Regulation of subcellular distribution and oncogenic potential of nucleophosmin by plakoglobin.

Nucleophosmin (NPM) is a nucleolar phosphoprotein that is involved in many cellular processes and has both oncogenic and growth suppressing activities. NPM is localized primarily in nucleoli but shuttles between the nucleus and the cytoplasm, and sustained cytoplasmic distribution contributes to its tumor promoting activities. Plakoglobin (PG, γ-catenin) is a homolog of ß-catenin with dual adhesive and signaling functions. These proteins interact with cadherins and mediate adhesion, while their signaling activities are regulated by association with various intracellular partners. Despite these similarities, ß-catenin has a well-defined oncogenic activity, whereas PG acts as a tumor/metastasis suppressor through unknown mechanisms. Comparison of the proteomic profiles of carcinoma cell lines with low- or no PG expression with their PG-expressing transfectants has identified NPM as being upregulated upon PG expression. Here, we examined NPM subcellular distribution and in vitro tumorigenesis/metastasis in the highly invasive and very low PG expressing MDA-MB-231 (MDA-231) breast cancer cells and their transfectants expressing increased PG (MDA-231-PG) or NPM shRNA (MDA-231-NPM-KD) or both (MDA-231-NPM-KD+PG). Increased PG expression increased the levels of nucleolar NPM and coimmunoprecipitation studies showed that NPM interacts with PG. PG expression or NPM knockdown decreased the growth rate of MDA-231 cells substantially and this reduction was decreased further in MDA-231-NPM-KD+PG cells. In in vitro tumorigenesis/metastasis assays, MDA-231-PG cells showed substantially lower and MDA-231-NPM-KD cells substantially higher invasiveness relative to the MDA-231 parental cells, and the co-expression of PG and NPM shRNA led to even further reduction of the invasiveness of MDA-231-PG cells. Furthermore, examination of the levels and localization of PG and NPM in primary biopsies of metastatic infiltrating ductal carcinomas revealed coordinated expression of PG and NPM. Together, the data suggest that PG may regulate NPM subcellular distribution, which may potentially change the function of the NPM protein from oncogenic to tumor suppression.

Plakoglobin interacts with and increases the protein levels of metastasis suppressor Nm23-H2 and regulates the expression of Nm23-H1.

Plakoglobin (gamma-catenin) is a homolog of beta-catenin with similar dual adhesive and signaling functions. The adhesive function of these proteins is mediated by their interactions with cadherins, whereas their signaling activity is regulated by association with various intracellular partners. In this respect, beta-catenin has a well-defined oncogenic activity through its role in the Wnt signaling pathway, whereas plakoglobin acts as a tumor/metastasis suppressor through mechanisms that remain unclear. We previously expressed plakoglobin in SCC9 squamous carcinoma cells (SCC9-P) and observed a mesenchymal-to-epidermoid transition. Comparison of the protein and RNA profiles of parental SCC9 cells and SCC9-P transfectants identified various differentially expressed proteins and transcripts, including the nonmetastatic protein 23 (Nm23). In this study, we show that Nm23-H1 mRNA and Nm23-H2 protein are increased after plakoglobin expression. Coimmunoprecipitation and confocal microscopy studies using SCC9-P and various epithelial cell lines with endogenous plakoglobin expression revealed that Nm23 interacts with plakoglobin, cadherins and alpha-catenin. Furthermore, Nm23-H2 is the primary isoform involved in these interactions, which occur prominently in the cytoskeleton-associated pool of cellular proteins. In addition, we show that plakoglobin-Nm23 interaction requires the N-terminal (alpha-catenin interacting) domain of plakoglobin. Our data suggest that by increasing the expression and stability of Nm23, plakoglobin has a role in regulating the metastasis suppressor activity of Nm23, which may further provide a potential mechanism for the tumor/metastasis suppressor function of plakoglobin itself.

