bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of acid/base homeostasis in the suppression of apoptosis in haemopoietic cells by v-Abl protein tyrosine kinase
Authors: Chen Quan , Benson Rod S. P. , Whetton Anthony D. , Brant Steven R. , Donowitz Mark , Montrose Marshall H. , Dive Caroline , Watson Alastair J. M. 
Published: April 2021

ABSTRACT
Removal of interleukin-3 from murine IC.DP pre-mast cells results in irreversible commitment to apoptosis within 18 hours. To identify early events necessary for the engagement of apoptosis we examined the regulation of intracellular pH (pHi). IC.DP cells acidified 2 hours after removal of interleukin-3 (before discernible signs of apoptosis) and by 18 hours pHi had decreased by 0.15 units. The acidification was due to both an increase in an acid-loading process which only occurs when intracellular pH is above 6.8 and a slight reduction in H+ efflux via Na+/H+ exchange. Activation of a temperature sensitive mutant of v-Abl protein tyrosine kinase suppressed apoptosis of IC.DP cells in the absence of interleukin-3 but did not stimulate proliferation, and moreover prevented cellular acidification. Acidification of the cells by 0.2 units to pH 6.86 by complete inhibition of Na+/H+ exchange by 10 µM 5’-(N-methyl-N-isobutyl)-amiloride prevented the suppression of apoptosis by v-abl protein tyrosine kinase following IL 3 withdrawal. However in the presence of interleukin-3, addition of 10 µM 5’-(N-methyl-N-isobutyl)-amiloride only resulted in a fall of pHi to 7.17. Apoptosis did not occur and the cells continued to proliferate. Thus, in this model intracellular pH must fall below a critical value for apoptosis to occur. Together these data point to a step in cytokine deprivation induced apoptosis (at least in some haemopoietic cell types) which is either enhanced by or dependent upon an acidic intracellular environment which is the result of an increase in acid loading and inhibition of Na+/H+ exchange activity. One of the mechanisms by which activation of v-Abl protein tyrosine kinase suppresses apoptosis is by prevention of intracellular acidification.
Dimples, pores, star-rings, and thin rings on growing nuclear envelopes: evidence for structural intermediates in nuclear pore complex assembly
Authors: Goldberg Martin W. , Wiese Christiane , Allen Terence D. , Wilson Katherine L. 
Published: April 2021

ABSTRACT
We used field emission in-lens scanning electron microscopy to examine newly-assembled, growing nuclear envelopes in Xenopus egg extracts. Scattered among nuclear pore complexes were rare ‘dimples’ (outer membrane depressions, 5-35 nm diameter), more abundant holes (pores) with a variety of edge geometries (35-45 nm diameter; 3.3% of structures), pores containing one to eight triangular ‘star-ring’ subunits (2.1% of total), and more complicated structures. Neither mature complexes, nor these novel structures, formed when wheat germ agglutinin (which binds O-glycosylated nucleoporins) was added at high concentrations (&gt;500 µg/ml) directly to the assembly reaction; low concentrations (10 µg/ml) had no effect. However at intermediate concentrations (50-100 µg/ml), wheat germ agglutinin caused a dramatic, sugar-reversible accumulation of ‘empty’ pores, and other structures; this effect correlated with the lectininduced precipitation of a variable proportion of each major Xenopus wheat-germ-agglutinin-binding nucleoporin. Another inhibitor, dibromo-BAPTA (5,5‛-dibromo1,2-bis[o-aminophenoxy]ethane-N,N,N‛,N‛-tetraacetic acid), had different effects depending on its time of addition to the assembly reaction. When 1 mM dibromo-BAPTA was added at time zero, no pore-related structures formed. However, when dibromo-BAPTA was added to growing nuclei 40-45 minutes after initiating assembly, star-rings and other structures accumulated, suggesting that dibromo-BAPTA can inhibit multiple stages in pore complex assembly. We propose that assembly begins with the formation and stabilization of a hole (pore) through the nuclear envelope, and that dimples, pores, star-rings, and thin rings are structural intermediates in nuclear pore complex assembly.
Nuclear domains of the RNA subunit of RNase P
Authors: Jacobson Marty R. , Cao Long-Guang , Taneja Krishan , Singer Robert H. , Wang Yu-li , Pederson Thoru 
Published: April 2021

