Rat small intestinal epithelial cell lines have been established in vitro and subcultured serially for periods up to 6 mo. These cells have an epithelioid morphology, grow as monolayers of closely opposed polygonal cells, and during the logarithmic phase of growth have a population doubling time of 19-22 h. Ultrastructural studies revealed the presence of microvilli, tight junctions, an extensive Golgi complex, and the presence of extracellular amorphous material similar in appearance to isolated basement membrane. These cells exhibit a number of features characteristic of normal cells in culture; namely, a normal rat diploid karyotype, strong density inhibition of growth, lack of growth in soft agar, and a low plating efficiency when seeded at low density. They did not produce tumors when injected in syngeneic animals. Immunochemical studies were performed to determine their origin using antisera prepared against rat small intestinal crypt cell plasma membrane, brush border membrane of villus cells and isolated sucrase-isomaltase complex. Antigenic determinants specific for small intestinal epithelial (crypt and villus) cells were demonstrated on the surface of the epithelioid cells, but they lacked immunological determinants specific for differentiated villus cells. An antiserum specifically staining extracellular material surrounding the cells cultured in vitro demonstrated cross-reactivity to basement membrane in rat intestinal frozen sections. It is concluded that the cultured epithelioid cells have features of undifferentiated small intestinal crypt cells. KEY WORDS small intestine epithelioid cell cultures cell-specific antigensSmall intestinal epithelial cells represent a rapidly renewing cell population characterized by a precise segregation between mitotically active cells, present in the crypt region, and mature differentiated villus cells, mitotically inactive. Intestinal cells have a rapid cell turnover, with a mean cell duration time of 2-3 d in most animals (5,6,8,27). The differentiation of the mitotically active crypt cells is accompanied by dramatic changes in enzyme and transport activities and in cell morphology, including the appearance of a well-organized brush border at the luminal surface and a more columnar cell shape. It is of interest that, although intestinal crypt cells have one of the shortest cell cycle times in vivo (5), the occurrence 248 J, CELL BIOLOGY 9 The Rockefeller University Press

Abstract. We studied the sorting and surface delivery of three apical and three basolateral proteins in the polarized epithelial cell line Caco-2, using pulse-chase radiolabeling and surface domain-selective biotinylation (Le Bivic, A., E X. Real, and E. RodriguezBoulan. 1989. Proc. Natl. Acad. Sci. . While the basolateral proteins (antigen 525, HLA-I, and transferrin receptor) were targeted directly and efficiently to the basolateral membrane, the apical markers (sucrase-isomaltase [SI], aminopeptidase N [APN], and alkaline phosphatase [ALP]) reached the apical membrane by different routes. The large majority (80%) of newly synthesized ALP was directly targeted to the apical surface and the missorted basolateral pool was very inefficiently transcytosed. SI was more efficiently targeted to the apical membrane (>90%) but, in contrast to ALP, the missorted basolateral pool was rapidly transcytosed. Surprisingly, a distinct peak of APN was detected on the basolateral domain before its accumulation in the apical membrane; this transient basolateral pool (at least 60-70% of the enzyme reaching the apical surface, as measured by continuous basal addition of antibodies) was efficiently transcytosed. In contrast with their transient basolateral expression, apical proteins were more stably localized on the apical surface, apparently because of their low endocytic capability in this membrane. Thus, compared with two other wellcharacterized epithelial models, MDCK cells and the hepatocyte, Caco-2 cells have an intermediate sorting phenotype, with apical proteins using both direct and indirect pathways, and basolateral proteins using only direct pathways, during biogenesis.T HE polarized distribution of epithelial plasma membrane proteins into apical and basolateral domains is a fascinating paradigm of molecular sorting in cell biology. Work in the last ten years has defined the general aspects of the biogenesis of plasma membrane proteins but has generated contradictory findings on the pathways followed by apical proteins during intracellular transport (for reviews see references 1,8,39,47). Experiments carried out in native intestinal cells by Hauri et al. (16) and Massey et al. (27) and in hepatocytes by Bartles et al. (2) indicated that apical and basal glycoproteins are initially delivered to the basolateral cell surface, where sorting takes place: basolateral proteins remain there whereas apical proteins are transcytosed to the apical surface. On the other hand, experiments with the model epithelial cell line MDCK (39-42) have shown that viral envelope proteins introduced by infection, namely influenza HA (apical) and vesicular stomatitis virus G protein (basolateral) are sorted in the trans-Golgi Network and are vectorially delivered to the respective surface domain (14,28,32,35,38).Because radically diverse methods were used, doubts arose as to whether the different biogenetic pathways observed in native and cultured cells represent real differences between epithelial cell types, or are the consequence of the var...

