bluuebunny/crossref_metadata_2025_split huggingface dataset:
The nuclear pore complex: mediator of translocation between nucleus and cytoplasm
Authors: Allen T. D. , Cronshaw J. M. , Bagley S. , Kiseleva E. , Goldberg M. W. 
Published: April 2021

ABSTRACT
The enclosure of nuclear contents in eukaryotes means that cells require sites in the boundary that mediate exchange of material between nucleus and cytoplasm. These sites, termed nuclear pore complexes (NPCs), number 100-200 in yeast, a few thousand in mammalian cells and ∼50 million in the giant nuclei of amphibian oocytes. NPCs are large (125 MDa) macromolecular complexes that comprise 50-100 different proteins in vertebrates. In spite of their size and complex structure, NPCs undergo complete breakdown and reformation at cell division. Transport through NPCs can be rapid (estimated at several hundred molecules/pore/second) and accommodates both passive diffusion of relatively small molecules, and active transport of complexes up to several megadaltons in molecular mass. Each pore can facilitate both import and export. The two processes apparently involve multiple pathways for different cargoes, and their transport signals, transport receptors and adapters, and the molecules (and their regulators) that underpin the transport mechanisms. Over the past few years there has been an increasing interest in the pore complex: structural studies have been followed by elucidation of the biochemical aspects of nuclear import, and subsequent investigations into nuclear export. The current challenge is to understand the interactions between the structural elements of the pore complex and the mechanisms that drive the physical processes of translocation through it.
Movies available on-line: http://www.biologists.com/JCS/movies/jcs0712.html & Video 2
The amino- and carboxyl-terminal tails of β-catenin reduce its affinity for desmoglein 2
Authors: Wahl James K. , Nieset Jill E. , Sacco-Bubulya Paula A. , Sadler Tammy M. , Johnson Keith R. , Wheelock Margaret J. 
Published: April 2021

ABSTRACT
Beta-catenin and plakoglobin are members of the armadillo family of proteins and were first identified as components of intercellular adhering junctions. In the adherens junction β-catenin and plakoglobin serve to link classical cadherins to the actin-based cytoskeleton. In the desmosome plakoglobin links the desmosomal cadherins, the desmogleins and the desmocollins, to the intermediate filament cytoskeleton. β-catenin is not a component of the desmosome. Previously we have shown that the central armadillo repeat region of plakoglobin is the site for desmosomal cadherin binding. We hypothesized that the unique amino- and/or carboxyl-terminal ends of β-catenin may regulate its exclusion from the desmosomal plaque. To test this hypothesis we used chimeras between β-catenin and plakoglobin to identify domain(s) that modulate association with desmoglein 2. Chimeric constructs, each capable of associating with classical cadherins, were assayed for association with the desmosomal cadherin desmoglein 2. Addition of either the N- or C-terminal tail of β-catenin to the armadillo repeats of plakoglobin did not interfere with desmoglein 2 association. However, when both β-catenin amino terminus and carboxyl terminus were added to the plakoglobin armadillo repeats, association with desmoglein 2 was diminished. Removal of the first 26 amino acids from this construct restored association. We show evidence for direct protein-protein interactions between the amino- and carboxyl-terminal tails of β-catenin and propose that a sequence in the first 26 amino acids of β-catenin along with its carboxyl-terminal tail decrease its affinity for desmoglein and prevent its inclusion in the desmosome.
Tight junctions are membrane microdomains
Authors: Nusrat A. , Parkos C. A. , Verkade P. , Foley C. S. , Liang T. W. , Innis-Whitehouse W. , Eastburn K. K. , Madara J. L. 
Published: April 2021

