bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sustained ERK phosphorylation is necessary but not sufficient for MMP-9 regulation in endothelial cells: involvement of Ras-dependent and-independent pathways
Authors: Genersch Elke , Hayeß Katrin , Neuenfeld Yvette , Haller Hermann 
Published: April 2021

ABSTRACT
Endothelial expression of matrix metalloproteinase-9 (MMP-9), which degrades native type IV collagen, was implicated as a prerequisite for angiogenesis. Therefore, the aim of this study was to determine signaling requirements that regulate MMP-9 expression in endothelial cells. Both, primary and permanent human umbilical vein endothelial cells (HUVEC and ECV304, respectively) were stimulated with phorbol 12-myristate 13-acetate (PMA) and the cytokine tumor necrosis factor-α (TNFα) to induce MMP-9 expression. While both cell types responded to PMA at the protein, mRNA and promoter level by induction of MMP-9, TNFα caused this response only in ECV304. Inhibitors specific for mitogen-activated protein/ERK kinase 1/2 (MEK1/2), protein kinase C (PKC), and Ras and co-transfections of wild-type and mutant Raf were used to elucidate the signaling cascades involved. Thus, we could show that the Raf/MEK/ERK cascade is mainly responsible for MMP-9 induction in endothelial cells and that this cascade is regulated independently of PKC and Ras subsequent to TNFα stimulation and in a PKC-dependent manner as a result of PMA treatment. In addition, PMA triggers a Ras-dependent signal transduction pathway bypassing the phosphorylation of ERK. Finally, we provide evidence that sustained phosphorylation of ERK1/2 is necessary but not sufficient for expression of MMP-9.
The SHR3 homologue from S. pombe demonstrates a conserved function of ER packaging chaperones
Authors: Martínez Paula , Ljungdahl Per O. 
Published: April 2021

ABSTRACT
In Saccharomyces cerevisiae cells lacking SHR3, amino acid permeases do not enter into COPII transport vesicles and specifically accumulate in the membrane of the endoplasmic reticulum. Shr3p functions as a packaging chaperone to prime transport vesicle formation in the proximity of amino acid permeases. A genetic screen was developed that enabled the Schizosaccharomyces pombe SHR3 functional homologue, designated psh3+ (pombe SHR3), to be cloned. The psh3+ gene encodes a protein of 215 amino acids, which shares a high degree of structural and functional similarity with Shr3p. The heterologous expression of psh3+ complements many, but not all, shr3 null mutant phenotypes in S. cerevisiae in a temperature-dependent manner. Psh3p is localised to the endoplasmic reticulum of S. pombe cells, and strains lacking the psh3+gene exhibit decreased rates of amino acid uptake due to reduced levels of functional permeases in the plasma membrane. No packaging chaperones, or proteins exhibiting homology with packaging chaperones, have so far been identified in other eukayotic organisms. The findings reported here are the first to establish that specific packaging chaperones exist in divergent organisms, and demonstrate a conserved function of packaging chaperones in facilitating the export of large polytopic membrane proteins from the endoplasmic reticulum.
Association of cortactin with dynamic actin in lamellipodia and on endosomal vesicles
Authors: Kaksonen Marko , Peng H. Benjamin , Rauvala Heikki 
Published: April 2021

ABSTRACT
We have used fluorescent protein tagging to study the localization and dynamics of the actin-binding protein cortactin in living NIH 3T3 fibroblast cells. Cortactin was localized to active lamellipodia and to small cytoplasmic spots. Time-lapse imaging revealed that these cortactin labeled structures were very dynamic. In the lamellipodia, cortactin labeled structures formed at the leading edge and then moved toward the cell center. Experiments with green fluorescent protein (GFP)-tagged actin showed that cortactin movement was coincident with the actin retrograde flow in the lamellipodia. Cytoplasmic cortactin spots also contained F-actin and were propelled by actin polymerization. Arp3, a component of the arp2/3 complex which is a key regulator of actin polymerization, co-localized with cortactin. Cytoplasmic cortactin-labeled spots were found to be associated with endosomal vesicles. Association was asymmetric and approximately half of the endosomes were associated with cortactin spots. Time-lapse imaging suggested that these cortactin and F-actin-containing spots propelled endosomes. Actin polymerization based propulsion may be a common mechanism for endomembrane trafficking in the same manner as used in the plasma membrane protrusions. As cortactin is known to interact with membrane-associated signaling proteins it could have a role in linking signaling complexes with dynamic actin on endosomes and in lamellipodia.
Movies available on-line: http://www.biologists.com/JCS/movies/jcs1893.html
Unique properties of lamp2a compared to other lamp2 isoforms
Authors: Cuervo A. M. , Dice J. F. 
Published: April 2021

