bluuebunny/crossref_metadata_2025 huggingface dataset:
Downregulation of amyloid precursor protein inhibits neurite outgrowth in vitro.
Authors: Allinquant B , Hantraye P , Mailleux P , Moya K , Bouillot C , Prochiantz A
Published: May 2004

The amyloid precursor protein (APP) is a transmembrane protein expressed in several cell types. In the nervous system, APP is expressed by glial and neuronal cells, and several lines of evidence suggest that it plays a role in normal and pathological phenomena. To address the question of the actual function of APP in normal developing neurons, we undertook a study aimed at blocking APP expression using antisense oligonucleotides. Oligonucleotide internalization was achieved by linking them to a vector peptide that translocates through biological membranes. This original technique, which is very efficient and gives direct access to the cell cytosol and nucleus, allowed us to work with extracellular oligonucleotide concentrations between 40 and 200 nM. Internalization of antisense oligonucleotides overlapping the origin of translation resulted in a marked but transient decrease in APP neosynthesis that was not observed with the vector peptide alone, or with sense oligonucleotides. Although transient, the decrease in APP neosynthesis was sufficient to provoke a distinct decrease in axon and dendrite outgrowth by embryonic cortical neurons developing in vitro. The latter decrease was not accompanied by changes in the spreading of the cell bodies. A single exposure to coupled antisense oligonucleotides at the onset of the culture was sufficient to produce significant morphological effects 6, 18, and 24 h later, but by 42 h, there were no remaining significant morphologic changes. This report thus demonstrates that amyloid precursor protein plays an important function in the morphological differentiation of cortical neurons in primary culture.
Intracellular domain of desmoglein 3 (pemphigus vulgaris antigen) confers adhesive function on the extracellular domain of E-cadherin without binding catenins.
Authors: Roh J Y , Stanley J R
Published: May 2004

For the extracellular (EC) domain of E-cadherin to function in homophilic adhesion it is thought that its intracytoplasmic (IC) domain must bind alpha- and beta-catenins, which link it to the actin cytoskeleton. However, the IC domain of pemphigus vulgaris antigen (PVA or Dsg3), which is in the desmoglein subfamily of the cadherin gene superfamily, does not bind alpha- or beta-catenins. Because desmogleins have also been predicted to function in the cell adhesion of desmosomes, we speculated that the PVA IC domain might be able to act in a novel way in conferring adhesive function on the EC domain of cadherins. To test this hypothesis we studied aggregation of mouse fibroblast L cell clones that expressed chimeric cDNAs encoding the EC domain of E-cadherin with various IC domains. We show here that the full IC domain of PVA as well as an IC subdomain containing only 40 amino acids of the PVA intracellular anchor (IA) region confer adhesive function on the E-cadherin EC domain without catenin-like associations with cytoplasmic molecules or fractionation with the cell cytoskeleton. This IA region subdomain is evolutionarily conserved in desmogleins, but not classical cadherins. These findings suggest an important cell biologic function for the IA region of desmogleins and demonstrate that strong cytoplasmic interactions are not absolutely necessary for E-cadherin-mediated adhesion.
Association of p120, a tyrosine kinase substrate, with E-cadherin/catenin complexes.
Authors: Shibamoto S , Hayakawa M , Takeuchi K , Hori T , Miyazawa K , Kitamura N , Johnson K R , Wheelock M J , Matsuyoshi N , Takeichi M
Published: May 2004

p120 was originally identified as a substrate of pp60src and several receptor tyrosine kinases, but its function is not known. Recent studies revealed that this protein shows homology to a group of proteins, beta-catenin/Armadillo and plakoglobin (gamma-catenin), which are associated with the cell adhesion molecules cadherins. In this study, we examined whether p120 is associated with E-cadherin using the human carcinoma cell line HT29, as well as other cell lines, which express both of these proteins. When proteins that copurified with E-cadherin were analyzed, not only alpha-catenin, beta-catenin, and plakoglobin but also p120 were detected. Conversely, immunoprecipitates of p120 contained E-cadherin and all the catenins, although a large subpopulation of p120 was not associated with E-cadherin. Analysis of these immunoprecipitates suggests that 20% or less of the extractable E-cadherin is associated with p120. When p120 immunoprecipitation was performed with cell lysates depleted of E-cadherin, beta-catenin was no longer coprecipitated, and the amount of plakoglobin copurified was greatly reduced. This finding suggests that there are various forms of p120 complexes, including p120/E-cadherin/beta-catenin and p120/E-cadherin/plakoglobin complexes; this association profile contrasts with the mutually exclusive association of beta-catenin and plakoglobin with cadherins. When the COOH-terminal catenin binding site was truncated from E-cadherin, not only beta-catenin but also p120 did not coprecipitate with this mutated E-cadherin. Immunocytological studies showed that p120 colocalized with E-cadherin at cell-cell contact sites, even after non-ionic detergent extraction. Treatment of cells with hepatocyte growth factor/scatter factor altered the level of tyrosine phosphorylation of p120 as well as of beta-catenin and plakoglobin. These results suggest that p120 associates with E-cadherin at its COOH-terminal region, but the mechanism for this association differs from that for the association of beta-catenin and plakoglobin with E-cadherin, and thus, that p120, whose function could be modulated by growth factors, may play a unique role in regulation of the cadherin-catenin adhesion system.
Embryonic axis induction by the armadillo repeat domain of beta-catenin: evidence for intracellular signaling.
Authors: Funayama N , Fagotto F , McCrea P , Gumbiner B M
Published: May 2004

