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A L Hubbard

Publications and source records attributed to A L Hubbard.

At least 37 records · Page 2Linked to original sources
PubMed1994

Biosynthesis and intracellular transport of a bile canalicular plasma membrane protein: studies in vivo and in the perfused rat liver.

B10 is an integral glycoprotein of the plasma membrane that is exclusively localized to the canalicular (apical) domain in normal rat hepatocytes but may be expressed on the basolateral (sinusoidal and lateral) membrane in pathophysiological situations. To understand how B10 may be localized to the basolateral surface, we studied the biosynthesis and transport of this apical protein. In vivo pulse-chase experiments, followed by subcellular fractionation of the liver and immunoprecipitation, showed that B10 is first synthesized as a high-mannose form of 123 kD and then matured to a complex glycosylated form of 130 kD, which peaks in the Golgi apparatus after 15 min of chase and reaches the plasma membrane with a half-time of 30 to 45 min. Analysis of the protein in plasma membrane domain fractions showed that most of the newly synthesized molecule was localized in basolateral fractions after 30 min of chase and subsequently appeared in apical fractions. After 90 min of chase, most of the radiolabeled protein had reached its steady-state apical distribution. The same experiments performed in the perfused rat liver, in which the chase can be improved, gave similar results, except that the apical distribution of the radioactive molecule was attained more quickly. Thus B10, like all apical plasma membrane proteins studied so far in hepatocytes, is first transported to the basolateral surface and then reaches the membrane of the bile canaliculi. Alterations of the transcytotic step from the basolateral to the apical surfaces may result in abnormal basolateral localization.

Animals↗
PubMed1994

Critical determination of the frequency of c-erbB-2 amplification in breast cancer.

Tissues from 323 methacarn-fixed and paraffin-embedded breast cancers were assessed for c-erbB-2 gene amplification by differential polymerase chain reaction (dPCR). The sensitivity of dPCR was ascertained using cell lines with c-erbB-2 amplification, and the relationship between dPCR ratio value and gene copy number was established. In clinical material the technique was not affected by the DNA contribution of normal tissue elements or by cancer DNA ploidy change. c-erbB-2 gene amplification was detected in 55% of invasive cancers and in 66% of in situ cancers. c-erbB-2 protein overexpression in breast cancer cells, as determined by specific immunohistochemistry, was only detected in 11% of invasive cancers and 43% of in situ cancers. Comparisons show that a substantial number of cancers with c-erbB-2 amplification lack detectable protein overexpression. This illustrates the complex nature of c-erbB-2 gene disregulation in cancer and suggests that multiple combinations of biological events and consequences are possible.

Aged↗
PubMed1994

An improved polarized rat hepatoma hybrid cell line. Generation and comparison with its hepatoma relatives and hepatocytes in vivo.

Studies of hepatocyte polarity, an important property of liver epithelial cells, have been hampered by the lack of valid in vitro models. We report here that a new polarized hepatoma-derived hybrid cell line, called WIF-B, has improved characteristics to those of its parent, WIF12-1. This latter line originated from the fusion of non-polarized rat hepatoma Fao cells with human fibroblasts (WI-38) and selection for a polarized phenotype. We generated the WIF-B line by growing WIF12-1 cells as unattached aggregates for three weeks and selecting for survivors. Karyotype analysis showed a broad chromosome pattern in the initial WIF-B population, but this pattern stabilized after a few passages. The growth and phenotypic properties of these cells were quite different from those of their polarized WIF12-1 parent. WIF-B cells attained a 4-fold higher maximal density in monolayer culture, survived at this density for > 5 days rather than 1 day, and exhibited two to three times more apical structures during this period (80 to 95%). We compared several parameters of liver differentiation in the WIF-B cells with those of a related hybrid clone, WIF12-E, which is extinguished for most liver-specific functions, and with the common hepatoma parent, Fao. By immunoblot analysis, the levels of expression of eight plasma membrane proteins were higher in the WIF-B cells than in either of the other two cell lines and ranged from 10 to 200% of those in vivo. Two plasma membrane proteins were not detected in WIF12-E cells. By immunofluorescence, the apical membrane proteins in WIF-B displayed different cellular localizations than in either of the other two cell lines. In WIF-B cells, apical proteins were confined to a plasma membrane region that we have identified as the apical domain by several criteria (Ihrke, G., Neufeld, E.D., Meads, T., Shanks, M.R., Cassio, D., Laurent, M., Schroer, T.A., Pagano, R. E. and Hubbard, A. L. J. Cell Biol., 123, 1761-1765). The same molecules were distributed over the entire plasma membrane of Fao and WIF12-E cells and also (for Fao cells) in intracellular punctate structures that did not colocalize with the majority of structures containing a secretory protein, albumin. Our results indicate that the WIF-B cells are more highly differentiated than any of their ancestors (Fao or WIF12-1 cells) and thus, are promising candidates for in vitro studies of hepatocyte polarity.