ABSTRACT
Lamp2a acts as a receptor in the lysosomal membrane for substrate proteins of chaperone-mediated autophagy. Using antibodies specific for the cytosolic tail of lamp2a and others recognizing all lamp2 isoforms, we found that in rat liver lamp2a represents 25% of lamp2s in the lysosome. We show that lamp2a levels in the lysosomal membrane in rat liver and fibroblasts in culture directly correlate with rates of chaperone-mediated autophagy in a variety of physiological and pathological conditions. The concentration of other lamp2s in the lysosomal membrane show no correlation under the same conditions. Furthermore, substrate proteins bind to lamp2a but not to other lamp2s. Four positively-charged amino acids uniquely present in the cytosolic tail of lamp2a are required for the binding of substrate proteins. Lamp2a also distributes to an unique subpopulation of perinuclear lysosomes in cultured fibroblasts in response to serum withdrawal, and lamp2a, more than other lamp2s, tends to multimerize. These characteristics may be important for lamp2a to act as a receptor for chaperone-mediated autophagy.
Characterization of the amino-terminal regions in the human multidrug resistance protein (MRP1)
Authors: Bakos Éva , Evers Raymond , Calenda Giulia , E. Tusnády Gábor , Szakács Gergely , Váradi András , Sarkadi Balázs 
Published: April 2021

ABSTRACT
The human multidrug resistance protein (MRP1) contributes to drug resistance in cancer cells. In addition to an MDR1-like core, MRP1 contains an N-terminal membrane-bound (TMD0) region and a cytoplasmic linker (L0), both characteristic of several members of the MRP family. In order to study the role of the TMD0 and L0 regions, we constructed various truncated and mutated MRP1, and chimeric MRP1-MDR1 molecules, which were expressed in insect (Sf9) and polarized mammalian (MDCKII) cells. The function of the various proteins was examined in isolated membrane vesicles by measuring the transport of leukotriene C4 and other glutathione conjugates, and by vanadate-dependent nucleotide occlusion. Cellular localization, and glutathione-conjugate and drug transport, were also studied in MDCKII cells. We found that chimeric proteins consisting of N-terminal fragments of MRP1 fused to the N terminus of MDR1 preserved the transport, nucleotide occlusion and apical membrane routing of wild-type MDR1. As shown before,MRP1 without TMD0L0 (ΔMRP1), was non-functional and localized intracellularly, so we investigated the coexpression of ΔMRP1 with the isolated L0 region. Coexpression yielded a functional MRP1 molecule in Sf9 cells and routing to the lateral membrane in MDCKII cells. Interestingly, the L0 peptide was found to be associated with membranes in Sf9 cells and could only be solubilized by urea or detergent. A 10-amino-acid deletion in a predicted amphipathic region of L0 abolished its attachment to the membrane and eliminated MRP1 transport function, but did not affect membrane routing. Taken together, these experiments suggest that the L0 region forms a distinct domain within MRP1, which interacts with hydrophobic membrane regions and with the core region of MRP1.
The role of transient ERK2 signals in fibronectin- and insulin-mediated DNA synthesis
Authors: Asthagiri Anand R. , Reinhart Cynthia A. , Horwitz Alan F. , Lauffenburger Douglas A. 
Published: April 2021

