Oct 10, 1989 - lateral plasma membranes of adjacent hepatocytes. The microvillar membrane surrounding the bile canaliculus con- stitutes the cell's apical ...
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Membrane compartmentation and trafficking in hepatocytes W. HOWARD EVANS,* NAWAB ALI* and CARLOS ENRICH$ *National Institute for Medical Research, Mill Hill, London NW7 I A A , U.K. and $Department of Cell Biology and Histology, University of Barcelona, Spain The plasma membrane of a polarized epithelial cell is differentiated into basolateral and apical domains. In hepatocytes, the basolateral domain comprises a microvillar sinusoidal membrane curving into a flattened lateral membrane that is closely aligned and attached by intercellular junctions to the lateral plasma membranes of adjacent hepatocytes. The microvillar membrane surrounding the bile canaliculus constitutes the cell's apical domain and this accounts for up to one-fifth of the hepatocyte's surface area. Sealing and separating the canalicular domain from the basolateral plasma membrane are tight junctions. An often considered question is how epithelial cells generate a continuous, yet functionally asymmetric, plasma membrane containing a polarized distribution of proteins and, to a lesser extent, lipids. Biochemical analysis of the hepatocyte's plasma membrane has shown that many receptors, channels, pumps and enzymes are distributed in a polarized fashion on the cell surface [ l , 21. The signal transduction apparati are mainly functional at the receptor-containing, blood-facing basolateral surface. However, although the distribution in the domains of some membrane components is absolute, e.g. the asialoglycoprotein and mannosc-6-phosphate receptors and Na+, K + -ATPase are confined to the basolateral plasma membrane. others are located o n both hasolateral and apical membranes. with the highest levels found at the apical membrane, e.g. 5'-nucleotidase, phosphoinositidascs and Gproteins [ 3-5 I. Central to the resolution o f the mechanisms generating the polarized distribution o f plasma membrane componcnts is advancing knowledge of the role of the truris-Golgi networks in directing outward traffic and the endocytic compartments in directing inward traffic, both involving primarily the blood sinusoidal plasma membrane domain. To analyse the compartmentation in the Golgi, endocytic, lysosomal and plasma membranes of various enzymes, receptors. and especially key components featuring in signal transduction, we have adopted a combined subcellular fractionation and immuno-
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cytochemical approach to study their distribution in normal and regenerating liver tissue.
Siihcellirlar dirsection of hepatic trtrffickirtg pathways Methods have been developed for isolating by subcellular fractionation the membrane networks comprising the exocytic and endocytic pathways operational in hepatocytes. On the basis of the analysis of the distribution of various receptors and receptor-transducing components in these subcellular fractions, information on the trafficking pathways operational in hepatocytes can be deduced. The methods used to fractionate rat liver homogenates are summarized in Fig. 1. The techniques used to prepare the hepatocyte's plasma membrane domains are well established [2,6],but recent methodological advances in the preparation of highly enriched bile canalicular and lateral plasma membranes are worth noting. Subfractionation of a sonicated 'light' liver plasma membrane fraction yielded by isopycnic centrifugation in iso-osmotic Nycodenz gradients a vesicular bile canalicular and a sheet-like lateral plasma membrane fraction. This new bile canalicular plasma membrane fraction was enriched over a 100-fold realtive to the homogenate in five markers (alkaline phosphatasc. alkaline phosphodiesterase, leucylnaphthylamidase S'-nucleotidasc and Ca'+ATPase), thus attesting to the substantially higher purity of these vesicles when compared with those prepared by current methods (N. Ali & W. H. Evans, unpublished work). Antibodies to proteins in these bilc canalicular vesicles immunolocalized exclusively to the bile canaliculus in frozen liver sections. The bile canalicular vesicles contained no detectable Na', I(+-ATPase activity. but this enzyme was highly enriched especially in the lateral plasma membranes obtained by this procedure. The isolation of endocytic vesicles from liver homogenatcs has already been described 17-YI. The designation of endosomcs as either 'early' o r 'late' was based on the recovery in membrane vesicle5 of intact radio-iodinated ligands at 2-3 min or I S min intervals, respectively, following uptake by liver 17 I. Whereas many ligands, e.g. insulin and asialoglycopro~eins.were recovered sequentially in 'carly' and 'late' endocytic vesicles 17 1, other ligands binding t o plasma membrane channels, e.g. apamin [ 101. or receptors. e.g. transferrin ( H . Goldenberg & W. H.
