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Biomedical subjects

L Orci

Publications and source records attributed to L Orci.

At least 37 records · Page 2Linked to original sources
PubMed1998

Coatomer, Arf1p, and nucleotide are required to bud coat protein complex I-coated vesicles from large synthetic liposomes.

Synthetic coat protein complex I (COPI)-coated vesicles form spontaneously from large ( approximately 300 nm in diameter), chemically defined liposomes incubated with coatomer, Arf1p, and guanosine 5'-[gamma-thio]triphosphate. Coated vesicles are 40-70 nm in diameter, approximately the size of COPI vesicles formed from native membranes. The formation of COPI-coated buds and vesicles and the binding of Arf1p to donor liposomes depends on guanosine 5'-[gamma-thio]triphosphate. In contrast to the behavior of the COPII coat, coatomer binds to liposomes containing a variety of charged or neutral phospholipids. However, the formation of COPI buds and vesicles is stimulated by acidic phospholipids. In the absence of Arf1p, coatomer binds to liposomes containing dioleoylphosphatidic acid as a sole acidic phospholipid to form large coated surfaces without forming COPI-coated buds or vesicles. We conclude that Arf1p-GTP and coatomer comprise the minimum apparatus necessary to create a COPI-coated vesicle.

ADP-Ribosylation Factor 1↗
PubMed1998

Two transgenic approaches to define the cell lineages in endocrine pancreas development.

Ontogenic relationships between the different endocrine cell types of the islets of Langerhans were explored by generating transgenic mice, in which cells transcribing the glucagon, insulin, or pancreatic polypeptide genes were destroyed through the promoter-targeted expression of the diphtheria toxin A chain. In an alternate approach, to assess whether insulin cells are derived from precursors producing glucagon or PP, transgenic mice were generated bearing an insulin promoter-driven, and loxP-containing ('floxed') reporter transgene that can be irreversibly 'tagged' by recombination. They were crossed with mice expressing another transgene ('tagger') encoding Cre (cyclization recombination) recombinase in either glucagon or PP cells. The results obtained using both approaches indicate that neither glucagon nor insulin gene-expressing cells are the precursors to the other islet cells; also, they suggest that PP gene-expressing cells are necessary for the differentiation of islet insulin and somatostatin cells, through a cell lineage or a paracrine relationship.

Animals↗
PubMed1998

COPII-coated vesicle formation reconstituted with purified coat proteins and chemically defined liposomes.

COPII vesicle formation requires only three coat assembly subunits: Sar1p, Sec13/31p, and Sec23/24p. PI 4-phosphate or PI 4,5-bisphosphate is required for the binding of these proteins to liposomes. The GTP-bound form of Sar1p recruits Sec23/24p to the liposomes as well as to the ER membranes, and this Sar1p-Sec23/24p complex is required for the binding of Sec13/31p. Ultrastructural analysis shows that the binding of COPII coat proteins to liposomes results in coated patches, coated buds, and coated vesicles of 50-90 nm in diameter. Budding proceeds without rupture of the donor liposome or vesicle product. These observations suggest that the assembly of the COPII coat on the ER occurs by a sequential binding of coat proteins to specific lipids and that this assembly promotes the budding of COPII-coated vesicles.

COP-Coated Vesicles↗
PubMed1998

Vesicles on strings: morphological evidence for processive transport within the Golgi stack.

Cis-Golgi cisternae have a higher freeze-fracture particle density than trans-cisternae. Transport vesicles neighboring cis or trans positions of the Golgi stack have a particle concentration comparable to that of the adjacent cisterna and the buds emerging from it. This implies that transport vesicles remain locally within the stack during their lifetime, near their origin, favoring a processive pattern of transport in which vesicle transfers occur preferentially between adjacent cisternae in the stack. A "string theory" is proposed to account for processive transport, in which a carpet of fibrous attachment proteins located at the surface of cisternae (the strings) prevent budded vesicles from diffusing away but still allow them to diffuse laterally, effectively limiting transfers to adjoining cisternae in the stack. Fibrous elements that multivalently connect otherwise free COPI-coated vesicles and uncoated transport vesicles to one or two cisternae simultaneously are discerned readily by electron microscopy. It is suggested that long, coiled coil, motif-rich, Golgi-specific proteins including p115, GM130, and possibly giantin, among others, function as the proposed strings.

Adenosine Triphosphate↗
PubMed1998

Incomplete processing of proinsulin to insulin accompanied by elevation of Des-31,32 proinsulin intermediates in islets of mice lacking active PC2.