ABSTRACT
Both the extracellular matrix and growth factors jointly regulate cell cycle progression via a complex network of signaling pathways. Applying quantitative assays and analysis, we demonstrate here that concurrent stimulation of Chinese hamster ovary (CHO) cells with fibronectin (Fn) and insulin elicits a DNA synthesis response that reveals a synergy far more complex than a simple additive enhancement of response magnitude. CHO cell adhesion to higher Fn density shifts the sensitivity of the DNA synthesis response to insulin concentration from smoothly graded to sharply ‘switch-like’ and dramatically decreases the insulin concentration required for half-maximal response by about 1000-fold. Conversely, treatment with insulin has a milder and less complex effect on the response to varying Fn concentrations. Governing this DNA synthesis response is a common requirement for a transient, cell area-independent extracellular signal-regulated kinase 2 (ERK2) signal. Moreover, we show that the time-integrated value of this ‘pulse’ signal provides an appropriate metric for quantifying the dependence of DNA synthesis on the degree of ERK2 activation. Indeed, in the absence of insulin, the adhesion-mediated response is linearly proportional to ERK2 activation over a broad range of stimulatory Fn and MEK inhibitor amounts. However, in the presence of both Fn and insulin, total integrated ERK2 activity (the sum of Fn- and insulin-mediated signals) no longer serves as a predictor of DNA synthesis, demonstrating that the signaling crosstalk underlying response synergism does not converge at ERK2 activation. Instead, adhesion to higher Fn density enhances insulin stimulation of DNA synthesis, not by increasing insulin-mediated ERK2 activation, but via parallel elevation of at least one other insulin-mediated signal such as IRS-1 phosphorylation.
The developmentally regulated ECM glycoprotein ISG plays an essential role in organizing the ECM and orienting the cells of Volvox
Authors: Hallmann Armin , Kirk David L. 
Published: April 2021

ABSTRACT
Volvox is one of the simplest multicellular organisms with only two cell types, yet it has a surprisingly complex extracellular matrix (ECM) containing many regionspecific morphological components, making Volvox suitable as a model system for ECM investigations. ECM deposition begins shortly after inversion, which is the process by which the embryo turns itself right-side-out at the end of embryogenesis. It was previously shown that the gene encoding an ECM glycoprotein called ISG is transcribed very transiently during inversion. Here we show that the developmentally controlled ISG accumulates at the bases of the flagella right after inversion, before any morphologically recognizable ECM structures have yet developed. Later, ISG is abundant in the ‘flagellar hillocks’ that encircle the basal ends of all flagella, and in the adjacent ‘boundary zone’ that delimits the spheroid. Transgenic Volvox were generated which express a truncated form of ISG. These transgenics exhibit a severely disorganized ECM within which the cells are embedded in a highly chaotic manner that precludes motility. A synthetic version of the C-terminal decapeptide of ISG has a similar disorganizing effect, but only when it is applied during or shortly after inversion. We postulate that ISG plays a critical role in morphogenesis and acts as a key organizer of ECM architecture; at the very beginning of ECM formation ISG establishes an essential initial framework that both holds the somatic cells in an adaptive orientation and acts as the scaffold upon which the rest of the ECM can be properly assembled, assuring that somatic cells of post-inversion spheroids are held in orientations and locations that makes adaptive swimming behavior possible.
Reversible programming of pluripotent cell differentiation
Authors: Lake Julie-anne , Rathjen Joy , Remiszewski Jackie , Rathjen Peter D. 
Published: April 2021

ABSTRACT
We have undertaken an in vitro differentiation analysis of two related, interconvertible, pluripotent cell populations, ES and early primitive ectoderm-like (EPL) cells, which are most similar in morphology, gene expression, cytokine responsiveness and differentiation potential in vivo to ICM and early primitive ectoderm, respectively. Pluripotent cells were differentiated in vitro as aggregates (embryoid bodies) and the appearance and abundance of cell lineages were assessed by morphology and gene expression. Differentiation in EPL cell embryoid bodies recapitulated normal developmental progression in vivo, but was advanced in comparison to ES cell embryoid bodies, with the rapid establishment of late primitive ectoderm specific gene expression, and subsequent loss of pluripotent cell markers. Nascent mesoderm was formed earlier and more extensively in EPL cell embryoid bodies, and resulted in the appearance of terminally differentiated mesodermal cell types prior to and at higher levels than in ES cell embryoid bodies. Nascent mesoderm in EPL cell embryoid bodies was not specified but could be programmed to alternative fates by the addition of exogenous factors.
EPL cells remained competent to form primitive endoderm even though this is not the normal fate of primitive ectoderm in vivo. The establishment of primitive ectoderm-like gene expression and inability to participate in embryogenesis following blastocyst injection is therefore not directly associated with restriction in the ability to form extra-embryonic lineages. However, the EPL cell embryoid body environment did not support differentiation of primitive endoderm to visceral endoderm, indicating the lack of an inductive signal for visceral endoderm formation deduced to originate from the pluripotent cells. Similarly, the inability of EPL cells to form neurons when differentiated as embryoid bodies was attributable to perturbation of the differentiation environment and loss of inductive signals rather than a restricted differentiation potential. Reversion of EPL cells to ES cells was accompanied by restoration of ES cell-like differentiation potential. These results demonstrate the ability of pluripotent cells to adopt developmentally distinct, stable cell states with altered differentiation potentials.
The Dictyostelium RasS protein is required for macropinocytosis, phagocytosis and the control of cell movement
Authors: Chubb Jonathan R. , Wilkins Andrew , Thomas Geraint M. , Insall Robert H. 
Published: April 2021