ABSTRACT
Tight junctions (TJ) of polarized epithelial cells regulate barrier function at mucosal surfaces. Structural proteins of TJs include hyperphosphorylated occludin (HO) and the peripheral membrane protein, ZO-1. Since TJs are dynamically regulated, and lipid-modified signal transduction proteins localize to TJs, we considered the possibility that the TJ itself is composed of microdomains with unique structure. Differential detergent extraction and isopycnic sucrose density gradients were utilized to isolate TJ-enriched membranes from a polarized intestinal epithelial cell line, T84. Here we report that major pools of hyperphosphorylated occludin (HO) and ZO-1 are found in raft-like membrane microdomains with characteristics of the previously described detergent-insoluble glycolipid rafts (DIGs). Properties of such gradient fractions included Triton X-100 (TX-100) insolubility, light scattering at 600 nm, buoyant density of approximately 1.08 g/cm3 and increased cholesterol content compared to high density fractions. Similar results were obtained using natural epithelium. Unlike the TJ proteins HO and ZO-1, other basolateral transmembrane proteins including E-cadherin, c-met and beta 1 integrin were not increased in DIG-like fractions. Immunoprecipitation studies revealed coprecipitation of a pool of occludin with caveolin-1, a scaffolding protein abundant in DIGs. Coprecipitation results were supported by immunofluorescence and immunogold labeling studies demonstrating caveolin-1 localization in the apical membrane and focal colocalization with occludin in TJs. TJ disassembly by calcium chelation resulted in displacement of TJ proteins from the ‘raft-like’ compartment. Our findings suggest that raft-like compartments play an important role in the spatial organization of TJs and probably in regulation of paracellular permeability in epithelial cells.
Disassembly of membrane-associated NSF 20S complexes is slow relative to vesicle fusion and is Ca2+-independent
Authors: Swanton Eileithyia , Bishop Naomi , Sheehan John , High Stephen , Woodman Philip 
Published: April 2021

ABSTRACT
N-ethylmaleimide-sensitive fusion protein (NSF) and its co-factor soluble NSF attachment protein (α-SNAP) are essential components of the synaptic vesicle fusion machinery and form part of a structurally-conserved 20S protein complex. However, their precise function, relative to fusion itself, is not clear. Using a UV-activated cross-linking approach, we have measured the rate at which a single round of NSF-driven ATP hydrolysis leads to 20S complex disassembly within synaptic membranes. Although this rate is substantially faster than previous
estimates of NSF-dependent ATP hydrolysis, it remains much lower than published rates for fusion of synaptic vesicles. Furthermore, the stability of 20S complexes is unaffected by Ca2+ at concentrations that elicit rapid membrane fusion. We conclude that the ATPase activity of NSF does not contribute directly to vesicle fusion, but more likely plays an earlier role in the synaptic vesicle cycle.
Expression of Eph receptors and ephrins is differentially regulated by E-cadherin
Authors: Orsulic Sandra , Kemler Rolf 
Published: April 2021

ABSTRACT
E-cadherin is the main cell adhesion molecule of early embryonic and adult epithelial cells. Downregulation of E-cadherin is associated with epithelial-mesenchymal transition during embryonic mesoderm formation and tumor progression. To identify genes whose expression is affected by the loss of E-cadherin, we compared mRNA expression patterns between wild-type and E-cadherin null mutant embryonic stem (ES) cells. We found that expression of several Eph receptors and ephrins is dependent on E-cadherin. Rescue of E-cadherin null ES cells with E-cadherin cDNA restores the wild-type expression pattern of Eph family members. Rescue of E-cadherin null ES cells with N-cadherin cDNA does not restore the wild-type expression pattern, indicating that the
regulation of differential expression of Eph family members is specific to E-cadherin. Constitutive ectopic expression of E-cadherin in non-epithelial NIH3T3 cells results in the production of the EphA2 receptor. In epithelial cells, E-cadherin is required for EphA2 receptor localization at cell-cell contacts; in the absence of functional E-cadherin, EphA2 localizes to the perinuclear region. Our results indicate that E-cadherin may be directly or indirectly required for the membrane localization of Eph receptors and their membrane-bound ligands.
Requirement for C-terminal end of fibroblast growth factor receptor 4 in translocation of acidic fibroblast growth factor to cytosol and nucleus
Authors: Klingenberg Olav , Więdłocha Antoni , Rapak Andrzej , Khnykin Denis , Citores Lucia , Olsnes Sjur 
Published: April 2021

