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1.
Proc Natl Acad Sci U S A ; 113(19): 5287-92, 2016 May 10.
Article in English | MEDLINE | ID: mdl-27114531

ABSTRACT

Retinoschisin (RS1) is involved in cell-cell junctions in the retina, but is unique among known cell-adhesion proteins in that it is a soluble secreted protein. Loss-of-function mutations in RS1 lead to early vision impairment in young males, called X-linked retinoschisis. The disease is characterized by separation of inner retinal layers and disruption of synaptic signaling. Using cryo-electron microscopy, we report the structure at 4.1 Å, revealing double octamer rings not observed before. Each subunit is composed of a discoidin domain and a small N-terminal (RS1) domain. The RS1 domains occupy the centers of the rings, but are not required for ring formation and are less clearly defined, suggesting mobility. We determined the structure of the discoidin rings, consistent with known intramolecular and intermolecular disulfides. The interfaces internal to and between rings feature residues implicated in X-linked retinoschisis, indicating the importance of correct assembly. Based on this structure, we propose that RS1 couples neighboring membranes together through octamer-octamer contacts, perhaps modulated by interactions with other membrane components.


Subject(s)
Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/ultrastructure , Cell Adhesion , Eye Proteins/chemistry , Eye Proteins/ultrastructure , Intercellular Junctions/ultrastructure , Retina/chemistry , Retina/ultrastructure , Amino Acid Sequence , Animals , Computer Simulation , Dimerization , Intercellular Junctions/chemistry , Mice , Models, Biological , Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/ultrastructure , Protein Conformation
2.
Biochem Biophys Res Commun ; 469(2): 257-62, 2016 Jan 08.
Article in English | MEDLINE | ID: mdl-26642954

ABSTRACT

Neocarzinostatin (NCS) a potent DNA-damaging, anti-tumor toxin extracted from Streptomyces carzinostaticus that recognizes double-stranded DNA bulge and induces DNA damage. 2 Fluoro (2F) Modified EpCAM RNA aptamer is a 23-mer that targets EpCAM protein, expressed on the surface of epithelial tumor cells. Understanding the interaction between NCS and the ligand is important for carrying out the targeted tumor therapy. In this study, we have investigated the biophysical interactions between NCS and 2-fluro Modified EpCAM RNA aptamer using Circular Dichroism (CD) and Infra-Red (IR) spectroscopy. The aromatic amino acid residues spanning the ß sheets of NCS are found to participate in intermolecular interactions with 2 F Modified EpCAM RNA aptamer. In-silico modeling and simulation studies corroborate with CD spectra data. Furthermore, it reinforces the involvement of C and D1 strand of NCS in intermolecular interactions with EpCAM RNA aptamer. This the first report on interactions involved in the stabilization of NCS-EpCAM aptamer complex and will aid in the development of therapeutic modalities towards targeted cancer therapy.


Subject(s)
Antigens, Neoplasm/chemistry , Antigens, Neoplasm/ultrastructure , Aptamers, Nucleotide/chemistry , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/ultrastructure , Models, Chemical , Molecular Docking Simulation , Zinostatin/chemistry , Binding Sites , Cytotoxins , Epithelial Cell Adhesion Molecule , Molecular Conformation , Protein Binding , Protein Interaction Mapping/methods
3.
J Recept Signal Transduct Res ; 35(3): 220-3, 2015 Jun.
Article in English | MEDLINE | ID: mdl-26366621

ABSTRACT

The family of adhesion G protein-coupled receptors (aGPCRs) comprises 33 members in the human genome, which are subdivided into nine subclasses. Many aGPCRs undergo an autoproteolytic process via their GPCR Autoproteolysis-INducing (GAIN) domain during protein maturation to generate an N- and a C-terminal fragments, NTF and CTF, respectively. The NTF and CTF are non-covalently reassociated on the plasma membrane to form a single receptor unit. How aGPCRs are activated upon ligand binding remains one of the leading questions in the field of aGPCR research. Recent work from our labs and others shows that ligand binding can remove the NTF from the plasma membrane-bound CTF, exposing a tethered agonist which potently activates downstream signaling.


Subject(s)
Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/physiology , Membrane Proteins/chemistry , Membrane Proteins/physiology , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/physiology , Animals , Cell Adhesion Molecules/ultrastructure , Computer Simulation , Humans , Membrane Proteins/ultrastructure , Models, Biological , Models, Chemical , Receptors, G-Protein-Coupled/ultrastructure , Structure-Activity Relationship
4.
Biol Reprod ; 86(5): 153, 1-14, 2012 May.
Article in English | MEDLINE | ID: mdl-22321830

