Epidermal growth factor receptors associated to cytoskeletal elements of epidermoid carcinoma (A431) cells.

The structural interaction of the epidermal growth factor (EGF) receptor and the cytoskeleton of A431 cells has been studied using a monoclonal anti-EGF receptor antibody. This has been done with immunogold labeling using a variety of electron microscopical preparation procedures and EGF binding studies. By providing an image of the membrane-associated cytoskeleton, the dry cleavage method reveals a preferential localization of EGF receptors superimposed upon cytoskeletal filaments. The colocalization of gold particles with cytoskeletal filaments is not affected when pre-labeled cells are extracted with the non-ionic detergent Triton X-100, as visualized by dry cleavage. Using surface replication, this treatment results in visualization of the cytoskeleton. In these latter preparations, it is also observed that EGF receptor-coupled gold particles remain associated with cytoskeletal elements. Moreover, Triton extraction performed before immunogold labeling of EGF receptors demonstrates that isolated cytoskeletons contained binding sites for anti-EGF receptor antibodies. Using stereo micrographs of replica's obtained from these isolated cytoskeletons, it is shown that gold-labeled EGF receptors are exclusively present on the cortical membrane-associated region of the cytoskeleton and not on more intracellular-located filaments. Scatchard analysis of EGF binding to cells fixed with glutaraldehyde and treated with Triton X-100 before and after EGF binding indicates that a high affinity EGF binding site is associated with the Triton X-100 insoluble cytoskeleton.

Parathyroid hormone-related peptide as an endogenous inducer of parietal endoderm differentiation.

Parathyroid hormone related peptide (PTHrP), first identified in tumors from patients with the syndrome of "Humoral Hypercalcemia of Malignancy," can replace parathyroid hormone (PTH) in activating the PTH-receptor in responsive cells. Although PTHrP expression is widespread in various adult and fetal tissues, its normal biological function is as yet unknown. We have examined the possible role of PTHrP and the PTH/PTHrP-receptor in early mouse embryo development. Using F9 embryonal carcinoma (EC) cells and ES-5 embryonic stem (ES) cells as in vitro models, we demonstrate that during the differentiation of these cells towards primitive and parietal endoderm-like phenotypes, PTH/PTHrP-receptor mRNA is induced. This phenomenon is correlated with the appearance of functional adenylate cyclase coupled PTH/PTHrP-receptors. These receptors are the mouse homologues of the recently cloned rat bone and opossum kidney PTH/PTHrP-receptors. Addition of exogenous PTH or PTHrP to RA-treated EC or ES cells is an efficient replacement for dBcAMP in inducing full parietal endoderm differentiation. Endogenous PTHrP is detectable at very low levels in undifferentiated EC and ES cells, and is upregulated in their primitive and parietal endoderm-like derivatives as assessed by immunofluorescence. Using confocal laser scanning microscopy on preimplantation mouse embryos, PTHrP is detected from the late morula stage onwards in developing trophectoderm cells, but not in inner cell mass cells. In blastocyst stages PTHrP is in addition found in the first endoderm derivatives of the inner cell mass. Together these results indicate that the PTH/PTHrP-receptor signalling system serves as a para- or autocrine mechanism for parietal endoderm differentiation in the early mouse embryo, thus constituting the earliest hormone receptor system involved in embryogenesis defined to date.

A431 cell variants lacking the blood group A antigen display increased high affinity epidermal growth factor-receptor number, protein-tyrosine kinase activity, and receptor turnover.