beta-catenin was identified as a cytoplasmic cadherin-associated protein required for cadherin adhesive function (Nagafuchi, A., and M. Takeichi. 1989. Cell Regul. 1:37-44; Ozawa, M., H. Baribault, and R. Kemler. 1989. EMBO [Eur. Mol. Biol. Organ.] J. 8:1711-1717). Subsequently, it was found to be the vertebrate homologue of the Drosophila segment polarity gene product Armadillo (McCrea, P. D., C. W. Turck, and B. Gumbiner. 1991. Science [Wash. DC]. 254:1359-1361; Peifer, M., and E. Wieschaus. 1990. Cell. 63:1167-1178). Also, antibody perturbation experiments implicated beta-catenin in axial patterning of the early Xenopus embryo (McCrea, P. D., W. M. Brieher, and B. M. Gumbiner. 1993. J. Cell Biol. 123:477-484). Here we report that overexpression of beta-catenin in the ventral side of the early Xenopus embryo, by injection of synthetic beta-catenin mRNA, induces the formation of a complete secondary body axis. Furthermore, an analysis of beta-catenin deletion constructs demonstrates that the internal armadillo repeat region is both necessary and sufficient to induce axis duplication. This region interacts with C-cadherin and with the APC tumor suppressor protein, but not with alpha-catenin, that requires the amino-terminal region of beta-catenin to bind to the complex. Since alpha-catenin is required for cadherin-mediated adhesion, the armadillo repeat region alone probably cannot promote cell adhesion, making it unlikely that beta-catenin induces axis duplication by increasing cell adhesion. We propose, rather, that beta-catenin acts in this circumstance as an intracellular signaling molecule. Subcellular fractionation demonstrated that all of the beta-catenin constructs that contain the armadillo repeat domain were present in both the soluble cytosolic and the membrane fraction. Immunofluorescence staining confirmed the plasma membrane and cytoplasmic localization of the constructs containing the armadillo repeat region, but revealed that they also accumulate in the nucleus, especially the construct containing only the armadillo repeat domain. These findings and the beta-catenin protein interaction data offer several intriguing possibilities for the site of action or the protein targets of beta-catenin signaling activity.
Dual pathways of internalization of the cholecystokinin receptor.
Authors: Roettger B F , Rentsch R U , Pinon D , Holicky E , Hadac E , Larkin J M , Miller L J
Published: May 2004

Receptor molecules play a major role in the desensitization of agonist-stimulated cellular responses. For G protein-coupled receptors, rapid desensitization occurs via receptor phosphorylation, sequestration, and internalization, yet the cellular compartments in which these events occur and their interrelationships are unclear. In this work, we focus on the cholecystokinin (CCK) receptor, which has been well characterized with respect to phosphorylation. We have used novel fluorescent and electron-dense CCK receptor ligands and an antibody to probe receptor localization in a CCK receptor-bearing CHO cell line. In the unstimulated state, receptors were diffusely distributed over the plasmalemma. Agonist occupation stimulated endocytosis via both clathrin-dependent and independent pathways. The former was predominant, leading to endosomal and lysosomal compartments, as well as recycling to the plasmalemma. The clathrin-independent processes led to a smooth vesicular compartment adjacent to the plasmalemma resembling caveolae, which did not transport ligand deeper within the cell. Potassium depletion largely eliminated clathrin-dependent endocytosis, while not interfering with agonist-stimulated receptor movement into subplasmalemmal smooth vesicle compartments. These cellular endocytic events can be related to the established cycle of CCK receptor phosphorylation and dephosphorylation, which we have previously described (Klueppelberg, U. G., L. K. Gates, F. S. Gorelick, and L. J. Miller. 1991. J. Biol. Chem. 266:2403-2408; Lutz, M. P., D. I. Pinon, L. K. Gates, S. Shenolikar, and L. J. Miller. 1993. J. Biol. Chem. 268:12136-12142). The rapid onset and peak of receptor phosphorylation after agonist occupation correlates best with a plasmalemmal localization, while stimulated receptor phosphatase activity correlates best with receptor residence in intracellular compartments. We postulate that the smooth vesicular compartment adjacent to the plasmalemma functions for the rapid resensitization of the receptor, while the classical clathrin-mediated endocytotic pathway is key for receptor downregulation via lysosomal degradation, as well as less rapid resensitization.
Expression of functional domains of beta G-spectrin disrupts epithelial morphology in cultured cells.
Authors: Hu R J , Moorthy S , Bennett V
Published: May 2004