ABSTRACT

In the ovary, initiation of follicle growth is marked by cuboidalization of flattened granulosa cells (GCs). The regulation and cell biology of this shape change remains poorly understood. We propose that characterization of intercellular junctions and associated proteins is key to identifying as yet unknown regulators of this important transition. As GCs are conventionally described as epithelial cells, this study used mouse ovaries and isolated follicles to investigate epithelial junctional complexes (tight junctions [TJ], adherens junctions [AJ], and desmosomes) and associated molecules, as well as classic epithelial markers, by quantitative PCR and immunofluorescence. These junctions were further characterized using ultrastructural, calcium depletion and biotin tracer studies. Junctions observed by transmission electron microscopy between GCs and between GCs and oocyte were identified as AJs by expression of N-cadherin and nectin 2 and by the lack of TJ and desmosome-associated proteins. Follicles were also permeable to biotin, confirming a lack of functional TJs. Surprisingly, GCs lacked all epithelial markers analyzed, including E-cadherin, cytokeratin 8, and zonula occludens (ZO)-1alpha+. Furthermore, vimentin was expressed by GCs, suggesting a more mesenchymal phenotype. Under calcium-free conditions, small follicles maintained oocyte-GC contact, confirming the importance of calcium-independent nectin at this stage. However, in primary and multilayered follicles, lack of calcium resulted in loss of contact between GCs and oocyte, showing that nectin alone cannot maintain attachment between these two cell types. Lack of classic markers suggests that GCs are not epithelial. Identification of AJs during GC cuboidalization highlights the importance of AJs in regulating initiation of follicle growth.


Subject(s)
Cell Adhesion Molecules/physiology , Connexins/physiology , Ovarian Follicle/physiology , Adherens Junctions/physiology , Adherens Junctions/ultrastructure , Animals , Calcium/physiology , Cell Adhesion Molecules/ultrastructure , Connexins/ultrastructure , Epithelial Cells/physiology , Epithelial Cells/ultrastructure , Female , Gap Junctions/physiology , Gap Junctions/ultrastructure , Mice , Ovarian Follicle/ultrastructure , Tight Junctions/physiology , Tight Junctions/ultrastructure
5.
Curr Opin Cell Biol ; 1(5): 892-7, 1989 Oct.
Article in English | MEDLINE | ID: mdl-2697291

ABSTRACT

The adhesive function of Ca2(+)-dependent CAMS has in the past been studied only indirectly, mainly using immunological techniques. The molecular cloning and information about the primary structure of several CAMs has been an important step in a more detailed molecular analysis. If there is a homophilic interaction between CAMs of neighbouring cells, an important question concerns the specificity of each CAM-mediated adhesiveness. Has each CAM a unique specificity and can this specificity be linked to a defined amino acid sequence? It will be important to elucidate the molecular mechanism of how each CAM interacts with the other. The experiments of Volk et al. (1987) suggest that an interaction of two different CAMs can occur. Since during development a given cell can express more than one CAM such an heterophilic interaction could play some regulatory role. Alternative splicing mechanisms or different protein forms during development or on different cell types have not yet been observed for Ca2(+)-dependent CAMs. However, uvomorulin is assumed to have a slightly different function during development and in adult tissues. During development uvomorulin is involved in the condensation, the pattern formation, and the sorting out of cells. In these processes the uvomorulin-mediated adhesiveness should be controlled, since cells reorganize and migrate during development. For the maintenance of the histoarchitecture in adult tissues uvomorulin might act more as a glue. This argues for the existence of mechanisms to regulate the strength of adhesiveness, and the cytoplasmic domain might be involved in these processes. The association of the cytoplasmic domain of uvomorulin with catenins could be an important observation in this respect.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Calcium/physiology , Cell Adhesion Molecules , Cell Adhesion , Animals , Cell Adhesion Molecules/physiology , Cell Adhesion Molecules/ultrastructure , Humans , Models, Biological , Protein Conformation
6.
Cell Motil Cytoskeleton ; 66(11): 1017-29, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19598236

ABSTRACT

Focal adhesions (FAs) are large clusters of transmembrane receptors of the integrin family and a multitude of associated cytoplasmic "plaque" proteins, which connect the extracellular matrix-bound receptors with the actin cytoskeleton. The formation of nearly stationary FAs defines a boundary between the dense and highly dynamic actin network in lamellipodium and the sparser and more diverse cytoskeletal organization in the lamella proper, creating a template for the organization of the entire actin network. The major "mechanical" and "sensory" functions of FAs; namely, the nucleation and regulation of the contractile, myosin-II-containing stress fibers and the mechanosensing of external surfaces depend, to a major extent, on the dynamics of molecular components within FAs. A central element in FA regulation concerns the positive feedback loop, based on the most intriguing feature of FAs; that is, their dependence on mechanical tension developing by the growing stress fibers. FAs grow in response to such tension, and rapidly disassemble upon its relaxation. In this article, we address the mechanistic relationships between the process of FA development, maturation and dissociation and the dynamic molecular events, which take place in different regions of the FA, primarily in the distal end of this structure (the "toe") and the proximal "heel," and discuss the central role of local mechanical forces in orchestrating the complex interplay between FAs and the actin system.


