Organization of Desmosomal Plaque Proteins in Cells Growing at Low Calcium Concentrations Rainer Duden and Werner W. Franke Division of Membrane Biology and Biochemistry, Institute of Cell and Tumor Biology, German Cancer Research Center, D-6900 Heidelberg, Federal Republic of Germany Abstract. Desmosomes are not formed in epithelial for long times (2-4 wk) in LCM are practically free cell cultures growing in media with low (x<0.1 mM) from such vesicles. In addition, we demonstrate that concentrations of Ca 2+ (LCM) but appear rapidly certain cells such as A-431 cells, when passaged in upon shift to media of normal calcium concentrations LCM and in the absence of stable junctions, are able (NCM). Previous authors using immunolocalization of to continually assemble "half-desmosomes" on the desmoplakin, a marker protein for the desmosomai plasma membrane which in turn can be endocytosed plaque, in LCM-grown cells have interpreted posi- as plaque-bearing vesicles. We also show that in LCM tively stained, dense, cytoplasmic aggregates on inter- the synthesis of several desmosomal proteins (des- mediate filaments (IF) bundles as preformed plaque moplakins I and II, plakoglobin, desmoglein, "band 6 units which upon NCM shift would move to the plas- protein") continues and that most of the plaque pro- ma membrane and contribute to desmosome forma- tein, desmoplakin, is diffusely spread over the cyto- tion. Studying various cell cultures, including primary plasm, apparently in a soluble monodisperse form of mouse keratinocytes and human A-431 cells, we show ~9S. From our results we propose that the plaque that most, probably all, desmoplakin-positive aggre- proteins occur in small, discrete, diffusible entities in gates in LCM-grown cells are associated with mem- the cytoplasm, in concentrations that are relatively branous structures, mostly vesicles, and also contain high in LCM and low in NCM, from which they as- other desmosomal markers, including desmoglein, a semble directly, i.e., without intermediate precursor transmembrane glycoprotein. We interpret such vesi- aggregates on IFs in the cytoplasm, on certain plasma cles as residual desmosome-derived domains endocy- membrane domains in a Ca 2+ dependent process. tosed upon cell dissociation. Only keratinocytes grown NTERACTION and communication between vertebrate filaments (22, 61). Primarily due to the availability of proce- cells, notably the formation and maintenance of tissues, dures for isolating sufficient quantities of desmosomes from I is largely dependent on semi-stable linkage structures, certain stratified epithelia (13, 17, 19, 25, 59), special pro- the intercellular junctions. Among the major categories of gress has been made in the identification of major desmo- junctions (gap junctions, adhering junctions, tight junctions) somal constituent proteins, including obligatory ones such as the junctions of the adhaerens category are of particular in- the plaque proteins, desmoplakin I (Mr ~ 250,000; 18, 46), terest in our understanding of cell and tissue morphology, as and plakoglobin (Mr * 83,000; 10, 26; this protein also oc- they are involved not only in cell-cell connections but also curs in plaques of nondesmosomal adhering junctions; 11), in the cell type-specific spatial organization of the filamen- and the transmembrane glycoprotein, desmoglein (Mr * tous cytoskeleton. Cytoplasmic plaques of adhering junc- 165,000; "band 3" polypeptide of isolated desmosomes from tions of the desmosomal kind provide anchorage sites for the bovine muzzle epidermis; 6, 17, 25, 55, 56, 62), and "acces- attachment of intermediate filaments (IFs) whereas the non- sory" components not found in desmosomes of all tissues, in- desmosomal junctions (zonulae and fasciae adhaerentes, cluding plaque proteins such as desmoplakin II and the basic puncta adhaerentia) are usually associated with actin micro- "band 6 polypeptide" (cf. 9, 10, 35) as well as glycoproteins (6, 7, 8, 17, 24, 25, 43, 52, 53). Further minor and/or cell type-specific desmosome-associated components have been Rainer Dudeffs current address is European Molecular Biology Laboratory, Meyerhofstr. 