Spectrin is a major structural protein associated with the cytoplasmic surface of plasma membranes of many types of cells. To study the functions of spectrin, we transfected Caco-2 intestinal epithelial cells with a plasmid conferring neomycin resistance and encoding either actin-binding or ankyrin-binding domains of beta G-spectrin fused with beta-galactosidase. These polypeptides, in principle, could interfere with the interaction of spectrin with actin or ankyrin, as well as block normal assembly of alpha- and beta-spectrin subunits. Cells expressing the fusion proteins represented only a small fraction of neomycin-resistant cells, but they could be detected based on expression of beta-galactosidase. Cells expressing spectrin domains exhibited a progressive decrease in amounts of endogenous beta G-spectrin, although alpha-spectrin was still present. Beta G-spectrin-deficient cells lost epithelial cell morphology, became multinucleated, and eventually disappeared after 10-14 d in culture. Spectrin-associated membrane proteins, ankyrin and adducin, as well as the Na+,K(+)-ATPase, which binds to ankyrin, exhibited altered distributions in cells transfected with beta G-spectrin domains. E-cadherin and F-actin, in contrast to ankyrin, adducin, and the Na+,K(+)-ATPase, were expressed, and they exhibited unaltered distribution in beta G-spectrin-deficient cells. Cells transfected with the same plasmid encoding beta-galactosidase alone survived in culture as the major population of neomycin-resistant cells, and they exhibited no change in morphology or in the distribution of spectrin-associated membrane proteins. These results establish that beta G-spectrin is essential for the normal morphology of epithelial cells, as well as for their maintenance in monolayer culture.
Molecular cloning, expression, and mapping of the high affinity actin-capping domain of chicken cardiac tensin.
Authors: Chuang J Z , Lin D C , Lin S
Published: May 2004

Tensin, an actin filament capping protein first purified from chicken gizzard, is localized to various types of adherens junctions in muscle and nonmuscle cells. In this paper, we describe the isolation and sequencing of tensin cDNA from a chicken cardiac library. The 6.3-kb chicken cardiac tensin cDNA encodes an open reading frame of 1,792 amino acids. Mammalian cells transfected with the chicken tensin cDNA expressed a polypeptide of approximately 200 kD recognizable by antibodies to chicken gizzard tensin. The expressed protein was incorporated into focal adhesions and other actin-containing structures in the transfected cells. To map the domain associated with tensin's high affinity, barbed-end F-actin-capping activity, bacterially expressed recombinant fusion proteins containing various segments of tensin were prepared and assayed for activity. The results of these experiments show that the high affinity capping domain (kD = 1.3 nM) lies within amino acid residues R1037-V1169. Additional studies on a shorter construct, S1061-H1145, showed that these 85 residues were sufficient for producing complete inhibition of actin polymerization and depolymerization. While this active domain is located within that of the "insertin" sequence (Weigt, C., A. Gaertner, A. Wegner, H. Korte, and H. E. Meyer. 1992. J. Mol. Biol. 227:593-595), our data showing complete inhibition of polymerization and shift in critical concentration are consistent with a simple barbed-end capping mechanism rather than the "insertin model." Our results also differ from those of a recent report (Lo, S. H., P. A. Janmey, J. H. Hartwig, and L. B. Chen. 1994. J. Cell Biol. 125:1067-1075), which concluded that their recombinant tensin has an "insertin-like" inhibitory effect on barbed-end actin polymerization, and that this activity is attributed to residues T936-R1037 (residues 888-989 in their numbering system). In our study, a fusion construct (N790-K1060) encompassing T936-R1037 had no significant effect on actin polymerization and depolymerization, even at high concentrations.
Cytoskeletal control of gene expression: depolymerization of microtubules activates NF-kappa B.
Authors: Rosette C , Karin M
Published: May 2004

Cell shape changes exert specific effects on gene expression. It has been speculated that the cytoskeleton is responsible for converting changes in the cytoarchitecture to effects on gene transcription. However, the signal transduction pathways responsible for cytoskeletal-nuclear communication remained unknown. We now provide evidence that a variety of agents and conditions that depolymerize microtubules activate the sequence-specific transcription factor NF-kappa B and induce NF kappa B-dependent gene expression. These effects are caused by depolymerization of microtubule because they are blocked by the microtubule-stabilizing agent taxol. In nonstimulated cells, the majority of NF-kappa B resides in the cytosplasm as a complex with its inhibitor I kappa B. Upon cell stimulation, NF-kappa B translocates to the nucleus with concomitant degradation of I kappa B. We show that cold-induced depolymerization of microtubules also leads to I kappa B degradation and activation of NF-kappa B. However, the activated factor remains in the cytoplasm and translocates to the nucleus only upon warming to 37 degrees C, thus revealing two distinct steps in NF-kappa B activation. These findings establish a new role for NF-kappa B in sensing changes in the state of the cytoskeleton and converting them to changes in gene activity.
Regulation of the interaction of nicotinic acetylcholine receptors with the cytoskeleton by agrin-activated protein tyrosine kinase.
Authors: Wallace B G
Published: May 2004

Agrin induces the accumulation of nicotinic acetylcholine receptors (AChRs) in the myofiber membrane at synaptic sites in vertebrate skeletal muscle and causes an increase in tyrosine phosphorylation of the AChR beta subunit. To examine further the mechanism of agrin-induced AChR phosphorylation and the relationship between changes in protein phosphorylation and AChR aggregation, the effect of the protein tyrosine phosphatase inhibitor sodium pervanadate was tested on chick myotubes in culture. Pervanadate caused an increase in the phosphotyrosine content of a variety of proteins, including the AChR. Pervanadate also prevented agrin-induced AChR aggregation and slowed the rate at which AChRs were extracted from intact myotubes by mild detergent treatment. The rate at which phosphorylation of the AChR beta subunit and receptor detergent extractability changed following pervanadate-induced phosphatase inhibition was increased by agrin, indicating that agrin activates a protein tyrosine kinase rather than inhibiting a protein tyrosine phosphatase. The present results, taken together with previous findings on the inhibition of agrin-induced AChR aggregation by protein kinase inhibitors, demonstrate that protein tyrosine phosphorylation regulates the formation and stability of AChR aggregates, apparently by strengthening the interaction between AChRs and the cytoskelton.
A novel pool of protein phosphatase 2A is associated with microtubules and is regulated during the cell cycle.
Authors: Sontag E , Nunbhakdi-Craig V , Bloom G S , Mumby M C
Published: May 2004

