bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tie-1-directed expression of Cre recombinase in endothelial cells of embryoid bodies and transgenic mice
Authors: Gustafsson Erika , Brakebusch Cord , Hietanen Kristiina , Fässler Reinhard 
Published: April 2021

ABSTRACT
Tissue-specific gene inactivation using the Cre-loxP system has become an important tool to unravel functions of genes when the conventional null mutation is lethal. We report here the generation of a transgenic mouse line expressing Cre recombinase in endothelial cells. In order to avoid the production and screening of multiple transgenic lines we used embryonic stem cell and embryoid body technology to identify recombinant embryonic stem cell clones with high, endothelial-specific Cre activity. One embryonic stem cell clone that showed high Cre activity in endothelial cells was used to generate germline chimeras. The in vivo efficiency and specificity of the transgenic Cre was analysed by intercrossing the tie-1-Cre line with the ROSA26R reporter mice. At initial stages of vascular formation (E8-9), LacZ staining was detected in almost all cells of the forming vasculature. Between E10 and birth, LacZ activity was detected in most endothelial cells within the embryo and of extra-embryonic tissues such as yolk sac and chorioallantoic placenta. Ectopic expression of Cre was observed in approximately 12-20% of the adult erythroid, myeloid and lymphoid cells and in subregions of the adult brain. These results show that the tie-1-Cre transgenic strain can efficiently direct deletion of floxed genes in endothelial cells in vivo.
Protective role of Bcl2 in metabolic oxidative stress-induced cell death
Authors: Lee Yong J. , Chen Jenn C. , Amoscato Andrew A. , Bennouna Jaafar , Spitz Douglas R. , Suntharalingam Mohan , Rhee Juong G. 
Published: April 2021

ABSTRACT
Previous studies have shown that overexpression of Bcl2 protects cells from glucose deprivation-induced cell death in multidrug-resistant human breast carcinoma, MCF-7/ADR cells. In this study, we further investigated the protective role of Bcl2 in glucose deprivation-induced cytotoxicity. Although Bcl2 did not prevent a 3.2-fold increase in the level of hydroperoxide during glucose deprivation, it led to a compartmentalization of hydroperoxide molecules in the mitochondria. It also inhibited glucose deprivation-induced cytochrome c release from the mitochondria. It is possible that overexpression of Bcl2 prevents glucose deprivation-induced ceramide generation, probably by preventing the leakage of hydroperoxide from the mitochondria. We also observed that glucose deprivation induced a sixfold increase in oxidized glutathione content, as well as in thiol precursor content. Overexpression of Bcl2 suppressed an increase in oxidized glutathione content and thiol precursor content. Our results indicate that Bcl2 protects cells from metabolic oxidative stress-induced damage by inhibiting the leakage of hydroperoxide from the mitochondria and subsequently preventing ceramide generation. Preventing ceramide generation inhibits the signal transduction pathway and results in the suppression of cytochrome c release from the mitochondria.
Plakophilin 1 interferes with plakoglobin binding to desmoplakin, yet together with plakoglobin promotes clustering of desmosomal plaque complexes at cell-cell borders
Authors: Bornslaeger Elayne A. , Godsel Lisa M. , Corcoran Connie M. , Park Jung K. , Hatzfeld Mechthild , Kowalczyk Andrew P. , Green Kathleen J. 
Published: April 2021

ABSTRACT
Desmosomes are adhesive junctions that link intermediate filament networks to sites of strong intercellular adhesion. These junctions play an important role in providing strength to tissues that experience mechanical stress such as heart and epidermis. The basic structural elements of desmosomes are similar to those of the better-characterized adherens junctions, which anchor actin-containing microfilaments to cadherins at the plasma membrane. This linkage of actin to classic cadherins is thought to occur through an indirect mechanism requiring the associated proteins, α- and β-catenin. In the case of desmosomes, both linear and lateral interactions have been proposed as playing an important role in formation of the plaque and linkage to the cytoskeleton. However, the precise nature of these interactions and how they cooperate in desmosome assembly are poorly understood.
Here we employ a reconstitution system to examine the assembly of macromolecular complexes from components found in desmosomes of the differentiated layers of complex tissues. We demonstrate the existence of a Triton-soluble complex of proteins containing full length desmoplakin (DP), the arm protein plakoglobin, and the cytoplasmic domain of the desmosomal cadherin, desmoglein 1 (Dsg1). In addition, full length DP, but not an N-terminal plakoglobin binding domain of DP, co-immunoprecipitated with the Dsg1 tail in the absence of plakoglobin in HT1080 cells. The relative roles of the arm proteins plakoglobin and plakophilin 1 (PKP1) were also investigated. Our results suggest that, in the Triton soluble pool, PKP1 interferes with binding of plakoglobin to full length DP when these proteins are co-expressed. Nevertheless, both plakoglobin and PKP1 are required for the formation of clustered structures containing DP and the Dsg1 tail that ultrastructurally appear similar to desmosomal plaques found in the epidermis. These findings suggest that more than one armadillo family member is required for normal assembly and clustering of the desmosomal plaque in the upper layers of the epidermis.
Cellular membranes that undergo cyclic changes in tension: Direct measurement of force generation by an in vitro contractile vacuole of Paramecium multimicronucleatum
Authors: Tani Tomomi , Allen Richard D. , Naitoh Yutaka 
Published: April 2021

