Ocular albinism: evidence for a defect in an intracellular signal transduction system.

G protein-coupled receptors (GPCRs) participate in the most common signal transduction system at the plasma membrane. The wide distribution of heterotrimeric G proteins in the internal membranes suggests that a similar signalling mechanism might also be used at intracellular locations. We provide here structural evidence that the protein product of the ocular albinism type 1 gene (OA1), a pigment cell-specific integral membrane glycoprotein, represents a novel member of the GPCR superfamily and demonstrate that it binds heterotrimeric G proteins. Moreover, we show that OA1 is not found at the plasma membrane, being instead targeted to specialized intracellular organelles, the melanosomes. Our data suggest that OA1 represents the first example of an exclusively intracellular GPCR and support the hypothesis that GPCR-mediated signal transduction systems also operate at the internal membranes in mammalian cells.

The Eps15 C. elegans homologue EHS-1 is implicated in synaptic vesicle recycling.

Eps15 represents the prototype of a family of evolutionarily conserved proteins that are characterized by the presence of the EH domain, a protein-protein interaction module, and that are involved in many aspects of intracellular vesicular sorting. Although biochemical and functional studies have implicated Eps15 in endocytosis, its function in the endocytic machinery remains unclear. Here we show that the Caenorhabditis elegans gene, zk1248.3 (ehs-1), is the orthologue of Eps15 in nematodes, and that its product, EHS-1, localizes to synaptic-rich regions. ehs-1-impaired worms showed temperature-dependent depletion of synaptic vesicles and uncoordinated movement. These phenotypes could be correlated with a presynaptic defect in neurotransmission. Impairment of EHS-1 function in dyn-1(ky51) worms, which express a mutant form of dynamin and display a temperature-sensitive locomotion defect, resulted in a worsening of the dyn-1 phenotype and uncoordination at the permissive temperature. Thus, ehs-1 and dyn-1 interact genetically. Moreover, mammalian Eps15 and dynamin protein were shown to interact in vivo. Taken together, our results indicate that EHS-1 acts in synaptic vesicle recycling and that its function might be linked to that of dynamin.

Tyrosine phosphorylation of Eps15 is required for ligand-regulated, but not constitutive, endocytosis.

Membrane receptors are internalized either constitutively or upon ligand engagement. Whereas there is evidence for differential regulation of the two processes, little is known about the molecular machinery involved. Previous studies have shown that an unidentified kinase substrate is required for endocytosis of the epidermal growth factor receptor (EGFR), the prototypical ligand-inducible receptor, but not of the transferrin receptor (TfR), the prototypical constitutively internalized receptor. Eps15, an endocytic protein that is tyrosine phosphorylated by EGFR, is a candidate for such a function. Here, we show that tyrosine phosphorylation of Eps15 is necessary for internalization of the EGFR, but not of the TfR. We mapped Tyr 850 as the major in vivo tyrosine phosphorylation site of Eps15. A phosphorylation-negative mutant of Eps15 acted as a dominant negative on the internalization of the EGFR, but not of the TfR. A phosphopeptide, corresponding to the phosphorylated sequence of Eps15, inhibited EGFR endocytosis, suggesting that phosphotyrosine in Eps15 serves as a docking site for a phosphotyrosine binding protein. Thus, tyrosine phosphorylation of Eps15 represents the first molecular determinant, other than those contained in the receptors themselves, which is involved in the differential regulation of constitutive vs. regulated endocytosis.

In vitro morphogenesis of chick embryo hypertrophic cartilage.

Dedifferentiated chick embryo chondrocytes (Castagnola, P., G. Moro, F. Descalzi-Cancedda, and R. Cancedda, 1986, J. Cell Biol., 102:2310-2317), when transferred to suspension culture on agarose-coated dishes in the presence of ascorbic acid, aggregate and remain clustered. With time in culture, clusters grow in size and adhere to each other, forming structures that may be several millimeters in dimension. These structures after 7 d of culture have the histologic appearance of mature hypertrophic cartilage partially surrounded by a layer of elongated cells resembling the perichondrium. Cells inside the aggregates have ultrastructural features of stage I (proliferating) or stage II (hypertrophic) chondrocytes depending on their location. Occurrence and distribution of type I, II, and X collagens in the in vitro-formed cartilage at different times of culture, show a temporal and spatial distribution of these antigens reminiscent of the maturation events occurring in the cartilage in vivo. A comparable histologic appearance is shown also by cell aggregates obtained starting with a population of cells derived from a single, cloned, dedifferentiated chondrocyte.