ABSTRACT
The ribonucleoprotein enzyme RNase P catalyzes the 5′ processing of pre-transfer RNA, and has also recently been implicated in pre-ribosomal RNA processing. In the present investigation, in situ hybridization revealed that RNase P RNA is present throughout the nucleus of mammalian cells. However, rhodamine-labeled human RNase P RNA microinjected into the nucleus of rat kidney (NRK) epithelial cells or human (HeLa) cells initially localized in nucleoli, and subsequently became more evenly distributed throughout the nucleus, similar to the steadystate distribution of endogenous RNase P RNA. Parallel microinjection and immunocytochemical experiments revealed that initially nucleus-microinjected RNase P RNA localized specifically in the dense fibrillar component of the nucleolus, the site of pre-rRNA processing. A mutant RNase P RNA lacking the To antigen binding domain (nucleotides 25-75) did not localize in nucleoli after nuclear microinjection. In contrast, a truncated RNase P RNA containing the To binding domain but lacking nucleotides 89341 became rapidly localized in nucleoli following nuclear microinjection. However, unlike the full-length RNase P RNA, this 3′ truncated RNA remained stably associated with the nucleoli and did not translocate to the nucleoplasm. These results suggest a nucleolar phase in the maturation, ribonucleoprotein assembly or function of RNase P RNA, mediated at least in part by the nucleolar To antigen. These and other recent findings raise the intriguing possibility of a bifunctional role of RNase P in the nucleus: catalyzing pre-ribosomal RNA processing in the nucleolus and pre-transfer RNA processing in the nucleoplasm.
Recruitment of the LIM protein hic-5 to focal contacts of human platelets
Authors: Hagmann Jorg , Grob Marianne , Welman Arkadiusz , Willigen Gijsbert van , Burger Max M. 
Published: April 2021

ABSTRACT
Platelets are anuclear, membrane-bounded fragments derived from megakaryocytes which, upon stimulation, assemble an actin skeleton including stress fibres and focal contacts. The focal contacts resemble those of tissue culture cells. However, they lack paxillin, a conspicuous component of these organelles. We found that instead of paxillin, platelets contain a related protein with a molecular mass of 55 kDa that crossreacts with a monoclonal antibody against paxillin. The gene for the 55 kDa protein was cloned from a bone marrow cDNA library and turned out to be identical to a recently discovered gene encoding hic-5. Like paxillin, hic-5 is a cytoskeletal protein containing four carboxyterminal LIM domains and LD motifs in the aminoterminal half. The LIM domains of both hic-5 and paxillin are capable of targetting green fluorescent protein to focal contacts. In addition, GST-hic-5 precipitates the focal adhesion kinase pp125FAK and talin from platelet extracts. Only trace amounts of hic-5 occur in DAMI cells, a megakaryocytic cell line, and in megakaryocytes cultured from CD34+ cells obtained from umbilical cord blood. However, RT-polymerase chain reactions performed with RNA obtained from platelets gave a positive result when primers specific for hic-5 were used, but were negative with paxillin-specific primers, indicating that a switch from paxillin expression to hic-5 expression must occur late in the maturation of megakaryocytes into platelets.
Nuclear envelope remodelling during rat spermiogenesis: distribution and expression pattern of LAP2/thymopoietins
Authors: Alsheimer Manfred , Fecher Ellen , Benavente Ricardo 
Published: April 2021

ABSTRACT
Lamina-associated polypeptide 2 (LAP2) and the thymopoietins (TPs) are a family of proteins described in somatic cells of mammals, which are derived by alternative splicing from a single gene. For one of the members of the family (LAP2 = TPβ) it has been shown that this integral membrane protein locates to the inner membrane of the nuclear envelope, and that it binds to chromatin and B-type lamins. In the present study, we observed that during the third phase of spermatogenesis (i.e. spermiogenesis), TP-labelling shifted progressively to one half of the nuclear periphery in round spermatids. In the elongating spermatid the signal then becomes restricted to one spot located at the posterior (centriolar) pole of the nucleus. Changes in localization are accompanied by the disappearance, first of TPγ, and later on of LAP2/TPβ. TPα is the only member of the family detectable in the mature sperm. Concomitantly, lamin B1, the only nuclear lamina protein known to be expressed in mammalian spermatids, showed a similar behaviour, i.e. shifted progressively to the centriolar pole of spermatid nuclei before it became undetectable in fully differentiated mature sperms. These results are the first demonstration that expression and localization patterns of TPs are coordinately and differentially regulated with lamins during a differentiation process.
p21WAF1 is dynamically associated with JNK in human T-lymphocytes during cell cycle progression
Authors: Patel Rajnikant , Bartosch Birke , Blank Jonathan L. 
Published: April 2021