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Fig. 1. Schernaiic representation of the methods used to prepare the varioir.s liver siihcellular~ructiorlsdisciissed in the text Vol. 18
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BIOCHEMICAL SOCIETY TRANSACTIONS
Evans, unpublished work), were confined to the ‘early’ endocytic vesicles. The major marker used for endocytic vesicles was their capacity to acidify in the presence of ATP and to neutralize in the presence of ionophores and the high enrichment of various radio-iodinated ligands relative to the levels in liver homogenates [ 1 1 I. A by-product of the procedure for the isolation of endosomes was the preparation from the same homogenate of ‘endosome-depleted’ Golgi subfractions 191. Lysosomal fractions were prepared by modifying a standard method involving the separation of lysosomes from a ‘light’ mitochondrial-lysosomal fraction by density gradients [ 121. The mitochondrial-lysosomal pellet was washed extensively by centrifugation to minimize Contamination by endosomal vesicles and the final separation o f lysosomes was then carried out on Nycodenz gradients. The distribution among the subcellular fractions was assessed by enzymc analysis and the use of specific antibodies. Thus an analysis of the localization of D-myoinositol 1,4,5-trisphosphate and 1,3,4,5-tetrakisphosphate-Sphosphatase activities indicated that, although this enzyme involved in signal transduction was present at the blood sinusoidal plasma membrane, the highest activities were found in ‘late’ endosomal and bile canalicular plasma membranes (31. This distribution corresponded to that of 5’nucleotidase, a plasma membrane enzyme of unknown function. The composition of the subcellular fractions can be analysed by Western blotting of the polypeptides resolved by gel electrophoresis, using various appropriate antibodies as illustrated in Fig. 2 . The distribution of the a - and p-subunits of the nucleotide-binding G-proteins was assessed in this way and it was demonstrated that, although two subunits of G-proteins were found in the three plasma membrane domains, Golgi membranes and endosomes (but not in lysosomes), the highest relative enrichment was in the bile canalicular membranes [4, S ] . A different relative localization of a newly identified family in liver of low molecular mass GTPbinding proteins that corresponded to rus polypeptides was found, for these were enriched in the ‘early’ and ‘late’ endosome fractions (N. Ali & w. H. Evans, unpublished work). The availability of specific antibodies allows this dual approach to be applied to a wide range of hepatic receptors and antigens. For example, the asialoglycoprotein and mannose-6-phosphate receptors were demonstrated to be confined to the basolateral plasma membranes and endosomes and were absent from the bile canalicular plasma membrane (C. Enrich, P. Tabona & W. H. Evans, unpublished work). A group of proteins bound to the plasma membrane by a phospholipase-sensitive lipid anchor was confined to ‘late’ endosomes and bile canalicular plasma membranes ( N . Ali & W. H. Evans, unpublished work). To complement such studies, the antibodies are being used to stain by indirect immunofluorescence liver sections to identify the primary location of the antigens. Such studies reinforce the compartmentation and interaction of the membrane components described above and illustrate the apparent specificity of the trafficking networks that involve the plasma membrane domains and the Golgi and endocytic compart ment. Endocytic compartment: regenerating liver
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The role of the endocytic compartment in directing inward-moving intracellular trafficking to the various plasma membrane domains and to lysosomes is becoming more evident, as described above. In liver, although the endocytic membranes can be isolated and biochemically characterized [ 2 , 7-11, 131, knowledge of the molecular mechanisms underlying the specific direction of trafficking is sparse. Important questions to pose concern the geographical local-
Fig. 2. Anulysis by SDSlpolyucrylurnide-gel electrophoresis of vurioirs rut liver sirhcellirlurfructions Abbreviations: Can., canalicular; Lat., lateral; Sin., sinusoidal plasma membranes; E, ‘early’; L, ‘late’; Rec., ‘heavy’ endosomes. In the lower two panels are shown autoradiographs of Western blots of the fractions transferred from the polyacrylamide gel to nitrocellulose and probed with antibodies to the 41 kDa a-inhibitory subunit of the G-protein ( G , a ) o r exposed t o 7%-labelled GTP[S](guanosine S’-[ y-thioltriphosphate) which identifies in the liver subcellular fractions a series of GTP-binding polypeptides of molecular mass 21-27 kDa. The two lower panels illustrate the specific but different distribution of two types of GTP-binding proteins in the liver fractions believed to influence membrane trafficking.