The prohormone convertases PC2 (SPC2) and PC3/PC1 (SPC3) are the major precursor processing endoproteases in a wide variety of neural and endocrine tissues. Both enzymes are normally expressed in the islet beta cells and participate in proinsulin processing. Recently we generated mice lacking active PC2 due to a disruption of the PC2 gene (Furuta, M., Yano, H., Zhou, A., Rouillé, Y., Holst, J. J., Carroll, R. J., Ravazzola, M., Orci, L., Furuta, H., and Steiner, D. F. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6646-6651). Here we report that these PC2 mutant mice have elevated circulating proinsulin, comprising 60% of immunoreactive insulin-like components. Acid ethanol extractable proinsulin from pancreas is also significantly elevated, representing about 35% of total immunoreactive insulin-like components. These increased amounts of proinsulin are mainly stored in secretory granules, giving rise to an altered appearance on electron microscopy. In pulse-chase experiments, the mutant islets incorporate lesser amounts of isotopic amino acids into insulin-related components than normal islets. In both wild-type and mutant islets, proinsulin I was processed more rapidly to insulin, reflecting the preference of both PC2 and PC3 for substrates having a basic amino acid positioned four residues upstream of the cleavage site. The overall half-time for the conversion of proinsulin to insulin is increased approximately 3-fold in the mutant islets and is associated with a 4-5-fold greater elevation of des-31,32 proinsulin, an intermediate that is formed by the preferential cleavage of proinsulin at the B chain-C-peptide junction by PC3 and is C-terminally processed to remove Arg31 and Arg32 by carboxypeptidase E. The constitutive release of newly synthesized proinsulin from both mutant and wild-type islets during the first 1-2 h of chase was normal (<2% of total). These results demonstrate that PC2 plays an essential role in proinsulin processing in vivo, but is quantitatively less important in this regard than PC3, and that its absence does not influence the efficient sorting of proinsulin into the regulated secretory pathway.

Amino Acid Sequence↗
PubMed1998

Isolation of EpH4 mammary epithelial cell subpopulations which differ in their morphogenetic properties.

EpH4 is a nontumorigenic cell line derived from spontaneously immortalized mouse mammary gland epithelial cells (Fialka et al., 1996). When grown in collagen gels, EpH4 cells give rise to different types of structures, e.g., solid cords or branching tubes. By removing and subsequently dissociating single three-dimensional colonies of defined morphology, we have isolated six clonal subpopulations of EpH4 cells which display distinct morphogenetic properties in collagen gel cultures. Thus, cells from the H1B clone form branching cords devoid of a central lumen, K3A3 cells form cords enclosing small multifocal lumina, and J3B1 cells form large cavitary structures containing a wide lumen. I3G2 cells form either cords or tubes, depending on the type of serum added to the culture medium. Finally, when grown in serum-free medium, Bela cells form spherical cysts, whereas Be4a cells form long, extensively branched tubes. In additional assays of morphogenesis, i.e., cell sandwiching between two collagen gels or culture on a thick layer of Matrigel (a laminin-rich extracellular matrix), all clones form epithelial-cell lined cavitary structures, except H1B cells which are unable to generate lumina under these conditions. The EpH4 sublines we have isolated provide an in vitro system for studying the mechanisms responsible for lumen formation and branching morphogenesis, as well as for identifying the factors which subvert these developmental processes during mammary carcinogenesis.

Animals↗
PubMed1998

Roles of hepatocyte growth factor/scatter factor and transforming growth factor-beta1 in mammary gland ductal morphogenesis.

Epithelial-mesenchymal interactions are responsible for the unique pattern of ductal branching morphogenesis characteristic of the mammary gland. To investigate the factors which control the elongation and branching of lactiferous ducts, we developed an in vitro model of ductal morphogenesis in which clonal mouse mammary epithelial cells (TAC-2 cells) are grown in collagen gels. In this experimental system, fibroblast conditioned medium (CM)3 stimulates the formation of extensively arborized tubules. The molecule responsible for this tubulogenic effect was identified as hepatocyte growth factor/scatter factor (HGF/SF). To determine whether HGF/SF plays a role in mammary gland morphogenesis in vivo, the expression of HGF/SF and its receptor, c-Met, were analyzed in the rat mammary gland during pregnancy, lactation, and involution. Levels of HGF/SF and c-Met transcripts were progressively reduced during pregnancy, were virtually undetectable during lactation, and increased again during involution. Collectively, these in vitro and in vivo findings suggest that HGF/SF is a paracrine mediator of mammary gland ductal morphogenesis. We subsequently investigated the effect of another multifunctional cytokine, namely TGF-beta1, on branching morphogenesis of TAC-2 cells. TGF-beta1 had a striking biphasic effect: whereas relatively high concentrations of this cytokine inhibited colony formation, lower concentrations stimulated extensive elongation and branching of epithelial cords. Taken together, these studies indicate that HGF/SF is a stromal-derived paracrine mediator of mammary ductal morphogenesis, and that when present at low concentrations, TGF-beta1 can contribute to this process.

