bluuebunny/crossref_metadata_2025 huggingface dataset:
The combination of epidermal growth factor and transforming growth factor-β induces novel phenotypic changes in mouse liver stem cell lines
Authors: Isfort Robert J. , Cody David B. , Stuard Sharon B. , Randall Catherine J. , Miller Catherine , Ridder Gregg M. , Doersen Claus-Jens , Richards William G. , Yoder Bradley K. , Wilkinson J. Erby , Woychik Richard P.
Published: April 2021

Mouse liver stem cell (oval cell) lines were investigated in order to determine the role which two families of growth and differentiation factors (GDFs), epidermal growth factor (EGF) family and transforming growth factor beta (TGF-β) family, play in liver regeneration. EGF family members, including EGF, amphiregulin, betacellulin, heparin-binding epidermal growth factor, and TGF-alpha, were mitogenic for oval cell lines while TGF-β family members, including TGF-β1, TGF-β2 and TGF-β3, inhibited mitogenesis and induced apoptosis in oval cell lines. Surprisingly, the combination of EGF family members and TGF-β family members resulted in neither proliferation nor apoptosis but instead in a novel cellular response, cellular scattering in tissue culture and morphological differentiation in Matrigel. Analysis of the signal transduction pathways activated by exposure of oval cell lines to either EGF, EGF+TGF-β, or TGF-β indicated that novel combinations of intracellular signals result following stimulation of the cells with the combination of EGF+TGF-β. These data reveal that the dynamics of synergistic GDF action following tissue injury and regeneration results in a new level of complexity not obvious from the study of indi-vidual GDFs.
Centrosome-microtubule nucleation
Authors: Pereira Gislene , Schiebel Elmar
Published: April 2021

In many cell types the formation of microtubules from tubulin subunits is initiated at defined nucleation sites at the centrosome. Thesse sites contain the conserved γ-tubulin which is in association with additional not very well characterised proteins, identified as components of a γ-tubulin ring complex from Xenopus egg extracts or from suppressor screens in the yeast Saccharomyces cerevisiae. In this review we discuss two recently proposed models of how the γ-tubulin complex assists in the assembly of tubulin to form microtubules. These models propose different roles for γ-tubulin and the other proteins in the complex in tubulin assembly. While the structure and composition of a microtubule nucleation site is becoming clearer, it is still unknown how the cell-cycle dependent regulation of microtubule nucleation sites is achieved and whether they disassemble after microtubule formation in order to allow microtubule fluxes towards the centrosome which have been observed in mitotic cells.
E-cadherin mediates adhesion and suppresses cell motility via distinct mechanisms
Authors: Chen Haiyan , Paradies Nancy E. , Fedor-Chaiken Mary , Brackenbury Robert
Published: April 2021

Expression of the calcium-dependent adhesion molecule E-cadherin suppresses the invasion of cells in vitro, but the mechanism of this effect is unknown. To investigate this mechanism, we analyzed the effects of expressing E-cadherin in mouse L-cells and rat astrocyte-like WC5 cells. Increased cellular adhesion mediated by E-cadherin reduced invasion in WC5 cells and in some L-cells, but not in others. In all cases, suppression of invasion was correlated with decreased cell movement as assessed in an in vitro wound-filling assay and a transwell motility assay. To define the relationship between adhesion mediated by E-cadherin and suppression of motility, we analyzed the effects of deleting different regions of the E-cadherin cytoplasmic domain. E-cadherin lacking the entire cytoplasmic domain did not mediate calcium-dependent adhesion and did not reduce cell motility when expressed in WC5 cells. E-cadherin lacking a portion of the catenin-binding domain did not associate with the cytoskeleton and did not promote adhesion, yet still suppressed the motility of WC5 cells. In addition, E-cadherin that retains an intact catenin-binding domain, but lacks a juxtamembrane portion of the cytoplasmic domain, mediated effective adhesion, but did not suppress motility. These results indicate E-cadherin mediates adhesion and suppresses cell motility via distinct mechanisms, and suggest that the juxtamembrane domain of E-cadherin plays a key role in suppressing motility.
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

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

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 (>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.
The E-selectin-ligand ESL-1 is located in the Golgi as well as on microvilli on the cell surface
Authors: Steegmaier M. , Borges E. , Berger J. , Schwarz H. , Vestweber D.
Published: April 2021