Immunofluorescence microscopy revealed the presence of protein phosphatase 2A (PP2A) on microtubules in neuronal and nonneuronal cells. Interphase and mitotic spindle microtubules, as well as centrosomes, were all labeled with antibodies against individual PP2A subunits, showing that the AB alpha C holoenzyme is associated with microtubules. Biochemical analysis showed that PP2A could be reversibly bound to microtubules in vitro and that approximately 75% of the PP2A in cytosolic extracts could interact with microtubules. The activity of microtubule-associated PP2A was differentially regulated during the cell cycle. Enzymatic activity was high during S phase and intermediate during G1, while the activity in G2 and M was 20-fold lower than during S phase. The amount of microtubule-bound PP2A remained constant throughout the cell cycle, implying that cell cycle regulation of its enzymatic activity involves factors other than microtubules. These results raise the possibility that PP2A regulates cell cycle-dependent microtubule functions, such as karyokinesis and membrane transport.
The intracellular distribution and pattern of expression of Mcl-1 overlap with, but are not identical to, those of Bcl-2.
Authors: Yang T , Kozopas K M , Craig R W
Published: May 2004

A family of genes related to the bcl-2 protooncogene has recently emerged. One member of this family, mcl-1, was cloned from a human myeloblastic leukemia cell line (ML-1) undergoing differentiation. The intracellular localization of mcl-1, as well as the kinetics of its expression during differentiation, have now been studied. These studies show that the intracellular distribution of mcl-1 overlaps with, but is not identical to, that of bcl-2: mcl-1 is similar to bcl-2 in that the mcl-1 protein has a prominent mitochondrial localization, and in that it associates with membranes through its carboxyl hydrophobic tail. mcl-1 differs from bcl-2, however, in its relative distribution among other (nonmitochondrial/heavy membrane) compartments, mcl-1 also being abundant in the light membrane fraction of immature ML-1 cells while bcl-2 is abundant in the nuclear fraction. Similarly, in differentiating ML-1 cells, the timing of expression of mcl-1 overlaps with, but is not identical to, that of bcl-2: the mcl-1 protein increases rapidly as cells initiate differentiation, and mcl-1 is a labile protein. In contrast, bcl-2 decreases gradually as cells complete differentiation. Overall, the mcl-1 and bcl-2 proteins have some properties in common and others tht are distinct. A burst of expression of mcl-1, prominently associated with mitochondria, complements the continued expression of bcl-2 in ML-1 cells differentiating along the monocyte/macrophage pathway.
p53-dependent and p53-independent activation of apoptosis in mammary epithelial cells reveals a survival function of EGF and insulin.
Authors: Merlo G R , Basolo F , Fiore L , Duboc L , Hynes N E
Published: May 2004

The p53 tumor suppressor protein has been implicated as a mediator of programmed cell death (PCD). A series of nontransformed mammary epithelial cell (MEC) lines were used to correlate p53 function with activation of PCD. Treatment of MECs expressing mutant, inactive, or no p53 with DNA-damaging agents did not induce apoptosis. Upon introduction of temperature-sensitive p53 into HC11 cells, which lack wild-type (wt) p53, PCD was observed after mitomycin treatment at 32 degrees, when the ts p53 protein is in wt conformation. Thus, wt p53 mediates activation of PCD in response to mitomycin in HC11 cells. Treatment of the MCF10-A cells, which express wt p53, with various DNA-damaging agents led to nuclear accumulation of p53. Only mitomycin treatment led to an increase in the number of apoptotic nuclei. ErbB-2-transformed MCF10-A cells responded to mitomycin, cisplatin, and 5-Fl-uracil, suggesting that signaling from activated ErbB-2 enhances the cells ability to respond to DNA damage. A combination of high cell density and serum-free medium induces apoptosis in all MECs tested, irrespective of their p53 status. Under these conditions, EGF or insulin act as survival factors in preventing PCD. These data might elucidate some aspects of breast involution and tumorigenesis.
Expression of the mad gene during cell differentiation in vivo and its inhibition of cell growth in vitro.
Authors: Västrik I , Kaipainen A , Penttilä T L , Lymboussakis A , Alitalo R , Parvinen M , Alitalo K
Published: May 2004

Mad is a basic region helix-loop-helix leucine zipper transcription factor which can dimerize with the Max protein and antagonize transcriptional activation by the Myc-Max transcription factor heterodimer. While the expression of Myc is necessary for cell proliferation, the expression of Mad is induced upon differentiation of at least some leukemia cell lines. Here, the expression of the mad gene has been explored in developing mouse tissues. During organogenesis in mouse embryos mad mRNA was predominantly expressed in the liver and in the mantle layer of the developing brain. At later stages mad expression was detected in neuroretina, epidermis, and whisker follicles, and in adult mice mad was expressed at variable levels in most organs analyzed. Interestingly, in the skin mad was highly expressed in the differentiating epidermal keratinocytes, but not in the underlying proliferating basal keratinocyte layer. Also, in the gut mad mRNA was abundant in the intestinal villi, where cells cease proliferation and differentiate, but not in the crypts, where the intestinal epithelial cells proliferate. In the testis, mad expression was associated with the completion of meiosis and early development of haploid cells. In cell culture, Mad inhibited colony formation of a mouse keratinocyte cell line and rat embryo fibroblast transformation by Myc and Ras. The pattern of mad expression in tissues and its ability to inhibit cell growth in vitro suggests that Mad can cause the cessation of cell proliferation associated with cell differentiation in vivo.
Analysis of nuclear lamin isoprenylation in Xenopus oocytes: isoprenylation of lamin B3 precedes its uptake into the nucleus.
Authors: Firmbach-Kraft I , Stick R
Published: May 2004