Animals↗
PubMed1997

Bidirectional transport by distinct populations of COPI-coated vesicles.

Electron microscope immunocytochemistry reveals that both anterograde-directed (proinsulin and VSV G protein) and retrograde-directed (the KDEL receptor) cargo are present in COPI-coated vesicles budding from every level of the Golgi stack in whole cells; however, they comprise two distinct populations that together can account for at least 80% of the vesicles budding from Golgi cisternae. Segregation of anterograde- from retrograde-directed cargo into distinct sets of COPI-coated vesicles is faithfully reproduced in the cell-free Golgi transport system, in which VSV G protein and KDEL receptor are packaged into separable vesicles, even when budding is driven by highly purified coatomer and a recombinant ARF protein.

Animals↗
PubMed1997

Defective prohormone processing and altered pancreatic islet morphology in mice lacking active SPC2.

The prohormone convertase SPC2 (PC2) participates in the processing of proinsulin, proglucagon, and a variety of other neuroendocrine precursors, acting either alone or in conjunction with the structurally related dense-core granule convertase SPC3 (PC3/PC1). We have generated a strain of mice lacking active SPC2 by introducing the neomycin resistance gene (Neor) into the third exon of the mSPC2 gene. This gene insertion results in the synthesis of an exon 3-deleted form of SPC2 that does not undergo autoactivation and is not secreted. The homozygous mutant mice appear to be normal at birth. However, they exhibit a small decrease in rate of growth. They also have chronic fasting hypoglycemia and a reduced rise in blood glucose levels during an intraperitoneal glucose tolerance test, which is consistent with a deficiency of circulating glucagon. The processing of proglucagon, prosomatostatin, and proinsulin in the alpha, delta, and beta cells, respectively, of the pancreatic islets is severely impaired. The islets in mutant mice at 3 months of age show marked hyperplasia of alpha and delta cells and a relative diminution of beta cells. SPC2-defective mice offer many possibilities for further delineating neuroendocrine precursor processing mechanisms and for exploring more fully the physiological roles of many neuropeptides and peptide hormones.

Animals↗
PubMed1997

ERS-24, a mammalian v-SNARE implicated in vesicle traffic between the ER and the Golgi.

We report the identification and characterization of ERS-24 (Endoplasmic Reticulum SNARE of 24 kD), a new mammalian v-SNARE implicated in vesicular transport between the ER and the Golgi. ERS24 is incorporated into 20S docking and fusion particles and disassembles from this complex in an ATP-dependent manner. ERS-24 has significant sequence homology to Sec22p, a v-SNARE in Saccharomyces cerevisiae required for transport between the ER and the Golgi. ERS-24 is localized to the ER and to the Golgi, and it is enriched in transport vesicles associated with these organelles.

Adenosine Triphosphatases↗
PubMed1997

Vascular endothelial growth factor is increased in devascularized rat islets of Langerhans in vitro.

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen with potent angiogenic and vascular permeability-inducing properties, both of which may be important for the function of islets of Langerhans. In this study, we have examined the expression of VEGF and its tyrosine kinase receptors (flt and flk-1) in isolated rat islets of Langerhans in vitro. When analyzed by in situ hybridization, islet tissue showed a significant 4.6-fold increase in VEGF mRNA expression over time in culture from 0 to 7 days. Islet tissue exposed to hypoxic/anoxic conditions for a period of 8 hr showed a 3.7-fold increase in VEGF mRNA when analyzed by Northern blot hybridization. Reverse transcriptase-polymerase chain reaction revealed the presence of both flt and flk-1 in freshly isolated islets, and two VEGF isoforms, namely VEGF120 and VEGF164. Three rodent beta-cell lines derived from insulinomas (RINm5F-2A, INS-1, and MIN6) were also found to express VEGF by Northern blot hybridization. However, neither hypoxia/anoxia nor low (0.3 g/L)- or high (3.0 g/L)-glucose culture conditions modulated their expression of VEGF. VEGF derived from RINm5F-2A cells was bioactive in a three-dimensional in vitro model of angiogenesis, which assays for endothelial cell invasion and capillary morphogenesis. These findings demonstrate, first, that devascularization increases VEGF expression in isolated islet tissue, and they point to VEGF as a potentially important endogenous angiogenic stimulus for subsequent revascularization in vivo. Second, our observations raise the possibility that survival of transplanted islets may be improved by increasing VEGF expression before transplantation.