Subject(s)
Cell Movement , Cytoskeletal Proteins/ultrastructure , Focal Adhesions/metabolism , Pseudopodia/metabolism , Animals , Cell Adhesion Molecules/metabolism , Cell Adhesion Molecules/ultrastructure , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/metabolism , Extracellular Matrix Proteins/chemistry , Extracellular Matrix Proteins/metabolism , Extracellular Matrix Proteins/ultrastructure , Focal Adhesions/chemistry , Focal Adhesions/ultrastructure , Humans , Pseudopodia/chemistry , Pseudopodia/ultrastructure , Stress, Mechanical
7.
J Neurosci Res ; 87(15): 3492-501, 2009 Nov 15.
Article in English | MEDLINE | ID: mdl-19565653

ABSTRACT

Formation of the paranodal axoglial junction (PNJ) requires the presence of three cell adhesion molecules: the 155-kDa isoform of neurofascin (NF155) on the glial membrane and a complex of Caspr and contactin found on the axolemma. Here we report that the clustering of Caspr along myelinated axons during development differs fundamentally between the central (CNS) and peripheral (PNS) nervous systems. In cultures of Schwann cells (SC) and dorsal root ganglion (DRG) neurons, membrane accumulation of Caspr was detected only after myelination. In contrast, in oligodendrocytes (OL)/DRG neurons cocultures, Caspr was clustered upon initial glial cell contact already before myelination had begun. Premyelination clustering of Caspr was detected in cultures of oligodendrocytes and retinal ganglion cells, motor neurons, and DRG neurons as well as in mixed cell cultures of rat forebrain and spinal cords. Cocultures of oligodendrocyte precursor cells isolated from contactin- or neurofascin-deficient mice with wild-type DRG neurons showed that clustering of Caspr at initial contact sites between OL processes and the axon requires glial expression of NF155 but not of contactin. These results demonstrate that the expression of membrane proteins along the axolemma is determined by the type of the contacting glial cells and is not an intrinsic characteristic of the axon.


Subject(s)
Cell Adhesion Molecules, Neuronal/metabolism , Ganglia, Spinal/metabolism , Oligodendroglia/metabolism , Schwann Cells/metabolism , Sensory Receptor Cells/metabolism , Animals , Cell Adhesion Molecules/metabolism , Cell Adhesion Molecules/ultrastructure , Cell Adhesion Molecules, Neuronal/genetics , Cell Communication/physiology , Cells, Cultured , Coculture Techniques , Ganglia, Spinal/cytology , Intercellular Junctions/metabolism , Intercellular Junctions/ultrastructure , Mice , Mice, Inbred ICR , Mice, Knockout , Motor Neurons/metabolism , Motor Neurons/ultrastructure , Myelin Sheath/metabolism , Myelin Sheath/ultrastructure , Nerve Fibers, Myelinated/metabolism , Nerve Fibers, Myelinated/ultrastructure , Nerve Growth Factors/metabolism , Nerve Growth Factors/ultrastructure , Oligodendroglia/cytology , Prosencephalon/metabolism , Prosencephalon/ultrastructure , Ranvier's Nodes/metabolism , Ranvier's Nodes/ultrastructure , Rats , Rats, Wistar , Retinal Ganglion Cells/cytology , Retinal Ganglion Cells/metabolism , Schwann Cells/cytology , Sensory Receptor Cells/cytology , Spinal Cord/metabolism , Spinal Cord/ultrastructure
8.
J Cell Biol ; 116(5): 1283-9, 1992 Mar.
Article in English | MEDLINE | ID: mdl-1371289

ABSTRACT

Hyalin is a large (ca. 350 x 10(3) kD by gel electrophoresis) molecule that contributes to the hyalin layer surrounding the sea urchin embryo. In previous work a mAb (McA Tg-HYL), specific for hyalin, was found to inhibit cell-hyalin adhesion and block morphogenesis of whole embryos (Adelson, D. L., and T. D. Humphreys. 1988. Development. 104:391-402). In this report, hyalin ultrastructure was examined via rotary shadowing. Hyalin appeared to be a filamentous molecule approximately 75-nm long with a globular "head" about 12 nm in diameter that tended to form aggregates by associating head to head. Hyalin molecules tended to associate with a distinct high molecular weight globular particle ("core"). In fractions containing the core particle often more than one hyalin molecule were seen to be associated with the core. The core particle maintained a tenacious association with hyalin throughout purification procedures. The site(s) of McA Tg-HYL binding to the hyalin molecule were visualized by decorating purified hyalin with the antibody and then rotary shadowing the complex. In these experiments, McA Tg-HYL attached to the hyalin filament near the head region in a pattern suggesting that more than one antibody binding site exists on the hyalin filament. From the ultrastructural data and from the cell adhesion data presented earlier we conclude that hyalin is a filamentous molecule that binds to other hyalin molecules and contains multiple cell binding sites. Attempts were made to demonstrate the existence of lower molecular weight hyalin precursors. Whilst no such precursors could be identified by immunoprecipitation of in vivo labeled embryo lysates, immunoprecipitation of in vitro translation products suggested such precursors (ca 40 x 10(3) kD) might exist.