Neutrophils and subsets of lymphocytes bind to E-selectin, a cytokine inducible adhesion molecule on endothelial cells. The E-selectin-ligand-1 (ESL-1) is a high affinity glycoprotein ligand which participates in the binding of mouse myeloid cells to E-selectin. The sequence of mouse ESL-1 is highly homologous to the cysteine rich FGF receptor (CFR) in chicken and the rat Golgi protein MG160. We have analysed the subcellular distribution of ESL-1 by indirect immunofluorescence, flow cytometry, various biochemical techniques and by immunogold scanning electron microscopy. We could localize ESL-1 in the Golgi as well as on the cell surface of 32Dc13 cells and neutrophils. Cell surface staining was confirmed by cell surface biotinylation and by cell surface immunoprecipitations in which antibodies only had access to surface proteins on intact cells. In addition, ESL-1(high) and ESL-1(low) expressing cells, sorted by flow cytometry, gave rise to high and low immunoprecipitation signals for ESL-1, respectively. Based on immunogold labeling of intact cells, we localized ESL-1 on microvilli of 32Dc13 cells and of the lymphoma cell line K46. Quantitative evaluation determined 80% of the total labeling for ESL-1 on microvilli of K46 cells while 69% of the labeling for the control antigen B220 was found on the planar cell surface. These data indicate that ESL-1 occurs at sites on the leukocyte cell surface which are destined for the initiation of cell contacts to the endothelium.
Localization of integral membrane peptidylglycine alpha-amidating monooxygenase in neuroendocrine cells
Authors: Milgram S.L. , Kho S.T. , Martin G.V. , Mains R.E. , Eipper B.A.
Published: April 2021

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the amidation of glycine-extended peptides in neuroendocrine cells. At steady state, membrane PAM is accumulated in a perinuclear compartment. We examined the distribution of membrane PAM in stably transfected AtT-20 cells and compared its localization to markers for the trans-Golgi network (TGN), endosomes, and lysosomes. At the light microscopic level, the distribution of membrane PAM does not overlap extensively with lysosomal markers but does overlap with TGN38 and with SCAMP, a component of post-Golgi membranes involved in recycling pathways. By immunoelectron microscopy, membrane PAM is present in tubulovesicular structures which constitute the TGN; some of these PAM-containing tubulovesicular structures are more distal to the Golgi stacks and do not contain TGN38. While some POMC-derived peptides are present in tubulovesicular structures like those that contain membrane PAM, the majority of the POMC-derived peptides are present in secretory granules. There is little overlap between the steady state distribution of membrane PAM and internalized FITC-transferrin in the early endosomes. Few of the perinuclear PAM-containing structures are labeled with HRP or WGA-HRP even following long incubations. Therefore, membrane PAM is localized to perinuclear tubulovesicular structures which are partially devoid of TGN38 and are not all endosomal in origin.
Rho, Rac and Cdc42 regulate actin organization and cell adhesion in macrophages
Authors: Allen W.E. , Jones G.E. , Pollard J.W. , Ridley A.J.
Published: April 2021

Rho family proteins are known to regulate actin organization in fibroblasts, but their functions in cells of haematopoietic origin have not been studied in detail. Bac1.2F5 cells are a colony-stimulating factor-1 (CSF-1)-dependent murine macrophage cell line; CSF-1 stimulates their proliferation and motility, and acts as a chemoattractant. CSF-1 rapidly induced actin reorganization in Bac1 cells: it stimulated the formation of filopodia, lamellipodia and membrane ruffles at the plasma membrane, as well as the appearance of fine actin cables within the cell interior. Microinjection of constitutively activated (V12)Rac1 stimulated lamellipodium formation and membrane ruffling. The dominant inhibitory Rac mutant, N17Rac1, inhibited CSF-1-induced lamellipodium formation, and also induced cell rounding. V12Cdc42 induced the formation of long filopodia, while the dominant inhibitory mutant N17Cdc42 prevented CSF-1-induced formation of filopodia but not lamellipodia. V14RhoA stimulated actin cable assembly and cell contraction, while the Rho inhibitor, C3 transferase, induced the loss of actin cables. Bac1 cells had cell-to-substratum adhesion sites containing beta1 integrin, pp125FAK, paxillin, vinculin, and tyrosine phosphorylated proteins. These ‘focal complexes’ were present in growing and CSF-1-starved cells, but were disassembled in cells injected with N17Cdc42 or N17Rac1. Interestingly, beta1 integrin did not disperse until long after focal phosphotyrosine and vinculin staining had disappeared. We conclude that in Bac1 macrophages Cdc42, Rac and Rho regulate the formation of distinct actin filament-based structures, and that Cdc42 and Rac are also required for the assembly of adhesion sites to the extracellular matrix.
Cdc6p establishes and maintains a state of replication competence during G1 phase
Authors: Detweiler C.S. , Li J.J.
Published: April 2021