Animals↗
PubMed1993

Newly synthesized hepatocyte plasma membrane proteins are transported in transcytotic vesicles in the bile duct-ligated rat.

BACKGROUND: Newly synthesized apical membrane proteins in hepatocytes go first to the basolateral membrane, from which they are retrieved and delivered to the apical domain. The goal of the present study was to identify the vesicular carriers of these molecules. METHODS: The common bile duct of rats was ligated for 10-72 hours, and then various plasma membrane proteins were localized using immunofluorescence and quantitative immuno-electron microscopy of fixed liver tissue. RESULTS: By immunofluorescence, we found intracellular punctate staining near the bile canalicular membrane of polymeric immunoglobulin A (IgA) receptor and several apical membrane proteins, but not basolateral proteins. This compartment was membrane bounded and pleiomorphic by immunoelectron microscopy. Colocalization at the electron microscopic level showed that the apical protein, dipeptidyl peptidase IV, was in the same structures as aminopeptidase N, polymeric IgA receptor, or intravenously injected horseradish peroxidase. This intracellular immunolabeling decreased after cycloheximide treatment (t1/2 = 2-2.5 hours) or reversal of the ligation for 1 hour. In the latter case, bile canalicular labeling increased. Furthermore, polymeric IgA receptor was delivered to the bile canaliculi. CONCLUSIONS: Bile duct ligation leads to an intracellular accumulation of vesicles carrying polymeric IgA receptor, several apical membrane proteins, and a fluid phase marker. These vesicles continue to fuse with the apical membrane, even during ligation.

Animals↗
PubMed1993

WIF-B cells: an in vitro model for studies of hepatocyte polarity.

We have evaluated the utility of the hepatoma-derived hybrid cell line, WIF-B, for in vitro studies of polarized hepatocyte functions. The majority (> 70%) of cells in confluent culture formed closed spaces with adjacent cells. These bile canalicular-like spaces (BC) accumulated fluorescein, a property of bile canaliculi in vivo. By indirect immunofluorescence, six plasma membrane (PM) proteins showed polarized distributions similar to rat hepatocytes in situ. Four apical PM proteins were concentrated in the BC membrane of WIF-B cells. Microtubules radiated from the BC (apical) membrane, and actin and foci of gamma-tubulin were concentrated in this region. The tight junction-associated protein ZO-1 was present in belts marking the boundary between apical and basolateral PM domains. We explored the functional properties of this boundary in living cells using fluorescent membrane lipid analogs and soluble tracers. When cells were incubated at 4 degrees C with a fluorescent analog of sphingomyelin, only the basolateral PM was labeled. In contrast, when both PM domains were labeled by de novo synthesis of fluorescent sphingomyelin from ceramide, fluorescent lipid could only be removed from the basolateral domain. These data demonstrate the presence of a barrier to the lateral diffusion of lipids between the PM domains. However, small soluble FITC-dextrans (4,400 mol wt) were able to diffuse into BC, while larger FITC-dextrans were restricted to various degrees depending on their size and incubation temperature. At 4 degrees C, the surface labeling reagent sNHS-LC-biotin (557 mol wt) had access to the entire PM, but streptavidin (60,000 mol wt), which binds to biotinylated molecules, was restricted to only the basolateral domain. Such differential accessibility of well-characterized probes can be used to mark each membrane domain separately. These results show that WIF-B cells are a suitable model to study membrane trafficking and targeting in hepatocytes in vitro.

Animals↗
PubMed1992

Rat liver dipeptidylpeptidase IV contains competing apical and basolateral targeting information.

Madin-Darby canine kidney (MDCK) cells deliver endogenous apical and basolateral proteins directly to the appropriate domains. We are investigating the molecular signals on a model plasma membrane hydrolase, dipeptidylpeptidase IV (DPPIV). Most newly synthesized rat liver DPPIV is delivered directly to the apical surface of transfected MDCK cells; however, about 20% is delivered first to the basolateral surface and reaches the apical surface via transcytosis (Casanova, J. E., Mishumi, Y., Ikehara, Y., Hubbard, A. L., and Mostov, K. E. (1991) J. Biol. Chem. 266, 24428-24432). A soluble form of DPPIV (solDPPIV) containing only the lumenal domain of the protein was efficiently transported and secreted by stably transfected MDCK cells. If this domain contains apical sorting information, we would expect 80% of the soluble protein to be secreted apically. Surprisingly, 95% of the secreted solDPPIV was found in the apical medium. The high efficiency of apical secretion suggested that the transmembrane domain and cytoplasmic tail of DPPIV might contain competing basolateral targeting information. To test this hypothesis, we investigated the trafficking of a chimera in which the cytoplasmic tail and transmembrane domains of DPPIV were joined to lysozyme, an exogenous protein which should not contain sorting information. This protein was delivered predominantly to the basolateral surface. Our results suggest that the lumenal domain of DPPIV carries dominant apical sorting information while the transmembrane domain and cytoplasmic tail of the molecule contains competing basolateral sorting information.