Developmentally regulated synthesis of a low molecular weight protein (Ch 21) by differentiating chondrocytes.

When transferred to suspension culture on agarose-coated dishes, dedifferentiated chick embryo chondrocytes resume the chondrocyte phenotype and continue their maturation to hypertrophic chondrocytes (Castagnola, P., G. Moro, F. Descalzi Cancedda, and R. Cancedda. 1986. J. Cell Biol. 102:2310-2317). In this paper we report the identification, purification, and characterization of a low molecular weight protein, named Ch 21, expressed and secreted by in vitro differentiating chondrocytes at a late stage of development. This protein is detectable in the cells after a short pulse labeling and is directly secreted in the culture medium. The Ch 21 protein has a peculiar resistance to limited pepsin digestion; nevertheless it is not collagenous in nature as revealed by its unaltered mobility when isolated from cells grown in the presence of alpha-alpha' dipyridyl, its resistance to bacterial collagenase, and its amino acid composition. By metabolic labeling of tissue slices and by immunohistochemistry, we show that in the chick embryo tibia the Ch 21 protein first appears at the boundary of the cone of hypertrophic cartilage and in the newly formed bone between the 6 and 10 d of embryo development and localizes in calcifying hypertrophic cartilage thereafter. The Ch 21 protein synthesized by the cultured chondrocytes is closely related and possibly identical to a 21K transformation-sensitive protein associated to the cell substratum of chick embryo fibroblasts.

In vitro development of hypertrophic chondrocytes starting from selected clones of dedifferentiated cells.

Single cells from enzymatically dissociated chick embryo tibiae have been cloned and expanded in fresh or conditioned culture media. A cloning efficiency of approximately 13% was obtained using medium conditioned by dedifferentiated chondrocytes. A cloning efficiency of only 1.4% was obtained when conditioned medium from hypertrophic chondrocytes was used, and efficiencies of essentially 0 were found with fresh medium or medium conditioned by J2-3T3 mouse fibroblasts. Cell clones were selected by morphological criteria and clones showing a dedifferentiated phenotype (fibroblast-like) were further characterized. Out of 38 clones analyzed, 17 were able to differentiate to the hypertrophic chondrocyte stage and reconstitute hypertrophic cartilage when placed in the appropriate culture conditions. Cells from these clones expressed the typical markers of chondrocyte differentiation, i.e., type II and type X collagens. Clones not undergoing differentiation continued to express only type I collagen. Hypertrophic chondrocytes from differentiating clones were analyzed at the single cell level by immunofluorescence; all the cells were positive for type X collagen, while approximately 50% of them showed positivity for type II collagen.

Numb is an endocytic protein.

Numb is a protein that in Drosophila determines cell fate as a result of its asymmetric partitioning at mitosis. The function of Numb has been linked to its ability to bind and to biologically antagonize Notch, a membrane receptor that also specifies cell fate. The biochemical mechanisms underlying the action of Numb, however, are still largely unknown. The wide pattern of expression of Numb suggests a general function in cellular homeostasis that could be additional to, or part of, its action in fate determination. Such a function could be endocytosis, as suggested by the interaction of Numb with Eps15, a component of the endocytic machinery. Here, we demonstrate that Numb is an endocytic protein. We found that Numb localizes to endocytic organelles and is cotrafficked with internalizing receptors. Moreover, it associates with the appendage domain of alpha adaptin, a subunit of AP2, a major component of clathrin-coated pits. Finally, fragments of Numb act as dominant negatives on both constitutive and ligand-regulated receptor-mediated internalization, suggesting a general role for Numb in the endocytic process.