ABSTRACT
The contractile vacuole of the fresh water protozoan Paramecium is a membrane-bound vesicle that expels excess cytosolic water, acquired osmotically, through its periodic exocytotic activity. The in vitro contractile vacuole, isolated in a small amount of cytosol from the Paramecium cell and confined under mineral oil, showed periodic rounding and slackening at regular intervals for an extended time. The contractile vacuole rounded against the cytosol-mineral oil boundary tension. The tension at the surface of the contractile vacuole is, therefore, assumed to increase during the rounding phase. We first estimated the tension relative to the boundary tension from the degree of compression of the contractile vacuole by the boundary. We then determined the absolute value for the tension at the surface of the contractile vacuole from the degree of bending of an elastic carbon fiber microcantilever (8 μm thick; 2 mm long), whose free end was placed at the surface of an in vitro contractile vacuole. The tension was found to increase to its maximum value of approximately 5 mN m−1 when the contractile vacuole rounded. This value was more than 35 times higher than that for the slackened contractile vacuole. Electron micrographs of conventional thin sections of chemically fixed in vitro contractile vacuoles as well as those of in vivo contractile vacuoles obtained from rapid frozen and cryosubstituted cells revealed the lack of any ultrastructural evidence for the presence of a fibrous network system surrounding the contractile vacuole. Thus we conclude that the mechanism(s) by which tension is developed at the surface of the contractile vacuole membrane resides in the contractile vacuole membrane itself. We propose a hypothesis that periodic changes in the spontaneous curvature of the contractile vacuole’s lipid bilayer membrane is involved in the periodic development of higher contractile vacuole membrane tension. The isolated CV promises to be an excellent model system for understanding the molecular mechanisms of the dynamics of biological membrane.
Kendrin/pericentrin-B, a centrosome protein with homology to pericentrin that complexes with PCM-1
Authors: Li Qingqin , Hansen Darcy , Killilea Alison , Joshi Harish C. , Palazzo R. E. , Balczon Ron 
Published: April 2021

ABSTRACT
The centrosome is responsible for nucleating microtubules and performing other cellular roles. To define the organization of the centrosome more completely, a human anti-centrosome serum was used to screen a human cDNA library, and a cDNA encoding a &gt;350 kDa centrosome protein was identified. Sequence analyses revealed that this novel centrosome protein contains two coiled-coil domains bounded by non-coiled regions. The N-terminal region of the protein, named pericentrin-B, shares 61% identity (75% similarity) with pericentrin, suggesting an evolutionary relationship between these proteins. Antibodies against pericentrin-B stain centrosomes at all stages of the cell cycle, and pericentrin-B remains associated with centrosomes following microtubule depolymerization. Immunodepletion of neither pericentrin-B nor PCM-1 from cellular extracts inhibited the ability of salt-stripped centrosomes to recover microtubule nucleation potential, demonstrating that neither protein plays a key role in microtubule nucleation processes. Moreover, the binding of both PCM-1 and pericentrin-B with salt-stripped centrosomes required intact microtubules, demonstrating that the association of PCM-1 and pericentrin-B with centrosomes is a late event in the centrosome maturation process. Finally, pericentrin-B and PCM-1 coimmunoprecipitate, suggesting that PCM-1 and pericentrin-B form a functional complex in cells. This observation may help to explain the generation of anti-centrosome autoantibodies in certain autoimmune patients and may be important for centrosome function.
The NITY motif of the beta-chain cytoplasmic domain is involved in stimulated internalization of the β3 integrin A isoform
Authors: Gawaz Meinrad , Besta Felicitas , Ylänne Jari , Knorr Thomas , Dierks Henning , Böhm Thomas , Kolanus Waldemar 
Published: April 2021