Amino Acid Sequence↗
PubMed1992

Lumenal labeling of rat hepatocyte endocytic compartments. Distribution of several acid hydrolases and membrane receptors.

We used a combination of subcellular fractionation and lactoperoxidase-mediated iodination to examine the polypeptide compositions of three hepatocyte endocytic compartments: early endosomes, late endosomes, and lysosomes. A chemical conjugate of asialoorosomucoid and lactoperoxidase which binds specifically to asialoglycoprotein receptors was perfused through isolated rat livers at 37 degrees C. Subcellular fractions enriched in various endocytic compartments were then isolated by differential and isopycnic centrifugation, and the lactoperoxidase moiety of the internalized conjugate was used to catalyze the iodination of lumenal-facing proteins. The 125I profiles of early and late endosomes were strikingly similar after gel electrophoresis. Using immunoprecipitation, we directly identified and compared the relative amounts of the Na+,K(+)-ATPase and several different acid hydrolases and membrane receptors in all three fractions. The asialoglycoprotein receptor and the low density lipoprotein related protein were approximately nine times more abundant in early endosomes than late endosomes, suggesting that they recycle from early endosomes. In addition, cathepsin D, but not cathepsin L, beta-glucuronidase, and lgp 120, was detected in early endosomes; however, all of these molecules were detected in lysosomes. Our findings provide strong evidence that early endosomes mature into late endosomes and that there is either selective delivery or selective retention of hydrolases at discrete points in the endocytic pathway.

Animals↗
PubMed1992

Lumenal labeling of rat hepatocyte early endosomes. Presence of multiple membrane receptors and the Na+,K(+)-ATPase.

We used lactoperoxidase-mediated iodination to investigate the lumenal polypeptide composition of rat hepatocyte endosomes. A chemical conjugate of asialoorosomucoid and lactoperoxidase that binds specifically to hepatocyte asialoglycoprotein receptors was perfused through isolated rat livers at 16 degrees C in the presence of mannan, resulting in the accumulation of ligand in early endosomes. Endosome containing low density vesicle fractions were subsequently isolated from sucrose gradients of microsomes, and the lactoperoxidase moiety was used to catalyze the iodination of lumenal-facing proteins. After gel electrophoresis, 125I-labeled early endosomes reproducibly showed a distinct 125I-polypeptide profile containing prominently labeled bands migrating at 43, 52, 58, 90, 110, 135, 230, and greater than 300 kDa. The asialoglycoprotein receptor (43-, 52-, and 58-kDa subunits) was by far the predominantly labeled protein even when iodinations were performed under conditions of receptor-ligand dissociation, and we conclude that it is the most abundant hepatocyte early endosomal protein. Furthermore, the iodination profile of the three asialoglycoprotein receptor subunits differed strikingly from their chemical amounts. Using immunoprecipitation, we directly identified the Na+,K(+)-ATPase; to our knowledge, this is the first biochemical evidence for the Na+,K(+)-ATPase in rat hepatocyte early endosomes. We also directly identified receptors for mannose 6-phosphate, epidermal growth factor, transferrin, and polymeric IgA in 125I-labeled early endosomes.

Animals↗
PubMed1992

Dynamics of four rat liver plasma membrane proteins and polymeric IgA receptor. Rates of synthesis and selective loss into the bile.