Abstract. We characterized the three-dimensional organization of microtubules in the human intestinal epithelial cell line Caco-2 by laser scanning confocal microscopy. Microtubules formed a dense network ",,4-/zm thick parallel to the cell surface in the apical pole and a loose network 1-/zm thick in the basal pole. Between the apical and the basal bundles, microtubules run parallel to the major cell axis, concentrated in the vicinity of the lateral membrane. Colchicine treatment for 4 h depolymerized 99.4 % of microtubular tubulin. Metabolic pulse chase, in combination with domain-selective biotinylation, immune and streptavidin precipitation was used to study the role of microtubules in the sorting and targeting of four apical and one basolateral markers. Apical proteins have been recently shown to use both direct and transcytotic (via the basolateral membrane) routes to the apical surface of Caco-2 cells. Colchicine treatment slowed down the transport to the cell surface of apical and basolateral proteins, but the effect on the apical proteins was much more drastic and affected both direct and indirect pathways. The final effect of microtubular disruption on the distribution of apical proteins depended on the degree of steady-state polarization of the individual markers in control cells. Aminopeptidase N (APN) and sucrase-isomaltase (SI), which normally reach a highly polarized distribution (110 and 75 times higher on the apical than on the basolateral side) were still relatively polarized (9 times) after colchicine treatment. The decrease in the polarity of APN and SI was mostly due to an increase in the residual basolateral expression (10% of control total surface expression) since 80% of the newly synthesized APN was still transported, although at a slower rate, to the apical surface in the absence of microtubules. Alkaline phosphatase and dipeptidylpeptidase IV, which normally reach only low levels of apical polarity (four times and six times after 20 h chase, nine times and eight times at steady state) did not polarize at all in the presence of colchicine due to slower delivery to the apical surface and increased residence time in the basolateral surface. Colchicinetreated cells displayed an ectopic localization of microvilli or other apical markers in the basolateral surface and large intracellular vacuoles. Polarized secretion into apical and basolateral media was also affected by microtubular disruption. Thus, an intact microtubular network facilitates apical protein transport to the cell surface of Caco-2 cells via direct and indirect routes; this role appears to be crucial for the final polarity of some apical plasma membrane proteins but only an enhancement factor for others.PITHELIAL cells characteristically display two plasma membrane domains, apical and basolateral, with different protein and lipid compositions, separated by tight junctions (for reviews, see references 55, 63). Both the cytoplasmic and submembrane cytoskeletons are also asymmetrically distributed. The polarized organization of ...

To investigate the biosynthetic basis for the mosaic expression of brush border enzymes in confluent Caco-2 cells, a human colon carcinoma cell line exhibiting characteristics of adult small intestinal enterocytes, we have obtained a series of clones differing markedly in their growth rates, amounts of transforming growth factor-a/epidermal growth factor-like activity released into the culture medium, and sucrase-isomaltase (SI) activity. Other intestinal markers (aminopeptidase N, dipeptidylpeptidase IV, lactase, alkaline phosphatase and 'crypt cell antigen') displayed a much more limited variability in expression, suggesting that the Caco-2 cell clones we have obtained did not differ in their overall ability to differentiate.Immunofluorescence staining, metabolic labelling with radioactive methionine and hybridization analysis of SI mRNA abundance were used to investigate SI synthesis and its regulation in clones endowed with low, intermediate or high sucrase activity. The results obtained have demonstrated heterogeneous SI expression, even in clonal cell lines, and a negative correlation between SI expression and growth factor concentrations in the culture medium, suggesting an autocrine regulation of cell proliferation and differentiation in confluent Caco-2 cells. Pulse-chase experiments using the two clones endowed with the lowest and highest levels of SI activity, followed by immunoprecipitation of labelled SI with epitope-specific antibodies and SDS/PAGE analysis, suggested that both transcriptional and post-translational mechanisms play a role in the regulation of SI expression in intestinal cells.

The biosynthesis in vivo of rat intestinal sucrase-isomaltase [a complex of sucrose a-glucohydrolase, EC 3.2.1.48, and oligo-1,6-glucosidase (dextrin 6-a-D-glucanohydrolase), EC 3.2.1.10] has been studied by following the incorporation of L46-3H]fucose into the enzyme with time. Immunoprecip ion of sucrase-isomaltase from Triton-X-100-solubilized Golgi or basolateral membranes and subsequent polyacrylamide gel electrophoresis revealed the presence of an immunoreactive glycoprotein with an apparent molecular weight approximately twice that of the separated sucrase-isomaltase subunits, but no active subunits were found in these membranes. This glycoprotein was also found in the microvillus membrane in addition to the subunits of sucrase-isomaltase. Kinetic studies showed a maximal labeling of this glycoprotein in Golgi membranes at 15 min, in basolateral membranes at 30 min, and in microvillus membranes at 45 min and a half-life of less than 30 min in each membrane. However, the radioactivity of the sucrase-isomaltase subunits in the microvillus membrane reached a plateau after 60 min. These data suggest that sucrase-isomaltase is synthesized as a one-chain polypeptide precursor that is split into the subunits after its transfer to the microvillus membrane. Elastase (EC 3.4.21.11), but not trypsin (EC 3.4.21.4) or a-chymotrypsin (EC 3.4.21.1), split the putative precursor into two polypeptides that had electrophoretic behaviors similar to those ofthe active enzyme subunits. These studies suggest that pancreatic proteases may play an important role in the late posttranslational processing of sucrase-isomaltase in vivo. The membrane flow theory for the biogenesis of the plasma membrane assumes that there is a physical transfer of membrane components from the endoplasmic reticulum to the Golgi complex and subsequently to the cell surface (2). This concept, however, is still controversial, mainly due to the considerable differences in liquid and protein composition among the different membrane compartments (3). Much insight into membrane biogenesis has been provided recently by studies concerning the early posttranslational events in the biogenesis of viral glycoproteins (4-6), but the knowledge of the posttranslational fate of eukaryotic membrane proteins is still limited. In order to elucidate some of these mechanisms we have studied the biosynthesis of the sucrase-isomaltase complex [SI; sucrose a-glucohydrolase, EC 3.2.1.48, and oligo-1,6-glucosidase (dextrin 6-a-D-glucanohydrolase), EC 3.2.1.10] of the rat small intestinal microvillus membrane (MVM).SI is an intrinsic membrane glycoprotein consisting of two similar subunits with apparent molecular weights of approximately 140,000 to 160,000 each; one subunit splits sucrose and maltose and the other hydrolyzes isomaltose and maltose (7). Studies of rabbit SI have shown that this enzyme is anchored to the MVM via a hydrophobic amino acid sequence near the NH2 terminus of the isomaltase subunit with the sucrase subunit occupying a more peripheral position (...