Expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins in the developing pancreas: roles in the adhesion and migration of putative endocrine progenitor cells.

Cell-cell and cell-matrix interactions play a critical role in tissue morphogenesis and in homeostasis of adult tissues. The integrin family of adhesion receptors regulates cellular interactions with the extracellular matrix, which provides three-dimensional information for tissue organization. It is currently thought that pancreatic islet cells develop from undifferentiated progenitors residing within the ductal epithelium of the fetal pancreas. This process involves cell budding from the duct, migration into the surrounding mesenchyme, differentiation, and clustering into the highly organized islet of Langerhans. Here we report that alpha(v)beta(3) and alpha(v)beta(5), two integrins known to coordinate epithelial cell adhesion and movement, are expressed in pancreatic ductal cells and clusters of undifferentiated cells emerging from the ductal epithelium. We show that expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins are developmentally regulated during pancreatic islet ontogeny, and mediate adhesion and migration of putative endocrine progenitor cells both in vitro and in vivo in a model of pancreatic islet development. Moreover, we demonstrate the expression of fibronectin and collagen IV in the basal membrane of pancreatic ducts and of cell clusters budding from the ductal epithelium. Conversely, expression of vitronectin marks a population of epithelial cells adjacent to, or emerging from, pancreatic ducts. Thus, these data provide the first evidence for the contribution of integrins alpha(v)beta(3) and alpha(v)beta(5) and their ligands to morphogenetic events in the human endocrine pancreas.

Disruption of alpha3 connexin gene leads to proteolysis and cataractogenesis in mice.

Gap junction channels formed by alpha3 (Cx46) and alpha8 (Cx50) connexin provide pathways for communication between the fiber cells in the normal transparent lens. To determine the specific role of alpha3 connexin in vivo, the alpha3 connexin gene was disrupted in mice. Although the absence of alpha3 connexin had no obvious influence on the early stages of lens formation and the differentiation of lens fibers, mice homozygous for the disrupted alpha3 gene developed nuclear cataracts that were associated with the proteolysis of crystallins. This study establishes the importance of gap junctions in maintaining normal lens transparency by providing a cell-cell signaling pathway or structural component for the proper organization of lens membrane and cytoplasmic proteins.

Vascular cell adhesion molecule-1 is expressed by cortical thymic epithelial cells and mediates thymocyte adhesion. Implications for the function of alpha4beta1 (VLA4) integrin in T-cell development.

T-cell development requires a series of discrete selection and activation signals delivered to maturing progenitors in the thymic cortex and medulla. We have previously shown the constitutive activity of the integrin, alpha4beta1 (VLA4), on a unique subpopulation of immature cortical thymocytes and proposed a role for integrin-mediated adhesion in positive selection by cortical epithelium. In the present report we show that thymic epithelial cell lines express vascular cell adhesion molecule-1 (VCAM-1) a high-affinity ligand for apha4beta1, and that VCAM-1 mediates thymocyte binding to these lines. Immunohistochemistry and confocal microscopy show that VCAM-1 is selectively expressed in situ by thymic epithelium in the cortex and corticomedullary junction, two locations at which VCAM-1 could determine the interaction between immature thymocytes and selecting elements on epithelial cells. In parallel, we confirmed that fibronectin (FN), the alternative ligand for alpha4beta1, is expressed predominantly in the medulla. These results suggest that VCAM-1 is an adhesive ligand in the thymic cortex for the activated form of alpha4beta1 constitutively expressed during development by immature double positive thymocytes. The structural segregation of the alternative ligand, FN, to the medulla suggests that medullary FN may regulate the migration, development, and export of more mature thymocytes.

Structural analysis of the p62 complex, an assembly of O-linked glycoproteins that localizes near the central gated channel of the nuclear pore complex.

The p62 complex is an oligomeric assembly of O-linked glycoproteins of the nuclear pore complex that interacts with cytosolic transport factors and is part of the machinery for nuclear protein import. In this study we have purified the p62 complex from rat liver nuclear envelopes and analyzed its structure and composition. The p62 complex consists of four distinct polypeptides (p62, p58, p54, and p45) and has a mass of approximately 234 kDa, calculated from its hydrodynamic properties and supported by chemical cross-linking and scanning transmission electron microscopy. These data suggest that the p62 complex contains one copy of each constituent polypeptide. Analysis of preparations of the p62 complex by electron microscopy using rotary metal shadowing and negative staining revealed donut-shaped particles with a diameter of approximately 15 nm. Immunogold electron microscopy of isolated rat liver nuclear envelopes demonstrated that p62 occurs on both the nucleoplasmic and cytoplasmic sides of the pore complex near the central gated channel involved in active transport of proteins and RNAs. The properties and localization of the p62 complex suggest that it may be involved in binding transport ligands near the center of the nuclear pore complex and in subsequently transferring them to the gated transport channel.

Trophinin and tastin, a novel cell adhesion molecule complex with potential involvement in embryo implantation.