We have determined the half-lives and amounts per hepatocyte of the polymeric IgA receptor (pIgA-R) and four rat hepatocyte plasma membrane proteins and subsequently have predicted their rates of synthesis and possible routes of degradation. Using in vivo pulse-chase metabolic labeling with L-[35S]cysteine, we found that the pIgA-R had an apparent half-life of 1.1 h. Additional metabolic labeling experiments showed that CE9, HA4, and HA321 had apparent half-lives of 4-5 days, and dipeptidyl peptidase IV had an apparent half-life of 9 days. To quantify the amount of each protein per hepatocyte, homogenates and a standard curve of purified protein were compared by immunoblotting. We found that these proteins were present at 1-8 x 10(6) molecules/hepatocyte. The calculated rate of synthesis for pIgA-R was 1.6 x 10(6) molecules/hepatocyte/h, whereas the others were synthesized at much lower rates (0.9-5 x 10(4) molecules/hepatocyte/h). Using immunoblot analysis, we found that pIgA-R was released into bile at a rate of 30%/h (700%/day), whereas dipeptidyl peptidase IV and HA4 were released at a rate of 2-3%/day. While the majority of the loss of pIgA-R from hepatocytes occurred by release into the bile, less than 30% of the degradation of dipeptidyl peptidase IV and HA4 could be accounted for by this pathway, suggesting that the remaining molecules must be retrieved from the apical surface before degradation.

Animals↗
PubMed1992

5'nucleotidase is sorted to the apical domain of hepatocytes via an indirect route.

In hepatocytes, all newly synthesized plasma membrane (PM) proteins so far studied arrive first at the basolateral domain; apically destined proteins are subsequently endocytosed and sorted to the apical domain via transcytosis. A mechanism for the sorting of newly synthesized glycophosphatidylinositol (GPI)-linked proteins has been proposed whereby they associate in lipid microdomains in the trans-Golgi network and then arrive at the apical domain directly. Such a mechanism poses a potential exception to the hepatocyte rule. We have used pulse-chase techniques in conjunction with subcellular fractionation to compare the trafficking of 5' nucleotidase (5NT), an endogenous GPI-anchored protein of hepatocytes, with two transmembrane proteins. Using a one-step fractionation technique to separate a highly enriched fraction of Golgi-derived membranes from ER and PM, we find that both 5NT and the polymeric IgA receptor (pIgAR) traverse the ER and Golgi apparatus with high efficiency. Using a method that resolves PM vesicles derived from the apical and basolateral domains, we find that 5NT first appears at the basolateral domain as early as 30 min of chase. However the subsequent redistribution to the apical domain requires > 3.5 h of chase to reach steady state. This rate of transcytosis is much slower than that observed for dipeptidylpeptidase IV, an apical protein anchored via a single transmembrane domain. We propose that the slow rate of transcytosis is related to the fact that GPI-linked proteins are excluded from clathrin-coated pits/vesicles, and instead must be endocytosed via a slower nonclathrin pathway.

5'-Nucleotidase↗
PubMed1992

Effects of tannic acid on antigenicity and membrane contrast in ultrastructural immunocytochemistry.

We have modified our previous method for immunogold staining of unosmicated, plastic-embedded tissue by addition of tannic acid as a post-fixative to increase membrane contrast. Overall cell ultrastructure and organelle membranes, in particular, appeared well preserved after this treatment. We evaluated quantitatively the effect of tannic acid on the antigenicity of several membrane proteins in rat liver and intestine. For all antigens tested, significant antigenicity was retained on both intracellular and plasma membranes. However, the level of antigenicity decreased with increased concentrations of tannic acid. This effect was most apparent on the apical and basolateral membranes of hepatocytes and on the apical membrane of enterocytes, surfaces that had been in direct contact with the tannic acid fixative. The results indicate that when low concentrations of tannic acid are employed, this method yields greatly enhanced membrane contrast while preserving sufficient antigenicity to facilitate the ultrastructural localization of many membrane and other antigens.

Animals↗
PubMed1991

Direct apical sorting of rat liver dipeptidylpeptidase IV expressed in Madin-Darby canine kidney cells.

In Madin-Darby canine kidney (MDCK) cells, apical and basolateral membrane proteins are segregated from each other in the trans-Golgi network (TGN) and are transported to the appropriate membrane domain via separate vesicle populations. In hepatocytes, however, all plasma membrane proteins are delivered basolaterally. Apical proteins are then selectively retrieved and reach the apical surface by transcytosis. The sorting of apical proteins in different cell types may be the result of differences in the cellular sorting machinery, or alternatively, due to expression of cell-specific sorting signals on the proteins themselves. To test this directly, we have stably expressed cDNA encoding an apical protein from rat liver, dipeptidylpeptidase IV (DPPIV), in MDCK cells. We found that approximately 90% of the exogenous DPPIV is expressed on the apical cell surface at steady state. Furthermore, we demonstrate that this distribution is primarily due to vectorial transport from the TGN to the apical plasma membrane. The small pool of mis-sorted DPPIV that appears basolaterally is slowly endocytosed (t1/2 approximately 60 min) and is subsequently transcytosed. These data are consistent with the notion that both hepatocytes and MDCK cells are capable of correctly sorting rat liver DPPIV, but that this sorting occurs at different sites in the two cell types.