The importance of MUC1 cellular localization in patients with breast carcinoma: an immunohistologic study of 71 patients and review of the literature.

BACKGROUND: The MUC1 mucin is present on the apical surface of normal secretory epithelia. In breast carcinoma, MUC1 expression is variable in amount and cellular localization, the significance of which is controversial. The authors undertook a detailed analysis of staining pattern combined with a comprehensive literature review to better understand the role of MUC1 in breast carcinoma. METHODS: Seventy-one patients with breast carcinoma were examined for MUC1, beta-catenin, and E-cadherin staining patterns. These data were compared with data from 25 articles from the literature examining the expression of MUC1 in breast carcinoma. RESULTS: All invasive carcinomas showed some MUC1 staining. In invasive ductal carcinomas, MUC1 was detected in the apical membrane (15%), cytoplasm (93%), or circumferential membrane (13%), with 81% of tumors showing a mixture of patterns. Tumors with low overall MUC1 expression (< or = 50% positive tumor cells) had a higher nuclear grade than tumors with high overall MUC1 expression (> 50%; P = 0.01). Tumors with high and low cytoplasmic expression had no difference in nuclear grade (P > 0.3). Circumferential membrane staining was correlated with positive lymph node status (P = 0.011). CONCLUSIONS: In the literature, similar findings prevailed in which overall MUC1 expression was increased in lower grade (10 of 14 studies), estrogen receptor positive (8 of 13 studies) tumors and was associated with a better prognosis (8 of 13 studies). High cytoplasmic staining was associated with a worse prognosis, an association that was not explained by differences in histologic grade. Thus, the presence of MUC1 in the majority of tumor cells is associated with better differentiated tumors and with an improved prognosis. However, aberrantly localized MUC1 in the tumor cell cytoplasm or nonapical membrane is associated with a worse prognosis.

Plasticity of cadherin-catenin expression in the melanocyte lineage.

Cadherins are calcium-dependent cell adhesion receptors with strong morphoregulatory functions. To mediate functional adhesion, cadherins must interact with actin cytoskeleton. Catenins are cytoplasmic proteins that mediate the interactions between cadherins and the cytoskeleton. In addition to their role in cell-cell adhesion, catenins also participate in signaling pathways that regulate cell growth and differentiation. Cadherins and catenins appear to be involved in melanocyte development and transformation. Here, we investigated the function of cadherin-catenin complexes in the normal development and transformation of melanocytes by studying the patterns of expression of the cell-cell adhesion molecules, E-, N- and P-cadherin, and the expression of their cytoplasmic partners, alpha-, beta- and gamma-catenin during murine development. Similar analyses were performed in vitro using murine melanoblast, melanocyte, and melanoma cell lines in the presence and absence of keratinocytes, the cells with which melanocytes interact in vivo. Overall, the results suggest that the expression of cadherins and catenins is very plastic and depends on their environment as well as the transformation status of the cells. This plasticity is important in fundamental cellular mechanisms associated with normal and pathological ontogenesis, as well as with tumorigenesis.

Plakoglobin regulates the expression of the anti-apoptotic protein BCL-2.

Plakoglobin is a cytoplasmic protein and a homologue of beta-catenin and Armadillo of Drosophila with similar adhesive and signaling functions. These proteins interact with cadherins to mediate cell-cell adhesion and associate with transcription factors to induce changes in the expression of genes involved in cell fate determination and proliferation. Unlike the relatively well characterized role of beta-catenin in cell proliferation via activation of c-MYC and cyclin D1 gene expression, the signaling function of plakoglobin in regulation of cell growth is undefined. Here, we show that high levels of plakoglobin expression in plakoglobin-deficient human SCC9 cells leads to uncontrolled growth and foci formation. Concurrent with the change in growth characteristics we observe a pronounced inhibition of apoptosis. This correlates with an induction of expression of BCL-2, a prototypic member of apoptosis-regulating proteins. The BCL-2 expression coincides with decreased proteolytic processing and activation of caspase-3, an executor of programmed cell death. Our data suggest that the growth regulatory function of plakoglobin is independent of its role in mediating cell-cell adhesion. These observations clearly implicate plakoglobin in pathways regulating cell growth and provide initial evidence of its role as a pivotal molecular link between pathways regulating cell adherence and cell death.