ABSTRACT
The ability of COS cells to bind and internalise acidic fibroblast growth factor (aFGF) was studied after transient transfection of the cells with wild-type and mutated fibroblast growth factor receptor 4. In one case the tyrosine kinase of the receptor was inactivated by a point mutation in the active site, whereas in other cases parts of the receptor were deleted to remove various parts of the cytoplasmic domain. In all cases the receptors were expressed at the cell surface at a high level and the cells bound labelled growth factor efficiently and internalised it by endocytosis. Translocation of externally added aFGF across cellular membranes to reach the cytosol and nucleus was measured as transport of labelled growth factor to the nuclear fraction obtained by centrifugation, by farnesylation of growth factor modified to carry a CAAX motif, and by phosphorylation of the growth factor at a site specific for protein kinase C. Whereas both full-length receptors (with and without an active kinase domain) facilitated translocation of the growth factor to the cytosol and nucleus, as assessed by these methods, the mutants of the receptor where the C terminus was deleted, were unable to do so. In contrast, a receptor containing only the 57 most C-terminal amino acids of the cytoplasmic domain in addition to the juxtamembrane, transmembrane and extracellular domains, was in fact able to mediate translocation of aFGF to the cytosol. These data indicate that information contained in the C terminus of the receptor is required for translocation.
Galectin-8 binding to integrins inhibits cell adhesion and induces apoptosis
Authors: Hadari Yaron R. , Goren Rinat Arbel- , Levy Yifat , Amsterdam Avraham , Alon Ronen , Zakut Rina , Zick Yehiel 
Published: April 2021

ABSTRACT
The interaction of cells with the extracellular matrix regulates cell adhesion, motility, growth, survival and differentiation through integrinmediated signal transduction. Here we demonstrate that galectin-8, a secreted mammalian β-galactoside binding protein, inhibits adhesion of human carcinoma (1299) cells to plates coated with integrin ligands, and induces cell apoptosis. Pretreatment of the cells with Mn2+, which increases the affinity of integrins for their ligands, abolished the inhibitory effects of galectin-8. The inhibitory effects of galectin-8 were specific and were not mimicked by plant lectins or other galectins (galectin-1 and galectin-3). In accordance with its antiadhesive effects, transfection of galectin-8 cDNA into 1299 cells significantly reduced (by 75%) colony formation, when compared to the number of colonies formed by cells transfected with an empty vector. Affinity chromatography over immobilized galectin-8 indicated that few membrane proteins interacted with galectin-8 in a sugardependent manner. Microsequencing and western immunoblotting revealed that α3β1 integrin derived from 1299 as well as other cells (e.g. HeLa and human endothelial cells) is a major galectin-8 bindingprotein. Furthermore, immunoprecipitation and immunohistochemical studies suggested that endogenous galectin-8, secreted from 1299 cells, forms complexes with α3β1 integrins expressed on the surface of 1299 cells. Galectin-8 also interacts with other members of the integrin family, like α6β1 integrins. In contrast, galectin-8 only minimally interacts with α4 or β3 integrins. We propose that galectin-8 is an integrin bindingprotein that interacts to a different extent with several, but not all members of the integrin family. Binding of galectin-8 modulates integrin interactions with the extracellular matrix and thus regulates cell adhesion and cell survival.
Toxoplasma gondii catalase: are there peroxisomes in Toxoplasma?
Authors: Ding M. , Clayton C. , Soldati D. 
Published: April 2021

ABSTRACT
The intracellular protozoan parasite Toxoplasma gondii, like all members of the phylum Apicomplexa, is known to possess many organelles: in addition to mitochondria and the compartments of the secretory pathway, there is a reduced chloroplast (the apicoplast) and the phylumspecific components of the apical complex: dense granules, micronemes and rhoptries. Conspicuously missing so far are microbodies, organelles that can be found in nearly all eukaryotic organisms. Microbodies show a large variation with regard to their size, number and contents, depending on the organism and cell type. One marker enzyme of this single membranebound organelle is catalase, which is responsible for the degradation of hydrogen peroxide to water and oxygen. The EST project in T. gondii revealed the existence of two overlapping clones which showed similarity with catalase, and these were used to clone the corresponding gene. The predicted sequence of T. gondii catalase has -AKM at the C terminus, which falls within the consensus of the PTS1 peroxisomal targeting signal. Southern blot analysis confirmed the presence of a single copy gene. Northern and western blot analyses showed that the catalase gene is transcribed and translated. Immunofluorescence assays using an antibody raised against a catalase peptide identified a distinct structure towards the apical end, but other catalasespecific antibodies failed to confirm this localisation. Cell fractionations indicated that the majority of the enzyme was in the cytosol. The fusion of the C-terminal twelve amino acids, including AKM, or the canonical peroxisomal targeting signal, -SKL, to GFP resulted in predominantly cytosolic localization in T. gondii. There was therefore no evidence for membranebound peroxisomes in Toxoplasma.
Intranuclear trafficking of transcription factors: implications for biological control
Authors: Stein Gary S. , André J. van Wijnen , Stein Janet L. , Lian Jane B. , Montecino Martin , Choi Je-Yong , Zaidi Kaleem , Javed Amjad 
Published: April 2021