Subject(s)
Cell Adhesion Molecules/isolation & purification , Hyalin/chemistry , Sea Urchins/embryology , Animals , Blotting, Southern , Cell Adhesion Molecules/ultrastructure , Cell-Free System , Fractional Precipitation , Molecular Structure , Morphogenesis
9.
J Cell Biol ; 124(4): 601-8, 1994 Feb.
Article in English | MEDLINE | ID: mdl-7508942

ABSTRACT

Interaction of the vascular cell adhesion molecule (VCAM-1) with its counter-receptor very late antigen-4 (VLA-4) (integrin alpha 4 beta 1) is important for a number of developmental pathways and inflammatory functions. We are investigating the molecular mechanism of this binding, in the interest of developing new anti-inflammatory drugs that block it. In a previous report, we showed that the predominant form of VCAM-1 on stimulated endothelial cells, seven-domain VCAM (VCAM-7D), is a functionally bivalent molecule. One binding site requires the first and the other requires the homologous immunoglobulin-like domain. Rotary shadowing and electron microscopy of recombinant soluble VCAM-7D molecules suggests that the seven Ig-like domains are extended in a slightly bent linear array, rather than compactly folded together. We have systematically mutagenized the first domain of VCAM-6D (a monovalent, alternately spliced version mission domain 4) by replacing 3-4 amino acids of the VCAM sequence with corresponding portions of the related ICAM-1 molecule. Specific amino acids, important for binding VLA-4 include aspartate 40 (D40), which corresponds to the acidic ICAM-1 residue glutamate 34 (E34) previously reported to be essential for binding of ICAM-1 to its integrin counter-receptor LFA-1. A small region of VCAM including D40, QIDS, can be replaced by the similar ICAM-1 sequence, GIET, without affecting function or epitopes, indicating that this region is part of a general integrin-binding structure rather than a determinant of binding specificity for a particular integrin. The VCAM-1 sequence G65NEH also appears to be involved in binding VLA-4.


Subject(s)
Cell Adhesion Molecules/metabolism , Receptors, Very Late Antigen/metabolism , Amino Acid Sequence , Amino Acids/chemistry , Animals , Antibodies, Monoclonal , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/ultrastructure , Cell Line , Cloning, Molecular , Epitopes , Humans , Microscopy, Electron , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Binding , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Vascular Cell Adhesion Molecule-1
10.
J Cell Biol ; 115(1): 209-21, 1991 Oct.
Article in English | MEDLINE | ID: mdl-1918137

ABSTRACT

Extracellular matrix molecules are generally categorized as collagens, elastin, proteoglycans, or other noncollagenous structural/cell interaction proteins. Many of these extracellular proteins contain distinctive repetitive modules, which can sometimes be found in other proteins. We describe the complete primary structure of an alpha 1 chain of type XII collagen from chick embryonic fibroblasts. This large, structurally chimeric molecule identified by cDNA analysis combines previously unrelated molecular domains into a single large protein 3,124 residues long (approximately 340 kD). The deduced chicken type XII collagen sequence starts at the amino terminus with one unit of the type III motif of fibronectin, which is followed by one unit homologous to the von Willebrand factor A domain, then one more fibronectin type III module, a second A domain from von Willebrand factor, 6 units of type III motif and a third A domain, 10 consecutive units of type III motif and a fourth A domain, a domain homologous to the NC4 domain peptide of type IX collagen, and finally two short collagenous regions previously described as part of the partially sequenced collagen type XII molecule; an Arg-Gly-Asp potential cell adhesive recognition sequence is present in a hydrophilic region at the terminus of one collagenous domain. Antibodies raised to type XII collagen synthesized in a bacterial expression system recognized not only previously reported bands (220 kD et cetera) in tendons, but also bands with apparently different molecular sizes in fibroblasts and 4-d embryos. The antibodies stained a wide variety of extracellular matrices in embryos in patterns distinct from those of fibronectin or interstitial collagens. They prominently stained extracellular matrix associated with certain neuronal tissues, such as axons from dorsal root ganglia and neural tube. These studies identify a novel chimeric type of molecule that contains both adhesion molecule and collagen motifs in one protein. Its structure blurs current classification schemes for extracellular proteins and underscores the potentially large diversity possible in these molecules.


Subject(s)
Collagen/chemistry , Amino Acid Sequence , Animals , Base Sequence , Cell Adhesion , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/ultrastructure , Chick Embryo , Cloning, Molecular , Collagen/genetics , Collagen/metabolism , Collagen/ultrastructure , DNA/genetics , Fibronectins/chemistry , Fibronectins/ultrastructure , Molecular Sequence Data , Oligonucleotides/chemistry , Oligopeptides , Polymerase Chain Reaction , Recombinant Proteins/immunology , Sequence Alignment , Tissue Distribution , von Willebrand Factor/chemistry , von Willebrand Factor/ultrastructure
11.
J Cell Biol ; 164(1): 145-55, 2004 Jan 05.
Article in English | MEDLINE | ID: mdl-14709546