Dense core secretory vesicles revealed as a dynamic Ca(2+) store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera.

The role of dense core secretory vesicles in the control of cytosolic-free Ca(2+) concentrations ([Ca(2+)](c)) in neuronal and neuroendocrine cells is enigmatic. By constructing a vesicle-associated membrane protein 2-synaptobrevin.aequorin chimera, we show that in clonal pancreatic islet beta-cells: (a) increases in [Ca(2+)](c) cause a prompt increase in intravesicular-free Ca(2+) concentration ([Ca(2+)]SV), which is mediated by a P-type Ca(2+)-ATPase distinct from the sarco(endo) plasmic reticulum Ca(2+)-ATPase, but which may be related to the PMR1/ATP2C1 family of Ca(2+) pumps; (b) steady state Ca(2+) concentrations are 3-5-fold lower in secretory vesicles than in the endoplasmic reticulum (ER) or Golgi apparatus, suggesting the existence of tightly bound and more rapidly exchanging pools of Ca(2+); (c) inositol (1,4,5) trisphosphate has no impact on [Ca(2+)](SV) in intact or permeabilized cells; and (d) ryanodine receptor (RyR) activation with caffeine or 4-chloro-3-ethylphenol in intact cells, or cyclic ADPribose in permeabilized cells, causes a dramatic fall in [Ca(2+)](SV). Thus, secretory vesicles represent a dynamic Ca(2+) store in neuroendocrine cells, whose characteristics are in part distinct from the ER/Golgi apparatus. The presence of RyRs on secretory vesicles suggests that local Ca(2+)-induced Ca(2+) release from vesicles docked at the plasma membrane could participate in triggering exocytosis.

Angiogenic potential in vivo by Kaposi's sarcoma cell-free supernatants and HIV-1 tat product: inhibition of KS-like lesions by tissue inhibitor of metalloproteinase-2.

OBJECTIVE: To determine the neoplastic nature of Kaposi's sarcoma (KS). A highly vascularized lesion, KS is frequently associated with AIDS, indicating HIV products may be involved. DESIGN AND METHODS: We determined the angiogenic properties of KS cell-secreted products and the HIV-1-tat gene product in vivo. Cell-free secreted products (KS-CM) from cultured epidemic and sporadic KS spindle cells or recombinant (r) HIV-1 tat protein were injected into mice with a matrix support (Matrigel). RESULTS: KS-CM produced lesions carrying all the phenotypic hallmarks of KS, as observed by light and electron microscopy: spindle-shaped cells, haemorrhages and an inflammatory infiltrate, as well as Factor VIII-positive endothelial cells lining new blood vessels. Electron microscopy indicated an initial granulocyte invasion, with spindle-cell migration and neocapillary formation in the centre of the matrix. These lesions required the cofactor heparin; KS-CM or heparin alone were poorly angiogenic. A less intense angiogenesis, with lower cellularity and few granulocytes, was observed in basic fibroblast growth factor (bFGF)/heparin lesions, indicating that factors other than bFGF are present in the KS spindle-cell products. When the collagenase inhibitor tissue inhibitor of metalloproteinases (TIMP)-2 was added to the sponges, KS-CM-induced angiogenesis was reduced by approximately 65% and bFGF-induced angiogenesis inhibited completely. Recombinant HIV-1 tat protein, a growth factor for KS cells, induced vascularization that was also enhanced by heparin, implying that HIV-1 tat could contribute to the aetiology of HIV-associated KS. CONCLUSIONS: KS-like lesions were obtained by injecting cell-free secreted products, suggesting that KS is a 'self-propagating' proliferative lesion caused by a cytokine imbalance and not a true neoplasm. Heparin-binding factors appear to be involved, and HIV-1 tat angiogenic properties implicate this molecule in AIDS-associated KS. Inhibition of KS-CM-induced KS-like lesions by TIMP-2 suggests that metalloproteinase inhibitors could be potential therapeutic agents for KS.

Monocyte-derived dendritic cells and monocytes migrate to HIV-Tat RGD and basic peptides.