The epidermal growth factor receptor (EGF-R) of human A431 cells bears an antigenic determinant that is closely related to the human blood group A carbohydrate structure. Labeling studies with blood group A reactive anti-EGF-R monoclonal antibodies and various lectins revealed that A431 cultures are heterogeneous with respect to blood group A expression. We have isolated clonal variants of these cells that either express (A431A+ cells) or completely lack (A431A- cells) the blood group A specific N-acetyl-D-galactosamine (GalNAc) residue. We show that this difference is due to the absence of a UDP-GalNAc:Gal transferase activity in A431A- cells. Subsequently, we have compared EGF-R functioning in these cell lines. Scatchard analysis of EGF-binding shows that in A431A- cells 6.3% of the EGF-R belongs to a high affinity subclass (Kd = 0.4 nM) while in A431A+ this subclass represents only 3.2% of the total receptor pool. The elevated level of high affinity receptors in A431A- cells is accompanied by a parallel increase in receptor protein- tyrosine kinase activity. In membrane preparations of A431A- cells, receptor autophosphorylation as well as phosphorylation of a tyrosine-containing peptide substrate is 2-3-fold higher as compared with A431A+ cells. In intact A431A-cells, the difference in receptor activity is measured as a 2-3-fold elevated level of receptor phosphorylation and a 2-3-fold higher abundance of phosphotyrosine in total cellular protein in A431A- cells. In addition, [35S]methionine pulse-chase experiments showed a ligand-independent increase in turnover of EGF-R in A431A- cells: the receptor's half life in these cells is 10 h as compared with 17 h in A431A+ cells. Our results suggest a possible involvement of GalNAc residue(s) in determining EGF-R affinity, protein-tyrosine kinase activity and turnover in A431 cells. Furthermore, our results indicate that high affinity EGF-R are the biologically active species with respect to protein-tyrosine kinase activity.

Signal transduction by epidermal growth factor occurs through the subclass of high affinity receptors.

Many cell types display two classes of epidermal growth factor receptor (EGFR) as judged from EGF binding studies; i.e., a major class of low affinity EGFR and a minor class of high affinity EGFR. We have studied their respective contribution to the cascade of events elicited by EGF in human A431 carcinoma cells, using anti-EGFR mAb 2E9. This antibody specifically blocks EGF binding to low affinity EGFR, without activating receptors in intact cells, and thus enables us to study the effects of exclusive EGF binding to high affinity EGFR. We show that blocking of low affinity EGFR by mAb 2E9 has almost no effect on the activation of the receptor protein-tyrosine kinase by EGF, suggesting that EGFR kinase activation occurs exclusively through the subclass of high affinity EGFR (5-10%). In addition, we provide evidence that high affinity EGFR exists both in monomeric and dimeric forms, and that cross-phosphorylation of low affinity EGFR by high affinity EGFR may take place in dimers of both receptor types. We demonstrate that the following early cellular response to EGF are also unimpaired in the presence of mAb 2E9: (a) inositol phosphate production, (b) release of Ca2+ from intracellular stores, (c) rise in intracellular pH, (d) phosphorylation of EGF on threonine residue 654, (e) induction of c-fos gene expression, and (f) alteration in cell morphology. As possible nonspecific side effects, we observed that the EGF induced Ca2+ influx and fluid-phase pinocytosis were inhibited in A431 cells in the presence of mAb 2E9. We conclude, therefore, that the activation of the EGFR signal transduction cascade can occur completely through exclusive binding of EGF to the subclass of high affinity EGFR.

PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth.

The PTH/PTHrP receptor binds to two ligands with distinct functions: the calcium-regulating hormone, parathyroid hormone (PTH), and the paracrine factor, PTH-related protein (PTHrP). Each ligand, in turn, is likely to activate more than one receptor. The functions of the PTH/PTHrP receptor were investigated by deletion of the murine gene by homologous recombination. Most PTH/PTHrP receptor (-/-) mutant mice died in mid-gestation, a phenotype not observed in PTHrP (-/-) mice, perhaps because of the effects of maternal PTHrP. Mice that survived exhibited accelerated differentiation of chondrocytes in bone, and their bones, grown in explant culture, were resistant to the effects of PTHrP and Sonic hedgehog. These results suggest that the PTH/PTHrP receptor mediates the effects of Indian Hedgehog and PTHrP on chondrocyte differentiation.

Glycogen synthase kinase 3 phosphorylates Jun family members in vitro and negatively regulates their transactivating potential in intact cells.