CDC6 is essential for the initiation of DNA replication in the budding yeast Saccharomyces cerevisiae. Here we examine the timing of Cdc6p expression and function during the cell cycle. Cdc6p is expressed primarily between mitosis and Start. This pattern of expression is due in part to posttranscriptional controls, since it is maintained when CDC6 is driven by a constitutively induced promoter. Transcriptional repression of CDC6 or exposure of cdc6-1(ts) cells to the restrictive temperature at mitosis blocks subsequent S phase, demonstrating that the activity of newly synthesized Cdc6p is required each cell cycle for DNA replication. In contrast, similar perturbations imposed on cells arrested in G(1) before Start have moderate or no effects on DNA replication. This suggests that, between mitosis and Start, Cdc6p functions in an early step of initiation, effectively making cells competent for replication. Prolonged exposure of cdc6-1(ts) cells to the restrictive temperature at the pre-Start arrest eventually does cripple S phase, indicating that Cdc6p also functions to maintain this initiation competence during G(1). The requirement for Cdc6p to establish and maintain initiation competence tightly correlates with the requirement for Cdc6p to establish and maintain the pre-replicative complex at a replication origin, strongly suggesting that the pre-replicative complex is an important intermediate for the initiation of DNA replication. Confining assembly of the complex to G(1) by restricting expression of Cdc6p to this period may be one way of ensuring precisely one round of replication per cell cycle.
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

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

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

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

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

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

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

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

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

Plectin and its isoforms are versatile cytoskeletal linker proteins of very large size (>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

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

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.
Depolarization of the actin cytoskeleton is a specific phenotype in Saccharomyces cerevisiae
Authors: Karpova Tatiana S. , Moltz Samuel L. , Riles Linda E. , Güldener Ulrich , Hegemann Johannes H. , Veronneau Steeve , Bussey Howard , Cooper John A.
Published: April 2021

The yeast actin cytoskeleton is polarized during most of the cell cycle. Certain environmental factors and mutations are associated with depolarization of the actin cytoskeleton. Is depolarization of the actin cytoskeleton a specific response, or is it a nonspecific reaction to harsh conditions or poor metabolism? If depolarization is a nonspecific response, then any mutation that slows growth should induce depolarization. In addition, the number of genes with the depolarization phenotype should constitute a relatively large part of the genome. To address this question, we determined the effect of slow growth on the actin cytoskeleton, and we determined the frequency of mutations that affect the actin cytoskeleton. Eight mutants with slow growth showed no defect in actin polarization, indicating that slow growth alone is not sufficient to cause depolarization. Among 273 viable haploids disrupted for ORFs of chromosome I and VIII and 950 viable haploids with random genome disruptions, none had depolarization of the cytoskeleton. We conclude that depolarization of the actin cytoskeleton is a specific phenotype.
Flagellar mitochondrial association of the male-specific Don Juan protein in Drosophila spermatozoa
Authors: Santel Ansgar , Blümer Nicole , Kämpfer Matthias , Renkawitz-Pohl Renate
Published: April 2021