ABSTRACT

The C-type lectin dendritic cell (DC)-specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN; CD209) facilitates binding and internalization of several viruses, including HIV-1, on DCs, but the underlying mechanism for being such an efficient phagocytic pathogen-recognition receptor is poorly understood. By high resolution electron microscopy, we demonstrate a direct relation between DC-SIGN function as viral receptor and its microlocalization on the plasma membrane. During development of human monocyte-derived DCs, DC-SIGN becomes organized in well-defined microdomains, with an average diameter of 200 nm. Biochemical experiments and confocal microscopy indicate that DC-SIGN microdomains reside within lipid rafts. Finally, we show that the organization of DC-SIGN in microdomains on the plasma membrane is important for binding and internalization of virus particles, suggesting that these multimolecular assemblies of DC-SIGN act as a docking site for pathogens like HIV-1 to invade the host.


Subject(s)
Cell Adhesion Molecules/metabolism , Cell Membrane/metabolism , Dendritic Cells/metabolism , Lectins, C-Type/metabolism , RNA Virus Infections/metabolism , Receptors, Cell Surface/metabolism , Receptors, Virus/metabolism , Cell Adhesion Molecules/immunology , Cell Adhesion Molecules/ultrastructure , Cell Membrane/ultrastructure , Cell Membrane/virology , Cells, Cultured , Dendritic Cells/ultrastructure , Dendritic Cells/virology , Endocytosis/physiology , HIV Infections/immunology , HIV Infections/metabolism , HIV-1/pathogenicity , HIV-1/physiology , Humans , Immunohistochemistry , Lectins, C-Type/immunology , Lectins, C-Type/ultrastructure , Membrane Microdomains/metabolism , Membrane Microdomains/ultrastructure , Microscopy, Electron , Monocytes/metabolism , Monocytes/ultrastructure , Monocytes/virology , Protein Structure, Tertiary/physiology , RNA Virus Infections/immunology , Receptors, Cell Surface/immunology , Receptors, Cell Surface/ultrastructure , Receptors, Virus/immunology , Receptors, Virus/ultrastructure
12.
Eukaryot Cell ; 7(5): 776-82, 2008 May.
Article in English | MEDLINE | ID: mdl-18083824

ABSTRACT

The cell wall-bound Als adhesins of Candida albicans mediate both yeast-to-host tissue adherence and yeast aggregation. This aggregation is amyloid-like, with self-propagating secondary-structure changes, amyloid-characteristic dye binding, and induced birefringence (J. M. Rauceo, N. K. Gaur, K. G. Lee, J. E. Edwards, S. A. Klotz, and P. N. Lipke, Infect. Immun. 72:4948-4955, 2004). Therefore, we determined whether Als proteins could form amyloid fibers with properties like those in cellular aggregation. The beta-aggregation predictor TANGO identified a heptapeptide sequence present in a highly conserved sequence with amyloid-forming potential in Als1p, Als3p, and Als5p. A tridecapeptide containing this sequence formed fibers that bound Congo red and thioflavin T and had characteristic amyloid morphology. Als5p(20-431) and Als5p(20-664), large fragments of Als5p containing the amyloid sequence, also formed amyloid-like fibers and bound Congo red under native conditions. K(a)/K(s) analysis showed that the amyloid-forming sequences are highly conserved in Als proteins and evolve more slowly than other regions of the proteins. Therefore, amyloid-forming ability itself is conserved in these proteins.


Subject(s)
Amyloid/chemistry , Candida albicans/chemistry , Cell Adhesion Molecules/chemistry , Fungal Proteins/chemistry , Amino Acid Sequence , Amyloid/ultrastructure , Cell Adhesion Molecules/ultrastructure , Conserved Sequence , Evolution, Molecular , Fungal Proteins/ultrastructure , Microscopy, Electron, Transmission , Peptides/chemical synthesis , Peptides/chemistry , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/ultrastructure
13.
Neuron ; 101(2): 224-231.e5, 2019 01 16.
Article in English | MEDLINE | ID: mdl-30551998

ABSTRACT

The initiation of axoglial contact is considered a prerequisite for myelination, yet the role cell adhesion molecules (CAMs) play in mediating such interactions remains unclear. To examine the function of axoglial CAMs, we tested whether enhanced CAM-mediated adhesion between OLs and neurons could affect myelination. Here we show that increased expression of a membrane-bound extracellular domain of Cadm4 (Cadm4dCT) in cultured oligodendrocytes results in the production of numerous axoglial contact sites that fail to elongate and generate mature myelin. Transgenic mice expressing Cadm4dCT were hypomyelinated and exhibit multiple myelin abnormalities, including myelination of neuronal somata. These abnormalities depend on specific neuron-glial interaction as they were not observed when these OLs were cultured alone, on nanofibers, or on neurons isolated from mice lacking the axonal receptors of Cadm4. Our results demonstrate that tightly regulated axon-glia adhesion is essential for proper myelin targeting and subsequent membrane wrapping and lateral extension.


