bluuebunny/crossref_metadata_2025_split huggingface dataset:
Vascular-endothelial-cadherin modulates endothelial monolayer permeability
Authors: Hordijk Peter L. , Anthony Eloise , Mul Frederik P. J. , Rientsma Ronald , Oomen Lauran C. J. M. , Roos Dirk 
Published: April 2021

ABSTRACT
Vascular endothelial (VE)-cadherin is the endothelium-specific member of the cadherin family of homotypic cell adhesion molecules. VE-cadherin, but not the cell adhesion molecule platelet/endothelial cell adhesion molecule (PECAM-1), markedly colocalizes with actin stress fibers at cell-cell junctions between human umbilical vein endothelial cells. Inhibition of VE-cadherin-mediated, but not PECAM-1-mediated, adhesion induced reorganization of the actin cytoskeleton, loss of junctional VE-cadherin staining and loss of cell-cell adhesion. In functional assays, inhibition of VE-cadherin caused increased monolayer permeability and enhanced neutrophil transendothelial migration.
In a complementary set of experiments, modulation of the actin cytoskeleton was found to strongly affect VE-cadherin distribution. Brief stimulation of the β2-adrenergic receptor with isoproterenol induced a loss of actin stress fibers resulting in a linear, rather than ‘jagged’, VE-cadherin distribution. The concomitant, isoproterenol-induced, reduction in monolayer permeability was alleviated by a VE-cadherin-blocking antibody. Finally, cytoskeletal reorganization resulting from the inactivation of p21Rho caused a diffuse localization of VE-cadherin, which was accompanied by reduced cell-cell adhesion. Together, these data show that monolayer permeability and neutrophil transendothelial migration are modulated by VE-cadherin-mediated cell-cell adhesion, which is in turn controlled by the dynamics of the actin cytoskeleton.
Human sweat gland myoepithelial cells express a unique set of cytokeratins and reveal the potential for alternative epithelial and mesenchymal differentiation states in culture
Authors: Schön Margarete , Benwood Jennifer , O’Connell-Willstaedt Therese , Rheinwald James G. 
Published: April 2021

ABSTRACT
We have characterized precisely the cytokeratin expression pattern of sweat gland myoepithelial cells and have identified conditions for propagating this cell type and modulating its differentiation in culture. Rare, unstratified epithelioid colonies were identified in cultures initiated from several specimens of full-thickness human skin. These cells divided rapidly in medium containing serum, epidermal growth factor (EGF), and hydrocortisone, and maintained a closely packed, epithelioid morphology when co-cultured with 3T3 feeder cells. Immunocytochemical and immunoblot analysis disclosed that the cells differed from keratinocytes in that they were E-cadherin-negative, vimentin-positive, and expressed an unusual set of cytokeratins, K5, K7, K14, and K17. When subcultured without feeder cells, they converted reversibly to a spindle morphology and ceased K5 and K14 expression. Under these conditions, EGF deprivation induced flattening, growth arrest, and expression of α-smooth muscle actin (α-sma). Coexpression of keratins and α-sma is a hallmark of myoepithelial cells, a constituent of secretory glands. Immunostaining of skin sections revealed that only sweat gland myoepithelial cells expressed the same pattern of keratins and α-sma and lack of E-cadherin as the cell type we had cultured. Interestingly, our immunocytochemical analysis of ndk, a skin-derived cell line of uncertain identity, suggests that this line is of myoepithelial origin. Earlier immunohistochemical studies by others had found myoepithelial cells to be K7-negative. We tested five K7-specific antibodies that can recognize this protein in western blots and in the assembled keratin filaments of mesothelial cells. Three of these antibodies did not recognize the K7 present in myoepithelial cell filaments or in HeLa cell filaments, indicating that some K7 epitopes are masked when K7 pairs with K17 instead of with its usual keratin filament partner, K19.
Sto1p, a fission yeast protein similar to tubulin folding Cofactor E, plays an essential role in mitotic microtubule assembly
Authors: Grishchuk Ekaterina L. , McIntosh J. Richard 
Published: April 2021