ABSTRACT
β3 integrin adhesion molecules play important roles in wound repair and the regulation of vascular development and three β3 integrin isoforms (β3-A, -B, -C) have been described so far. Surface expression of β3 integrins is dynamically regulated through internalization of β3 integrins, however, the molecular mechanisms are understood incompletely. To evaluate the role of the cytoplasmic domain of β3 integrins for internalization, we have generated single chain chimeras with variant and mutated forms of β3 cytoplasmic domains. Upon transient transfection into chinese hamster ovary cells, it was found that the β3-A chimera had strongly reduced cell surface expression compared with the corresponding β3-B, or β3-C fusion proteins, or the tail-less constructs, whereas steady state levels of all chimeras were near identical. Studies employing cytoplasmic domain mutants showed that the NITY motif at β3-A 756-759 is critical for plasma membrane expression of β3-A. Furthermore, delivery of β3-A to the cell surface was specifically modulated by the cytoplasmic protein β3-endonexin, a previously described intracellular protein. Coexpression of the native, long form of β3-endonexin, which does not interact with the β3 tail, acted as a dominant negative inhibitor of β3-A-internalization and enhanced steady-state surface expression of the β3-A-chimera. Furthermore, anti-β3 antibody-induced internalization of the native β3 integrin (αIIbβ3) was dramatically reduced for the Tyr759-Ala substitution mutant αIIbβ3 (Y759A) and expression of the long isoform of β3-endonexin substantially decreased the internalization of wild-type αIIbβ3. Thus, the NITY motif of the beta-chain cytoplasmic domain is involved in stimulated internalization of the β3 integrin A isoform and β3-endonexin appears to couple the β3-A isoform to a specific receptor-recycling pathway.
Distinct roles for dystroglycan, β1 integrin and perlecan in cell surface laminin organization
Authors: Henry Michael D. , Satz Jakob S. , Brakebusch Cord , Costell Mercedes , Gustafsson Erika , Fässler Reinhard , Campbell Kevin P. 
Published: April 2021

ABSTRACT
Dystroglycan (DG) is a cell surface receptor for several extracellular matrix (ECM) molecules including laminins, agrin and perlecan. Recent data indicate that DG function is required for the formation of basement membranes in early development and the organization of laminin on the cell surface. Here we show that DG-mediated laminin clustering on mouse embryonic stem (ES) cells is a dynamic process in which clusters are consolidated over time into increasingly more complex structures. Utilizing various null-mutant ES cell lines, we define roles for other molecules in this process. In β1 integrin-deficient ES cells, laminin-1 binds to the cell surface, but fails to organize into more morphologically complex structures. This result indicates that β1 integrin function is required after DG function in the cell surface-mediated laminin assembly process. In perlecan-deficient ES cells, the formation of complex laminin-1 structures is defective, implicating perlecan in the laminin matrix assembly process. Moreover, laminin and perlecan reciprocally modulate the organization of the other on the cell surface. Taken together, the data support a model whereby DG serves as a receptor essential for the initial binding of laminin on the cell surface, whereas β1 integrins and perlecan are required for laminin matrix assembly processes after it binds to the cell.
Involvement of CD44 in cytoskeleton rearrangement and raft reorganization in T cells
Authors: Föger Niko , Marhaba Rachid , Zöller Margot 
Published: April 2021

ABSTRACT
T cell activation is accompanied by actin-mediated receptor clustering and reorganization of lipid rafts. It has been suggested that costimulatory molecules might be involved in these processes. We here provide evidence that engagement of the adhesion molecule CD44 initiates cytoskeletal rearrangement and membrane reorganization in T cells.
Cross-linking of CD44 on a T helper line was accompanied by adhesion, spreading and actin bundle formation. These processes were energy dependent and required an intact actin and microtubule system. They involved the small GTPase Rac as evidenced by the absence of spreading in cells overexpressing a dominant negative form of Rac. The CD44 initiated reorganization of the cytoskeleton was associated with the recruitment of CD44 and the associated tyrosine phosphokinases p56lck and p59fyn into glycolipid enriched membrane microdomains (GEM). We interpret the data in the sense that CD44 functions as a costimulatory molecule in T cell activation by inducing actin cytoskeletal rearrangements and membrane protein and lipid reorganization including its association with GEMs. Due to the association of CD44 with lck and fyn this colocalization with the TCR allows an abundant provision of these kinases, which are essential to initiate the TCR signaling cascade.
Inhibition of neuronal maturation in primary hippocampal neurons from tau deficient mice
Authors: Dawson Hana N. , Ferreira Adriana , Eyster Michele V. , Ghoshal Nupur , Binder Lester I. , Vitek Michael P. 
Published: April 2021