Expression of immediate-early genes involving the 12-O-tetradecanoyl phorbol 13-acetate (TPA)-responsive element (TRE) is modulated by post-translational modification of pre-existing activator protein 1 (AP-1) constituents. One of the components of AP-1, c-Jun, has been shown to be phosphorylated by glycogen synthase kinase 3 (GSK-3) in vitro in a region proximal to the DNA-binding domain, resulting in decreased DNA binding. Here, we have used transient transfection to show that AP-1 activity is inhibitable by coexpression of GSK-3 in intact cells. Furthermore, we show that the c-Jun-related proteins JunD and JunB are subject to similar regulation by GSK-3 in intact cells. Comparison of tryptic phosphopeptide maps of the three Jun proteins incubated with GSK-3 in vitro with maps of the same proteins immunoprecipitated from 32P-labelled cells indicates similar sites of phosphorylation. Together, these data support the hypothesis that GSK-3 is an important regulator of AP-1 activity in vivo.

Interdependent action of RalGEF and Erk in Ras-induced primitive endoderm differentiation of F9 embryonal carcinoma cells.

Previous work by us and others has implicated a role for Ral guanine exchange factors (RalGEFs) in Ras-induced cell growth and oncogenic transformation. Here we show for the first time that RalGEFs are involved in Ras-induced differentiation as well. Expression of oncogenic Ras in F9 embryonal carcinoma (EC) cells is known to induce differentiation to a primitive endoderm (PrE)-like phenotype, but the downstream signal transduction mechanisms involved are unclear. We found that PrE differentiation is induced by the Ras effector domain mutants, RasV12G37 and RasV12E38, but not by RasV12C40. Accordingly, expression of constitutively active forms of RalGEF (Rlf-CAAX) or Rafl (Raf-CAAX) is sufficient to induce differentiation. Inhibition of RalGEF activity by expression of dominant negative Ral completely abolishes Rlf-CAAX- and RasV12G37-induced differentiation, while it reduces differentiation by RasV12 and Raf-CAAX. Finally, while Rlf-CAAX does not increase Erk activity, inhibition of MEK blocks both Ras- as well as Rlf-CAAX-induced differentiation, suggesting that RalGEFs induce PrE differentiation in a manner depending on basal MEK or Erk activity. Based on these results we conclude that Ras induces PrE differentiation of F9 EC cells via an interplay of Erk-and RalGEF-mediated pathways.

Transfer properties of the bovine brain phospholipid transfer protein. Specificity towards phosphatidylcholine analogs and the inhibitory effect of sphingomyelin.

A coupled transport of phosphatidylinositol from the monolayer to phosphatidylcholine vesicles, and a phosphatidylcholine transport in the reverse direction in the presence of bovine brain transfer protein is demonstrated. No significant amounts of protein accumulate at the interface during the transfer reaction. The transfer protein from bovine brain shows a lower specificity for phosphatidylcholine than does the transfer protein from bovine liver. Relative to egg phosphatidylcholine a low transfer rate is found for derivatives with a chain length of 14 carbon atoms and a distance between phosphorus and nitrogen of 6 carbon atoms. The gel state of phosphatidylcholine does not reduce the transfer reaction as catalyzed by the bovine brain protein. The transfer of phosphatidylinositol is inhibited by sphingomyelin. The presence of 200 mM K+ or 1mM Ca2+ does not affect the transfer activity of the bovine brain protein. Divalent ions at concentrations higher than 5 mM cause a fusion of vesicles with monolayers. The pH optimum of the phosphatidylinositol transfer reaction is 8.

Transcriptional regulation of retinoic acid receptor beta in retinoic acid-sensitive and -resistant P19 embryocarcinoma cells.