OBJECTIVE AND DESIGN: Extracellular Tat released from HIV-1-infected cells is a mitogenic and motogenic factor for endothelial and Kaposi's sarcoma (KS)-derived cells and is angiogenic in vivo. Here we show for the first time that Tat induces migration of human dendritic cells in a concentration-dependent manner and that the Arg-Gly-Asp (RGD) and basic Tat peptides contribute to dendritic and monocyte cell migration. In vivo, Tat stimulates invasion of macrophages into a matrigel sponge. METHODS: Monocyte and dendritic cell chemotaxis was assessed using the Boyden chamber assay. RESULTS: Tat induced migration of monocyte-derived dendritic cells at the same levels as the N-formyl-Met-Leu-Phe peptide, and of monocytes at levels comparable to RANTES. Peptide mapping of the chemotactic activity of Tat showed that the RGD domain, which has been shown to support integrin-mediated cell migration, and the basic domain which binds and activates the tyrosine kinase receptor KDR on endothelial cells, both had activity. Antibody-blocking experiments indicate that responses to the RGD domain was inhibited by beta1 and alpha vbeta3 integrin blocking antibodies. Combination of the Tat RGD and basic peptides did not show additive effects; however, Tat co-operated with macrophage-chemotactic protein or RANTES in inducing monocyte migration. CONCLUSIONS: Our results show that Tat can act as a chemoattractant for dendritic cells, and that both the RGD and basic domains are involved in this response. These same domains attract monocytes. The alpha vbeta3 and beta1 integrins are equally involved in Tat-induced monocyte migration, while the alpha vbeta3 integrin largely mediates the dendritic cell response to Tat.

Study of the function and regulation of liver N-CAM in Xenopus laevis.

The liver of Xenopus laevis is a unique exception in terms of the cell adhesion molecules (CAM) which it expresses. In most species, hepatocytes are characterized by the expression of the epithelial Ca(2+)-dependent CAM E-cadherin or of closely related variants of this molecule (e.g., L-CAM); in Xenopus liver, however, the levels of expression of epithelial cadherins is very low while a thyroxine-inducible isoform of N-CAM is expressed in postmetamorphic hepatocytes. Since Xenopus liver N-CAM is localized in regions of contact between hepatocytes, it has been proposed that it might be involved in mediating hepatocyte adhesion in this species. In this study, we demonstrate that N-CAM can indeed act as a functional adhesion molecule in the liver of Xenopus and that its expression is correlated with a number of profound morphological changes of this organ. After thyroxine treatment, hepatocytes are no longer organized in long loose cords but in compact lobules of cells. Furthermore, at the ultrastructural level, plasma membranes are in much closer proximity with the appearance of electron-dense material in areas of closer contact. We have established two novel culture systems for premetamorphic Xenopus hepatocytes as adherent and non-adherent cells, and we describe the induction of expression of N-CAM in these cells. Given the difference in the profile of adhesion molecules present in the liver of Xenopus and of other species, our results are discussed in view of the importance of the expression of a specific set of cell adhesion molecules in defining the development of homologous organs in different species.

Synthesis and secretion of Ch 21 protein in embryonic chick skeletal tissues.

We reported the identification, purification and characterization of a low molecular weight protein (Ch 21) expressed in vitro by differentiating chondrocytes at a late stage of development and observed in vivo in the growth plate region of the long bones at the border between hypertrophic cartilage and newly formed bone (Descalzi Cancedda, F., P. Manduca, C. Tacchetti, P. Fossa, R. Quarto, R. Cancedda, J. Cell Biol. 107, 2455-2463 (1988]. In this article, the synthesis and location of Ch 21 protein in the chick embryo tibia at late stage of development were further investigated. Ch 21 was observed in the cartilage matrix surrounding marrow cavities and in the prearticular outer layer by immunolocalization. In addition, the timing of Ch 21 appearance during the tibia development and its distribution in the growth plate region was better defined. We first observed presence of Ch 21 in the perichondral mid-diaphyseal sleeve of 7-day-old tibia. Ch 21 antibodies stained also the newly formed bone. Synthesis and secretion in the culture medium of Ch 21 protein was observed when bone fragments or cultured osteoblasts isolated from 19-day-old embryo tibiae were labeled in vitro. A search for the presence of Ch 21 in the chick embryo sternum was performed. The synthesis of Ch 21, both in the presumptive calcification cranial portion and in the permanent cartilaginous caudal portion of the sternum, was shown by metabolic labeling of tissue slices.(ABSTRACT TRUNCATED AT 250 WORDS)