ABSTRACT
Conflicting evidence supports a role for tau as an essential neuronal cytoskeletal protein or as a redundant protein whose function can be fulfilled by other microtubule-associated proteins. To investigate the function of tau in axonogenesis, we created tau deficient mice by disrupting the TAU gene. The engineered mice do not express the tau protein, appear physically normal and are able to reproduce. In contrast to a previously reported tau knockout mouse, embryonic hippocampal cultures from tau deficient mice show a significant delay in maturation as measured by axonal and neuritic extensions. The classic technique of selectively enhancing axonal growth by growth on laminin substrates failed to restore normal neuronal maturation of tau knockout neurons. By mating human TAU-gene transgenic and tau knockout mice, we reconstituted tau-deficient neurons with human tau proteins and restored a normal pattern of axonal growth and neuronal maturation. The ability of human tau proteins to rescue tau-deficient mouse neurons confirms that tau expression affects the rate of neurite extension.
Role of aquaporin-4 water channel in the development and integrity of the blood-brain barrier
Authors: Nico Beatrice , Frigeri Antonio , Nicchia Grazia Paola , Quondamatteo Fabio , Herken Rainer , Errede Mariella , Ribatti Domenico , Svelto Maria , Roncali Luisa 
Published: April 2021

ABSTRACT
In this study, we have investigated the expression of aquaporin 4 during blood-brain barrier development in the optic tectum of chick embryos and newly hatched chicks, by means of western-blot, reverse transcriptase-polymerase chain reaction, immunohistochemistry, and freeze-fracture and high-resolution immunogold electron microscopy.
In the optic tecta of day-14 embryos, western blot analysis revealed an approx. 30 kDa band, immunoreactive for aquaporin-4, which was increased in day-20 embryos and in chicks. Semi-quantitative reverse transcriptase chain reaction experiments showed that there was already a high level of aquaporin-4 mRNA in day-9 embryos as well as in the subsequent stages and in newly hatched chicks. Immunohistochemically, reactivity for aquaporin-4 was detected in the optic tectum of day-14 embryos; similar results were obtained in telencephalon and cerebellum. Ultrastructurally, the microvessels of the tectum showed immunoreactivity for aquaporin-4 on the astroglial endfeet, which discontinuously surrounded endothelial cells joined by immature tight junctions. In the tectum, telencephalon and cerebellum of 20-day embryos and chicks, aquaporin-4 strongly labeled the ependymal cells and the subpial glial membranes, as well as the bodies and processes of astroglial cells. A continuous aquaporin-4 staining was found around the microvessel endothelial cells, which were sealed off from one another by extensive tight junctions. A complete astrocytic sheath, labeled by anti-aquaporin-4 gold particles, enveloped the endothelium-pericyte layer. Orthogonal arrays of particles were observed on fractured astrocytic membranes, starting from embryonic day 14 when the aquaporin-4 immunogold staining revealed clusters of gold particles, often forming square or rectangular clusters. The results showed that aquaporin-4 expression and organization of the intramembrane particles in orthogonal arrays followed the same temporal sequence. Finally, the lipopolysaccharide, a substance that induces blood-brain barrier distruption, determines a remarkable reduction in aquaporin-4 labeling, expressed by a few aquaporin-4 gold particles attached on swollen perivascular glial membranes.
All these data show that aquaporin-4 expression occurs in the chick embryonic brain, in parallel with maturation and functioning of the blood-brain barrier and suggest that there is a close relationship between water transport regulation and brain development.
Differential galactosylation of neuronal and haematopoietic signal regulatory protein-α determines its cellular binding-specificity
Authors: Nieuwenhof Ingrid M. van den , Lavalette Chantal Renardel de , Diaz Niubel , Die Irma van , van den Berg Timo K. 
Published: April 2021