L-CAM expression induces fibroblast-epidermoid transition in squamous carcinoma cells and down-regulates the endogenous N-cadherin.

SCC9 cells, derived from a squamous carcinoma of the tongue, were shown to lack E-cadherin but express alpha- and beta-catenins and N-cadherin. These cells also lack plakoglobin expression, do not assemble desmosomes and exhibit the typical morphology and growth properties of transformed cells. The N-cadherin expressed in SCC9 cells has properties similar to other classical cadherins, including interactions with the catenins. We transfected SCC9 cells with a full-length cDNA for L-CAM (liver cell adhesion molecule), the functional chicken homologue of E-cadherin. The exogenously expressed L-CAM formed complexes with catenins and the cytoskeleton and induced a morphological transition from fibroblastoid to epithelioid, conferred density-dependent growth inhibition, increased aggregation ability, and increased synthesis and stability of alpha- and beta-catenins. Coincident with these phenotypic changes, we detected a significant reduction in the level of endogenous N-cadherin, primarily as a result of rapid degradation of this protein in L-CAM-expressing cells. These results show the abnormal expression of N-cadherin in these transformed epidermoid cells, demonstrate the dynamics of the relationship between two cadherins, and provide a model system for the functional analysis of the tumor suppressor activity of E-cadherin in carcinomas.

Plakoglobin induces desmosome formation and epidermoid phenotype in N-cadherin-expressing squamous carcinoma cells deficient in plakoglobin and E-cadherin.

Pg is a homologue of beta-catenin and Armadillo, the product of the Drosophila segment polarity gene and has been shown to have both adhesive and signaling functions. It interacts with both classic and desmosomal cadherins. Pg interaction with the desmosomal cadherins is essential for desmosome assembly. Its precise role in the classic cadherin complexes is unclear, although Pg-E-cadherin interaction appears to be necessary for the formation of desmosomes. In addition to cadherins in adhesion complexes, Pg interacts with a number of proteins involved in regulation of cell differentiation and proliferation such as Lef-1/Tcf-1 transcription factors and the tumor suppressor protein APC. In this study, we have introduced Pg cDNA into SCC9 cells, a Pg- and E-cadherin-deficient squamous cell carcinoma line, which also lacks desmosomes. These cells have both alpha-catenin and beta-catenin, display unusual expression of N-cadherin, and have the typical fibroblastic phenotype of transformed cells. Pg-expressing SCC9 cells (SCC9P) formed desmosomes. Desmosome formation coincided with the appearance of an epidermoid phenotype, with increased adhesiveness and a contact-dependent decrease in growth. Biochemical characterization of SCC9P cells showed an increase in the expression and stability of N-cadherin and a decrease in level and stability of beta-catenin, without any apparent effects on alpha-catenin. These results show that, in the absence of E-cadherin, Pg can efficiently use N-cadherin to induce desmosome formation and epidermoid phenotype. They also suggest a role for Pg as one of the regulators of the intracellular beta-catenin levels and underscore the pivotal role of this protein in regulating cell adhesion and differentiation.

Inhibition of junction assembly in cultured epithelial cells by hepatocyte growth factor/scatter factor is concomitant with increased stability and altered phosphorylation of the soluble junctional molecules.

