Methods to estimate the polarized distribution of surface antigens in cultured epithelial cells.
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Animals↗
Biomedical subjects
M Sargiacomo
Publications and source records attributed to M Sargiacomo.
Steady-state distribution and biogenesis of endogenous Madin-Darby canine kidney glycoproteins: evidence for intracellular sorting and polarized cell surface delivery.
We used domain-selective biotinylation/125I-streptavidin blotting (Sargiacomo, M., M. P. Lisanti, L. Graeve, A. Le Bivic, and E. Rodriguez-Boulan. 1989 J. Membr. Biol. 107:277-286), in combination with lectin precipitation, to analyze the apical and basolateral glycoprotein composition of Madin-Darby canine kidney (MDCK) cells and to explore the role of glycosylation in the targeting of membrane glycoproteins. All six lectins used recognized both apical and basolateral glycoproteins, indicating that none of the sugar moieties detected were characteristic of the particular epithelial cell surface. Pulse-chase experiments coupled with domain-selective glycoprotein recovery were designed to detect the initial appearance of newly synthesized glycoproteins at the apical or basolateral cell surface. After a short pulse with a radioactive precursor, glycoproteins reaching each surface were biotinylated, extracted, and recovered via precipitation with immobilized streptavidin. Several basolateral glycoproteins (including two sulfated proteins) and at least two apical glycoproteins (one of them the major sulfated protein of MDCK cells) appeared at the corresponding surface after 20-40 min of chase, but were not detected in the opposite surface, suggesting that they were sorted intracellularly and vectorially delivered to their target membrane. Several "peripheral" apical proteins were detected at maximal levels on the apical surface immediately after the 15-min pulse, suggesting a very fast intracellular transit. Finally, domain-selective labeling of surface carbohydrates with biotin hydrazide (after periodate oxidation) revealed strikingly different integral and peripheral glycoprotein patterns, resembling the Con A pattern, after labeling with sulfo-N-hydroxy-succinimido-biotin. The approaches described here should be useful in characterizing the steady-state distribution and biogenesis of endogenous cell surface components in a variety of epithelial cell lines.
Animals↗
Polarized apical distribution of glycosyl-phosphatidylinositol-anchored proteins in a renal epithelial cell line.
Polarized epithelial cell monolayers contain two distinct plasma membrane domains as delineated by the presence of tight junctions--i.e., an apical surface that faces the external environment and a basolateral surface that functions both in cell-cell contact and cell-substrate attachment. Central to the understanding of epithelial cell polarity is the question of how such cell-surface specializations are generated. A different class of membrane glycoproteins has recently emerged that may yield new insight into the mechanism underlying the biogenesis of this polarity. Members of this class contain a large extracellular protein domain linked to the membrane via glycosyl-phosphatidylinositol. Using a polarized renal epithelial cell line (Madin-Darby canine kidney), we identified endogenous glycosyl-phosphatidylinositol-anchored proteins through release by a phosphatidylinositol-specific phospholipase C. Six glycosyl-phosphatidylinositol-anchored proteins of 110, 85, 70, 55, 38, and 35 kDa were identified and appeared to be restricted to the apical surface. Our data are consistent with the notion that the glycosyl-phosphatidylinositol membrane anchor may contain the necessary information for "targeting" to the apical surface.
Animals↗
Cytotoxicity acquired by ribosome-inactivating proteins carried by reconstituted Sendai virus envelopes.
Association of the ribosome-inactivating proteins (RIPs): pokeweed antiviral protein (PAP), gelonin, Momordica charantia inhibitor (MCI), with reconstituted Sendai virus envelopes (RSVE) was obtained without detectable loss of activities either of RIPs or of viral envelope glycoproteins. RIPs are inactive towards intact cells, but, once encapsulated in RSVE, they become cytotoxic. The concentration of RSVE-associated PAP, which causes 50% inhibition of protein synthesis by Friend erythroleukemic cells, is 0.5 ng/ml. Substances capable to inhibit the viral activities block the acquired cytotoxicity of RIPs associated to RSVE.
Animals↗
Interaction of ricin-sensitive and ricin-resistant cell lines with other carbohydrate-binding toxins.
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Animals↗
Cholera toxin B-subunit protects mammalian cells from ricin and abrin toxicity.
The glycoproteins ricin and abrin intoxicate cells by inhibiting protein synthesis. Pretreatment of HeLa cells with cholera toxin partially protects them from ricin and abrin activity. The involvement in this phenomenon of the various effects of cholera toxin, namely, redistribution of membrane receptors elicited from protomer B and increasing cyclic AMP concentrations induced by protomer A, were studied. Substances able to enhance cyclic AMP concentrations do not affect ricin and abrin activity, while protomer B alone protects cells. In addition, the effects of several lectins on ricin or abrin toxicity were examined. Almost complete prevention of ricin or abrin activity was obtained using concanavalin A (Con A) and wheat germ agglutinin (WGA). Conversely, neither succinyl Con A nor Ulex europeus agglutinin (UEA) affected the cellular response. Both protomer B of cholera toxin and Con A did not alter the binding of ricin or abrin; they seem to protect cells by altering membrane structure.
Abrin↗
A cytotoxic, photolabile cross-linking derivative of ricin. Action on various cells and application to the study of ricin toxicity.
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Animals↗
Replication and multiplication of hepatitis C virus genome in human foetal liver cells.
The ability to obtain primary long-term cultures of human foetal hepatocytes maintaining liver differentiation characteristics in serum-free medium prompted us to test their susceptibility to hepatitis C virus infection. Using PCR, we detected the presence of the HCV RNA-positive strand in the supernatants and in the cells of the virus-infected hepatocyte cultures, at various times post-infection. Evidence of effective virus genome replication and multiplication was also based on the time-dependent appearance of the putative HCV RNA-negative strand, the detection of virus replicative intermediates and an increase in HCV genomic templates in the HCV-infected cells.
Cells, Cultured↗
Caveolae, transmembrane signalling and cellular transformation.
Caveolae are approximately 50-100 nm membrane micro-invaginations associated with the plasma membrane of a wide variety of cells. Although they were first identified in transmission electron micrographs approximately 40 years ago, their exact function(s) has remained controversial. Two well-established functions include: (1) the transcytosis of both large and small molecules across capillary endothelial cells and (2) the utilization of GPI-linked proteins to concentrate small molecules in caveolae for translocation to the cytoplasm (termed potocytosis). Recently, interest in a 'third' proposed caveolar function, namely transmembrane signalling, has been revived by the identification of caveolin--a transformation-dependent v-Src substrate and caveolar marker protein--and the isolation of caveolin-rich membrane domains from cultured cells. Here we will discuss existing evidence that suggests a role for caveolae in signalling events.
Biomarkers↗