As in other embryocarcinoma (EC) cell lines retinoic acid (RA) rapidly induces expression of the nuclear retinoic acid receptor (RAR) beta in murine P19 EC cells, while RAR alpha is expressed constitutively. In the RA-resistant P19 EC-derived RAC65 cells, however, there is no such induction and an aberrant (smaller) RAR alpha transcript is expressed. RAR gamma 1 is expressed at low levels in both cell lines. To study the regulation of the RAR beta gene and the possible involvement of RAR alpha protein in transcriptional activation of the RAR beta gene we transfected these cells with a construct containing a 1.6 kb promoter fragment of the human RAR beta gene fused to the CAT gene. Upon transient assays in P19 EC cells CAT activity is enhanced rapidly by RA, to more than 100-fold in a concentration-dependent fashion. On the contrary no activity can be observed in the RA-resistant RAC65 cells; however, co-transfection of hRAR alpha, hRAR beta or hRAR gamma 1 restores the RA-dependent induction of CAT activity. These results clearly show that RAR alpha and RAR gamma 1 can transactivate the RAR beta gene; that RAR beta can stimulate its own expression and that resistance to RA in RAC65 cells is probably due to the altered RAR alpha transcript present in these cells.

Expression pattern of parathyroid hormone/parathyroid hormone related peptide receptor mRNA in mouse postimplantation embryos indicates involvement in multiple developmental processes.

In this paper we describe the cloning of the mouse Parathyroid Hormone/Parathyroid Hormone related Peptide Receptor (PTH/PTHrPR) cDNA and expression of its mRNA during mouse postimplantation development from day 5.5 until day 15.5 post coitum (p.c.). In support of a model from previous studies, in which parietal endoderm differentiation is regulated by the interaction of the PTH/PTHrPR and Parathyroid Hormone related Peptide (PTHrP), high levels of PTH/PTHrPR mRNA levels were detected in developing parietal endoderm from day 5.5 p.c. and onwards. In the embryo proper, PTH/PTHrPR mRNA expression was mainly detected at sites of epithelium/mesenchyme interactions, starting at day 9.5 p.c. in the epithelium of the intestine and later in the mesenchyme of the lung, the epithelium of meso- and metanephric tubuli, the dermis and at all sites where bone formation takes place. The complexity of the PTH/PTHrPR expression pattern suggests tight developmental regulation and indicates multiple roles in embryogenesis for the receptor and its ligands, not only in extraembryonic tissue but also in the formation of various organs.

Parathyroid hormone-related peptide (PTHrP) induces parietal endoderm formation exclusively via the type I PTH/PTHrP receptor.

A number of studies suggest a role for PTHrP and the classical PTH/PTHrP receptor (type I) in one of the first differentiation processes in mouse embryogenesis, i.e. the formation of parietal endoderm (PE). We previously reported that although in type I receptor (-/-) embryos PE formation seemed normal, the embryos were smaller from at least day 9.5 p.c. and 60% had died before day 12.5 p.c. Here we show that the observed growth defect commences even earlier, at day 8.5 p.c. Using two novel antibodies, we show that the expression of the type I receptor protein at this stage is confined to extraembryonic endoderm only. In addition, we show that large amounts of PTHrP protein are present in the adjacent trophoblast giant cells, suggesting a paracrine interaction of PTHrP and the type I PTH/PTHrP receptor in PE formation. The involvement in PE differentiation of other recently described receptors for PTHrP would explain a possible redundancy for the type I receptor in PE formation. However, deletion of the type I PTH/PTHrP receptor in ES cells by homologous recombination completely prevents PTHrP-induced PE differentiation. Based upon these observations, we propose that PTHrP and the type I PTH/PTHrP receptor, although not required for the initial formation of PE, are required for its proper differentiation and/or functioning.

Expression of the parathyroid hormone-related peptide gene in retinoic acid-induced differentiation: involvement of ETS and Sp1.