Hepatocyte growth factor/scatter factor (HGF/SF) is a mesenchymally derived glycoprotein with a strong scattering effect on epithelial cells. A receptor tyrosine kinase encoded by the met proto-oncogene has been identified as the cellular receptor for HGF/SF. Following stimulation with HGF/SF, cell scattering occurs concurrent with decreased cell-cell adhesion and disassembly of junctional components. In culture, junction formation is cell-cell contact dependent and can be regulated by modulating the Ca2+ concentrations of the growth media. Decreasing the Ca2+ concentrations below 50 microM causes rapid disassembly of junctions, whereas increasing the Ca2+ concentrations to 1.8 mM induces cell-cell contact and junction assembly. Although associated with decreased cell-cell adhesion and disassembly of the junctional complex, HGF/SF-induced scattering occurs under high extracellular Ca2+ concentrations. To gain insight into the mechanisms of HGF/SF-induced scattering of epithelial cells, we have studied the effect(s) of HGF/SF on junction assembly by examining the solubility, stability, phosphorylation, and subcellular localization of the major components of the adhering junctions, plakoglobin (Pg) and E-cadherin, in Madin-Darby canine kidney (MDCK) epithelial cells and in a MDCK cell line expressing an exogenous chimeric met receptor (CSF-MET) that scatters in response to colony-stimulating factor 1 (CSF-1). The results have shown that in HGF/SF-stimulated MDCK cells, adhering junctions were not assembled upon induction of cell-cell contact. Immunofluorescence analyses showed that larger amounts of Pg and E-cadherin were Triton X-100 extractable, and more significantly, these proteins were homogeneously distributed along the membrane and were not concentrated at the areas of cell-cell contact. Similar results were obtained for CSF-MET expressing MDCK cells in response to CSF-1. In contrast, none of the above effects were detected in MDCK cells expressing a mutant CSF-MET chimera containing a phenylalanine substitution at tyrosine 1356 in met, which fails to scatter in response to CSF-1. When compared with the unstimulated cells, the inhibition of cell adhesion promoted by HGF/SF correlated with an increased stability of the newly synthesized soluble E-cadherin and Pg and an altered phosphorylation pattern of E-cadherin, as determined by partial proteolytic peptide mapping.

Desmosome assembly and disassembly are regulated by reversible protein phosphorylation in cultured epithelial cells.

Desmosomes are one component of the intercellular junctional complex in epithelia. In cultures of epithelial cells, desmosome assembly can be regulated by modulating the calcium concentrations of the growth media. At present, very little is known about the intracellular signal transduction mechanisms that regulate desmosome assembly and disassembly in response to changing extracellular calcium concentrations. We have used inhibitors of protein kinases and phosphatases in a combined biochemical and morphological approach to analyze the role of protein phosphorylation in the assembly and disassembly of desmosomes in Madin-Darby canine kidney epithelial cells. Our results suggest that desmosomal proteins (desmoplakins I/II and desmoglein 1) are primarily phosphorylated on serine residues. Electron microscopic analyses of desmosome assembly upon induction of cell-cell contact, in the presence of protein kinase inhibitor, H-7, revealed an apparently normal assembly of desmosomes. However, complete disassembly of desmosomes was inhibited by H-7 upon removal of extracellular calcium. Under these conditions, although desmosomes split, desmosomal plaques and their associated cytokeratin filaments can not be internalized. In contrast, treatment of the cultures with okadaic acid (OA), an inhibitor of protein phosphatases, inhibited desmosome assembly but had no effect on disassembly. In addition, the inhibitory effect of okadaic acid on desmosome assembly was specific to this junction since we observed apparently normal tight junction and adherens junction in okadaic acid-treated cultures. These results suggest that assembly and disassembly of desmosomes may be regulated by extracellular Ca2+ via reversible protein phosphorylation involving both protein kinase and protein phosphatases.

Plakoglobin: kinetics of synthesis, phosphorylation, stability, and interactions with desmoglein and E-cadherin.