Animals↗
PubMed1996

Interleaflet clear space is reduced in the membrane of COP I and COP II-coated buds/vesicles.

Intracellular transfers between membrane-bound compartments occur through vesicles that bud from a donor compartment to fuse subsequently with an acceptor membrane. We report that the membrane that delimits COP I or COP II-coated buds/vesicles from the endoplasmic reticulum and the Golgi complex has a thinner interleaflet clear space as compared with the surrounding, noncoated parental membrane. This change is compatible with a compositional change of the membrane bilayer during the budding process.

COP-Coated Vesicles↗
PubMed1996

Inositol 1,4,5-trisphosphate receptor subtype 3 in pancreatic islet cell secretory granules revisited.

It has been reported that the inositol 1,4,5-trisphosphate receptor subtype 3 is expressed in islet cells and is localized to both insulin and somatostatin granules [Blondel, O., Moody, M. M., Depaoli, A. M., Sharp, A. H., Ross, C. A., Swift, H. & Bell, G. I. (1994) Proc. Natl. Acad. Sci. USA 91, 7777-7781]. This subcellular localization was based on electron microscope immunocytochemistry using antibodies (affinity-purified polyclonal antiserum AB3) directed to a 15-residue peptide of rat inositol trisphosphate receptor subtype 3. We now show that these antibodies cross-react with rat, but not human, insulin. Accordingly, the anti-inositol trisphosphate receptor subtype 3 (AB3) antibodies label electron dense cores of mature (insulin-rich) granules of rat pancreatic beta cells, and rat granule labeling was blocked by preabsorption of the AB3 antibodies with rat insulin. The immunostaining of immature, Golgi-associated proinsulin-rich granules with AB3 antibodies was very weak, indicating that cross-reactivity is limited to the hormone and not its precursor. Also, the AB3 antibodies labeled pure rat insulin crystals grown in vitro but failed to stain crystals grown from pure human insulin. By immunoprecipitation, the antibodies similarly displayed a higher affinity for rat than for human insulin. We could not confirm the labeling of somatostatin granules using AB3 antibodies.

Animals↗
PubMed1996

TGF-beta1 induces morphogenesis of branching cords by cloned mammary epithelial cells at subpicomolar concentrations.

The factors which control the formation of lactiferous ducts during mammary gland development are not yet fully understood. To address this issue, we assessed the effect of the multifunctional cytokine transforming growth factor-beta-1 (TGF-beta1) on the morphogenetic properties of TAC-2.1 mammary epithelial cells grown in collagen gels. Here we demonstrate that TGF-beta1, has a biphasic effect on these cells: whereas relatively high concentrations (0.5-5 ng/ml) inhibit colony formation, lower concentrations (5-100 pg/ml = 200fM-4pM) stimulate colony elongation and branching, resulting in the formation of an extensive network of epithelial cords. We suggest that, in concert with other regulatory factors, low concentrations of TGF-beta1 promote mammary gland ductal morphogenesis.

Animals↗
PubMed1996

Coat proteins and vesicle budding.

The trafficking of proteins within eukaryotic cells is achieved by the capture of cargo and targeting molecules into vesicles that bud from a donor membrane and deliver their contents to a receiving department. This process is bidirectional and may involve multiple organelles within a cell. Distinct coat proteins mediate each budding event, serving both to shape the transport vesicle and to select by direct or indirect interaction the desired set of cargo molecules. Secretion, which has been viewed as a default pathway, may require sorting and packaging signals on transported molecules to ensure their rapid delivery to the cell surface.

Biological Transport↗
PubMed1996

Traffic COPs and the formation of vesicle coats.

Forward and retrograde trafficking of secretory proteins between the endoplasmic reticulum and the Golgi apparatus is driven by two biochemically distinct vesicle coats, COPI and COPII. Assembly of the coats on their target membranes is thought to provide the driving force for membrane deformation and the selective packaging of cargo and targeting molecules into nascent transport vesicles. This review describes our current knowledge on these issues and discusses how the two coats may be differentially targeted and assembled to achieve protein sorting and transport within the early secretory pathway.

Journal Article↗