NAD+-dependent internalization of the transmembrane glycoprotein CD38 in human Namalwa B cells.

CD38 is a transmembrane glycoprotein involved as an orphan receptor in many physiological processes of lymphocytes. It is also a bifunctional enzyme that catalyzes at its ectocellular domain the synthesis from NAD+ (cyclase) and the hydrolysis (hydrolase) of the calcium-mobilizing metabolite cyclic ADP-ribose (cADPR). A still unexplained paradox concerns the relationship between ectocellular localization of CD38 and intracellular calcium-releasing activity of its intermediate product cADPR. Incubation of CD38+ human Namalwa B cells with external NAD+ elicited extensive membrane down-regulation of CD38 and its internalization in non-clathrin-coated vesicles. Since the internalized CD38 was demonstrated to be enzymatically active, this NAD+-dependent process is a hitherto unrecognized means for shifting cADPR metabolism from the cell surface to the intracellular environment.

Purification and partial characterization of Xenopus laevis tenascin from the XTC cell line.

We report here the purification of tenascin, an extracellular matrix molecule involved in the control of morphogenesis, from the conditioned medium of the Xenopus XTC cell line. Tenascin was purified by affinity chromatography on a column of the monoclonal antibody mAb TnM1; the molecule eluted from this column has a relative molecular mass of 210 kDa after reduction. Electrophoretic analysis under non-reducing conditions shows that the purified components are oligomeric disulfide-linked complexes which barely enter a 4% polyacrylamide gel. Upon rotary shadowing these molecules appear to possess a central globular domain to which pairs or triplets of arms are attached. Polyclonal antibodies have been raised against purified Xenopus tenascin. They recognise specifically the antigen on Western blots of XTC conditioned medium and adult brain, by immunofluorescence, these antibodies reveal large amounts of tenascin in the secretory vesicles as well as in the extracellular matrix of XTC cells. In the Xenopus tadpole, they stain the developing cartilage, the basal lamina of skin epidermis, myotendinous ligaments and restricted regions of the central nervous system.

Use of human chondrocyte cell cultures to identify and characterize reactive antibodies in rheumatoid arthritis sera.

OBJECTIVE: To study the reactivity of rheumatoid arthritis (RA) sera with human chondrocyte populations isolated from normal cartilage and expanded in vitro. METHODS: Human articular chondrocytes were cultured as adherent (non-differentiated) cells on plastic dishes or in suspension (differentiated) on dishes previously coated with a thin layer of 1% agarose. Sera from 28 RA patients and 5 paired synovial fluids were tested on lysates from chondrocytes and fibroblasts as control by immunoblot. Antigen expression on the cell membrane was evaluated by flow cytometry in a few sera. RESULTS: In 9/28 RA sera IgG antibodies specific for chondrocyte antigens (97 kDa, 74 kDa, 67 kDa, 60 kDa, 54 kDa, 48 kDa and 37 kDa) were detected. Twelve sera reacted with proteins expressed both on chondrocytes and fibroblasts and 7 with fibroblasts only; two sera had no reactivity. When lysates from adherent or suspension chondrocytes were compared, RA sera reacted with higher intensity and detected more antigens on chondrocytes cultured in suspension. Flow cytometry assay demonstrated that RA sera are able to recognize antigens expressed on the cell membrane of the human chondrocytes. CONCLUSION: Our data indicate that: a) 32% of the RA sera contain antibodies reactive with antigens expressed exclusively by chondrocytes, but this value rises to 75% if antigens expressed both by chondrocytes and fibroblasts are considered; b) the reactivity of fully differentiated chondrocytes in suspension culture is higher than the reactivity of chondrocytes cultured in monolayer; and c) some of the chondrocyte-specific antigens identified are associated with the chondrocyte membrane. Thus, in vitro cultured chondrocytes may be used to study both the specificity and the biological activity of autoantibodies in RA.