Differentiation of P19 embryonal carcinoma (EC) and embryonal stem (ES)-5 cells with retinoic acid (RA) induces expression of PTH-related peptide (PTHrP) mRNA. In this study we have characterized a region between nucleotide (nt) -88 and -58 relative to the transcription start site in the murine PTHrP gene that was involved in this expression. Sequence analysis identified two partially overlapping binding sites for the Ets family of transcription factors and an inverted Sp1-binding site. Two major specific bands were detected in a bandshift assay using an oligonucleotide spanning nt -88 and -58 as a probe and nuclear extracts from both undifferentiated and RA-differentiated P19 EC cells. The lower complex consisted of Ets-binding proteins as demonstrated by competition with consensus Ets-binding sites, while the upper complex contained Sp1-binding activity as demonstrated by competition with consensus Sp1-binding sites. The observed bandshift patterns using nuclear extracts of undifferentiated or RA-differentiated P19 cells were indistinguishable, suggesting that the differentiation-mediated expression was not caused by the induction of expression of new transcription factors. Mutations in either of the Ets-binding sites or the Sp1-binding site completely abolished RA-induced expression of PTHrP promoter reporter constructs, indicating that the RA effect was dependent on the simultaneous action of both Ets- and Sp1-like activities. Furthermore, these mutations also abolished promoter activity in cells that constitutively expressed PTHrP mRNA, suggesting a central role for the Ets and Sp1 families of transcription factors in the expression regulation of the mouse PTHrP gene.

Identification of a retinoic acid-inducible element in the murine PTH/PTHrP (parathyroid hormone/parathyroid hormone-related peptide) receptor gene.

We have shown previously that the PTH/PTHrP (PTH-related peptide) receptor mRNA becomes expressed very early in murine embryogenesis, i.e. during the formation of extraembryonic endoderm. Retinoic Acid (RA) is a potent inducer of extraembryonic endoderm formation and PTH/PTHrP-receptor expression in embryonal carcinoma (EC) and embryonal stem (ES) cells. Using the P19 EC cell line, we have characterized promoter elements of the murine PTH/PTHrP-receptor gene that are involved in this RA-induced expression. The data show that RA-induced expression of the PTH/ PTHrP-receptor gene is mediated by the downstream P2 promoter. Analysis of promoter reporter constructs in transiently transfected P19 cells treated with RA identified an enhancer region between nucleotides -2714 and -2702 upstream of the P2 transcription start site that is involved in the RA effect. This region matches a consensus hormone response element consisting of a direct repeat with an interspacing of 1 bp (R-DR1). The R-DR1 efficiently binds retinoic acid receptor-alpha (RARalpha)-retinoid X receptor-alpha (RXRalpha) and chicken ovalbumin upstream promoter (COUP)-transcription factor I (TFI)-RXRalpha heterodimers and RXRalpha and COUP-TFI homodimers in a bandshift assay using extracts of transiently transfected COS-7 cells. RA differentiation of P19 EC cells strongly increases protein binding to the R-DR1 in a band-shift assay. This is caused by increased expression of RXR (alpha, beta, or gamma) and by the induction of expression of RARbeta and COUP TFI/TFII, which bind to the R-DR1 as shown by supershifting antibodies. The presence of RXR (alpha, beta, or gamma) in the complexes binding to the R-DR1 suggests that RXR homodimers are involved in RA-induced expression of the PTH/PTHrP-receptor gene. The importance of the R-DR1 for RA-induced expression of PTH/ PTHrP-receptor was shown by an inactivating mutation of the R-DR1, which severely impairs RA-induced expression of PTH/PTHrP-receptor promoter reporter constructs. Since this mutation does not completely abolish RA-induced expression of PTH/PTHrP-receptor promoter reporter constructs, sequences other than the R-DR1 might also be involved in the RA effect. Finally, we show that the RA-responsive promoter region is also able to induce expression of a reporter gene in extraembryonic endoderm of 7.5 day-old transgenic mouse embryos.

Signals governing extraembryonic endoderm formation in the mouse: involvement of the type 1 parathyroid hormone-related peptide (PTHrP) receptor, p21Ras and cell adhesion molecules.

The formation of parietal endoderm (PE) from primitive endoderm (PrE) immediately after implantation of the early mouse embryo can be seen as the earliest example of an epithelio-mesenchyme transition (EMT) in murine development. Since EMT and EMI (epithelium-mesenchyme interactions) are at the very heart of morphogenesis, identifying molecular mechanisms governing these processes is of utmost importance. An excellent in vitro model system to study PE formation, i.e. F9 embryonal carcinoma cells, is available to this end. In the present paper we review our own recent results and those of others using these cells, and present our current view on the molecular mechanisms involved in PE formation.