Two human epithelial cell lines, trophoblastic teratocarcinoma HT-H and endometrial adenocarcinoma SNG-M cells, adhere to each other at their respective apical cell surfaces in a divalent cation-independent manner. Two novel molecules responsible for the adhesion between these two cell types were identified by expression cDNA cloning. One, named trophinin, is an intrinsic membrane protein and mediates homophilic self-binding. Another, named tastin, is a cytoplasmic protein and is necessary for trophinin to function as a cell adhesion molecule. Trophinin and tastin appear to be associated with the cytoskeleton in HT-H and SNG-M cells. These molecules are normally not expressed in various types of human cells in tissues, with the exception of macrophages. Strong expression of these molecules was detected in the trophectoderm surface of monkey blastocyst. These molecules are also expressed in human endometrial surface epithelium on day 16/17 at the early secretory phase of human endometrium, the time consistent with that expected for the "implantation window."

Gradient of integrin alpha 6A distribution in the myocardium during early heart development.

The interactions of cells with extracellular matrices (ECM)1 are likely to be key determinants of embryonic development. Integrin adhesion receptors are ideally positioned to mediate some of these interactions since, in addition to mechanical adhesion, they transduce signals affecting cell proliferation and differentiation. We investigated expression of the integrin alpha 6 beta 1, a receptor for the ECM component, laminin in the early mouse embryo. An intriguing feature of this integrin is the existence of alpha 6 subunit isoforms. The A and B isoforms, which differ in the cytoplasmic tails, are expressed in cell-type specific fashion, and are likely to implement distinct cellular interactions with laminin. By RT-PCR, alpha 6B but not alpha 6A mRNA was detectable in embryo extracts from fertilized oocytes to 6.5 d.p.c. In subsequent stages, up to 11.5 d.p.c., alpha 6A mRNA was observed in mRNA extracts from whole embryos, but still in significantly lower amounts than alpha 6B. However, in extracts from isolated heart (9.5 to 11.5 d.p.c.), alpha 6A was the predominant alpha 6 isoform, while in extracts from other embryo parts no alpha 6A mRNA was detectable. At the protein level, immunostaining with specific antibodies showed alpha 6A protein in myocardial cells, at the early stage of heart tube development (8.5 d.p.c.). Localization to the myocardium was tightly restricted, since other structures of the embryonic heart, e.g., endocardium, or of the remaining embryo did not stain with anti-alpha 6A antibody. In the ventricular myocardium, expression of alpha 6A appeared more intense than in the subendocardial layer. Quantitation by confocal microscopy unveiled a gradient of expression of alpha 6A, increasing from the outer to the inner layers of the myocardium. This is the first demonstration of a gradient distribution of integrin molecules in a tissue, which appears to be directly connected with the process of organogenesis. The mechanism underlying our observations is not the turning on of a gene, rather it is the activation of a splicing mechanism that substitutes the cytoplasmic domain of a laminin receptor. Because integrin cytoplasmic domains are thought to be an important functional end of the molecule, this may be a mechanism to modulate cellular responses to laminin.

Integrin alpha v beta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels.

A single intravascular injection of a cyclic peptide or monoclonal antibody antagonist of integrin alpha v beta 3 disrupts ongoing angiogenesis on the chick chorioallantoic membrane (CAM). This leads to the rapid regression of histologically distinct human tumors transplanted onto the CAM. Induction of angiogenesis by a tumor or cytokine promotes vascular cell entry into the cell cycle and expression of integrin alpha v beta 3. After angiogenesis is initiated, antagonists of this integrin induce apoptosis of the proliferative angiogenic vascular cells, leaving preexisting quiescent blood vessels unaffected. We demonstrate therefore that ligation of integrin alpha v beta 3 is required for the survival and maturation of newly forming blood vessels, an event essential for the proliferation of tumors.

Defective glycosylation of erythrocyte membrane glycoconjugates in a variant of congenital dyserythropoietic anemia type II: association of low level of membrane-bound form of galactosyltransferase.

Congenital dyserythropoietic anemia type II (CDA II) or HEMPAS is a genetic disease caused by plasma membrane abnormality. The enzymic defect of HEMPAS has been suggested to be the lowered activity of N-acetylglucosaminyltransferase II, resulting in lack of polylactosamine formation on proteins and leading to accumulation of polylactosaminyl lipids. In contrast to typical HEMPAS cases, cell-surface labeling of the erythrocytes of a HEMPAS variant G.K. showed an absence of polylactosamines either on proteins or on lipids. Fast-atom bombardment mass spectrometry analysis of G.K.'s erythrocyte glycopeptides detected a series of high mannose-type oligosaccharides, which were not detected in erythrocyte N-glycans of normal cells or of other HEMPAS cases: The former contains polylactosaminoglycans and the latter contains hybrid-type oligosaccharides. Keratansulfate (sulfated polylactosamines) in this patient's serum was abnormally low. The galactosyltransferase activity in microsomal membranes prepared from G.K.'s mononucleated cells was 24% of the normal level, whereas this enzyme activity in G.K.'s serum was comparatively higher than normal. Western blotting of G.K.'s membranes using antigalactosyltransferase antibodies showed that G.K. has reduced amounts of this enzyme present. The results collectively suggest that variant G.K. is defective in polylactosamine synthesis owing to the decreased quantity of the membrane-bound form of galactosyltransferase.