ABSTRACT
We have examined the regulation of the c-Jun NH2-terminal kinase (JNK) subfamily of mitogen-activated protein kinases (MAPKs) in response to inhibition of DNA replication during the cell cycle of human T-lymphocytes. In this study, we demonstrate that JNK is rapidly activated following release of T-lymphocytes from G1/S-phase arrest and that this activation precedes resumption of DNA synthesis upon S-phase progression. We also show that activation of JNK correlates with dissociation of the cyclindependent protein kinase (CDK) inhibitor, p21WAF1, from JNK1. Since JNK1 isolated from T-lymphocytes by immunoprecipitation can be inhibited by recombinant p21WAF1 in vitro, these data suggest that JNK activation may be regulated in part by its dissociation from p21WAF1. The observation of a dynamic, physical association of native JNK1 and p21WAF1 in vivo has not previously been described and suggests a novel mechanism for JNK-mediated regulation of the cell cycle of human T-lymphocytes.
Mouse myoblasts can fuse and form a normal sarcomere in the absence of β1 integrin expression
Authors: Hirsch Emilio , Lohikangas Lars , Gullberg Donald , Johansson Staffan , Fässler Reinhard 
Published: April 2021

ABSTRACT
Antibody perturbation experiments suggested that migration, terminal differentiation and fusion of myoblasts are dependent on β1 integrin expression. In addition, several studies have postulated that β1 integrins have a role in the formation of sarcomeres. In the present report we have analysed skeletal myogenesis in wild-type/β1-null chimeric mice and β1-null embryoid bodies. Trunk and limbs of β1-null chimeric mice contained muscle tissue composed of normal and β1-null myoblasts indicating that all myotomic sublineages can form, migrate to their peripheral targets and fuse in the absence of β1 integrin expression. Pure populations of β1-null myoblasts and satellite cells isolated from β1-null chimeric embryos and chimeric newborn mice, respectively, were able to differentiate in vitro and to fuse into multinucleated myotubes. Quantitative and qualitative comparisons between normal and β1-null myoblasts revealed no apparent difference in their capacity to terminally differentiate and fuse. Furthermore, β1-null myotubes developed sarcomeres which were indistinguishable from wild-type controls.
When normal and β1-null ES cells were differentiated into embryoid bodies, they contained fully differentiated myotubes with normal sarcomeres and normal deposition of costameric components. However, formation of β1-null myotubes was delayed and was less efficient in β1-null embryoid bodies than in wild-type controls. High expression of αv integrin subunit at the tips of normal as well as β1-null myotubes indicated that the lack of β1 integrins did not result in a significant redistribution of αv-containing receptors.
Phenotypic effects of the forced expression of HNF4 and HNF1α are conditioned by properties of the recipient cell
Authors: Bailly Alain , Späth Gerald , Bender Virginie , Weiss Mary C. 
Published: April 2021

ABSTRACT
Tagged versions of HNF4 or HNF1α cDNAs in expression vectors have been introduced by transient and stable transfection into three cell lines of hepatic origin that all fail to express these two liver-enriched transcription factors and hepatic functions. C2 and H5 cells are dedifferentiated rat hepatoma variants and WIF12-E cells are human fibroblast-rat hepatoma hybrids with a reduced complement of human chromosomes. Transfectants were analyzed for the expression state of the endogenous genes coding for these transcription factors and for hepatic functions. Each cell line showed a different response to the forced expression of the transcription factors. In C2 cells, no measurable effect was observed, either upon transitory or stable expression. H5 cells reexpressed the endogenous HNF4 gene only upon transient HNF1α transfection, and the endogenous HNF1α gene only in stable HNF4 transfectants. WIF12-E cells responded to the forced transient or stable expression of either HNF1α or HNF4 by cross-activation of the corresponding endogenous gene. In addition, the stable transfectants reexpress HNF3α and C/EBPα, as well as all of the hepatic functions examined. Hybrid cells similar to WIF12-E had previously been observed to show pleiotropic reexpression of the hepatic phenotype in parallel with loss of human chromosome 2. For the stable WIF12-E transfectants, it was verified that reexpression of the hepatic phenotype was not due to loss of human chromosome 2. The demonstration of reciprocal cross-regulation between HNF4 and HNF1α in transient as well as stable transfectants implies that direct effects are involved.
Epidermal growth factor alters fibroblast migration speed and directional persistence reciprocally and in a matrix-dependent manner
Authors: Ware Margaret F. , Wells Alan , Lauffenburger Douglas A. 
Published: April 2021