ABSTRACT
Signal regulatory protein-α (SIRPα) is a member of the Ig superfamily selectively expressed by neuronal and myeloid cells. The molecule mediates functional interactions with CD47/integrin-associated protein. Here we provide evidence for the tissue-specific glycosylation of neuronal and haematopoietic SIRPα. We demonstrate a major difference in the galactosylation of N-linked glycans isolated from neuronal (i.e. brain-derived) SIRPα as compared to myeloid (i.e. spleen-derived) SIRPα, with neuronal SIRPα almost completely lacking galactose. p4-galactosyltransferase assays demonstrated that this is most likely due to a low galactosylation capacity of the brain. In order to investigate the role of galactosylation of SIRPα in cellular interactions, soluble recombinant SIRPα glycoforms containing galactose (SIRPα-Fc) or lacking galactose (SIRPα(ΔGal)-Fc) were produced. Binding studies demonstrated superior binding of SIRPα(ΔGal)-Fc to cerebellar neurons and isolated lymphocytes. In contrast, SIRPα-Fc bound relatively strong to macrophages. These data show that the galactosylation of SIRPα determines its cellular binding specificity.
BMP-2 augments FGF-induced differentiation of PC12 cells through upregulation of FGF receptor-1 expression
Authors: Hayashi Hisaki , Ishisaki Akira , Suzuki Masashi , Imamura Toru 
Published: April 2021

ABSTRACT
When exposed to various neurotrophic factors, including fibroblast growth factors (FGF)-1 and -2, rat pheochromocytoma-derived PC12 cells differentiate into sympathetic neuron-like cells possessing elongated neurites. We found that while bone morphogenetic protein-2 (BMP-2) exerted little effect by itself on the differentiation of PC12 cells, in combination with FGF it strongly induced neurite outgrowth, even at subthreshold concentrations of FGF. Analysis of gene expression revealed that FGF receptor-1 (FGFR-1) mRNA was abundantly expressed in PC12 cells and that its expression was upregulated by pretreating the cells with BMP-2. Crosslinking the receptors with 125I-FGF-2 and then immunoprecipitating them confirmed that expression of FGFR-1, but not other FGF receptor types, was enhanced by BMP-2. Furthermore, Scatchard analyses revealed that the numbers of FGF-2 binding sites were increased by approximately 40% after BMP-2 treatment. Pretreatment with BMP-2 also enhanced peak and sustained levels of FGF-induced ERK1/2 phosphorylation in PC12 cells. Finally, the augmentation of neurotrophic activity by BMP-2 was inhibited by SU5402, an FGFR-1 inhibitor. These findings indicate that BMP-2 augments FGF-induced differentiation of PC12 cells through selective upregulation of FGFR-1 expression, and suggest that BMP-2 and FGF act in concert to regulate cell differentiation in the nervous system.
Structural analysis of the role of the p3 subunit of the αVβ3 integrin in IGF-I signaling
Authors: Maile Laura A. , Badley-Clarke Jane , Clemmons David R. 
Published: April 2021

ABSTRACT
The disintegrin echistatin inhibits ligand occupancy of the αVβ3 integrin and reduces Insulin-like growth factor I (IGF-I) stimulated migration, DNA synthesis, and receptor autophosphorylation in smooth muscle cells. This suggests that ligand occupancy of the αVp3 receptor is required for full activation of the IGF-I receptor.
Transfection of the full-length β3 subunit into CHO cells that have no endogenous p3 and do not migrate in response to IGF-I was sufficient for IGF-I to stimulate migration of these anchorage dependent cells. In contrast, transfection of either of two truncated mutant forms of p3 (terminating at W715 or E731) or a mutant with substitutions for Tyr747 Tyr759 (YY) into either CHO or into porcine smooth muscle cells did not restore the capacity of these cells to migrate across a surface in response to IGF-I. This effect was not due to loss of IGF-I receptor autophosphorylation since the response of the receptor to IGF-I was similar in cells expressing either the full-length or any of the mutant forms of the β3 subunit. Echistatin reduced IGF-I receptor phosphorylation in cells expressing the full-length or the YY mutant forms of β3 subunit, but it had no effect in cells expressing either of two truncated forms of β3. A cell-permeable peptide homologous to the C-terminal region of the β3 subunit (amino acids 747-762) reduced IGF-I stimulated migration and receptor autophosphorylation of non-transfected porcine smooth muscle cells. These results demonstrate that the full-length β3 with intact tyrosines at positions 747 and 759 is required for CHO cells to migrate in response to IGF-I. Furthermore, a region of critical amino acids between residues 742-762 is required for echistatin to induce its regulatory effect on receptor phosphorylation. Since the IGF-I receptor does not bind to αVβ3 the results suggest that specific but distinct regions of the β3 subunit interact with intermediary proteins to facilitate IGF-I stimulated cell migration and echistatin induced inhibition of IGF-I signal transduction.