Congenital dyserythropoietic anaemia type II (HEMPAS): characterization of aberrant intracellular organelles by immunogold electron microscopy.

Erythrocytes are reticulocytes obtained from peripheral blood of a congenital dyserythropoietic anaemia type II patient were examined by immunoelectron microscopy using anti-band 3 and anti-glycophorin A antibodies. Vacuoles which have membranous structures inside were stained heavily by these antibodies, indicating the presence of band 3 and glycophorin A in these vacuoles. Empty vacuoles which open to the cell surface and those present in cytoplasm were also stained by these antibodies at the inside of the wall. These observations suggest that the vacuoles are functioning for trapping and discarding the defective plasma membranes. On the other hand, small vesicles and ferritin-loaded vacuoles were not stained by these antibodies. In these experiments, peripheral cisternae and most of the intracellular membranous structures at blebs and clefts were not stained by the antibodies. Therefore, they are probably part of the endoplasmic reticulum or are derived from intracellular organelles but not from the plasma membranes.

Anomalous clustering of underglycosylated band 3 in erythrocytes and their precursor cells in congenital dyserythropoietic anemia type II.

Congenital dyserythropoietic anemia type II (CDA II or HEMPAS) is a genetic anemia caused by membrane abnormality. Our previous studies indicated that in HEMPAS, erythrocytes band 3 and band 4.5 are not glycosylated by polylactosaminoglycans. The present study was aimed at determining how such underglycosylated band 3 behaves in erythrocyte membranes. By using anti-band 3 antibodies, immunogold electron microscopy revealed that band 3s are clustered in HEMPAS erythrocyte membranes. By freeze-fracture electron microscopy, band 3s were also seen as lightly clumped intramembrane particles on a protoplasmic fracture face. Erythrocyte precursor cells stained by anti-band 3 antibodies showed that band 3s are present in the cytoplasmic area of the reticulocytes as scattered single particles. However, in young erythrocytes in which intracellular membranes are almost degenerated, band 3s were clustered in the cytoplasmic area of the cell. These observations suggest that band 3s cluster before they are incorporated into the plasma membranes of HEMPAS erythrocytes. In contrast to band 3, glycophorin A detected by anti-glycophorin A antibodies did not show a noticeable difference between normal and HEMPAS. Such a clustering of band 3 may cause abnormal localization of band 3-associated proteins and may thus result in the macroscopic membrane abnormality seen in HEMPAS erythrocytes.

Molecular assembly, secretion, and matrix deposition of type VI collagen.

Monoclonal antibodies reactive with the tissue form of type VI collagen were used to isolate the type VI collagen polypeptides from cultured fibroblasts and muscle cells. Two [35S]methionine-labeled polypeptides of 260 and 140 kD were found intracellularly, in the medium, and in the extracellular matrix of metabolically labeled cells. These polypeptides were disulfide cross-linked into very large complexes. The 260- and 140-kD polypeptides were intimately associated and could not be separated from each other by reduction without denaturation. In the absence of ascorbic acid, both polypeptides accumulated inside the cell, and their amounts in the medium and in the matrix were decreased. These results suggest that both the 260- and the 140-kD polypeptides are integral parts of the type VI collagen molecule. Examination of type VI collagen isolated from the intracellular pool by electron microscopy after rotary shadowing revealed structures corresponding to different stages of assembly of type VI collagen. Based on these images, a sequence for the intracellular assembly of type VI collagen could be discerned. Type VI collagen monomers are approximately 125 nm long and are composed of two globules separated by a thin strand. The monomers assemble into dimers and tetramers by lateral association. Only tetramers were present in culture media, whereas both tetramers and multimers were found in extracellular matrix extracts. The multimers appeared to have assembled from tetramers by end-to-end association into filaments that had prominent knobs and a periodicity of approximately 110 nm. These results show that, unlike other collagens, type VI collagen is assembled into tetramers before it is secreted from the cells, and they also suggest an extracellular aggregation mechanism that appears to be unique to this collagen.

Ultrastructural changes accompanying the induced differentiation of clonal rat nerve and glia.

Dibutyryladenosine 3'-5' monophosphate (DBcAMP) induces ultrastructural transformations in clonal rat nerve and glia. Cells cultured in the presence of DBcAMP contain large numbers of aligned microtubules and microfilaments in their elongated processes, while control cultures without DBcAMP are relatively devoid of these structures. In addition, the cells exposed to DBcAMP contain 2000 A to 6000 A vesicles which are not observed in control cells.