other membranes of the fraction show little binding. Competition experiments show that rat high density lipoproteins have the highest affinity for binding to the plasma membrane fraction. Radiolabeled plasma lipoprotein and the tissue-specific hormone, human chorionic gonadotropin, showed specific and saturable binding to the plasma membrane fraction, whereas other macromolecules used as controls did not. Radioautographic analyses of 125I-labeled lipoproteins and human chorionic gonadotropin indicate that binding occurs predominantly to the closely apposed plasma membranes (i.e. microvillar channels of the fraction). These studies show that microvillar channels of steroid-secreting cells entrap large numbers of plasma lipoproteins, particularly high density lipoproteins particles, presumably functioning in the delivery of cholesterol to these cells.     

Human teratoma cells share antigens with mouse teratoma cells.

Cytochalasin B inhibits the penetration of sperm nuclei into Urechis eggs without inhibiting sperm-induced egg activation. The acrosome reaction appears normal, and plasma membranes of the acrosomal tubule and egg become closely apposed. It is uncertain whether or not the drug blocks fusion of these membranes; however, sperm penetration cone formation is inhibited.     

Herpes simplex virus glycoprotein K is known to influence fusion of infected cells, yet is not on the cell surface.

Syncytial mutants of herpes simplex virus (HSV) cause extensive fusion of cultured cells, whereas wild-type HSV primarily causes cell rounding and aggregation. A large fraction of syncytial viruses contain mutations in the UL53 gene, which encodes glycoprotein K (gK). Previously, we demonstrated that wild-type and syncytial forms of gK are expressed at similar levels and possess identical electrophoretic mobilities. Using immunofluorescence, we show that gK is not transported to the surfaces of cells infected with either wild-type or syncytial HSV. Instead, gK accumulates in the perinuclear and nuclear membranes of cells. This finding is in contrast to the behavior of all other HSV glycoproteins described to date, which reach the cell surface. When gK was expressed in the absence of other HSV proteins, using a recombinant adenovirus vector, a similar perinuclear and nuclear pattern was observed. In addition, gK remained sensitive to endoglycosidase H, consistent with the hypothesis that gK does not reach the Golgi apparatus and is retained in the endoplasmic reticulum and nuclear envelope. Therefore, although gK mutations promote fusion between the surface membranes of HSV-infected cells, the glycoprotein does not reach the plasma membrane and, thus, must influence fusion indirectly.     

Cooperative elastic stresses, the hydrophobic effect, and lipid tilt in membrane remodeling

One of the fundamental properties of biological membranes is the high lateral integrity provided by the lipid bilayer, the structural core and the foundation of their barrier function. This tensile strength is due to the intrinsic properties of amphiphilic lipid molecules, which spontaneously self-assemble into a stable bilayer structure due to the hydrophobic effect. In the highly dynamic life of cellular membranes systems, however, this integrity has to be regularly compromised. One of the emerging puzzles is the mechanism of localized rupture of lipid monolayer, the formation of tiny hydrophobic patches and flipping of lipid tails between closely apposed monolayers. The energy cost of such processes is prohibitively high, unless cooperative deformations in a small membrane patch are carefully organized. Here we review the latest experimental and theoretical data on how such deformations can be conducted, specifically describing how elastic stresses yield tilting of lipids leading to cooperative restructuring of lipid monolayers. Proteins specializing in membrane remodeling assemble into closely packed circular complexes to arrange these deformations in time and space.     

Schwann cell myelination requires timely and precise targeting of P(0) protein

This report investigated mechanisms responsible for failed Schwann cell myelination in mice that overexpress P(0) (P(0)(tg)), the major structural protein of PNS myelin. Quantitative ultrastructural immunocytochemistry established that P(0) protein was mistargeted to abaxonal, periaxonal, and mesaxon membranes in P(0)(tg) Schwann cells with arrested myelination. The extracellular leaflets of P(0)-containing mesaxon membranes were closely apposed with periodicities of compact myelin. The myelin-associated glycoprotein was appropriately sorted in the Golgi apparatus and targeted to periaxonal membranes. In adult mice, occasional Schwann cells myelinated axons possibly with the aid of endocytic removal of mistargeted P(0). These results indicate that P(0) gene multiplication causes P(0) mistargeting to mesaxon membranes, and through obligate P(0) homophilic adhesion, renders these dynamic membranes inert and halts myelination.     

The gene encoding disabled-1 (DAB1), the intracellular adaptor of the Reelin pathway,reveals unusual complexity in human and mouse

The Disabled-1 (Dab1) gene encodes a key regulator of Reelin signaling. Reelin is a large glycoprotein secreted by neurons of the developing brain, particularly Cajal-Retzius cells. The DAB1 protein docks to the intracellular part of the Reelin very low density lipoprotein receptor and apoE receptor type 2 and becomes tyrosine-phosphorylated following binding of Reelin to cortical neurons. In mice, mutations of Dab1 and Reelin generate identical phenotypes. In humans, Reelin mutations are associated with brain malformations and mental retardation; mutations in DAB1 have not been identified. Here, we define the organization of Dab1, which is similar in human and mouse. The Dab1 gene spreads over 1100 kb of genomic DNA and is composed of 14 exons encoding the major protein form, some alternative internal exons, and multiple 5'-exons. Alternative polyadenylation and splicing events generate DAB1 isoforms. Several 5'-untranslated regions (UTRs) correspond to different promoters. Two 5'-UTRs (1A and 1B) are predominantly used in the developing brain. 5'-UTR 1B is composed of 10 small exons spread over 800 kb. With a genomic length of 1.1 Mbp for a coding region of 5.5 kb, Dab1 provides a rare example of genomic complexity, which will impede the identification of human mutations.     