ization in a polarized epithelial cell of the endocytic compartment, and whether one or more compartments are present. An extension of these questions concerns the changes in the organization and structure of the endocytic compartment that occur during cell division. Using rat liver, answers to these questions are beginning to emerge. Antibodies raised to the integral membrane proteins of hepatic endosomes were shown to label, by immunofluorescence and peroxidase techniques, two specific regions of the cytoplasm in hepatocytes [ 141. The region underpinning the sinusoidal plasma membrane was identified as one location of the endocytic compartment, but surprisingly, a high labelling intensity of endocytic structures was observed between the nucleus and the bile canalicular plasm? membrane. These results thus suggest that at least two endocytic compartments, that may be interconnected, exist in differentiated hepatocytes. However, these two geographical compartments cannot be equated with the kinetically identified 1990
DYNAMICS A N D RECEPTOR FUNCTIONS OF MEMBRANES and biochemically characterized endocytic membranes described above. The regenerating liver provides an attractive model system in which to study changes in organelles and membrane networks during the co-ordinated cell division of hepatocytes that occurs following partial hepatectomy. Using the antibodies raised to the integral membrane proteins of cndosomes to analyse by immunoperoxidase staining thin sections from regenerating livers, it was observed that the overall immunoreactivity of the two endocytic compartments was diminished ( C . Enrich & W. H. Evans, unpublished work). The major diminution was in the staining intensity of endocytic structures located below the blood sinusoidal plasma membrane domain, but the endocytic structures identified by the antibodies at the biliary axis were less affected. These results indicate that during the cell’s preparation for the first cycle of division ensuing 24 h after hepatectomy, the endocytic compartment participating in the uptake from the blood of nutrients, hormones, growth factors, etc. may undergo remodelling, whereas the compartment located at the biliary axis of the hepatocyte that is probably involved in storing intracellular receptors is maintained. The identification of a major endocytic compartment at the hepatocyte’s biliary axis highlights the metabolic importance of this area in the cell and agrees with the biochemical observations described above, showing that a number of components involved in signalling across the blood sinusoidal plasma membrane are also present at high levels in this region and are likely to have been transported to this region by the intermediation of the endocytic compartment. The functions of G-proteins, Ca? -ATPases and inositol phosphatases present at the bile canaliculus are unknown, but the possibility can be entertained that extensive ionic movements occur at this apical region in hepatocytes. The fact that drug excretory mechanisms have recently been shown to function at the bile canalicular plasma membrane, as exemplified by the identification of a 170 kDa product of a multi-drug+
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resistant gene requiring ATP as an energy source [ 151, would also support the view that extensive metabolic activity and transport occur at and around the bile canaliculus. N. Ali thanks the Wellcorne Trust for a Fellowship. C. Enrich is by thc Comision Asewra de Investigacion Cientifico y _supported . lecnica (PMXH-0044). Collahoration hetwcen London and