We have analyzed the kinetics of synthesis, phosphorylation, and stability of the soluble and insoluble plakoglobin (PG) and their interactions with Dsg1 and E-cadherin in Madin-Darby canine kidney (MDCK) epithelial cells in the absence of cell adhesion and after the induction of cell-cell contact. Using a combination of biochemical and morphological approaches, we show that newly synthesized PG enters a soluble:insoluble pool of proteins in a 60:40 ratio regardless of cell-cell contact. Following synthesis, PG is increasingly found in the insoluble pool. Although cell-cell contact does not effect either the size of each pool or the rate or efficiency of the transfer from the soluble into the insoluble pool, it results in a significant increase in the metabolic stability of the newly synthesized insoluble PG. The soluble PG initially forms separate complexes with E-cadherin and Dsg1. PG-Dsg1 complexes become insoluble and localize to the desmosome. PG-E-cadherin complexes remain soluble and are distributed intracellularly. The insoluble PG and E-cadherin detected at the cell periphery remain distinctly separate, as demonstrated previously [Hinck et al., 1994: J. Cell Biol. 125:1327-1340; Nathke et al., 1994: J. Cell Biol. 125:1341-1352]. In addition, we detected a separate pool of PG which is not associated with either Dsg1 or E-cadherin and after the induction of cell-cell contact becomes primarily insoluble and is distributed along the lateral membrane. Phosphorylation analysis showed that there is a significantly greater amount of phosphorylated PG in the soluble pool than in the insoluble pool. In addition the soluble pool is both serine and threonine phosphorylated, whereas the insoluble PG is primarily phosphorylated on serine residues.

Disorganization of microfilaments and intermediate filaments interferes with the assembly and stability of desmosomes in MDCK epithelial cells.

To investigate the possible role(s) of cytoskeletal elements in desmosome assembly we have studied the effects of cytostatic drugs on the assembly of desmosomes in MDCK epithelial cells. We showed previously [Pasdar et al.: Cell Motil. Cytoskeleton 23:201-213, 1992] that selective disruption of microtubules has no effect on desmosome assembly. Here, we have treated MDCK cells with cytochalasin B and a combination of cytochalasin B and nocodazole and analysed the effects of desmosome assembly. Immunofluorescence analysis of MDCK cultures following drug treatment indicated complete disruption of actin microfilaments and disorganization of cytokeratin intermediate filaments. Biochemical analysis of newly synthesized desmosomal membrane core glycoproteins as well as the cell adhesion protein E-cadherin revealed no effect of these drugs on the kinetics of synthesis, intracellular processing, or transport to the plasma membrane either in the presence or absence of cell-cell contact. However, morphological analyses revealed a significant disruption in the spatial organization of desmosomal proteins and E-cadherin. Drug treatment in the absence of cell-cell contact resulted in the disruption of the normally observed homogeneous punctate staining pattern and appearance of aggregate staining. Induction of cell-cell contact in these cultures resulted in redistribution of some of the aggregate staining to the plasma membrane. In contrast to control cultures, significant amount of intracellular staining was retained for all desmosomal proteins. Biochemical analyses of turnover rates of newly synthesized desmosomal proteins indicated a significant decrease in metabolic stability of these proteins while the turnover rate of E-cadherin was not significantly different among control and drug-treated cultures. Taken together, these results suggest that intact actin and cytokeratin filaments are necessary for the stability, efficient assembly, and spatial organization of the junctional components at the membrane. The regulatory role of cytokeratins and actin filaments in assembly and stability of desmosomes on the plasma membrane is discussed.

Desmosome assembly in MDCK epithelial cells does not require the presence of functional microtubules.

