Journal of cell science. Supplement最新文献

Cystic fibrosis is associated with defective epithelial sodium chloride and fluid secretion in epithelia. In addition, there is widespread reductions in sialylation of secreted proteins and increases in the sulfation and fucosylation of mucus glycoproteins. The major morbidity in the disease is due to the colonization of respiratory epithelia by Pseudomonas. The cystic fibrosis gene (CFTR) is a cyclic AMP activated Cl channel, which when mutated is retained in the endoplasmic reticulum. We postulate that this Cl channel is responsible for effective acidification of the Golgi. In CF cells, we demonstrate the Golgi pH is higher than in normal cells and suggest that the abnormalities in glycoprotein biosynthesis is due to changes in the kinetics of sialyl transferase, a pH sensitive enzyme. Defects in sialylation also result in decreased sialylation of glycolipids and asialogangliosides are potential Pseudomonas receptors.

The polarization of intestinal epithelial cells and the stereotypic arrangement of their actin-based cytoskeleton have made these epithelia an excellent system to explore the organization and formation of a cortical actin-based cytoskeleton. Through a combined morphological and biochemical analysis, the molecular arrangement of many of the components of the brush border has been elucidated. Study of brush border assembly in the Crypts of Lieberkühn suggests that cytoskeletal mRNA and protein expression, as well as morphological development, occur rapidly following cell differentiation. Protein kinases appear to be important regulators of intestinal cell growth, for differentiating cells in the crypts possess 15-fold higher levels of tyrosine phosphorylated proteins than differentiated cells of the villus. One of these kinases, pp60c-src, has a 4- to 7-fold higher activity in crypts and increased association with the cytoskeleton than it has in villus cells. The development and maintenance of polarization in epithelial cells require the targeting and transport of specific proteins to the apical and basolateral plasma membrane. It has been proposed that a dynein-like, microtubule-based motor is involved in the transport of apically directed materials from the trans-Golgi to the apical plasma membrane. However, microtubules do not reach the plasma membrane, but terminate below the actin-rich network of filaments comprising the terminal web. We propose that vesicles translocate from the Golgi to the apical cytoplasm along microtubules using dynein, and then move through the terminal web to reach the apical plasma membrane using the actin-based motor myosin-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Transepithelial transport of antigens by M cells in the epithelium associated with lymphoid follicles in the intestine delivers immunogens directly to organized mucosal lymphoid tissues, the inductive sites for mucosal immune responses. We have exploited M cell transport to generate and characterize specific monoclonal IgA antibodies that can prevent interaction of pathogens with epithelial surfaces. The relative protective capacities of specific monoclonal IgA antibodies have been tested in vivo by generation of hybridoma tumors that result in secretion of monoclonal IgA into the intestine. Using this method, we have established that secretion of IgA antibodies recognizing a single surface epitope on enteric pathogens can provide protection against colonization or invasion of the intestinal mucosa.

We have been interested in determining how epithelial cells generate and maintain their characteristically polarized distributions of membrane proteins. Our efforts to date strongly indicate that the polarized transport in MDCK cells may be due to a set of discrete targeting determinants often found on a membrane protein's cytoplasmic domain. Surprisingly, these determinants are widely distributed and are not specific to proteins expressed in polarized cells. They also appear to function in controlling polarized transport along both the biosynthetic and the endocytic (or transcytotic) pathways. Signals for basolateral transport have been characterized and, like the cytoplasmic domain signal used by plasma membrane receptors for accumulation at clathrin-coated pits, they often involve a critical tyrosine residue. Although the basolateral and coated pit signals may also be co-linear, they are not identical. The basolateral and apical transport determinants are also hierarchically arranged. Although a single protein may contain one or more signals specifying basolateral transport, inactivation of these signals appears to reveal a determinant that directs efficient apical transport. Given that the sequence determinants responsible for polarized transport are not restricted to epithelial cells and are related to determinants commonly utilized in all cells, it is possible that non-polarized cells contain cognate apical and basolateral pathways that are responsible for 'constitutive' transport from the Golgi to the plasma membrane. The presence of two cognate pathways might confer a high degree of plasticity to pre-differentiated cells, allowing them rapidly to begin assuming a polarized phenotype in response to extracellular stimuli without requiring the synthesis of epithelial cell-specific transport machinery.

A family of ankyrin-binding glycoproteins have been identified in adult rat brain that include alternatively spliced products of the same pre-mRNA. A composite sequence of ankyrin-binding glycoprotein (ABGP) shares 72% amino acid sequence identity with chicken neurofascin, a membrane-spanning neural cell adhesion molecule in the Ig super-family expressed in embryonic brain. ABGP polypeptides and ankyrin associate as pure proteins in a 1:1 molar stoichiometry at a site located in the predicted cytoplasmic domain. ABGP polypeptides are expressed late in postnatal development to approximately the same levels as ankyrin, and comprise a significant fraction of brain membrane proteins. Immunofluorescence studies have shown that ABGP polypeptides are co-localized with ankyrinB. Major differences in developmental expression have been reported for neurofascin in embryos compared with the late postnatal expression of ABGP, suggesting that ABGP and neurofascin represent products of gene duplication events that have subsequently evolved in parallel with distinct roles. Predicted cytoplasmic domains of rat ABGP and chicken neurofascin are nearly identical to each other and closely related to a group of nervous system cell adhesion molecules with variable extracellular domains, including L1, Nr-CAM and Ng-CAM of vertebrates, and neuroglian of Drosophila. A hypothesis to be evaluated is that ankyrin-binding activity is shared by all of these proteins.