Animals↗
PubMed1991

Degradation of epidermal growth factor receptor in rat liver. Membrane topology through the lysosomal pathway.

We have generated and characterized three rabbit polyclonal antibodies that recognize different regions of the epidermal growth factor receptor (EGF-R) and used them to study the degradation of the receptor in the isolated perfused rat liver. Quantitative immunoblot analyses of rat liver homogenates prepared from tissue biopsies collected at various times after epidermal growth factor (EGF) addition showed that both the ectoplasmic and cytoplasmic domains of rat liver EGF-Rs were degraded with similar kinetics (t1/2 = 3.5-3.8 h at 25 degrees C with cycloheximide). No immunoreactive intermediate breakdown products were detected. EGF-stimulated degradation of both receptor domains was inhibited by the thiol protease inhibitor leupeptin, suggesting lysosome involvement in the hydrolysis of the whole molecule. To study this further, protease protection experiments were performed on endosome- and lysosome-enriched fractions isolated from leupeptin-treated livers. We found that the cytoplasmic domains of greater than 90% of the EGF-Rs in endosomal fractions were accessible to digestion when proteinase K was added to the intact vesicle populations, while the ectoplasmic domain was unaltered. In contrast, both the ectoplasmic and cytoplasmic domains of approximately 55% of the EGF-Rs present in lysosome-enriched fractions were inaccessible to proteinase K digestion in the absence of detergent. These findings suggest that movement of EGF-Rs from the limiting membrane of endosomes to the lumen of lysosomes permits the degradation of the entire EGF-R molecule within lysosomes.

Amino Acid Sequence↗
PubMed1991

Hydrolases in intracellular compartments of rat liver cells. Evidence for selective activation and/or delivery.

We used perfused rat livers to investigate the role of endosomes versus lysosomes in the hydrolysis of endocytosed material. When perfusions were performed at 37 degrees C with 125I-asialoorosomucoid, 125I-galactosylated albumin, or 125I-mannosylated albumin, there was a 15-min lag before trichloroacetic acid-soluble degradation products were detected. Furthermore, no hydrolysis was detected at 16 degrees C, indicating that there was no significant prelysosomal degradation of these proteins. Since detection by this method depends on extensive hydrolysis, we subsequently used three small synthetic molecules from which fluorescent products are generated by a single cleavage. These were 4-methylumbelliferyl sulfate, 4-methylumbelliferyl phosphate, and 4-methylumbelliferyl-beta-D-glucosaminide, which are substrates for aryl sulfatase, acid phosphatase, and beta-hexosaminidase, respectively. Using the first two compounds, hydrolysis was detected after 3 min at 37 degrees C and still occurred, albeit to a reduced extent, at 16 and 4 degrees C. This indicates that aryl sulfatase and acid phosphatase are active prelysosomally. We found a different result with 4-methylumbelliferyl-beta-D-glucosaminide. At 37 degrees C, there was a greater than 15-min lag before hydrolysis products were measured; furthermore, hydrolysis ceased at 16 degrees C, indicating that beta-hexosaminidase is active lysosomally. Taken together, these findings show that there is selective activation and/or delivery of hydrolases along the endocytic pathway.

Acetylglucosamine↗
PubMed1991

Sequential processing of epidermal growth factor in early and late endosomes of rat liver.

We have used isolated perfused rat livers to examine the intracellular processing of 125I-epidermal growth factor (EGF) and to determine where in the endocytic pathway the hydrolases which degrade EGF are acting. Following uptake of 125I-EGF at 37 or 16 degrees C, subcellular fractions enriched in endosomes and lysosomes were isolated, and their 125I-EGF content was examined by reverse-phase high performance liquid chromatography. Three forms of EGF processed at their carboxyl termini are generated in endosomes. At 37 degrees C, EGF is first processed in early endosomes by a carboxypeptidase B-like protease and is further processed in late endosomes by a trypsin-like protease and then a carboxypeptidase B-like protease. At 16 degrees C, entry of EGF into late endosomes is slowed, and only the first processed form is generated over 60 min. Longer perfusions (180 min) at 16 degrees C result in some processing (7%) by proteases found in late endosomes. EGF-horseradish peroxidase cytochemistry confirmed that the additional processing detected at 180 min correlated with movement of EGF from tubulovesicular to multivesicular endosomes. These results, combined with in vitro incubations of EGF in isolated endosomal and lysosomal fractions, suggest that different proteases are active at selective points in the endocytic pathway and that the full complement of proteases needed for complete degradation of EGF is active only in lysosomes.

Amino Acid Sequence↗