Desmosomes, complex multisubunit structures that assemble at sites of cell-cell contact, are important components of the epithelial junctional complex. Desmosome assembly requires the coordinated interaction at the plasma membrane of at least 8 cytoplasmic and integral membrane proteins organized into two structurally and functionally distinct domains, the cytoplasmic plaque and membrane core. Previous studies (Pasdar et al., J. Cell Biol., 113:645-655) provided evidence that cytokeratin filaments and microtubules may regulate transfer and assembly of cytoplasmic plaque and membrane core proteins, respectively. To determine directly the role of microtubules in these processes, Madin-Darby canine kidney (MDCK) cells were treated with nocodazole or colchicine to disrupt the microtubular network. Biochemical analysis of the different components of the cytoplasmic plaque and membrane core domains revealed little or no effect of nocodazole or colchicine on the kinetics of synthesis, post-translational modifications, transfer of proteins to the plasma membrane or their metabolic stability in the presence or absence of cell-cell contact. Likewise, immunofluorescence analysis of desmosome formation demonstrated an apparently normal desmosome assembly in the presence of nocodazole or colchicine upon induction of cell-cell contact. These results indicate that an intact microtubular network is not necessary for the processing or transport of the desmosomal membrane core glycoproteins to the plasma membrane in the absence or presence of cell-cell contact. Furthermore, the integration of the cytoplasmic plaque and membrane core domains induced by cell-cell contact at the plasma membranes of adjacent cells does not require the presence of functional microtubules.

Regulation of desmosome assembly in MDCK epithelial cells: coordination of membrane core and cytoplasmic plaque domain assembly at the plasma membrane.

Desmosomes are major components of the intercellular junctional complex in epithelia. They consist of at least eight different cytoplasmic and integral membrane proteins that are organized into two biochemically and structurally distinct domains: the cytoplasmic plaque and membrane core. We showed previously that in MDCK epithelial cells major components of the cytoplasmic plaque (desmoplakin I and II; DPI/II) and membrane core domains (desmoglein I; DGI) initially enter a pool of proteins that is soluble in buffers containing Triton X-100, and then titrate into an insoluble pool before their arrival at the plasma membrane (Pasdar, M., and W. J. Nelson. 1988. J. Cell Biol. 106:677-685; Pasdar. M., and W. J. Nelson. 1989. J. Cell Biol. 109:163-177). We have now examined whether either the soluble or insoluble pool of these proteins represents an intracellular site for assembly and interactions between the domains before their assembly into desmosomes at the plasma membrane. Interactions between the Triton X-100-soluble pools of DPI/II and DGI were analyzed by sedimentation of extracted proteins in sucrose gradients. Results show distinct differences in the sedimentation profiles of these proteins, suggesting that they are not associated in the Triton X-100-soluble pool of proteins; this was also supported by the observation that DGI and DPI/II could not be coimmunoprecipitated in a complex with each other from sucrose gradient fractions. Immunofluorescence analysis of the insoluble pools of DPI/II and DGI, in cells in which desmosome assembly had been synchronized, showed distinct differences in the spatial distributions of these proteins. Furthermore, DPI/II and DGI were found to be associated with different elements of cytoskeleton; DPI/II were located along cytokeratin intermediate filaments, whereas DGI appeared to be associated with microtubules. The regulatory role of cytoskeletal elements in the intracellular organization and assembly of the cytoplasmic plaque and membrane core domains, and their integration into desmosomes on the plasma membrane is discussed.

Regulation of desmosome assembly in epithelial cells: kinetics of synthesis, transport, and stabilization of desmoglein I, a major protein of the membrane core domain.