Protein prenylation is a posttranslational modification involving the covalent attachment of a prenyl lipid to a cysteine at or near the COOH terminus of a protein. It is required for membrane localization and efficient function of a number of cytoplasmic as well as nuclear proteins including the proto-oncogenic and activated forms of Ras. Farnesylation in conjunction with a nuclear localization signal has been shown to be necessary to target newly synthesized nuclear lamins to the inner nuclear envelope membrane. It is, however, not clear where in the cell isoprenylation of nuclear lamins takes place. In this study we describe in vivo and in vitro experiments on the isoprenylation of the Xenopus oocyte nuclear lamin B3. We show by kinetic analysis that newly synthesized lamins are isoprenylated in the cytosol of oocytes before uptake into the nucleus. From our data it can be concluded that isoprenylation of lamins in the nucleus, as it is observed under certain conditions of isoprene starvation, represents a default pathway rather than the physiological situation. We further analyzed the capacity of isolated nuclei to carry out isoprenylation of B3. Our results are in line with a dual localization of a protein farnesyltransferase in the cytosol and nuclei of amphibian oocytes. Implications for the possible functions of a nuclear protein farnesyltransferase as well as possible mechanisms of the selective inhibition of farnesylation of cytoplasmic proteins by peptidomimetics are discussed.
A talin homologue of Dictyostelium rapidly assembles at the leading edge of cells in response to chemoattractant.
Authors: Kreitmeier M , Gerisch G , Heizer C , Müller-Taubenberger A
Published: May 2004

In an attempt to identify unknown actin-binding proteins in cells of Dictyostelium discoideum that may be involved in the control of cell motility and chemotaxis, monoclonal antibodies were raised against proteins that had been enriched on an F-actin affinity matrix. One antibody recognized a protein distinguished by its strong accumulation at the tips of filopods. These cell-surface extensions containing a core of bundled actin filaments are rapidly protruded and retracted by cells in the growth-phase stage. The protein of 269 kD turned out to resemble mouse fibroblast talin (Rees et al., 1990) in its primary structure. The fit is best among the first 400-amino acid residues of the NH2-terminal region where identity between the two proteins is 44% and the last 200-amino acid residues of the COOH-terminal region with 36% identity. In the elongated cells of the aggregation stage the Dictyostelium talin is accumulated at the entire front where also F-actin is enriched. Since this protein exists in a soluble state in the cytoplasm, mechanisms are predicted that cause accumulation at sites of the cell where a front is established. Evidence for receptor-mediated accumulation was obtained by local stimulation of cells with cAMP. When a new front was induced by the chemoattractant, the talin accumulated there within half a minute, indicating a signal cascade in Dictyostelium responsible for assembly of the talin beneath sites of the plasma membrane where chemoattractant receptors are strongly activated. The ordered assembly of the talin homologue together with actin and a series of other proteins is considered to play a key role in chemotactic orientation.
The molecular organization of endothelial cell to cell junctions: differential association of plakoglobin, beta-catenin, and alpha-catenin with vascular endothelial cadherin (VE-cadherin).
Authors: Lampugnani M G , Corada M , Caveda L , Breviario F , Ayalon O , Geiger B , Dejana E
Published: May 2004

In this paper we report that the assembly of interendothelial junctions containing the cell type-specific vascular endothelial cadherin (VE-cadherin or cadherin-5) is a dynamic process which is affected by the functional state of the cells. Immunofluorescence double labeling of endothelial cells (EC) cultures indicated that VE-cadherin, alpha-catenin, and beta-catenin colocalized in areas of cell to cell contact both in sparse and confluent EC monolayers. In contrast, plakoglobin became associated with cell-cell junctions only in tightly confluent cells concomitantly with an increase in its protein and mRNA levels. Furthermore, the amount of plakoglobin coimmunoprecipitated with VE-cadherin, increased in closely packed monolayers. Artificial wounding of confluent EC monolayers resulted in a major reorganization of VE-cadherin, alpha-catenin, beta-catenin, and plakoglobin. All these proteins decreased in intensity at the boundaries of EC migrating into the lesion. In contrast, EC located immediately behind the migrating front retained junctional VE-cadherin, alpha-catenin, and beta-catenin while plakoglobin was absent from these sites. In line with this observation, the amount of plakoglobin coimmunoprecipitated with VE-cadherin decreased in migrating EC. These data suggest that VE-cadherin, alpha-catenin, and beta-catenin are already associated with each other at early stages of intercellular adhesion and become readily organized at nascant cell contacts. Plakoglobin, on the other hand, associates with junctions only when cells approach confluence. When cells migrate, this order is reversed, namely, plakoglobin dissociates first and, then, VE-cadherin, alpha-catenin, and beta-catenin disassemble from the junctions. The late association of plakoglobin with junctions suggests that while VE-cadherin/alpha-catenin/beta-catenin complex can function as an early recognition mechanism between EC, the formation of mature, cytoskeleton-bound junctions requires plakoglobin synthesis and organization.
Cellubrevin and synaptobrevins: similar subcellular localization and biochemical properties in PC12 cells.
Authors: Chilcote T J , Galli T , Mundigl O , Edelmann L , McPherson P S , Takei K , De Camilli P
Published: May 2004