Barcelona is aided by a Travel Grant awarded by the British Council. 1. Evans, W. H. ( 1 980) Riochim. Riophys. Acta 604.27-64 2. Evans, W. H. & Enrich. C . ( 1 989) Biochwz. Joc. Trans. 17, 6 19-622 3. Shears, S. B., Evans, W. H., Kirk, C. J . & Michell, R. H. ( 1 988) Biochem. J. 256,363-369 4. Ali, N., Milligan, G. & Evans, W. H. ( 1 989) Biochem. J. 261, 905-9 I2 5. Ali, N., Milligan, G. & Evans, W. H. ( 1989) Mol. Cell Biocliem. 91,75-84 6. Wisher, M. H. & Evans, W. H. ( l Y 7 5 ) Riochem. J. 146, 375-388 7. Evans, W. H. & Flint, N. ( 1 985) Biochem. J. 232,25-32 8. Evans, W. H. & Hardison, W. G. M. (1985) Riochem. J. 232, 33-36 9. Evans, W. H. ( 1 985) Methods Enzymol. 109,246-257 10. Strong, P. N. & Evans, W. H. (1987) Eur. J. Biochem. 163, 267-273 1 1 . Saermark, T., Flint, N. & Evans, W. H. ( 1 985) Biochem. J. 225, 5 1-58 12. Wattiaux, R., Wattiaux, de Coninck, S., Ronveaux-Dupal, M. F. & Dubois, E ( I 978) J. Cell Biol. 78,349-368 13. Evans, W. H. & Enrich, C. (1989) in ffepulic Transport 01 Orgunic Subslances (Petzinger, E., Kinne, R. K. H. & Sies, H., eds.), pp. 35-44, Springer-Verlag, Berlin 14. Enrich, C. & Evans, W. H. (1989) Eur. J. Cell Riol. 48, 344-352 15. Kamimoto, Y., Gatmaitan. Z., Hsu, J. & Arias, I. M. ( 1989) J. R i d . (’hem. 264,11693- I I698
Received 10 October 1989
Endocytosis and recycling of CD4 MARK MARSH, JANE E. ARMES and ANNEGRET PELCHEN-MATTHEWS Itisritirte of Curicer Reseurch, C ’hesterHeutty Laboratories, Fiilhum R o d , Lotidoti S W 3 6JH, U.K . CD4 is a transmemhrane glycoprotein expressed primarily on T lymphocytes which interact with cells expressing major histocompatibility complex (MHC) class 11 molecules, and also on some macrophages [ I ] . On T cells, CD4 appears to function by increasing the avidity of T-cell antigen receptor (TcR)-dependent cell-cell interactions and in modulating the signals transmitted through the TcR/CD3 complex [ 2 ] .In addition, CD4 is used as a receptor by human immunodeficiency viruses (HIV-1 and -2) [ l , 3, 41. To understand further the role of CD4 in both T-cell activation and HIV entry, we have been studying its interaction with the endoeytic apparatus. T cells and T-cell lines have often proved difficult to use in studies of endocytosis; they are usually small cells and have low endocytic rates [5].For these studies we have made use of adherent cell lines (HeLa and NIH 3T3) that have been transfected with the human CD4 cDNA, and which constiAbbreviations used: MHC, major histocompatibility complex; TcR, T-cell antigen receptor; HIV, human immunodeficiency virus; Tf R, transferrin receptor; LDLR, low-density-lipoprotein receptor.
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tutively express cell surface CD4 [4]. We have used these cells to establish a series of assays to analyse the properties of CD4. Cell surface molecules can be followed by using ligands that interact specifically with a particular plasma membrane moiety or by covalently labelling the cell surface, allowing the labelled components to endocytose, and subsequently analysing the redistributed molecules with specific antibodies. We have used both of these approaches in our studies, though only experiments using ligands will be described here. To date, the only known ligands for CD4 are HIV-1 and 2 gp120 and the MHC class 11 antigens. As it is impractical to use either of these in a systematic analysis of CD4, we have chosen the anti-CD4 monoclonal antibody, Leu3a, as a soluble ligand. This antibody, which inhibits HIV infection, is believed to bind to an epitope on CD4 either overlapping, or close to, the HIV gp120 binding site 131. As multivalent ligands, such as IgG, are known to alter the intracellular trafficking of cell surface glycoproteins (see, for example [ 6 ] )we , have generated Fab‘ fragments of this antibody for our analysis.
I’repurutioii of /.‘ah’ fragniertu Carrier-frcc Leu3a was obtained from Becton Dickinson (Mountain View, California, U.S.A.). Fab‘ fragments were