Subject(s)
Axons/metabolism , Cell Adhesion Molecules/metabolism , Cell Adhesion/physiology , Central Nervous System/cytology , Myelin Sheath/physiology , Neurons/cytology , Oligodendrocyte Precursor Cells/physiology , 2',3'-Cyclic Nucleotide 3'-Phosphodiesterase/genetics , 2',3'-Cyclic Nucleotide 3'-Phosphodiesterase/metabolism , Animals , Animals, Newborn , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/ultrastructure , Cells, Cultured , Central Nervous System/metabolism , Coculture Techniques , Female , Ganglia, Spinal/cytology , Intermediate Filaments/metabolism , Intermediate Filaments/ultrastructure , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myelin Sheath/ultrastructure , Oligodendroglia/cytology , Rats, Wistar
14.
Invest Ophthalmol Vis Sci ; 48(3): 991-1000, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17325137

ABSTRACT

PURPOSE: Retinoschisin (RS) is a retina-specific, secreted protein implicated in X-linked juvenile retinoschisis and essential for the structural and functional integrity of the retina. This biochemical characterization and ultrastructural localization of RS in intact murine retina was performed to further understanding of the molecular basis of its function. METHODS: Subcellular fractions and fractions enriched in photoreceptor inner and outer segments were prepared from mouse retina by differential or density gradient ultracentrifugation. Immunoblot analysis was used to assess the expression of RS in various subcellular compartments and its fractionation into soluble phase on treatment of retinal cell membranes with several solubilizing reagents. RS-lipid interactions were evaluated by a protein-lipid overlay assay that used wild-type and mutant forms of RS discoidin domain glutathione S-transferase (GST) fusion proteins. The subcellular localization of RS in mouse retina was visualized by pre-embedding immunogold electron microscopy. Ultrastructure was evaluated by transmission electron microscopy. RESULTS: RS was intimately associated with cell membranes of the retina. It was found to cluster on the outer leaflet of the plasma membrane of the photoreceptor inner segments, which synthesize and secrete it. It was released from the membrane at high pH, which is characteristic of a peripheral membrane protein. It was extracted from the membrane by the nonionic detergent NP-40, together with glycerophospholipids. Protein-lipid overlay assays indicated a preferential interaction between RS and anioic phospholipids. Extraction of RS from the membrane was inhibited by divalent cations. Photoreceptor inner segment morphology was markedly affected in RS(-)(/y) mice, which failed to express RS protein. CONCLUSIONS: RS in intact retina is a peripheral membrane protein. Although distributed over the two membrane faces, RS is associated primarily with the outer leaflet of the inner segment plasma membrane through anionic phospholipids and divalent cations. RS's localization in photoreceptors and its biochemical properties suggest a functional role locally, at the site of secretion and membrane adhesion, in maintaining the photoreceptor inner segment stability and architecture.


Subject(s)
Cell Adhesion Molecules/metabolism , Cell Membrane/metabolism , Eye Proteins/metabolism , Membrane Lipids/metabolism , Membrane Proteins/metabolism , Phospholipids/metabolism , Photoreceptor Cells, Vertebrate/metabolism , Animals , Anions , Blotting, Western , Cell Adhesion Molecules/ultrastructure , Cell Membrane/ultrastructure , Centrifugation, Density Gradient , Chromatography, Thin Layer , Electrophoresis, Polyacrylamide Gel , Eye Proteins/ultrastructure , Glutathione Transferase/metabolism , Membrane Proteins/ultrastructure , Mice , Mice, Inbred C57BL , Microscopy, Immunoelectron , Photoreceptor Cells, Vertebrate/ultrastructure , Recombinant Fusion Proteins/metabolism , Retinoschisis/metabolism , Subcellular Fractions
15.
J Mol Biol ; 225(4): 1127-30, 1992 Jun 20.
Article in English | MEDLINE | ID: mdl-1351949

ABSTRACT

Crystals of the two amino-terminal domains of intercellular adhesion molecule-1, the receptor for the major group of human rhinovirus serotypes, diffract to 3.0 A resolution. The crystals are trigonal in space group P3(1)21 or P3(2)21 with cell dimensions of a = b = 55.7 A, c = 166.3 A, with probably six molecules per unit cell.


Subject(s)
Cell Adhesion Molecules/chemistry , Receptors, Virus/chemistry , Rhinovirus/physiology , Antigens, CD/chemistry , Cell Adhesion Molecules/physiology , Cell Adhesion Molecules/ultrastructure , Humans , Intercellular Adhesion Molecule-1 , Microscopy, Electron/methods , Protein Conformation , Receptors, Virus/physiology , Receptors, Virus/ultrastructure , Rhinovirus/ultrastructure , X-Ray Diffraction/methods
16.
Tissue Eng ; 11(9-10): 1379-91, 2005.
Article in English | MEDLINE | ID: mdl-16259593

