Endocytosis of Junctional Cadherins in Bovine Kidney Epithelial (MDBK) Cells Cultured in Low Ca e+ Ion Medium

The release of intercellular contacts in MDBK cells, initiated by the depletion of Ca 2+ ions from the culture medium, results in the endocytotic uptake of membrane vesicles containing specific membrane constituents of the zonula adhaerens (ZA). During this process the junction-derived, endocytosed vesicles remain associated with the ZA plaque components, while the plaque and its attached actin filaments retract as a whole in a ring-like fashion from the plasma membrane, often accumulating, usually in fragments, in the juxtanuclear cytoplasm. Double-label immunofluorescence microscopy with antiplakoglobin and antivinculin has indicated that both plaque proteins colocalize with the hallmark membrane glycoprotein of this junction type, E-cadherin (uvomorulin). When HRP used as a fluid phase marker is applied to the culture medium, simultaneously with the Ca 2+ ion-chelator EGTA, numerous HRP-positive vesicles are found in close association with the dislocated plaque material, suggesting that the HRP is contained in the vesicles formed upon EGTA-induced junction splitting. Immunoelectron microscopy with various cadherin-specific antibodies revealed vesicle-associated labeling, confirming the derivation of these plaqueassociated vesicles from the ZA. As the desmosomespecific cadherin, desmoglein, is recovered in another type of junction-derived vesicle, which is characterized by its association with a desmoplakin-plaque, we conclude that the membrane domains of both kinds of junction are endocytosed during Ca 2+ depletion but stay in different vesicle populations, emphasizing the selective interaction of the specific cadherins with their respective plaque and filament partners. T hE apical region of polarized epithelial cells contains two types of cytoskeletal filament-anchoring junctions, i.e., the desmosomes (maculae adhaerentes) connected to intermediate-sized filaments (IFs) 1 and the actin filament-associated zonula adhaerens (ZA), both contributing to cell-cell adhesion as well as to intracellular organization (for reviews see 15, 26, 27, 49, 50). The two kinds of junctions are distinct by biochemical and morphological criteria, yet they share some basic architectural principles and have at least one major constituent protein in common: their junctional membrane domain is intimately associated with plakoglobin (82 kD; 10, 19, 20, 36). In both types of junctions, the direct mediators of cell-tocell adhesion appear to be transmembrane glycoproteins that are concentrated, or exclusively located, in the specific junction membrane domains, and those that have been characterized so far belong to the cadherin superfamily of cell adhesion molecules: desmoglein and desmocollins I and II are localized in desmosomes (4-7, 29, 30, 35, 39, 47-49, 51), while the major cell adhesion molecule of the ZA is E-cadherin (uvomorulin, L-CAM; 2, 3, 14, 22, 33, 46, 53, 58). 1. Abbreviations used in this paper: IF, intermediate-sized filament; ZA, zonula adhaerens. It has also been shown that the cytoplasmic domains of the desmosomal glycoproteins and of the ZA-cadherin(s) contribute to the formation of the specific filament-anchoring cytoplasmic plaques (11, 33, 43, 44, 47, 48, 51). In addition to the COOH-terminal, cytoplasmic portions of their specific cadherins, these plaques contain, besides plakoglobin, a set of cytoplasmic proteins that are characteristic of either desmosomes or the ZA: in polarized epithelial cells, desmosomes have been shown to contain desmoplakin (5, 8, 9, 17-19, 32, 42, 49), whereas vinculin has been localized to ZA plaques (23-27; see 12 and 54 for other ZA proteins). Cadherins are Ca2+-binding proteins, and cadherin-mediated ceU-to-cell adhesion is dependent on a certain concentration of extraceUular Ca 2+ (for review see 53). In previous studies it has been shown that diverse types of cultured polar epithelial cells dissociate upon removal of Ca 2+ ions from the culture medium. During this process, half-desmosome equivalents are internalized in the form of endocytotic vesicles, including some desmosomal transmembrane glycoproteins, in association with the cytoplasmic plaque material and associated IF bundles (8, 13, 37, 38, 41, 48). The split-. ting of these cell-to-cell junctions in response to Ca 2+ depletion also results in the release of ZA plaque material from the plasma membrane: ring-like structures containing vincu© The Rockefeller University Press, 0021-9525/91/05/881/12 $2.00 The Journal of Cell Biology, Volume 113, Number 4, May 1991 881-892 881 on A uust 7, 2017 jcb.rress.org D ow nladed fom lin and actin have been identified deep in the cytoplasm, retracting centripetally to a juxtanuclear position (37, 57). However, the fate of the corresponding junctional membrane components, notably the cadherins, remained unresolved. In our previous study of Ca2+-depleted MDBK cell cultures (57) we have not recognized endocytotic ZA-derived membrane structures. More recently, however, we have applied several antibodies to ZA-specific cadherin(s), together with immunoelectron microscopy, to identify certain plasma membrane invaginations and vesicles containing fluid phase markers, and we can now show that during Ca 2+ depletion ZA membrane is also resumed into the cell interior, forming ZA plaque-associated vesicles. Therefore, we conclude that both kinds of junctions behave in a principally similar way, i.e., their membrane domains endocytose, but are kept separately, probably due to their remaining attachment to different filament systems. Materials and Methods