ABSTRACT
Endocytosis and cell migration both require transient localised remodelling of the cell cortex. Several lines of evidence suggest a key regulatory role in these activities for members of the Ras family of small GTPases. We have generated Dictyosteliumcells lacking one member of this family, RasS, and the mutant cells are perturbed in endocytosis and cell migration. Mutant amoebae are defective in phagocytosis and fluid-phase endocytosis and are impaired in growth. Conversely, the rasS−cells show an enhanced rate of cell migration, moving three times faster than wild-type controls. The mutant cells display an aberrant morphology, are highly polarised, carry many elongated actin protrusions and show a concomitant decrease in formation of pinocytic crowns on the cell surface. These morphological aberrations are paralleled by changes in the actin cytoskeleton, with a significant proportion of the cortical F-actin relocalised to prominent pseudopodia. Rapid migration and endocytosis appear to be mutually incompatible and it is likely that RasS protein is required to maintain the normal balance between these two actin-dependent processes.
TrioGEF1 controls Rac- and Cdc42-dependent cell structures through the direct activation of RhoG
Authors: Blangy A. , Vignal E. , Schmidt S. , Debant A. , Gauthier-Rouvière C. , Fort P. 
Published: April 2021

ABSTRACT
Rho GTPases regulate the morphology of cells stimulated by extracellular ligands. Their activation is controlled by guanine exchange factors (GEF) that catalyze their binding to GTP. The multidomain Trio protein represents an emerging class of Rho regulators that contain two GEF domains of distinct specificities. We report here the characterization of Rho signaling pathways activated by the N-terminal GEF domain of Trio (TrioD1). In fibroblasts, TrioD1 triggers the formation of particular cell structures, similar to those elicited by RhoG, a GTPase known to activate both Rac1 and Cdc42Hs. In addition, the activity of TrioD1 requires the microtubule network and relocalizes RhoG at the active sites of the plasma membrane. Using a classical in vitro exchange assay, TrioD1 displays a higher GEF activity on RhoG than on Rac1. In fibroblasts, expression of dominant negative RhoG mutants totally abolished TrioD1 signaling, whereas dominant negative Rac1 and Cdc42Hs only led to partial and complementary inhibitions. Finally, expression of a Rho Binding Domain that specifically binds RhoGGTPled to the complete abolition of TrioD1 signaling, which strongly supports Rac1 not being activated by TrioD1 in vivo. These data demonstrate that Trio controls a signaling cascade that activates RhoG, which in turn activates Rac1 and Cdc42Hs.
Regulation of protein sorting at the TGN by plasma membrane receptor activation
Authors: Baldassarre Massimiliano , Dragonetti Antonella , Marra Pierfrancesco , Luini Alberto , Isidoro Ciro , Buccione Roberto 
Published: April 2021

ABSTRACT
We show that in the rat basophilic leukemia cell line RBL, the physiological stimulation of the IgE receptor or direct activation of PKC leads to the missorting of proteins to the plasma membrane, diverting them from their normal intracellular destination. This is demonstrated for two classes of proteins that are normally targeted to the secretory lysosomes via completely different mechanisms, i.e. proteoglycans and the aspartic protease cathepsin D. In the latter case, normal processing of the enzyme is also affected, leading to secretion of the immature form of cathepsin. The present study shows how completely different sorting mechanisms, such as those for delivering proteoglycans and cathepsin D to secretory lysosomes, might share common regulatory signals and are similarly affected when the levels of these signals are perturbed. Finally, protein kinase C appears to be a major player in the signal transduction pathways, leading to proteoglycan and cathepsin D missorting.
Mammalian S-phase checkpoint integrity is dependent on transformation status and purine deoxyribonucleosides
Authors: Downes C. Stephen , Bachrati Csanád Z. , Devlin S. Jayne , Tommasino Massimo , Cutts Timothy J. R. , Watson James V. , Raskó stván , Johnson Robert T. 
Published: April 2021