Wnt genes encode secreted glycoproteins, and, because of their homology with the Drosophila segment polarity gene wingless, are likely to play important roles as modulators of local intercellular signalling during embryonic development. Although little is known of the mechanisms by which Wnts signal in an autocrine or paracrine manner, it is increasingly clear that cells can respond rapidly to Wnt signals in the absence of transcription, and that these responses may include changes in cell adhesion and cell movement. We review recent evidence from studies on Xenopus laevis and other systems, which demonstrate that (1) a subset of Wnts modulate gap junctional permeability, which may be a reflection of changes in cadherin-mediated cell adhesion, (2) embryos express beta-catenin and plakoglobin, which are homologs of the armadillo gene products, known to be involved in the wingless signalling pathway, and known to be found at cell junctions, and (3) overexpression of specific Wnts in Xenopus embryos leads to clear changes in cell behavior and movement.

The preimplantation embryo differentiates the trophectoderm epithelium which, from the 32-cell stage, generates the blastocoel of the blastocyst and, after implantation, gives rise to most extraembryonic lineages of the conceptus. Trophectoderm differentiation begins at compaction (8-cell stage) when cell-cell adhesion, mediated by uvomorulin, and epithelial cell polarisation first occur. Here, we review our work on the biogenesis of tight junctions and desmosomes during epithelial differentiation. Tight junction construction begins at compaction and appears to be a gradual process, both at morphological and molecular levels. This maturation pattern may be due in part to sequential expression of tight junction constituents from the embryonic genome. Tight junction formation is dependent upon uvomorulin adhesion but can be inhibited by different means without apparently disturbing cell adhesion or polarisation. Cell interactions appear to regulate tight junction tissue specificity, in part by controlling the level of synthesis of constituents. Desmosome formation begins at the 32-cell stage, particularly as the embryo initiates blastocoel accumulation, and, in contrast with tight junction formation, does not appear to be a gradual process. Thus, nascent desmosomes appear mature in terms of their molecular composition. Desmosomal proteins are synthesised well in advance of desmosome formation but the synthesis of the principal glycoprotein components begins at the blastocyst stage and may regulate the timing of junction assembly. Implications of these differing patterns of biogenesis for the embryo are discussed.

The catenins are polypeptides that bind to the conserved cytoplasmic tail of cadherins and are required for cadherin function. alpha-Catenin is related to vinculin and seems to be required for the interaction of cadherins with the actin cytoskeleton. beta-Catenin is homologous to armadillo, a segment polarity gene in Drosophila that participates in developmental signaling. Recent findings indicate that beta-catenin also participates in developmental signaling and embryonic patterning in Xenopus laevis. At least a portion of the electrophoretic band migrating at the position of gamma-catenin consists of plakoglobin, a desmosomal and zonula adherens protein that has high sequence similarity to beta-catenin and armadillo. The catenins may be involved in the regulation of cadherin function during tissue morphogenesis and tumorigenesis.

The cystic fibrosis transmembrane conductance regulator (CFTR) is mutated in patients with cystic fibrosis (CF). The most common CF-associated mutation is deletion of phenylalanine at residue 508, CFTR delta F508. When expressed in heterologous cells, CFTR bearing the delta F508 mutation fails to progress through the normal biosynthetic pathway and fails to traffic to the plasma membrane. As a result, CFTR delta F508 is mislocalized and is not present in the apical membrane of primary cultures of airway epithelia. Consequently, the apical membrane of CF airway epithelia is Cl- -impermeable, a defect that probably contributes to the pathogenesis of the disease.

In simple epithelia, specialized vectorial functions such as transport and secretion are made possible by the segregation of proteins and lipids into opposite surface domains. This polarized distribution results from selective delivery to and retention at the appropriate domain. In the case of direct delivery, the sorting site for apical and basolateral proteins is the trans-Golgi network (TGN) where they are incorporated into distinct apical and basolateral vesicles that are targeted to the respective surfaces. The machinery that controls this simple process is in fact rather complicated. It involves many different steps from the recognition event (between 'sorting signal(s)' and 'sorting receptor(s)' to the formation of the vesicles, their budding, and the docking to the specialized plasma membrane domain. Here we summarize the latest developments in the sorting of apical and basolateral proteins, focusing in particular on the signals that are involved in this process and the current hypotheses about the mechanisms responsible for it, in both epithelia and in non-polarized cells.