ABSTRACT
Growth factors stimulate sustained cell migration as well as inducing select acute motility-related events such as membrane ruffling and disruption of focal adhesions. However, an in-depth understanding of the characteristics of sustained migration that are regulated by growth factor signals is lacking: how the biochemical signals are related to physical processes underlying locomotion, and how these events are coordinately influenced by interplay between growth factor and matrix substratum signals. To address these issues, we studied sustained migration of NR6 fibroblasts on a complex human matrix substratum, Amgel, comparing effects of epidermal growth factor (EGF) treatment across a range of Amgel levels. In the absence of EGF, cell migration speed and directional persistence are relatively independent of Amgel level, whereas in the presence of EGF speed is increased at intermediate Amgel levels but not at low and high Amgel levels while directional persistence is decreased at intermediate but not at low and high Amgel levels. The net effect of EGF is to increase the frequency of changes in the cell direction, and at the same time to slightly increase the path-length and thereby greatly enhance random dispersion of cells. Despite increasing migration speed during long-term sustained migration EGF treatment does not lead to significantly increased absolute rates of membrane extension in contrast to its well-known elicitation of membrane ruffling in the short term. However, EGF treatment does decrease cell spread area, yielding an apparent enhancement of specific membrane extension rate, i.e. normalized to cell spread area. Cell movement speed and directional persistence are thus, respectively, directly related and indirectly related to the increase in specific membrane extension rate (alternatively, the decrease in cell spread area) induced by EGF treatment during sustained migration. These results indicate that growth factor and matrix substrata coordinately regulate sustained cell migration through combined governance of underlying physical processes.
SRC binding to the cytoskeleton, triggered by growth cone attachment to laminin, is protein tyrosine phosphatase-dependent
Authors: Helmke Steve , Lohse Kathryn , Mikule Keith , Wood Malcolm R. , Pfenninger Karl H. 
Published: April 2021

ABSTRACT
The interaction of the non-receptor tyrosine kinase, Src, with the cytoskeleton of adhesion sites was studied in nerve growth cones isolated from fetal rat brain. Of particular interest was the role of protein tyrosine phosphatases in the regulation of Src-cytoskeleton binding.
Growth cones were found to contain a high level of protein tryrosine phosphatase activity, most of it membrane-associated and forming large, multimeric and wheat germ agglutinin-binding complexes. The receptor tyrosine phosphatase PTPα seems to be the most prevalent species among the membrane-associated enzymes. As seen by immunofluorescence, PTPα is present throughout the plasmalemma of the growth cone including filopodia, and it forms a punctate pattern consistent with that of integrin β1. For adhesion site analysis, isolated growth cones were either plated onto the neurite growth substratum, laminin, or kept in suspension. Plating growth cones on laminin triggered an 8-fold increase in Src binding to the adherent cytoskeleton. This effect was blocked completely with the protein tyrosine phosphatase inhibitor, vanadate. Growth cone plating also increased the association with adhesion sites of tyrosine phosphatase activity (14-fold) and of PTPα immunoreactivity (6-fold). Vanadate blocked the enzyme activity but not the recruitment of PTPα to the adhesion sites.
In conjunction with our previous results on growth cones, these data suggest that integrin binding to laminin triggers the recruitment of PTPα (and perhaps other protein tyrosine phosphatases) to adhesion sites, resulting in de-phosphorylation of Src’s tyr 527. As a result Src unfolds, becomes kinase-active, and its SH2 domain can bind to an adhesion site protein. This implies a critical role for protein tyrosine phosphatase activity in the earliest phases of adhesion site assembly.
Role of plectin in cytoskeleton organization and dynamics
Authors: Wiche Gerhard 
Published: April 2021