ABSTRACT
The subnuclear organization of nucleic acids and cognate regulatory factors suggests that there are functional interrelationships between nuclear structure and gene expression. Nuclear proteins that are localized in discrete domains within the nucleus include the leukemiaassociated acute myelogenous leukemia (AML) and promyelocytic leukemia (PML) factors, the SC-35 RNA-processing factors, nucleolar proteins and components of both transcriptional and DNA replication complexes. Mechanisms that control the spatial distribution of transcription factors within the three-dimensional context of the nucleus may involve the sorting of regulatory information, as well as contribute to the assembly and activity of sites that support gene expression. Molecular, cellular, genetic and biochemical approaches have identified distinct protein segments, termed intranucleartargeting signals, that are responsible for directing regulatory factors to specific subnuclear sites. Gene rearrangements that remove or alter intranucleartargeting signals are prevalent in leukemias and have been linked to altered localization of regulatory factors within the nucleus. These modifications in the intranuclear targeting of transcription factors might abrogate fidelity of gene expression in tumor cells by influencing the spatial organization and/or assembly of machineries involved in the synthesis and processing of gene transcripts.
The role of retinoic acid receptors in neurite outgrowth from different populations of embryonic mouse dorsal root ganglia
Authors: Corcoran Jonathan , Shroot Braham , Pizzey John , Maden Malcolm 
Published: April 2021

ABSTRACT
Dorsal root ganglion (DRG) neurons can be categorised into at least three types, based upon their neurotrophin requirement for survival. We have analysed the expression of the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs) in NGF, NT-3 and BDNF dependent neurons isolated from embryonic day (E)13.5 mouse DRG. We show that each population of neurons expressed each of the three RXRs, α, β and γ; however, whilst the NGF and NT-3 dependent neurons expressed each of the RARs α, β and γ, the BDNF dependent neurons only expressed RAR α and β. When retinoic acid was added to each of the neuronal classes only the NGF and NT-3 dependent neurons responded by extending neurites, and this response involved the upregulation of RAR β2. This specificity was confirmed by the use of receptor-selective agonists as only a RAR β-selective compound stimulated neurite outgrowth. These results suggest a role for RA acting via RAR β2 in the outgrowth of neurites.
CD44 expression and regulation during mammary gland development and function
Authors: Hebbard Lionel , Steffen Anja , Zawadzki Volker , Fieber Christina , Howells Norma , Moll Jürgen , Ponta Helmut , Hofmann Martin , Sleeman Jonathan 
Published: April 2021

ABSTRACT
The CD44v6 epitope has been widely reported to be expressed in human mammary carcinomas, yet its prognostic significance is controversial and its function in mammary tumors and mammary glands is unknown. To begin to resolve these issues, we analysed in detail the normal postnatal expression patterns and regulation of the CD44v6 epitope in murine mammary glands. We demonstrate that significant CD44v6 epitope expression is first seen during puberty, and that after puberty CD44v6 epitope expression follows the estrous cycle. CD44v6 epitope expression is observed in the myoepithelium and also less widely in luminal epithelial cells. During lactation, CD44v6 epitope expression is turned off and reappears during involution. The CD44 variant isoform bearing the v6 epitope is CD44v1-v10. Using HC11, a mammary epithelial cell line with stem cell characteristics, and facilitated by the cloning of the murine CD44 promoter, we show that growth factors and hormones which regulate ductal growth and differentiation modulate CD44 transcription. Together our data suggest that the CD44v6 epitope is expressed in mammary epithelial stems cells and in lineages derived from these cells, and that CD44v6 expression is regulated in part by hormones and growth factors such as IGF-1 and EGF which regulate the growth and differentiation of the mammary epithelium. The function of these same growth factors and hormones is often perturbed in mammary carcinomas, and we suggest that CD44v6 expression in tumors reflects this perturbation. We conclude that the expression of the CD44v6 epitope observed in some mammary tumors reflects the stem cell origin of breast tumors, and that whether or not the CD44v6 epitope is expressed in a mammary tumor is determined by the differentiation status of the tumor cells.
Membrane gamma-glutamyl transpeptidase activity of melanoma cells: effects on cellular H2O2 production, cell surface protein thiol oxidation and NF-κB activation status
Authors: Maellaro Emilia , Dominici Silvia , Bello Barbara Del , Valentini Marta A. , Pieri Lisa , Perego Paola , Supino Rosanna , Zunino Franco , Lorenzini Evelina , Paolicchi Aldo , Comporti Mario , Pompella Alfonso 
Published: April 2021