There is strong evidence to indicate that proteins of the synaptobrevin family play a key role in exocytosis. Synaptobrevin 1 and 2 are expressed at high concentration in brain where they are localized on synaptic vesicles. Cellubrevin, a very similar protein, has a widespread tissue distribution and in fibroblasts is localized on endosome-derived, transferin receptor-positive vesicles. Since brain cellubrevin is not detectable in synaptic vesicles, we investigated whether cellubrevin and the synaptobrevins are differentially targeted when co-expressed in the same cell. We report that in the nervous system cellubrevin is expressed at significant levels only by glia and vascular cells. However, cellubrevin is coexpressed with the two synaptobrevins in PC12 cells, a neuroendocrine cell line which contains synaptic vesicle-like microvesicles. In PC12 cells, cellubrevin has a distribution very similar to that of synaptobrevin 1 and 2. The three proteins are targeted to neurites which exclude the transferrin receptor and are enriched in synaptic-like microvesicles and dense-core granules. They are recovered in the synaptic-like microvesicle peak of glycerol velocity gradients, have a similar distribution in isopycnic fractionation and are coprecipitated by anti-synaptobrevin 2 immunobeads. Finally, cellubrevin, like the synaptobrevins, interact with the neuronal t-SNAREs syntaxin 1 and SNAP-25. These results suggest that cellubrevin and the synaptobrevins have similar function and do not play a specialized role in constitutive and regulated exocytosis, respectively.
Differential modulation of cell phenotype by different molecular weight forms of basic fibroblast growth factor: possible intracellular signaling by the high molecular weight forms.
Authors: Bikfalvi A , Klein S , Pintucci G , Quarto N , Mignatti P , Rifkin D B
Published: May 2004

To study possible functional differences of the 18-kD and high molecular weight forms of basic fibroblast growth factor (bFGF), we have examined the effect of endogenous production of different bFGF forms on the phenotype of NIH 3T3 cells. Cells transfected with cDNAs coding for either 18-kD bFGF (18-kD bFGF) or all four molecular forms (18, 22, 22.5, 24 kD; wild type [WT] bFGF) exhibit increased migration and decreased FGF receptor number compared to parental cells. However, migration and FGF receptor number of cells transfected with a cDNA coding only for high molecular weight bFGF (22, 22.5, and 24 kD; HMW bFGF) were similar to that of parental cells transfected with vector alone. Cells expressing HMW, 18 kD, or WT bFGF grew to high saturation densities in 10% serum. However, only cells expressing HMW or WT bFGF grew in low serum. Cell surface or metabolic labeling of the different cell types followed by immunoprecipitation with anti-bFGF antibody showed primarily cell surface-associated 18-kD bFGF. In addition, when cells expressing exclusively HMW bFGF were transfected with a cDNA coding for 18-kD bFGF, migration was increased, bFGF receptors were down-regulated, and 18-kD bFGF was found on the cell surface. Cells expressing 18-kD bFGF transfected with a cDNA encoding FGF receptor-2 lacking the COOH-terminal domain (dominant negative bFGF receptor) exhibited a flat morphology and decreases in migration and saturation density. Cells expressing HMW bFGF transfected with the dominant negative bFGF receptor continued to grow to a high saturation density, proliferated in low serum, and exhibited no morphological changes. These results indicate that increased cell migration and FGF receptor down-regulation are mediated by the extracellular interaction of 18-kD bFGF with its cell surface receptor. Growth in low serum may be stimulated by the intracellular action of HMW bFGF through mechanisms independent of the presence of a cell surface receptor. Thus, the different molecular forms of bFGF may act through distinct but convergent pathways.
Mas37p, a novel receptor subunit for protein import into mitochondria.
Authors: Gratzer S , Lithgow T , Bauer R E , Lamping E , Paltauf F , Kohlwein S D , Haucke V , Junne T , Schatz G , Horst M
Published: May 2004

By screening a collection of Saccharomyces cerevisiae mutants temperature sensitive for growth on a nonfermentable carbon source, we have isolated a gene (termed MAS37) which encodes a novel receptor for protein import into mitochondria. Mas37p is a 37-kD outer membrane protein with two putative membrane-spanning regions. Inactivation of the MAS37 gene renders cells temperature-sensitive for respiration-driven growth, inhibits import of precursors into isolated mitochondria, and is synthetically lethal with a deletion of one of the genes encoding the import receptors Mas70p or Mas20p. Inactivation of Mas37p with specific antibodies inhibits import of different precursors to different extents; the precursor specificity of Mas37p resembles that of the previously described import receptor Mas70p. Mas70p and Mas37p form a 1:1 complex in detergent extracts of mitochondria and overexpression of one protein enhances that of the other. We suggest that the Mas37p/Mas70p heterodimer functions as a receptor for protein import into yeast mitochondria and that the mitochondrial receptor system consists of hetero-oligomeric subcomplexes with distinct binding activities, but overlapping precursor specificities.
Episialin (MUC1) overexpression inhibits integrin-mediated cell adhesion to extracellular matrix components.
Authors: Wesseling J , van der Valk S W , Vos H L , Sonnenberg A , Hilkens J
Published: May 2004

