Previous studies of fibroblasts have demonstrated that recycling of endocytic receptors occurs through a default mechanism of membrane-volume sorting. Epithelial cells require an additional level of polar membrane sorting, but there are conflicting models of polar sorting, some suggesting that it occurs in early endosomes, others suggesting it occurs in a specialized apical recycling endosome (ARE). The relationship between endocytic sorting to the lysosomal, recycling and transcytotic pathways in polarized cells was addressed by characterizing the endocytic itineraries of LDL, transferrin (Tf) and IgA, respectively, in polarized Madin-Darby canine kidney (MDCK) cells. Quantitative analyses of 3-dimensional images of living and fixed polarized cells demonstrate that endocytic sorting occurs sequentially. Initially internalized into lateral sorting endosomes, Tf and IgA are jointly sorted from LDL into apical and medial recycling endosomes, in a manner consistent with default sorting of membrane from volume. While Tf is recycled to the basolateral membrane from recycling endosomes, IgA is sorted to the ARE prior to apical delivery. Quantifications of the efficiency of sorting of IgA from Tf between the recycling endosomes and the ARE match biochemical measurements of transepithelial protein transport, indicating that all polar sorting occurs in this step. Unlike fibroblasts, rab11 is not associated with Tf recycling compartments in either polarized or glass-grown MDCK cells, rather it is associated with the compartments to which IgA is directed after sorting from Tf. These results complicate a suggested homology between the ARE and the fibroblast perinuclear recycling compartment and provide a framework that justifies previous conflicting models of polarized sorting.Key words: Endocytosis, endosome, epithelia, low density lipoprotein, MDCK, polarity, polymeric Ig receptor, transcytosis, transferrin Received 9 August 1999, revised and accepted for publication 26 October 1999The transport functions of an epithelium are determined by the distinct compositions of the apical and basolateral plasma membrane domains. This membrane polarity is maintained despite significant endocytic turnover, which in MadinDarby canine kidney (MDCK) cells can amount to 40% of the plasma membrane internalized per hour (1). Whereas early studies indicated that the apical and basolateral endocytic recycling pathways of MDCK cells are distinct (2,3), recent evidence indicates that the two pathways are interconnected (4 -6). With this continuous intermixing of apical and basolateral membranes, it is clear that endocytic sorting is crucial to maintaining the plasma membrane polarity of epithelial cells.

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Abstract. The 17-juxtamembrane cytoplasmic residues of the polymeric immunoglobulin receptor contain an autonomous basolateral targeting signal that does not mediate rapid endocytosis (Casanova, J. E., G. Apodaca, and K. E. . Alanine-scanning mutagenesis identifies three residues in this region, His656, Arg657, and Va1660, that are most essential for basolateral sorting and two residues, Arg655 and Tyr668, that play a lesser role in this process. Progressive truncations suggested that Ser664 and Ile665 might also play a role in basolateral sorting. However, mutation of these residues to Ala or internal deletions of these residues did not affect basolateral sorting, indicating that these residues are probably not required for basolateral sorting. Twodimensional NMR spectroscopy of a peptide corresponding to the 17-mer signal indicates that the sequence Arg658-Asn-Val-Asp661 has a propensity to adopt a/if-turn in solution. Residues COOH-terminal to the B-turn (Arg662 to Arg669) seem to take up a nascent helix structure in solution. Substitution of Va1660 with Ala destabilizes the turn, while mutation of Arg657 to Ala does not appear to affect the turn structure. Neither mutation detectably altered the stability of the nascent helix in the COOH-terminal portion of the peptide.p OLARIZED epithelial cells have two plasma membrane domains: the apical domain facing the external environment, and the basolateral domain facing the internal milieu. These domains display striking differences in protein and lipid composition (Caplan and Matlin, 1989;

Quantitative confocal microscopic analyses of living, polarized MDCK cells demonstrate different pH profiles for apical and basolateral endocytic pathways, despite a rapid and extensive intersection between the two. Three-dimensional characterizations of ligand trafficking demonstrate that the apical and basolateral endocytic pathways share early, acidic compartments distributed throughout the medial regions of the cell. Polar sorting for both pathways occurs in these common endosomes as IgA is sorted from transferrin to alkaline transcytotic vesicles. While transferrin is directly recycled from the common endosomes, IgA is transported to a downstream apical compartment that is nearly neutral in pH. By several criteria this compartment appears to be equivalent to the previously described apical recycling endosome. The functional significance of the abrupt increase in lumenal pH that accompanies IgA sorting is not clear, as disrupting endosome acidification has no effect on polar sorting. These studies provide the first detailed characterizations of endosome acidification in intact polarized cells and clarify the relationship between the apical and basolateral endocytic itineraries of polarized MDCK cells. The extensive mixing of apical and basolateral pathways underscores the importance of endocytic sorting in maintaining the polarity of the plasma membrane of MDCK cells.