Cell junctions acting as intestinal valves in nematodes

Serial sections of the rectal valve in Aphelenchoides blastophthorus Franklin and the oesophago-intestinal valve in Thornenema wickeni Yeates were examined electron microscopically. Each valve when closed appears as a convoluted path of closely apposed (10 nm) pairs of three-layered cell membranes. Both valves serve to stop intestinal leakage, open briefly and rapidly by forcible dilatation and are closed by pressure from surrounding tissues, helped perhaps by intermolecular forces.     

Characterization of the major mRNAs transcribed from the genes for glycoprotein B and DNA-binding protein ICP8 of herpes simplex virus type 1.

The structural genes encoding the herpes simplex virus type 1 glycoprotein B and the major DNA-binding protein ICP8 have been mapped previously within the EcoRI-F restriction fragment (map coordinates 0.314 to 0.420) of the viral genome. In this study the mRNAs transcribed from these DNA sequences were identified by hybridization selection of 32P-labeled RNA and by Northern blot analysis of polyadenylated cytoplasmic RNA. A 3.4-kilobase RNA was the major mRNA homologous to the DNA sequences between coordinates 0.343 and 0.386 in which mutations in the glycoprotein B gene have been mapped. A 4.5-kilobase RNA was the major mRNA homologous to the viral DNA sequences between coordinates 0.361 and 0.417 in which mutations in the ICP8 gene have been mapped. Hybridization-selected mRNAs were translated in vitro to determine the primary translation products encoded in each region. The glycoprotein B- and ICP8-specific polypeptides were identified by immunoprecipitation with specific antisera. The translation products encoded by the glycoprotein B gene were 103,000 and 99,000 in molecular weight. The translation products encoded by the ICP8 gene were 125,000 and 122,000 in molecular weight.     

Respective roles of protein tyrosine kinases and protein kinases C in the upregulation of beta-catenin distribution, and compaction in mouse preimplantation embryos: a pharmacological approach

The cellular distribution of beta-catenin was determined by western blotting and laser confocal scanning microscopy in both control and pharmacologically-manipulated mouse preimplantation embryos. Most of the stored maternal beta-catenin is Triton X-100-extractable and distributed throughout the cytoplasm. In 2-cell stage embryos, the remaining molecules are concentrated in regions of cell contact and, to a lesser extent, at non apposed surfaces. Association of beta-catenin with the cortex of non apposed membranes decreases as cleavage proceeds, and is lost at compaction. In contrast to the rapid cross-linking of cell surfaces induced by wheat germ agglutinin, the diacylglyceride-induced compaction-like adhesion of 2- and 4-cell embryos correlates with complete restriction of beta-catenin to the apposing membranes. On the contrary, tyrphostin B46, a specific protein tyrosine kinase inhibitor, fails to induce both premature beta-catenin relocalisation and compaction. In addition, we show that orthovanadate induces a dramatic increase in the level of phosphotyrosine labelling of cell-cell junctions in compacted 8-cell stage embryos without inducing their decompaction. However, most of these orthovanadate tyrosine-phosphorylated proteins are detergent-soluble, while beta-catenin restricted to the apposing membranes is not. In conclusion, our results confirm that diacylglycerol-dependent kinases upregulate both beta-catenin redistribution and compaction, and indicate that neither tyrosine kinases, nor tyrosine phosphatases are critical for the proper onset of compaction which seems, in addition, not causally linked to tyrosine dephosphorylation of beta-catenin.     

Eyespot placement and assembly in the green alga Chlamydomonas

The eyespot organelle of the green alga Chlamydomonas allows the cell to phototax toward (or away) from light to maximize the light intensity for photosynthesis and minimize photo-damage. At cytokinesis, the eyespot is resorbed at the cleavage furrow and two new eyespots form in the daughter cells 180 degrees from each other. The eyespots are positioned asymmetrically with respect to the microtubule cytoskeleton. Eyespots are assembled from all three chloroplast membranes and carotenoid-filled granules, which form a sandwich structure overlaid by the tightly apposed plasma membrane. This review describes (1) my interest in cellular asymmetry and organelle biology, (2) isolation of mutations that describe four genes governing eyespot placement and assembly, (3) the characterization of the EYE2 gene, which encodes a thioredoxin superfamily member, and (4) the characterization of the MIN1 gene, which is required for the layered organization of granules and membranes in the eyespot. BioEssays 25:410-416, 2003.     

A new occludens-like junction linking endothelial cells of small capillaries (probably venules) of rat jejunum.

Freeze-cleave replicas of small capillaries of rat jejunum have revealed the presence of a new type of junction linking endothelial cells. This new junction reveals tight junctions (zonulae occludentes) in that the adjacent plasma membranes are held closely together along lines of attachment organized in the form of a loose, but frequently discontinuous network. In contrast to tight junctions, the A-face ridges possess a very low profile, and only at low shadowing angles can a repeating, particulate substructure occasionally be resolved. The shallow B-face furrows lack any particulate components. Images of cross-fractured focal points of attachment suggest that the external leaflets of adjacent membranes are closely apposed but not actually fused, as is the case with zonulae occludentes. It appears that this new type of endothelial junction is characteristic of small venules. Thus we propose that it be termed small venule endothelial junction.