Episialin (MUC1) is a transmembrane molecule with a large mucin-like extracellular domain protruding high above the cell surface. The molecule is located at the apical side of most glandular epithelial cells, whereas in carcinoma cells it is often present at the entire surface and it is frequently expressed in abnormally large quantities. We have previously shown that overexpression of episialin reduces cell-cell interactions. Here we show that the integrin-mediated adhesion to extracellular matrix of transfectants of a melanoma cell line (A375), a transformed epithelial cell line (MDCK-ras-e) and a human breast epithelial cell line (HBL-100) is reduced by high levels of episialin. This reduction can be reversed by inducing high avidity of the beta 1 integrins by mAb TS2/16 (at least for beta 1-mediated adhesion). The adhesion can also be restored by redistribution of episialin on the cell surface by monoclonal antibodies into patches or caps. Similarly, capping of episialin on ZR-75-1 breast carcinoma cells, growing in suspension, caused adherence and spreading of these cells. We propose that there is a delicate balance between adhesion and anti-adhesion forces in episialin expressing cells, which can be shifted towards adhesion by strengthening the integrin-mediated adhesion, or towards anti-adhesion by increasing the level of expression of episialin.
Golgi and vacuolar membrane proteins reach the vacuole in vps1 mutant yeast cells via the plasma membrane.
Authors: Nothwehr S F , Conibear E , Stevens T H
Published: May 2004

The Vps1 protein of Saccharomyces cerevisiae is an 80-kD GTPase associated with the Golgi apparatus. Vps1p appears to play a direct role in the retention of late Golgi membrane proteins, which are mislocalized to the vacuolar membrane in its absence. The pathway by which late Golgi and vacuolar membrane proteins reach the vacuole in vps1 delta mutants was investigated by analyzing transport of these proteins in vps1 delta cells that also contained temperature sensitive mutations in either the SEC4 or END4 genes, which are required for a late step in secretion and the internalization step of endocytosis, respectively. Not only was vacuolar transport of a Golgi membrane protein blocked in the vps1 delta sec4-ts and vps1 delta end4-ts double mutant cells at the non-permissive temperature but vacuolar delivery of the vacuolar membrane protein, alkaline phosphatase was also blocked in these cells. Moreover, both proteins expressed in the vps1 delta end4-ts cells at the elevated temperature could be detected on the plasma membrane by a protease digestion assay indicating that these proteins are transported to the vacuole via the plasma membrane in vps1 mutant cells. These data strongly suggest that a loss of Vps1p function causes all membrane traffic departing from the late Golgi normally destined for the prevacuolar compartment to instead be diverted to the plasma membrane. We propose a model in which Vps1p is required for formation of vesicles from the late Golgi apparatus that carry vacuolar and Golgi membrane proteins bound for the prevacuolar compartment.
Recruitment of epidermal growth factor receptors into coated pits requires their activated tyrosine kinase.
Authors: Lamaze C , Schmid S L
Published: May 2004

EGF-receptor (EGF-R) tyrosine kinase is required for the down-regulation of activated EGF-R. However, controversy exists as to whether ligand-induced activation of the EGF-R tyrosine kinase is required for internalization or for lysosomal targeting. We have addressed this issue using a cell-free assay that selectively measures the recruitment of EGF-R into coated pits. Here we show that EGF bound to wild-type receptors is efficiently sequestered in coated pits. In contrast, sequestration of kinase-deficient receptors occurs inefficiently and at the same basal rate of endocytosis of unoccupied receptors or receptors lacking any cytoplasmic domain. Sequestration of deletion mutants of the EGF-R that lack autophosphorylation sites also requires an active tyrosine kinase. This suggests that a tyrosine kinase substrate(s) other than the EGF-R itself, is required for its efficient ligand-induced recruitment into coated pits. Addition of a soluble EGF-R tyrosine kinase fully and specifically restores the recruitment of kinase-deficient EGF-R into coated pits providing a powerful functional assay for identification of these substrate(s).
PEB1 (PAS7) in Saccharomyces cerevisiae encodes a hydrophilic, intra-peroxisomal protein that is a member of the WD repeat family and is essential for the import of thiolase into peroxisomes.
Authors: Zhang J W , Lazarow P B
Published: May 2004

We have previously described mutant S. cerevisiae that are defective in peroxisome biogenesis (peb mutants) (Zhang, J. W., Y. Han, and P. B. Lazarow. 1993. J. Cell Biol. 123:1133-1147.). In some mutants, peroxisomes are undetectable. Other mutants contain normal-looking peroxisomes but fail to package subsets of peroxisomal proteins into the organelle (Zhang, J. W., C. Luckey, and P. B. Lazarow. 1993. Mol. Biol. Cell. 4:1351-1359.). In peb1 (pas7) cells, for example, the peroxisomes contain proteins that are targeted by COOH-terminal tripeptides and contain acyl-CoA oxidase (which is probably targeted by internal oligopeptides), but fail to import thiolase (which is targeted by an NH(2)-terminal 16-amino acid sequence). These and other data suggest that there are three branches in the pathway for the import of proteins into peroxisomes, each of which contains a receptor for one type of peroxisomal topogenic information. Here, we report the cloning and characterization of the PEB1 gene, that encodes a 42,320-Da hydrophilic protein with no predicted transmembrane segment. The protein contains six WD repeats, a motif which has been found in 27 proteins involved in diverse cellular functions. The PEB1 gene product was tagged with the hemagglutinin epitope and found to rescue thiolase import in the peb1 null mutant. The epitope-tagged protein was shown to be inside of peroxisomes by immunofluorescence, digitonin permeabilization, equilibrium density centrifugation, immunoelectron microscopy, and proteinase K protection studies. The PEB1 gene product does not cleave the thiolase-targeting sequence. It may function to draw thiolase into peroxisomes.
Targeting of Salmonella typhimurium to vesicles containing lysosomal membrane glycoproteins bypasses compartments with mannose 6-phosphate receptors.
Authors: Garcia-del Portillo F , Finlay B B
Published: May 2004