ABSTRACT
The proper functioning of microtubules depends crucially on the availability of polymerizable α/β tubulin dimers. Their production occurs concomitant with the folding of the tubulin polypeptides and is accomplished in part by proteins known as Cofactors A through E. In the fission yeast, Schizosaccharomyces pombe, this tubulin folding pathway is essential. We have taken advantage of the excellent cytology available in S. pombe to examine the phenotypic consequences of a deletion of sto1+, a gene that encodes a protein similar to Cofactor E, which is required for the folding of α-tubulin. The interphase microtubule cytoskeleton in sto1-Δ cells is severely disrupted, and as cells enter mitosis their spindles fail to form. After a transient arrest with condensed chromosomes, the cells exit mitosis and resume DNA synthesis, whereupon they septate abnormally and die. Overexpression of Spo1p is toxic to cells carrying a cold-sensitive allele of the α-but not the β-tubulin gene, consistent with the suggestion that this protein plays a role like that of Cofactor E. Unlike its presumptive partner Cofactor D (Alp1p), however, Sto1p does not localize to microtubules but is found throughout the cell. Overexpression of Sto1p has no toxic effects in wild-type cells, suggesting that it is unable to disrupt α/β tubulin dimers in vivo.
Homophilic PECAM-1(CD31) interactions prevent endothelial cell apoptosis but do not support cell spreading or migration
Authors: Bird Ian N. , Taylor Vanessa , Newton Justin P. , Spragg Julia H. , Simmons David L. , Salmon Michael , Buckley Christopher D. 
Published: April 2021

ABSTRACT
PECAM-1 (CD31) is a highly abundant cell surface glycoprotein expressed on haemopoietic and endothelial cells. As well as mediating homophilic (PECAM-1/PECAM-1) adhesion, PECAM-1 can also bind the integrin αvβ3. Both PECAM-1 and αvβ3 have been shown to have roles in regulating angiogenesis, endothelial tube formation and in the case of αvβ3, endothelial cell apoptosis. In this study we show that despite being expressed at equivalent levels, endothelial αvβ3 is not a ligand for PECAM-1. Rather, PECAM-1 supports homophilic binding on HUVEC with similar characteristics to those we have previously reported for leukocytes and becomes tyrosine phosphorylated after homophilic PECAM-1 and integrin/fibronectin engagement. Immunoprecipitation studies show that in addition to SHP-2, tyrosine phosphorylated PECAM-1 can interact with at least four other phosphoproteins in pervanadate stimulated HUVEC. While PECAM-1/PECAM-1 interactions support robust endothelial cell adhesion, they do not support cell spreading or migration. In addition PECAM-1 homophilic adhesion rescues HUVEC from serum deprivation-induced apoptosis. Taken together our results indicate that PECAM-1 homophilic interactions play an important role in interendothelial cell adhesion, survival and signalling.
A nuclear localization signal can enhance both the nuclear transport and expression of 1 kb DNA
Authors: Ludtke James J. , Zhang Guofeng , Sebestyén Magdolna G. , Wolff Jon A. 
Published: April 2021

ABSTRACT
Although the entry of DNA into the nucleus is a crucial step of non-viral gene delivery, fundamental features of this transport process have remained unexplored. This study analyzed the effect of linear double stranded DNA size on its passive diffusion, its active transport and its NLS-assisted transport. The size limit for passive diffusion was found to be between 200 and 310 bp. DNA of 310-1500 bp entered the nuclei of digitonin treated cells in the absence of cytosolic extract by an active transport process. Both the size limit and the intensity of DNA nuclear transport could be increased by the attachment of strong nuclear localization signals. Conjugation of a 900 bp expression cassette to nuclear localization signals increased both its nuclear entry and expression in microinjected, living cells.
Insect cuticle, an in vivo model of protein trafficking
Authors: Csikós György , Molnár Kinga , Borhegyi Noémi H. , Talián Gábor Cs. , Sass Miklós 
Published: April 2021