Desmosomes are composed of two morphologically and biochemically distinct domains, a cytoplasmic plaque and membrane core. We have initiated a study of the synthesis and assembly of these domains in Madin-Darby canine kidney (MDCK) epithelial cells to understand the mechanisms involved in the formation of desmosomes. Previously, we reported the kinetics of assembly of two components of the cytoplasmic plaque domain, Desmoplakin I/II (Pasdar, M., and W. J. Nelson. 1988. J. Cell Biol. 106:677-685 and 106:687-699. We have now extended this analysis to include a major glycoprotein component of the membrane core domain, Desmoglein I (DGI; Mr = 150,000). Using metabolic labeling and inhibitors of glycoprotein processing and intracellular transport, we show that DGI biosynthesis is a sequential process with defined stages. In the absence of cell-cell contact, DGI enters a Triton X-100 soluble pool and is core glycosylated. The soluble DGI is then transported to the Golgi complex where it is first complex glycosylated and then titrated into an insoluble pool. The insoluble pool of DGI is subsequently transported to the plasma membrane and is degraded rapidly (t1/2 less than 4 h). Although this biosynthetic pathway occurs independently of cell-cell contact, induction of cell-cell contact results in dramatic increases in the efficiency and rate of titration of DGI from the soluble to the insoluble pool, and its transport to the plasma membrane where DGI becomes metabolically stable (t1/2 greater than 24 h). Taken together with our previous study of DPI/II, we conclude that newly synthesized components of the cytoplasmic plaque and membrane core domains are processed and assembled with different kinetics indicating that, at least initially, each domain is assembled separately in the cell. However, upon induction of cell-cell contact there is a rapid titration of both components into an insoluble and metabolically stable pool at the plasma membrane that is concurrent with desmosome assembly.

Kinetics of desmosome assembly in Madin-Darby canine kidney epithelial cells: temporal and spatial regulation of desmoplakin organization and stabilization upon cell-cell contact. II. Morphological analysis.

Biochemical analysis of the kinetics of assembly of two cytoplasmic plaque proteins of the desmosome, desmoplakins I (250,000 Mr) and II (215,000 Mr), in Madin-Darby canine kidney (MDCK) epithelial cells, demonstrated that these proteins exist in a soluble and insoluble pool, as defined by their extract ability in a Triton X-100 high salt buffer (CSK buffer). Upon cell-cell contact, there is a rapid increase in the capacity of the insoluble pool at the expense of the soluble pool; subsequently, the insoluble pool is stabilized, while proteins remaining in the soluble pool continue to be degraded rapidly (Pasdar, M., and W. J. Nelson. 1988. J. Cell Biol. 106:677-685). In this paper, we have sought to determine the spatial distribution of the soluble and insoluble pools of desmoplakins I and II, and their organization in the absence and presence of cell-cell contact by using differential extraction procedures and indirect immunofluorescence microscopy. In the absence of cell-cell contact, two morphologically and spatially distinct patterns of staining of desmoplakins I and II were observed: a pattern of discrete spots in the cytoplasm and perinuclear region, which is insoluble in CSK buffer; and a pattern of diffuse perinuclear staining, which is soluble in CSK buffer, but which is preserved when cells are fixed in 100% methanol at -20 degrees C. Upon cell-cell contact, in the absence or presence of protein synthesis, the punctate staining pattern of desmoplakins I and II is cleared rapidly and efficiently from the cytoplasm to the plasma membrane in areas of cell-cell contact (less than 180 min). The distribution of the diffuse perinuclear staining pattern remains relatively unchanged and becomes the principal form of desmoplakins I and II in the cytoplasm 180 min after induction of cell-cell contact. Thereafter, the relative intensity of staining of the diffuse pattern gradually diminishes and is completely absent 2-3 d after induction of cell-cell contact. Significantly, double immunofluorescence shows that during desmosome assembly on the plasma membrane both staining patterns coincide with a subpopulation of cytokeratin intermediate filaments. Taken together with the preceding biochemical analysis, we suggest that the assembly of desmoplakins I and II in MDCK epithelial cells is regulated at three discrete stages during the formation of desmosomes.

Kinetics of desmosome assembly in Madin-Darby canine kidney epithelial cells: temporal and spatial regulation of desmoplakin organization and stabilization upon cell-cell contact. I. Biochemical analysis.