ABSTRACT
The metabolism of glutathione by membrane-bound gamma-glutamyl transpeptidase (GGT) has been recently recognized as a basal source of hydrogen peroxide in the extracellular space. Significant levels of GGT activity are expressed by malignant tumours, and in melanoma cell lines they were found to correlate with the malignant behaviour. As hydrogen peroxide and other oxidants can affect signal transduction pathways at several levels, the present study was aimed to verify: (i) the occurrence of GGT-dependent production of hydrogen peroxide in melanoma cells; (ii) the effects of GGT-dependent prooxidant reactions on known redox-sensitive cellular targets, i.e. protein thiols, the nuclear transcription factor NF-κB and p53. Two melanoma Me665/2 cell clones, exhibiting traces of (clone 2/21) or high (clone 2/60) GGT activity, were studied. The occurrence of GGT-dependent production of hydrogen peroxide was apparent in 2/60 cells, in which it was accompanied by lower levels of cell surface protein thiols. In 2/60 cells, GGT expression was also associated with higher levels of NF-κB activation, as compared to GGT-poor 2/21 cell clone. Indeed, stimulation or inhibition of GGT activity in 2/60 cells resulted in progressive activation or inactivation of NF-κB, respectively. An analysis of the p53 gene product indicated lack of protein expression in 2/60 cells, whereas a mutant protein was highly expressed in 2/21 cells. Taken together, these results indicate that the expression of GGT activity can provide melanoma cells with an additional source of hydrogen peroxide, and that such prooxidant reactions are capable to modify protein thiols at the cell surface level. In addition, GGT expression results in an up-regulation of the transcription factor NF-κB, which could explain the higher metastatic behaviour reported for GGT-rich melanoma cells as compared to their GGT-poor counterparts.
Dynamic, Rho1p-dependent localization of Pkc1p to sites of polarized growth
Authors: Andrews Paul D. , Stark Michael J. R. 
Published: April 2021

ABSTRACT
In eukaryotes, the Rho GTPases and their effectors are key regulators of the actin cytoskeleton, membrane trafficking and secretion, cell growth, cell cycle progression and cytokinesis. Budding yeast Pkc1p, a protein kinase C-like enzyme involved in cell wall biosynthesis and cytoskeletal polarity, is structurally and functionally related to the Rho- associated kinases (PRK/ROCK) of mammalian cells. In this study, localization of Pkc1p was monitored in live cells using a GFP fusion (Pkc1p-GFP). Pkc1p-GFP showed dynamic spatial and temporal localization at sites of polarized growth. Early in the cell cycle, Pkc1p-GFP was found at the pre-bud site and bud tips, becoming delocalized as the cell progressed further and finally relocalizing around the mother-daughter bud neck in an incomplete ring, which persisted until cell separation. Bud localization was actin-dependent but stability of Pkc1p- GFP at the neck was actin-independent, although localization at both sites required functional Rho1p. In addition, Pkc1p-GFP showed rapid relocalization after cell wall damage. These results suggest that the roles of Pkc1p in both polarized growth and the response to cell wall stress are mediated by dynamic changes in its localization, and suggest an additional potential role in cytokinesis.
Movies available on-line: http://www.biologists.com/JCS/movies/jcs1621.html, Movie 2, Movie 3, Movie 4, Movie 5, Movie 6, Movie 7, Movie 8, Movie 9