Molecules that inhibit T-cell functions: cytochemical localization and shuttling.

Adaptive immune responses to antigens are mediated by specific receptors expressed on B cells (BCR's) and T cells (TCR's). Effector cells and memory cells are produced following a proliferative wave that accounts for clonal expansion. If not down-regulated, clonal expansion might lead to uncontrolled lymphoproliferation that would be harmful for the organism. Several mechanisms that account for the down-sizing of activated lymphocyte clones are briefly reviewed here. We next consider in detail one such mechanism that deals with the functional characterization and the immunocytochemical localization of two T-cell inhibitory molecules, namely the Cytotoxic T Lymphocyte Antigen-4 (CTLA-4) and the HP-F1 antigen, both present in all T lymphocytes. CTLA-4 and HP-F1 inhibit CD4+ T-helper cell proliferation and the lytic ability of CD8+ T-cytotoxic cells in non-specific and in antigen-specific cytolytic assays. Interestingly, a clonal distribution exists as for the ability of CTLA-4 and HP-F1 to inhibit T-cell functions. In resting and activated T cells, both molecules are largely confined in the endosomal compartment, as shown by immunofluorescence analyses. However, upon interaction of T cells with Antigen-Presenting Cells (APC's) or with target cells that must be killed, CTLA-4 molecules are transported to the plasma membrane, at the site of cell-to-cell contact where, following interaction with ligands, they trigger inhibitory signals.

[Bone metastasis of leiomyosarcoma. Apropos of a case]. / Métastase osseuse d'un leiomyosarcome. A propos d'un cas.

INTRODUCTION: Bone leiomyosarcoma is a rare tumor, whether it may be primary or secondary. The authors report on the case of a woman, aged 67, admitted in January 1992 complaining of pain in the left hip and the upper end of the femur. CASE REPORT: In 1985 the patient underwent surgical excision of a soft tissue tumor in the right thigh, histologically diagnosed as a benign fibrous tumor. This lesion recurred locally four times and repeated excisions were performed throughout the years, always with a histological diagnosis of a benign lesion. On admission to hospital, the physical examination as well as laboratory data and plain roentgenograms were unremarkable. Both tomography and MRI showed a lesion in the upper end of the left femur. An isotopic bone scan showed marked increased uptake in the left hip extending to the femoral diaphysis. An open biopsy was performed for histology, immunohistochemistry and electron microscopy. A diagnosis of metastatic leiomyosarcoma was made. The retrospective histological examination of specimens of the soft tissue tumor excised in 1985 showed the same immunohistochemical features of the contralateral leiomyosarcoma. On this basis, one stage resection of the left hip and the upper end of the femur was performed and a Kotz modular prosthesis was inserted. Postoperative healing was achieved without any complications and the function of the operated limb was satisfactory. Three months after the operation pulmonary lesions were noted on chest radiographs and CT scan. The patient died two years after the first admission for widespread metastasis. DISCUSSION: In the reported case, the bony metastasis appeared to be the presenting finding of the soft tissue tumor of the contralateral thigh. This presentation is rare in previously published series. The misdiagnosis of the primary tumor had caused local recurrences, and an increased malignity occurred. According to the literature, a soft tissue leiomyosarcoma can be easily confused with other spindle cell lesions. Therefore an accurate histological and ultrastructural diagnosis is necessary for adequate surgical treatment.

Functions and expression of liver N-CAM.

CONCLUSIONS: In conclusion we have shown that: (a) liver N-CAM is a functionally active adhesion molecule, (b) its expression during the morphogenetic processes occurring during Xenopus laevis metamorphosis correlates with morphological changes in the cell-to-cell interaction; (c) Thyroxine is not directely involved in the activation of the expression of liver N-CAM.