ABSTRACT
In the course of this study more than 20 proteins have been isolated from the larval cuticle of Manduca sexta. Synthesis, secretion, transport and accumulation of four particular proteins, representative members of four characteristic groups, were followed during metamorphosis by immunoblot and immuncytochemical methods and are described in detail in this paper. We established that only some of the proteins of the soft cuticle of Lepidopteran larvae are synthesized in epidermal cells at the beginning of the larval stages and are digested during the moulting period (MsCP29). Other proteins (MsCP30/11) are secreted into the cuticle by the epidermal cells in different forms during various developmental stages. Some proteins are secreted apically during the feeding period, but before ecdysis they are then taken up by epidermal cells and transported in a basolateral direction back into the hemolymph and saved in an immunologically intact form by the fat body cells (MsCP12.3). Some cuticle proteins have a non-epidermal origin. They are transported from the hemolymph into the cuticle. Before and during ecdysis these molecules reappear in the hemolymph and are detectable again in the pupal cuticle (MsCP78). Our data prove that the cuticle is not a non-living part of the insect body: it is not only an inert, protective armor, but maintains a continuous and dynamic metabolic connection with the other organs of the organism.
3-D organization of ribosomal transcription units after DRB inhibition of RNA polymerase II transcription
Authors: Panse Sophie Le , Masson Claude , Héliot Laurent , Chassery Jean-Marc , Junéra Henriette Roberte , Hernandez-Verdun Danièle 
Published: April 2021

ABSTRACT
In each bead of the nucleolar necklace, using adenosine analog DRB-treated PtK1 cells, we investigated the three components of rDNA transcription, i.e. the gene, transcription factor UBF and transcripts. In situ hybridization revealed the unraveling and 3-D dispersion of most of the rDNA coding sequences within the nucleus. The signals were small, of similar intensity and tandemly organized in the necklace. This observation is compatible with the fact that they might correspond to single gene units. Active transcription was visualized in these units, demonstrating that they were active functional units. Transcript labeling was not similar for each unit, contrary to UBF labeling. UBF and rRNA transcripts were only partially colocalized, as demonstrated by 3-D image analysis and quantification. As visualized by electron microscopy, the necklace was composed of a small fibrillar center partially surrounded by a dense fibrillar component. The 3-D arrangement of this individual unit in the necklace, investigated both by confocal and electron microscopy in the same cells, showed that the individual beads were linked by a dense fibrillar component. The reversibility of this organization after removal of DRB indicated that the beads in the necklace are certainly the elementary functional domain of the nucleolus. In addition, these results lead us to suggest that the organization of a functional domain, presumably corresponding to a single gene, can be studied by in situ approaches.
Receptor mediated and fluid phase pathways for internalization of the ER Hsp90 chaperone GRP94 in murine macrophages
Authors: Wassenberg James J. , Dezfulian Cameron , Nicchitta Christopher V. 
Published: April 2021

ABSTRACT
Immunization of mice with GRP94, the endoplasmic reticulum (ER) Hsp90, elicits cytotoxic T lymphocyte (CTL) responses to chaperone-bound, source cell-derived peptides. Elicitation of a CTL response requires that GRP94-associated peptides be transferred onto major histocompatability complex (MHC) class I molecules, a process that is postulated to accompany GRP94 internalization by antigen presenting cells, such as macrophages (MΦ) and dendritic cells (DC). In studies of GRP94 uptake in elicited MΦ, we report that MΦ display specific cell surface binding of GRP94, and that surface-bound GRP94 can be internalized via receptor mediated endocytosis. GRP94 internalized by this pathway co-localized predominately with transferrin-positive early endosomes. At time periods of up to 20 minutes, little trafficking of GRP94 to the lysosomal compartment was observed. When GRP94 was present in the medium, and thus accessible to both receptor-mediated and fluid phase internalization pathways, internalization was modestly inhibited in the presence of yeast mannan, a competitive inhibitor of mannose/fucose receptor activity, and substantially inhibited by dimethylamiloride, an inhibitor of macropinocytosis. GRP94 internalized via macropinocytosis did not display prominent co-staining with the lysosomal marker LAMP-2. These data identify multiple pathways of GRP94 internalization and indicate that receptor-dependent uptake of GRP94 is not dependent upon its high mannose oligosaccharide moiety. Most significantly, these data demonstrate the existence of cell surface receptor(s), apparently unique to antigen presenting cells, that function in the binding and internalization of the ER chaperone GRP94.
RHO-associated protein kinase α potentiates insulin-induced MAP kinase activation in Xenopus oocytes
Authors: Ohan Nicholas , Agazie Yehenew , Cummings Cathy , Booth Ronald , Bayaa Mustafa , Johné Liu X. 
Published: April 2021