ABSTRACT

Biomedical implants often exhibit poor clinical performance due to the formation of a periimplant avascular fibrous capsule. Surface modification of synthetic materials has been evaluated to accelerate the formation of functional microcirculation in association with implants. The current study used a flow-mediated protein deposition system to modify expanded polytetrafluoroethylene (ePTFE) with a laminin-5-rich conditioned growth medium and with medium from which laminin-5 had been selectively removed. An in vitro model of endothelial cell adherence determined that laminin-5 modification resulted in significantly increased adhesion of human microvessel endothelial cells to ePTFE. In vivo studies evaluating the periimplant vascular response to laminin-5-treated samples indicated that absorption of laminin-5-rich conditioned medium supported accelerated neovascularization of ePTFE implants. A flow system designed to treat porous implant materials facilitates laminin-5 modification of commercially available ePTFE, resulting in increased endothelial cell adhesion in vitro and increased vascularization in vivo.


Subject(s)
Biocompatible Materials , Cell Adhesion Molecules , Cell Adhesion , Endothelium, Vascular/cytology , Neovascularization, Physiologic , Polytetrafluoroethylene , Adipose Tissue/immunology , Adipose Tissue/physiology , Animals , Cell Adhesion Molecules/ultrastructure , Cell Line , Cells, Cultured , Culture Media, Conditioned/pharmacokinetics , Endothelium, Vascular/ultrastructure , Histological Techniques , Humans , Immunohistochemistry , Mice , Microcirculation , Prostheses and Implants , Umbilical Veins/cytology , Kalinin
17.
Neuron ; 88(6): 1165-1172, 2015 Dec 16.
Article in English | MEDLINE | ID: mdl-26687224

ABSTRACT

The cleft is an integral part of synapses, yet its macromolecular organization remains unclear. We show here that the cleft of excitatory synapses exhibits a distinct density profile as measured by cryoelectron tomography (cryo-ET). Aiming for molecular insights, we analyzed the synapse-organizing proteins Synaptic Cell Adhesion Molecule 1 (SynCAM 1) and EphB2. Cryo-ET of SynCAM 1 knockout and overexpressor synapses showed that this immunoglobulin protein shapes the cleft's edge. SynCAM 1 delineates the postsynaptic perimeter as determined by immunoelectron microscopy and super-resolution imaging. In contrast, the EphB2 receptor tyrosine kinase is enriched deeper within the postsynaptic area. Unexpectedly, SynCAM 1 can form ensembles proximal to postsynaptic densities, and synapses containing these ensembles were larger. Postsynaptic SynCAM 1 surface puncta were not static but became enlarged after a long-term depression paradigm. These results support that the synaptic cleft is organized on a nanoscale into sub-compartments marked by distinct trans-synaptic complexes.


Subject(s)
Cell Adhesion Molecules/physiology , Cell Adhesion Molecules/ultrastructure , Immunoglobulins/physiology , Immunoglobulins/ultrastructure , Synapses/physiology , Synapses/ultrastructure , Animals , Cell Adhesion Molecule-1 , Cell Adhesion Molecules, Neuronal/physiology , Cell Adhesion Molecules, Neuronal/ultrastructure , Cells, Cultured , Hippocampus/physiology , Hippocampus/ultrastructure , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Microscopy, Immunoelectron , Neurons/physiology , Neurons/ultrastructure
18.
Nanoscale ; 7(1): 171-8, 2015 Jan 07.
Article in English | MEDLINE | ID: mdl-25406726

ABSTRACT

The interaction of a designed bioactive lipopeptide C16-GGGRGDS, comprising a hexadecyl lipid chain attached to a functional heptapeptide, with the lipid-free apoliprotein, Apo-AI, is examined. This apolipoprotein is a major component of high density lipoprotein and it is involved in lipid metabolism and may serve as a biomarker for cardiovascular disease and Alzheimers' disease. We find via isothermal titration calorimetry that binding between the lipopeptide and Apo-AI occurs up to a saturation condition, just above equimolar for a 10.7 µM concentration of Apo-AI. A similar value is obtained from circular dichroism spectroscopy, which probes the reduction in α-helical secondary structure of Apo-AI upon addition of C16-GGGRGDS. Electron microscopy images show a persistence of fibrillar structures due to self-assembly of C16-GGGRGDS in mixtures with Apo-AI above the saturation binding condition. A small fraction of spheroidal or possibly "nanodisc" structures was observed. Small-angle X-ray scattering (SAXS) data for Apo-AI can be fitted using a published crystal structure of the Apo-AI dimer. The SAXS data for the lipopeptide/Apo-AI mixtures above the saturation binding conditions can be fitted to the contribution from fibrillar structures coexisting with flat discs corresponding to Apo-AI/lipopeptide aggregates.