Parathyroid hormone related peptide mRNA expression during murine postimplantation development: evidence for involvement in multiple differentiation processes.

In this study we describe the spatio-temporal expression of Parathyroid Hormone related Peptide (PTHrP) mRNA during murine postimplantation development from day 5.5 post coitum (pc) until day 12.5 pc. From day 5.5 pc and onwards PTHrP mRNA was detected in the trophoblast. In addition, at day 5.5 and 6.5 pc epithelial cells of the antimesometrial crypt and cells of the inner zone of the decidua directly adjacent to the implanted embryo expressed PTHrP mRNA. This supported a previous model in which parietal endoderm formation is regulated by a paracrine mechanism involving PTHrP expressing trophoblast cells and receptor expressing extra-embryonal endoderm cells. The first embryonal PTHrP mRNA expression was detected in the roof of the hindbrain at gestation day 10.5 pc. From day 11.5 pc and onwards PTHrP mRNA was detected in the otic vesicle, the semilateral channels, the roof of the hindbrain and later in the choroid plexus, in epithelial cells of the lung and heart ventricle, mesenchymal cells lining the nasal pit, the dermis of the snout and at all sites of endochondral bone formation. The widespread expression of PTHrP mRNA during embryogenesis in extra-embryonic and embryonic tissues suggests the involvement of the peptide in multiple growth and differentiation processes.

Growth factor signalling.

Signalling between cells in the developing vertebrate embryo is essential for normal embryonic development. In the mid 1970's, signal transduction research started at the Hubrecht Laboratory with special emphasis on analysis of the signalling mechanisms that direct cell proliferation and differentiation. The introduction of in vitro model systems contributed tremendously to the success of the signal transduction research at the Hubrecht Laboratory. Initially neuroblastoma cell lines, and later embryonal carcinoma and embryonal stem cells played an important role in identification of the molecular key players in developmental signalling. For instance, embryonal carcinoma cells were used to identify and characterise polypeptide growth factors. Growth factor signalling research was extended to analysis of growth factor receptor activation. Moreover, the second messenger systems that are linked to growth factor receptors were studied, as well as the nuclear responses to growth factor receptor activation. Finally, the role of growth factor signalling in differentiation was established using embryonal carcinoma cells. Here, we will review work that was characteristic for the growth factor receptor signalling research that was done at the Hubrecht Laboratory between 1980 and the early 1990's.

Snail is an immediate early target gene of parathyroid hormone related peptide signaling in parietal endoderm formation.

In mouse development, parietal endoderm (PE) is formed from both primitive endoderm (PrE) and visceral endoderm (VE). This process can be mimicked in vitro by using F9 embryonal carcinoma cells (EC) cells, differentiated to PrE or VE cells, and treating these with Parathyroid Hormone related Peptide (PTHrP). By means of differential display RT-PCR, we identified Snail (Sna) as a gene upregulated during the differentiation from F9 PrE to PE. We show that Sna is an immediate early target gene of PTHrP action in the formation of F9 PE cells. Using RT-PCR, we detected Sna transcripts in pre-implantation mouse embryos from the zygote-stage onwards. Sna was strongly upregulated in parallel with type 1 PTH/PTHrP Receptor (PTH(rP)-R1) mRNA in mouse blastocysts plated in culture, concomitant with detection of the PE-marker Follistatin and appearance of PE cells. By radioactive in situ hybridization on sections of mouse embryos, we found Sna expression in the earliest PE cells at E5.5. Sna remained expressed until at least E7.5. At this stage, we also observed clear expression in endoderm cells delaminating from the epithelial sheet of VE cells in the marginal zone. We conclude that PTH(rP)-R1 and Sna are expressed in endodermal cells that change from an epithelial to a mesenchymal phenotype. Since Sna expression has been described at other sites where epithelio-mesenchymal transitions (EMT) occur, such as the primitive streak at gastrulation and in pre-migratory neural crest cells, we hypothesize that Sna is instrumental in the action of PTHrP inducing PE formation, which we propose to be the first EMT in mouse development.