ABSTRACT
We recently identified Xenopus Rho-associated protein kinase α (xROKα) as a Xenopus insulin receptor substrate-1 binding protein and demonstrated that the non-catalytic carboxyl terminus of xROKα binds Xenopus insulin receptor substrate-1 and blocks insulin-induced MAP kinase activation and germinal vesicle breakdown in Xenopus oocytes. In the current study we further examined the role of xROKα in insulin signal transduction in Xenopus oocytes. We demonstrate that injection of mRNA encoding the xROKα kinase domain or full length xROKα enhanced insulin-induced MAP kinase activation and germinal vesicle breakdown. In contrast, injection of a kinase-dead mutant of xROKα or pre-incubation of oocytes with an xROKα inhibitor significantly reduced insulin-induced MAP kinase activation. To further dissect the mechanism by which xROKα may participate in insulin signalling, we explored a potential function of xROKα in regulating cellular Ras function, since insulin-induced MAP kinase activation and germinal vesicle breakdown is known to be a Ras-dependent process. We demonstrate that whereas injection of mRNA encoding c-H-Ras alone induced xMAP kinase activation and GVBD in a very low percentage (about 10%) of injected oocytes, co-injection of mRNA encoding xROKα and c-H-Ras induced xMAP kinase activation and germinal vesicle breakdown in a significantly higher percentage (50-60%) of injected oocytes. These results suggest a novel function for xROKα in insulin signal transduction upstream of cellular Ras function.
The Drosophila forked protein induces the formation of actin fiber bundles in vertebrate cells
Authors: Grieshaber Scott , Petersen Nancy S. 
Published: April 2021

ABSTRACT
The forked protein is an actin binding protein involved in the formation of large actin fiber bundles in developing Drosophila bristles. These are the largest example of a type of actin bundle characterized by parallel, hexagonally packed actin fibers, also found in intestinal microvilli, kidney proximal tubule microvilli, and stereocilia in the ear. Understanding how these structures are constructed and how that construction is regulated is an important question in cell and developmental biology. Because the timing of forked gene expression coincides with the formation of the actin fiber bundles, and since the forked protein is localized at the site of initiation of these bundles before they form, it has been proposed that the forked protein is an initiator of actin bundle formation. In this paper we show that the forked protein can induce the formation of bundles and increase actin polymerization in vertebrate cells. We use this system to identify regions of the forked protein which are essential for bundle formation and actin co-localization.
Induction of apoptosis by overexpression of the DNA-binding and DNA-PK-activating protein C1D
Authors: Rothbarth Karsten , Spiess Eberhard , Juodka Benediktas , Yavuzer Ugur , Nehls Peter , Stammer Hermann , Werner Dieter 
Published: April 2021

ABSTRACT
Apoptosis is induced in various tumor cell lines by vector-dependent overexpression of the conserved gene C1D that encodes a DNA-binding and DNA-PK-activating protein. C1D is physiologically expressed in 50 human tissues tested, which points to its basic cellular function. The expression of this gene must be tightly regulated because elevated levels of C1D protein, e.g. those induced by transient vector-dependent expression, result in apoptotic cell death. Cells transfected with C1D-expressing constructs show terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling of DNA ends. Transfections with constructs in which C1D is expressed in fusion with the (enhanced) green fluorescent protein from A. victoria (EGFP) allow the transfected cells to be identified and the morphological changes induced to be traced. Starting from intense nuclear spots, green fluorescence reflecting C1D expression increases dramatically at 12-24 hours post-transfection. Expression of C1D-EGFP protein is accompanied by morphological changes typical of apoptotic cell death, e.g. cytoplasmic vacuolation, membrane blebbing and nuclear disintegration. Cell shrinkage and detachment from extracellular matrix are observed in monolayer cultures while suspension cells become progressively flattened. The facility to differentiate between transfected and non-transfected cells reveals that non-transfected cells co-cultured with transfected cells also show the morphological changes of apoptosis, which points to a bystander effect.
C1D-dependent apoptosis is not induced in cells with non-functional p53. Accordingly, C1D-induced apoptosis is discussed in relation to its potential to activate DNA-PK, which has been considered to act as an upstream activator of p53.
Analysis of the roles of the head domains of type IV rat neuronal intermediate filament proteins in filament assembly using domain-swapped chimeric proteins
Authors: Ching Gee Y. , Liem Ronald K. H. 
Published: April 2021