The functional interaction of cells in the formation of tissues requires the establishment and maintenance of cell-cell contact by the junctional complex. However, little is known biochemically about the mechanism(s) that regulates junctional complex assembly. To address this problem, we have initiated a study of the regulation of assembly of one component of the junctional complex, the desmosome, during induction of cell-cell contact in cultures of Madin-Darby canine kidney epithelial cells. Here we have analyzed two major protein components of the desmosomal plaque, desmoplakins I (Mr of 250,000) and II (Mr of 215,000). Analysis of protein levels of desmoplakins I and II by immunoprecipitation with an antiserum that reacts specifically with an epitope common to both proteins revealed that desmoplakins I and II are synthesized and accumulate at steady state in a ratio of 3-4:1 (in the absence or presence of cell-cell contact). The kinetics of desmoplakins I and II stabilization and assembly were analyzed after partitioning of newly synthesized proteins into a soluble and insoluble protein fraction by extraction of whole cells in a Triton X-100 high salt buffer. In the absence of cell-cell contact, both the soluble and insoluble pools of desmoplakins I and II are unstable and are degraded rapidly (t1/2 approximately 8 h). Upon induction of cell-cell contact, the capacity of the insoluble pool increases approximately three-fold as a proportion of the soluble pool of newly synthesized desmoplakins I and II is titrated into the insoluble pool. The insoluble pool becomes relatively stable (t1/2 greater than 72 h), whereas proteins remaining in the soluble pool (approximately 25-40% of the total) are degraded rapidly (t1/2 approximately 8 h). Furthermore, we show that desmoplakins I and II can be recruited from this unstable soluble pool of protein to the stable insoluble pool upon induction of cell-cell contact 4 h after synthesis; significantly, the stabilization of this population of newly synthesized desmoplakins I and II is blocked by the addition of cycloheximide at the time of cell-cell contact, indicating that the coordinate synthesis of another protein(s) is required for protein stabilization.

Differences in tissue expressions of enzyme activities in interspecific sunfish (Centrarchidae) hybrids and their backcross progeny.

The extent of naturally occurring variations of enzyme locus expression was determined for three tissues (liver, muscle, and eye) in two species of sunfish (Centrarchidae), the green sunfish (Lepomis cyanellus) and the redear sunfish (L. microlophus). The genetic basis for species differences in tissue enzyme specific activities of malate dehydrogenase (EC 1.1.1.37), lactate dehydrogenase (EC 1.1.1.27), phosphoglucomutase (EC 2.7.5.1), and glucosephosphate isomerase (EC 5.3.1.9) was investigated by determining enzyme specific activities in the tissues of the reciprocal F1 hybrids and of their backcross progenies. The specific activities for most enzymes in hybrids were intermediate between those of the parental species. Significant differences in enzyme specific activity were detected among the F1 progeny as well as those of backcrosses. Variations in specific activity levels in one tissue were often independent of variations in specific activities in a different tissue. However, the changes in the specific activities of different enzymes within the same tissue were often positively correlated. The tissue glucosephosphate isomerase activity differences appear not to be due to different functional contributions of the glucosephosphate isomerase allelic isozymes. Cluster analysis of distributions of specific activities revealed no simple Mendelian pattern of inheritance for control of tissue enzyme activity. Our results suggest a polygenic control of tissue enzyme specific activity levels.

Linkage relationships of nine enzyme Loci in sunfishes (lepomis; centrarchidae).

Linkage relationships of nine enzyme loci; aconitase (Acon ), esterase (Est), glucosephosphate isomerase A and B ( Gpi), glycerate-2-dehydrogenase (G2dh), malic enzyme (Me ), phosphoglycerate kinase (Pgk), phosphoglucomutase (Pgm ) and superoxide dismutase (Sod), were investigated in sunfishes (Lepomis, Centrarchidae). Reciprocal F(1) hybrids produced from crosses between green sunfish (Lepomis cyanellus) and redear sunfish ( L. microlophus) were backcrossed with each of the two parental species. A three-point linkage map comprising G2dh, Pgk and Sod is reported. The frequencies of recombination between G2dh and Pgk and between Pgk and Sod are estimated as 45.3 and 24.7%. The remaining six loci assort independently. Possible linkage conservation and homology of this linkage group with those of other vertebrate species are discussed.