Subject(s)
Apolipoprotein A-I/chemistry , Cell Adhesion Molecules/chemistry , Lipopeptides/chemistry , Membrane Lipids/chemistry , Oligopeptides/chemistry , Amino Acid Motifs , Apolipoprotein A-I/ultrastructure , Binding Sites , Cell Adhesion Molecules/ultrastructure , Lipopeptides/ultrastructure , Materials Testing , Multiprotein Complexes/chemistry , Multiprotein Complexes/ultrastructure , Protein Binding , Protein Conformation , Protein Interaction Mapping
19.
J Comp Neurol ; 479(1): 15-29, 2004 Nov 01.
Article in English | MEDLINE | ID: mdl-15389614

ABSTRACT

p120 catenin (p120ctn) is involved in the regulation of cadherin-mediated adhesion and the dynamic organization of the actin cytoskeleton by modulating RhoGTPase activity. We have previously described the distribution of p120ctn during rat brain development and provided substantial evidence for the potential involvement of p120ctn in morphogenetic events and plasticity in the central nervous system. Here, we analyzed the cellular and ultrastructural distribution of p120ctn in glial cells of the adult rat forebrain. The highest intensity of immunostaining for p120ctn was found in cells of the choroid plexus and ependyma and was mainly restricted to the plasma membrane. However, p120ctn was almost absent from astrocytes. In contrast, in tanycytes, a particular glial cell exhibiting remarkable morphological plasticity, p120ctn, was localized at the plasma membrane and also in the cytoplasm. We show that a large subpopulation of oligodendrocytes expressed multiple isoforms, whereas other neural cells predominantly expressed isoform 1, and that p120ctn immunoreactivity was distributed through the cytoplasm and at certain portions of the plasma membrane. Finally, p120ctn was expressed by a small population of cortical NG2-expressing cells, whereas it was expressed by a large population of these cells in the white matter. However, in both regions, proliferating NG2-positive cells consistently expressed p120ctn. The expression of p120ctn by cells of the oligodendrocyte lineage suggests that p120ctn may participate in oligodendrogenesis and myelination. Moreover, the expression of p120ctn by various cell types and its differential subcellular distribution strongly suggest that p120ctn may serve multiple functions in the central nervous system.


Subject(s)
Cell Adhesion Molecules/metabolism , Cell Membrane/metabolism , Neuroglia/metabolism , Phosphoproteins/metabolism , Prosencephalon/metabolism , Animals , Antigens/metabolism , Catenins , Cell Adhesion Molecules/ultrastructure , Cell Membrane/ultrastructure , Choroid Plexus/metabolism , Choroid Plexus/ultrastructure , Ependyma/metabolism , Ependyma/ultrastructure , Female , Immunohistochemistry , Neuroglia/classification , Neuroglia/ultrastructure , Oligodendroglia/metabolism , Oligodendroglia/ultrastructure , Phosphoproteins/ultrastructure , Prosencephalon/ultrastructure , Protein Isoforms/metabolism , Protein Isoforms/ultrastructure , Proteoglycans/metabolism , Rats , Rats, Sprague-Dawley , Tissue Distribution , Delta Catenin
20.
J Histochem Cytochem ; 49(7): 809-19, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11410606

ABSTRACT

The aim of this study was to develop a model for the detection of individual cell adhesion molecules (CAMs) in the glycocalyx of spread human platelets using high-resolution cryo-field emission scanning electron microscopy (cryoFESEM). Three surface glycoprotein CAMs, P-selectin (CD62P), GPIba in the GPI-IX complex (CD42a/CD42b alpha,b beta), and the integrin GPIIbIIIa (CD41/CD61) in the human platelet were selected on the basis of their unique topographic shape. Spread human platelets were indirectly immunolabeled with 10-nm colloidal gold and then cryoimmobilized. After sublimation of water from the cryoimmobilized sample, partially freeze-dried platelets were coated unidirectionally with Pt, stabilized with carbon, and examined in an in-lens cryoFESEM using high-resolution backscattered electron imaging. CAMs were detected by indirect immunogold labeling and the length of each type of CAM was determined using analysis of differences in parallax as measured in the software program Sterecon. Our results demonstrate the efficacy of using high-resolution cryoFESEM to recognize and detect individual CAMs in the glycocalyx. Further advances in production of metal coatings with finer granularity, together with improvements in imaging (tilting and angle of stereo images), may provide better definition of the topography associated with glycosylation and formation of multimeric CAM complexes. (J Histochem Cytochem 49:809-819, 2001)


Subject(s)
Blood Platelets/metabolism , Cell Adhesion Molecules/metabolism , Glycocalyx/metabolism , Antigens, CD/metabolism , Antigens, CD/ultrastructure , Blood Platelets/ultrastructure , Cell Adhesion Molecules/ultrastructure , Cryoelectron Microscopy , Humans , Image Processing, Computer-Assisted , Immunohistochemistry , Integrin beta3 , Microscopy, Electron, Scanning , P-Selectin/metabolism , P-Selectin/ultrastructure , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Platelet Glycoprotein GPIIb-IIIa Complex/ultrastructure , Platelet Glycoprotein GPIb-IX Complex/metabolism , Platelet Glycoprotein GPIb-IX Complex/ultrastructure , Platelet Membrane Glycoproteins/metabolism , Platelet Membrane Glycoproteins/ultrastructure
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