ABSTRACT
Type IV neuronal intermediate filament proteins consist of α-internexin, which can self-assemble into filaments and the neurofilament triplet proteins, which are obligate heteropolymers, at least in rodents. These IF proteins therefore provide good systems for elucidating the mechanism of intermediate filament assembly. To analyze the roles of the head domains of these proteins in contributing to their differential assembly properties, we generated chimeric proteins by swapping the head domains between rat α-internexin and either rat NF-L or NF-M and examined their assembly properties in transfected cells that lack their own cytoplasmic intermediate filament network. Lαα and Mαα, the chimeric proteins generated by replacing the head domain of α-internexin with those of NF-L and NF-M, respectively, were unable to self-assemble into filaments. In contrast, αLL, a chimeric NF-L protein generated by replacing the head domain of NF-L with that of α-internexin, was able to self-assemble into filaments, whereas MLL, a chimeric NF-L protein containing the NF-M head domain, was unable to do so. These results demonstrate that the α-internexin head domain is essential for α-internexin’s ability to self-assemble. While coassembly of Lαα with NF-M and coassembly of Mαα with NF-L resulted in formation of filaments, coassembly of Lαα with NF-L and coassembly of Mαα with NF-M yielded punctate patterns. These coassembly results show that heteropolymeric filament formation requires that one partner has the NF-L head domain and the other partner has the NF-M head domain. Thus, the head domains of rat NF-L and NF-M play important roles in determining the obligate heteropolymeric nature of filament formation. The data obtained from these self-assembly and coassembly studies provide some new insights into the mechanism of intermediate filament assembly.
Contribution of plus and minus end pathways to microtubule turnover
Authors: Vorobjev I. A. , Rodionov V. I. , Maly I. V. , Borisy G. G. 
Published: April 2021

ABSTRACT
Turnover is important for the maintenance and remodeling of the cytoskeleton during the processes of cell morphogenesis, mitosis and motility. Microtubule (MT) turnover is thought to occur by dynamic instability, growth and shortening at distal (plus) ends. Recent observation of MT release from the centrosome and depolymerization from proximal (minus) ends indicates the existence of a minus end pathway. To evaluate the relative contributions of plus and minus end pathways to turnover, we analyzed MT dynamics in a model system, the fish melanophore, a large non-motile cell with a regular radial array of long MTs. MT ends were tracked in digital fluorescence time-lapse sequences and life histories of individual MTs were analyzed using random walk theory generalized to the case of diffusion with drift. Analysis of plus end dynamics gave an apparent diffusion coefficient of D=7.5 μm2/minute. The random walk model predicts that the half-time for turnover driven solely by plus end dynamics will depend strongly on position in the cell. Based on the experimentally determined value of D, turnover of MTs near the center of a typical melanophore of radius 70 μm was calculated to require over 5 hours, a paradoxically long time.
To examine MT behavior deep in the cytoplasm, we developed a novel, sequential subtraction mode of image analysis. This analysis revealed a subpopulation of MTs which shortened from their minus ends, presumably after constitutive release from the centrosome. Given the relative slowness of plus end dynamics to turn over the root of a long MT, the turnover of MTs near the cell center is determined primarily by the minus-end pathway. MTs released from the centrosome become replaced by newly nucleated ones. The relative contributions of plus and minus end pathways was estimated from the diffusion coefficient, D, for the plus end, the length distribution of MTs, t he frequency of free minus ends, and the rate of minus-end shortening. We conclude that, in large animal cells with a centrosomally focussed array of MTs, turnover occurs by a combination of plus and minus end pathways, the plus end dominating at the cell periphery and the minus end dominating near the cell center.
Innexin-3 forms connexin-like intercellular channels
Authors: Landesman Yosef , White Thomas W. , Starich Todd A. , Shaw Jocelyn E. , Goodenough Daniel A. , Paul David L. 
Published: April 2021

ABSTRACT
Innexins comprise a large family of genes that are believed to encode invertebrate gap junction channel-forming proteins. However, only two Drosophila innexins have been directly tested for the ability to form intercellular channels and only one of those was active. Here we tested the ability of Caenorhabditis elegans family members INX-3 and EAT-5 to form intercellular channels between paired Xenopus oocytes. We show that expression of INX-3 but not EAT-5, induces electrical coupling between the oocyte pairs. In addition, analysis of INX-3 voltage and pH gating reveals a striking degree of conservation in the functional properties of connexin and innnexin channels. These data strongly support the idea that innexin genes encode intercellular channels.