Visualization of epidermal growth factor receptor in cryosections of cultured A431 cells by immuno-gold labeling.

Cryo-ultramicrotomy in combination with immuno-gold labeling has been demonstrated to present a powerful tool in the visualization of extra- and intracellular located antigens. We have applied this method to localize epidermal growth factor (EGF) receptor in cultured A431 human epidermoid carcinoma cells. However, both the labeling efficiency, maintenance of antigenicity, and the recognizability of the ultrastructure in cryosections are highly dependent upon the fixation procedures. Using 125I-EGF or a consecutive labeling with a monoclonal anti EGF-receptor antibody, rabbit-anti-mouse antibody and 125I-protein A, it was shown that maintenance of antigenicity was optimal using 2% paraformaldehyde as a fixative, whereas under these conditions also the recognizability of ultrastructure was sufficient. After appropriate fixation and labeling, gold particles were observed associated with various regions of the plasma membrane, including coated pits, and with various types of vesicles, including coated vesicles, intracellular vesicular membranes, multi-vesicular bodies and lysosomes. The results indicate that this method allows a visualization of EGF-receptors and resolution of the EGF-receptor processing pathway at the electron microscopic level, independent of the internalization process of labeled ligands.

Dynamics of epidermal growth factor receptor internalization studied by Nanovid light microscopy and electron microscopy in combination with immunogold labeling.

Individual gold particles with a diameter of approximately 10 to 40 nm can be visualized using video-enhanced contrast microscopy (Nanovid) (De Brabander et al., Cell Motil. Cytoskel. 6, 105-113 (1986)). This technique allows a study of the dynamic properties of receptors and ligands in living cells at high resolution. We have studied epidermal growth factor (EGF) receptor internalization in human epidermoid carcinoma A431 cells, using a monoclonal anti-EGF-receptor antibody conjugated to 20-nm gold particles, referred to as 2E9-gold. Exposure of A431 cells to 2E9-gold at 37 degrees C resulted in binding of the complex at the cell surface. Most of the gold particles exhibit a Brownian type of movement, while a minority appeared immobile. Binding of the 2E9-gold complex is followed by internalization, as judged from Nanovid light microscopy studies in combination with electron microscopic observations. The internalized gold particles clearly cluster into large aggregates, most likely multivesicular bodies. Individual gold particles as well as aggregates are characterized by a saltatory movement, by which the gold particles eventually move from the cell periphery towards the cell center. Addition of EGF results in an increased rate of internalization of 2E9-gold, while Na-azide and nocodazole completely immobilize the intracellular gold particles, as has been demonstrated previously for the transferrin receptor.

Dynamic connexin43 expression and gap junctional communication during endoderm differentiation of F9 embryonal carcinoma cells.

Gap junctional communication permits the direct intercellular exchange of small molecules and ions. In vertebrates, gap junctions are formed by the conjunction of two connexons, each consisting of a hexamer of connexin proteins, and are either established or degraded depending on the nature of the tissue formed. Gap junction function has been implicated in both directing developmental cell fate decisions and in tissue homeostasis/metabolite exchange. In mouse development, formation of the extra embryonal parietal endoderm from visceral endoderm is the first epithelial-mesenchyme transition to occur. This transition can be mimicked in vitro, by F9 embryonal carcinoma (EC) cells treated with retinoic acid, to form (epithelial) primitive or visceral endoderm, and then with parathyroid hormone-related peptide (PTHrP) to induce the transition to (mesenchymal) parietal endoderm. Here, we demonstrate that connexin43 mRNA and protein expression levels, protein phosphorylation and subcellular localization are dynamically regulated during F9 EC cell differentiation. Dye injection showed that this complex regulation of connexin43 is correlated with functional gap junctional communication. Similar patterns of connexin43 expression, localization and communication were found in visceral and parietal endoderm isolated ex vivo from mouse embryos at day 8.5 of gestation. However, in F9 cells this tightly regulated gap junctional communication does not appear to be required for the differentiation process as such.