1, D-6900 Heidelberg, FRG. described (e.g., 62, 63). This compositional complexity sug- gests that the formation of the structure called desmosome 1. Abbreviations used in this paper: IF, intermediate filaments; LCM, low requires the coordination of the synthesis and assembly of calcium concentrations; NCM, normal calcium concentrations. various transmembrane proteins and proteins exclusive to the © The Rockefeller University Press, 0021-9525/88/09/1049/15 $2.00 The Journal of Cell Biology, Volume 107, September 1988 1049-1063 1049 cytoplasmic compartment as well as of cell-type specific ries, Richmond, CA), adjusted to 0.07 mM CaClr at this time-point and constituents. thrice weekly thereafter. LCM cultures could be maintained at near con- fluency for several months. Cell cultures were used for experiments at differ- Earlier electron microscopic studies have described the ent times after plating. advent of desmosome-like structures during early embryo- Human A-431 cells, a permanent line derived from an epidermoid carci- genesis (31, 40) and in cell cultures (e.g., 12; for reviews see noma of the vulva, and cells of Madin-Darby bovine kidney (MDBK) and references 9 and 39). It has also been observed, in certain Madin-Darby canine kidney (MDCK) lines were grown in normal medium (see 16, 18, 45) or LCM, with serial passages every third day. Routinely, tissues and in cell cultures depleted of calcium or treated cells on the third day after passage_ were used for experiments. with trypsin, that asymmetric plasma membrane domains The Ca r+ concentrations in the specific culture media were determined equivalent to "half-desmosomes" are formed and rapidly in- by atomic absorption spectrophotometry and adjusted to the desired level ternalized in the form of special plaque-bearing vesicles, by addition of the appropriate volume from a sterile 10 mM CaCl2 so- usually with IF bundles still attached, resulting in intracellu- lution. lar accumulations of desmosome-derived vesicles (17, 36, 37, 42, 49; for reviews see also 9, 39). These and other studies Antibodies have indicated that desmosomes, like other intercellular Four previously described mouse monoclonal antibodies were routinely junctions, are dependent on calcium, at least in cell culture used: anti-desmoglein DG3.10 (55, 56), anti-desmoplakin 1-2.17 (10), systems. Specifically, various epithelial cell cultures, when anti-plakoglobin PG5.1 (11) and anti-cytokeratin lu-5 (20). In some experi- ments we used a mixture of murine monoclonal desmoplakin antibodies grown in media with low calcium concentrations (LCM; DPI and I1-2.15, 2.17 and 2.19 (10). Affinity-purified guinea pig antibodies <0.1 mM), do not form desmosomes but do so within a rela- (IgG fraction) against the known major desmosomal proteins were used for tively short period of time upon shift to normal calcium con- comparison (10, 11, 17, 18, 35, 46, 55, 56). centrations (NCM; cf. 27-29, 32). Using mouse keratinocyte cultures grown in LCM and im- Immunofluorescence Microscopy munolocalization techniques Jones and Goldman (32) have Cells grown in NCM or LCM on uncoated (mouse keratinocytes) or on described reactions of desmoplakin antibodies with small poly-L-lysine-coated (A-431 cells in LCM) coverslips were fixed for 9 rain spheroidal structures ("dots") abundantly present in the in methanol (-20"C), followed by a brief immersion (1 rain) in acetone cytoplasm in association with IF bundles, and concluded that (-20°C), and air-dried. Alternatively, cells gently lysed by incubation with desmoplakin exists, in LCM-grown cells, in preformed ag- 0.01% saponin in PBS for 10 rain were washed three times, 5 min each, with PBS and incubated directly with the antibodies. Primary antibodies were gregate structures on IF bundles which then, upon shift to applied for 30 rain, followed by three 5-rain washes in PBS, 30 min of incu- normal calcium levels, move "as a package" to the cell sur- bation with Texas Red-coupled goat anti-mouse or anti-guinea pig Igs face, thus forming the typical desmosome-IF bundle ar- (Medac, Hamburg, FRG) diluted 1:150, and three 5-min washes in PBS. The chitecture. As this interpretation was at variance with several coverslips were dipped briefly in water, then in ethanol, and were mounted in Mowiol (Hoechst, Frankfurt, FRG). For double-label immunofluores- observations made by us and other authors (e.g. 3, 41) we cence microscopy, primary and, subsequently, secondary antibodies were studied the state of desmosomal proteins in various cell cul- applied as a cocktail. Texas red-coupled goat anti-mouse Igs were used in tures growing in LCM, including primary cultures of mouse cases of primary murine antibodies and FITC-coupled goat anti-guinea pig epidermal keratinocytes. We report here that for several days, Igs for primary antibodies of guinea pig origin. To prevent photobleaching desmoplakin, together with other desmosomal proteins, can of FITC, p-phenylendiamine (1 mg/ml) was added to the mounting medium. The cells were viewed, using epifluorescence illumination, with a Zeiss be identified in plaques attached to intracytoplasmic vesicles photomicroscope HI (Carl Zeiss, Oberkochen, FRG). and in association with IFs, probably resulting from en- Immunofluorescence microscopy of mouse keratinocytes grown for pro- docytotic uptake of previously existing desmosomal do- longed periods of time in LCM was