ABSTRACT
In eukaryotic cells arrested in S-phase, checkpoint controls normally restrain mitosis until after replication. We have identified an array of previously unsuspected factors that modulate this restraint, using transformed hamster cells in which cycle controls are known to be altered in S-phase arrest. Arrested cells accumulate cyclin B, the regulatory partner of the mitotic p34cdc2kinase, which is normally not abundant until late G2phase; treatment of arrested cells with caffeine produces rapid S-phase condensation. We show here that such S-phase checkpoint slippage, as visualised through caffeine-dependent S-phase condensation, correlates with rodent origin and transformed status, is opposed by reverse transformation, and is favoured by c-srcand opposed by wnt1overexpression. Slippage is also dependent on a prolonged replicative arrest, and is favoured by arrest with hydroxyurea, which inhibits ribonucleotide reductase. This last is a key enzyme in deoxyribonucleotide synthesis, recently identified as a determinant of malignancy. Addition of deoxyribonucleosides shows that rapid S-phase condensation is suppressed by a novel checkpoint mechanism: purine (but not pyrimidine) deoxyribonucleosides, like reverse transformation, suppress cyclin B/p34cdc2activation by caffeine, but not cyclin B accumulation. Thus, ribonucleotide reductase has an unexpectedly complex role in mammalian cell cycle regulation: not only is it regulated in response to cycle progression, but its products can also reciprocally influence cell cycle control kinase activation.
Cross-regulation of the Wnt signalling pathway: a role of MAP kinases
Authors: Behrens Jürgen 
Published: April 2021

ABSTRACT
The Wnt signal transduction pathway regulates various aspects of embryonal development and is involved in cancer formation. Wnts induce the stabilisation of cytosolic β-catenin, which then associates with TCF transcription factors to regulate expression of Wnt-target genes. At various levels the Wnt pathway is subject to cross-regulation by other components. Recent evidence suggests that a specific MAP kinase pathway involving the MAP kinase kinase kinase TAK1 and the MAP kinase NLK counteract Wnt signalling. In particular, homologues of TAK1 and NLK, MOM-4 and LIT-1, negatively regulate Wnt-controlled cell fate decision in the early Caenorhabditis elegansembryo. Moreover, TAK1 activates NLK, which phosphorylates TCFs bound to β-catenin. This blocks nuclear localization and DNA binding of TCFs. Since TAK1 is activated by TGF-β and various cytokines, it might provide an entry point for regulation of the Wnt system by other pathways. In addition, alterations in TAK1-NLK might play a role in cancer.
DNA-binding activity of the N-terminal cleavage product of poly(ADP-ribose) polymerase is required for UV mediated apoptosis
Authors: Kim Jin Woo , Won Jungyeon , Sohn Seonghyang , Joe Cheol O. 
Published: April 2021

ABSTRACT
The role of the N-terminal cleavage product of poly(ADP-ribose) polymerase (PARP) on UV mediated apoptosis was investigated in cultured HeLa cells. Ultrastructural analysis of cells expressing caspase-resistant PARP (PARPD214A) revealed the typical features of necrosis following UV treatment. However, cells co-expressing PARPD214Awith the N-terminal fragment of PARP containing the DNA-binding domain underwent apoptosis instead of necrosis. In this study, we have demonstrated that the DNA-binding activity of the N-terminal fragment of PARP is important for the execution of apoptosis. Point mutations were introduced in the DNA-binding sites of the N-terminal fragment. Cells co-expressing PARPD214Awith the mutated N-terminal fragments neither stimulated apoptosis nor prevented necrosis in response to UV irradiation. The present study proposes that the DNA-binding activity of the N-terminal fragment of PARP in UV treated cells prevents cellular ATP depletion, a mechanism by which necrotic cell death is triggered.