Salmonella typhimurium is an intracellular bacterial pathogen that remains enclosed in vacuoles (SCV) upon entry into the host cell. In this study we have examined the intracellular trafficking route of S. typhimurium within epithelial cells. Indirect immunofluorescence analysis showed that bacteria initiated fusion with lysosomal membrane glycoprotein (lgp)-containing compartments approximately 15 min after bacterial internalization. This process was completed approximately 75 min later and did not require microtubules. Cation-independent (CI)- or cation-dependent (CD)-mannose 6-phosphate receptors (M6PRs) were not observed at detectable levels in SCV. Lysosomal enzymes showed a different distribution in SCV: lysosomal-acid phosphatase (LAP) was incorporated into these vacuoles with the same kinetics as lgps, while cathepsin D was present in a low proportion (approximately 30%) of SCV. Uptake experiments with fluid endocytic tracers such as fluorescein-dextran sulphate (F-DX) or horseradish-peroxidase (HRP) showed that after 2 h of uptake, F-DX was present in approximately 75% of lgp-containing vesicles in uninfected cells, while only approximately 15% of SCV contained small amounts of the tracer during the same uptake period. SCV also showed only partial fusion with HRP-preloaded secondary lysosomes, with approximately 30% of SCV having detectable amounts of HRP at 6 h after infection. These results indicate that SCV show limited accessibility to fluid endocytic tracers and mature lysosomes, and are therefore functionally separated from the endocytic route. Moreover, the unusual intracellular trafficking route of S. typhimurium inside epithelial cells has allowed us to establish the existence of two different lgp-containing vesicles in Salmonella-infected cells: one population is separated from the endocytic route, fusogenic with incoming SCV and may arise from a secretory pathway, while the second involves the classical secondary or mature lysosomes.
The exocytotic fusion pore of small granules has a conductance similar to an ion channel.
Authors: Lollike K , Borregaard N , Lindau M
Published: May 2004

We measured capacitance changes in cell attached patches of human neutrophils using a high frequency lock-in method. With this technique the noise level is reduced to 0.025 fF such that capacitance steps of 0.1 fF are clearly detected corresponding to exo- and endocytosis of single 60 nm vesicles. It is thus possible to detect almost all known exocytotic and endocytotic processes including exocytosis of small neurotransmitter containing vesicles in most cell types as well as endocytosis of coated and uncoated pits. In neutrophils we demonstrate a stepwise capacitance decrease generated by 60-165 nm vesicles as expected for endocytosis of coated and non-coated pits. Following ionomycin stimulation a stepwise capacitance increase is observed consisting of 0.1-5 fF steps corresponding to the different granule types of human neutrophils from secretory vesicles to azurophil granules. The opening of individual fusion pores is resolved during exocytosis of 200 nm vesicles. The initial conductance has a mean value of 150 pS and can be as low as 35 pS which is similar to the conductance of many ion channels suggesting that the initial fusion pore is formed by a protein complex.
Transcription-dependent redistribution of the large subunit of RNA polymerase II to discrete nuclear domains.
Authors: Bregman D B , Du L , van der Zee S , Warren S L
Published: May 2004

A subpopulation of the largest subunit of RNA polymerase II (Pol II LS) is located in 20-50 discrete subnuclear domains that are closely linked to speckle domains, which store splicing proteins. The speckle-associated fraction of Pol II LS is hyperphosphorylated on the COOH-terminal domain (CTD), and it is highly resistant to extraction by detergents. A diffuse nucleoplasmic fraction of Pol II LS is relatively hypophosphorylated on the CTD, and it is easily extracted by detergents. In transcriptionally active nuclei, speckle bound hyperphosphorylated Pol II LS molecules are distributed in irregularly shaped speckle domains, which appear to be interconnected via a reticular network. When transcription is inhibited, hyperphosphorylated Pol II LS and splicing protein SC35 accumulate in speckle domains, which are transformed into enlarged, dot-like structures lacking interconnections. When cells are released from transcriptional inhibition, Pol IIO and SC35 redistribute back to the interconnected speckle pattern of transcriptionally active cells. The redistribution of Pol II and SC35 is synchronous, reversible, and temperature dependent. It is concluded that: (a) hyperphosphorylation of Pol II LS's CTD is a better indicator of its tight association to discrete subnuclear domains than its transcriptional activity; (b) during states of transcriptional inhibition, hyperphosphorylated Pol II LS can be stored in enlarged speckle domains, which under the light microscope appear to coincide with the storage sites for splicing proteins; and (c) Pol II and splicing proteins redistribute simultaneously according to the overall transcriptional activity of the nucleus.