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ] are phosphoinositides (PIs) present in small amounts in the inner leaflet of the plasma membrane (PM) lipid bilayer of host target cells. They are thought to modulate the activity of proteins involved in enteropathogenic Escherichia coli (EPEC) infection.However, the role of PI(4,5)P 2 and PI(3,4,5)P 3 in EPEC pathogenesis remains obscure. Here we show that EPEC induces a transient PI(4,5)P 2 accumulation at bacterial infection sites. Simultaneous actin accumulation, likely involved in the construction of the actin-rich pedestal, is also observed at these sites. Acute PI(4,5)P 2 depletion partially diminishes EPEC adherence to the cell surface and actin pedestal formation. These findings are consistent with a bimodal role, whereby PI(4,5)P 2 contributes to EPEC association with the cell surface and to the maximal induction of actin pedestals. Finally, we show that EPEC induces PI(3,4,5)P 3 clustering at bacterial infection sites, in a translocated intimin receptor (Tir)-dependent manner. Tir phosphorylated on tyrosine 454, but not on tyrosine 474, forms complexes with an active phosphatidylinositol 3-kinase (PI3K), suggesting that PI3K recruited by Tir prompts the production of PI(3,4,5)P 3 beneath EPEC attachment sites. The functional significance of this event may be related to the ability of EPEC to modulate cell death and innate immunity. INTRODUCTIONEnteropathogenic Escherichia coli (EPEC) is a major cause of a severe infantile diarrhea in developing countries. Studies performed on infected humans and animal models have shown that after ingestion, EPEC intimately adheres to the mucosal surface of the intestinal epithelium. Bacterial adhesion elicits a localized collapse of microvilli and a dramatic reorganization of the actin cytoskeleton, eventually leading to the establishment of a pedestal-like actin structure located underneath the adhering bacteria. These histopathological alterations, also termed attaching and effacing (A/E) lesions, are essential to promote successful EPEC colonization, but they also induce tissue damage and fluid loss, which may eventually lead to diarrhea.A/E lesion formation requires a type III secretion system (T3SS) of EPEC that mediates the delivery of bacterial effector proteins directly into the host cell cytoplasm. Upon contact with the host cell, the T3SS translocates the intimin receptor, Tir, which is inserted into the host cell plasma membrane (PM), and interacts with intimin, a bacterial surface protein. Tir-intimin interaction leads to intimate attachment of the bacterium to the host cell surface and triggers signaling cascades that lead to polymerization of F-actin and pedestal formation. Clustering of Tir by intimin enhances the activity of cellular tyrosine kinases that phosphorylate two C-terminal tyrosines on the Tir molecule: tyrosine 474 (Y474) and tyrosine 454 (Y454) (Kenny, 1999; Campellone and Leong, 2005). This results in direct recruitment of th...

Polarized epithelial cells can sort plasma membrane proteins to the apical or basolateral domain either by direct targeting from the trans‐Golgi network (TGN) or by targeting to one surface, followed by endocytosis and transcytosis to the opposite surface. In Madin‐Darby canine kidney (MDCK) cells, targeting of the polymeric immunoglobulin receptor (pIgR) to the basolateral surface is controlled by a sorting signal residing in the membrane proximal 17 amino acids of the cytoplasmic domain of this receptor. We have recently found that individual mutations at any of three residues in this signal, His656, Arg657 and Val660, substantially decrease targeting from the TGN to the basolateral surface and correspondingly increase targeting from the TGN to the apical surface. Here we report that these mutations decrease the recycling of basolaterally endocytosed pIgR to that surface, and correspondingly increase its transcytosis to the apical surface. This effect occurred in mutant pIgRs that either contained the full‐length cytoplasmic domain or were truncated to contain only the 17‐residue basolateral targeting signal, and was independent of phosphorylation of pIgR at Ser664. Our results indicate that polarized sorting of the pIgR in the endocytotic and exocytotic pathways are controlled by the same amino acids.

We developed a novel surface plasmon resonance (SPR) method, based on Fourier transform infrared (FTIR) spectroscopy, as a label-free technique for studying dynamic processes occurring within living cells in real time. With this method, the long (micrometer) infrared wavelength produced by the FTIR generates an evanescent wave that penetrates deep into the sample. In this way, it enables increased depth of sensing changes, covering significant portions of the cell-height volumes. HeLa cells cultivated on a gold-coated prism were subjected to acute cholesterol enrichment or depletion using cyclodextrins. Cholesterol insertion into the cell plasma membrane resulted in an exponential shift of the SPR signal toward longer wavelengths over time, whereas cholesterol depletion caused a shift in the opposite direction. Upon application of the inactive analog alpha-cyclodextrin (alpha-CD), the effects were minimal. A similar trend in the SPR signal shifts was observed on a model membrane system. Our data suggest that FTIR-SPR can be implemented as a sensitive technique for monitoring in real time dynamic changes taking place in living cells.

We discuss the Surface-Plasmon-Resonance (SPR) technique based on Fourier -Transform -In-fraRed (FTIR) spectrometry. We explore the potential of the infrared surface plasmon resonance technique for biological studies in aqueous solutions and compare it to the conventional surface plasmon technique operating in the visible range. We demonstrate that the sensitivity of the SPR technique in the infrared range is not lower and in fact is even higher. We show several examples of applying FTIR-SPR for biological studies: (i) monitoring D-glucose concentration in solution, and (ii) measuring D-glucose uptake by erythrocytes in suspension. We emphasize the advantages of infrared SPR for studying living cell cultures and show how this technique can be used for characterization of (i) cholesterol penetration into plasma membrane, and (ii) transferrin-induced clathrin-mediated endocytosis. * electronic address: golos@vms.huji.ac.il