ABSTRACT
Plectin and its isoforms are versatile cytoskeletal linker proteins of very large size (&gt;500 kDa) that are abundantly expressed in a wide variety of mammalian tissues and cell types. Earlier studies indicated that plectin molecules were associated with and/or directly bound to subcomponents of all three major cytoskeletal filament networks, the subplasma membrane protein skeleton, and a variety of plasma membrane-cytoskeleton junctional complexes, including those found in epithelia, various types of muscle, and fibroblasts. In conjunction with biochemical data, this led to the concept that plectin plays an important role in cytoskeleton network organization, with consequences for viscoelastic properties of the cytoplasm and the mechanical integrity and resistance of cells and tissues. Several recent findings lent strong support to this concept. One was that a hereditary disease, epidermolysis bullosa simplex (EBS)-MD, characterized by severe skin blistering combined with muscular dystrophy, is caused by defects in the plectin gene. Another was the generation of plectin-deficient mice by targeted inactivation of the gene. Dying shortly after birth, these animals exhibited severe defects in skin, skeletal muscle and heart. Moreover, in vitro studies with cells derived from such animals unmasked an essential new role of plectin as regulator of cellular processes involving actin stress fibers dynamics. Comprehensive analyses of the gene locus in man, mouse, and rat point towards a complex gene expression machinery, comprising an unprecedented diversity of differentially spliced transcripts with distinct 5′ starting exons, probably regulated by different promoters. This could provide a basis for cell type-dependent and/or developmentally-controlled expression of plectin isoforms, exerting different functions through binding to distinct partners. Based on its versatile functions and structural diversification plectin emerges as a prototype cytolinker protein among a family of proteins sharing partial structural homology and functions.
Aggregation of mononucleated precursors triggers cell surface expression of αvβ3 integrin, essential to formation of osteoclast-like multinucleated cells
Authors: Boissy Patrice , Machuca Irma , Pfaff Martin , Ficheux Damien , Jurdic Pierre 
Published: April 2021

ABSTRACT
αvβ3 is a key integrin mediating adhesion of multinucleated osteoclasts during bone resorption. 1,25-dihydroxyvitamin D3 upregulates αvβ3 integrin expression in mononucleated osteoclast precursors and concomitantly stimulates their differentiation into osteoclasts. This suggests that this integrin could play a major role during osteoclast differentiation.We have developed an in vitro model, in which 1,25-dihydroxyvitamin D3 sequentially modifies the behavior of macrophages: Ìt first induces rounding up of these cells, then their subsequent aggregation and spreading, which finally leads to cell fusion and the formation of osteoclast-like multinucleated giant cells. We show that, while 1,25-dihydroxyvitamin D3 stimulates the de novo synthesis of αvβ3 in macrophages early in this process, its accumulation on the surface is triggered by cell aggregation. A high level of integrin αvβ3 cell surface expression correlates with macrophage spreading preceding fusion. This was confirmed by means of novel cell permeable peptides containing the C-terminal sequence of the integrin β3 tail to specifically block αvβ3 function. Although this peptide has no effect on the aggregation step, it disrupts the spreading of osteoclast precursors and consequently inhibits their fusion. These findings suggest a novel role of the integrin αvβ3 in a discrete step of osteoclast differentiation.
Capturing novel mouse genes encoding chromosomal and other nuclear proteins
Authors: Tate Peri , Lee Muriel , Tweedie Susan , Skarnes William C. , Bickmore Wendy A. 
Published: April 2021

ABSTRACT
The burgeoning wealth of gene sequences contrasts with our ignorance of gene function. One route to assigning function is by determining the sub-cellular location of proteins. We describe the identification of mouse genes encoding proteins that are confined to nuclear compartments by splicing endogeneous gene sequences to a promoterless βgeo reporter, using a gene trap approach. Mouse ES (embryonic stem) cell lines were identified that express βgeo fusions located within sub-nuclear compartments, including chromosomes, the nucleolus and foci containing splicing factors. The sequences of 11 trapped genes were ascertained, and characterisation of endogenous protein distribution in two cases confirmed the validity of the approach. Three novel proteins concentrated within distinct chromosomal domains were identified, one of which appears to be a serine/threonine kinase. The sequence of a gene whose product co-localises with splicesome components suggests that this protein may be an E3 ubiquitin-protein ligase. The majority of the other genes isolated represent novel genes.
This approach is shown to be a powerful tool for identifying genes encoding novel proteins with specific sub-nuclear localisations and exposes our ignorance of the protein composition of the nucleus. Motifs in two of the isolated genes suggest new links between cellular regulatory mechanisms (ubiquitination and phosphorylation) and mRNA splicing and chromosome structure/function.