The Drosophila don juan gene encodes a basic protein (Don Juan protein), which is solely expressed postmeiotically during spermiogenesis in elongated spermatids and in mature sperm. Transgenic expression of a GFP-tagged Don Juan protein (DJ-GFP) in the male germ line showed an association of the fusion protein with the sperm tail. Detailed examination of DJ-GFP localization revealed novel insights into its distinct temporal and spatial distribution along the sperm tail during the last phase of spermatid maturation. Co-localization of DJ-GFP with actin-labeled cysts demonstrated its emergence in elongated spermatids during individualization. Additionally, the endogenous Don Juan protein was detected with epitope-specific antibodies in finally elongated nuclei of spermatids. After completion of nuclear shaping Don Juan is no longer detectable in the sperm heads with the onset of individualization. Mislocalization of the DJ-GFP protein in flagella of a mutant with defective mitochondrial differentiation provides evidence of mitochondrial association of the fusion protein with flagellar mitochondrial arrays. Ectopically expressed DJ-GFP in premeiotic germ cells as well as salivary gland cells confirmed the capability of the fusion protein to associate with mitochondria. Therefore we suppose that Don Juan is a nuclear-encoded, germ-cell specifically expressed mitochondrial protein, which might be involved in the final steps of mitochondrial differentiation within the flagellum.
Actin ‘purse string’ filaments are anchored by E-cadherin-mediated adherens junctions at the leading edge of the epithelial wound, providing coordinated cell movement
Authors: Danjo Yukitaka , Gipson Ilene K.
Published: April 2021

At the leading edge of healing embryonic epithelium, cables of actin filaments appear to extend from cell to cell, forming a ring around the wound circumference. It has been hypothesized that this actin filament cable functions as a contractile ‘purse string’ to facilitate wound closure. We have observed this cable in large, circular healing epithelial wounds in corneas of adult mice. To elucidate the role of the actin filament cable, we characterized the molecular components associated with the cell-cell junction where the actin filament cable inserts and with the actin filament cable itself, and we studied the effect of disruption of the cable using an E-cadherin function-blocking antibody, ECCD-1. Localization of E-cadherin and the direct association of catenins with actin filament cable at the cell-cell interface of the actin cable confirmed that the cell-cell junction associated with the actin filament cable is an adherens junction. The E-cadherin function-blocking antibody caused disruption of the actin filament cable and induction of prominent lamellipodial extensions on cells at the leading edge, leading to a ragged uneven epithelial wound margin. These data demonstrate that cell-to-cell associated E-cadherin molecules link the actin filament cable, forming a functional adherens junction, and that the actin filament cable plays a role in coordinating cell movement.
Conventional and novel PKC isoenzymes modify the heat-induced stress response but are not activated by heat shock
Authors: Holmberg Carina I. , Roos Pia M. K. , Lord Janet M. , Eriksson John E. , Sistonen Lea
Published: April 2021

In mammalian cells, the heat-induced stress response is mediated by the constitutively expressed heat shock transcription factor 1 (HSF1). Upon exposure to elevated temperatures, HSF1 undergoes several post-translational modifications, including inducible phosphorylation or hyperphosphorylation. To date, neither the role of HSF1 hyperphosphorylation in regulation of the transcriptional activity of HSF1 nor the signaling pathways involved have been characterized. We have previously shown that the protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol 13-acetate (TPA), markedly enhances the heat-induced stress response, and in the present study we elucidate the mechanism by which PKC activation affects the heat shock response in human cells. Our results show that several conventional and novel PKC isoenzymes are activated during the TPA-mediated enhancement of the heat shock response and that the enhancement can be inhibited by the specific PKC inhibitor bisindolylmaleimide I. Furthermore, the potentiating effect of TPA on the heat-induced stress response requires an intact heat shock element in the hsp70 promoter, indicating that PKC-responsive pathways are able to modulate the activity of HSF1. We also demonstrate that PKC is not activated by heat stress per se. These results reveal that PKC exhibits a significant modulatory role of the heat-induced stress response, but is not directly involved in regulation of the heat shock response.
Entry into mitosis without Cdc2 kinase activation
Authors: Gowdy Patrick M. , Anderson Hilary J. , Roberge Michel
Published: April 2021

Mouse FT210 cells at 39°C cannot enter mitosis but arrest in G2 phase, because they lack Cdc2 kinase activity as a result of a temperature-sensitive lesion in the cdc2 gene. Incubation of arrested cells with the protein phosphatase 1 and 2A inhibitor okadaic acid induces morphologically normal chromosome condensation. We now show that okadaic acid also induces two other landmark events of early mitosis, nuclear lamina depolymerization and centrosome separation, in the absence of Cdc2 kinase activity. Okadaic acid-induced entry into mitosis is accompanied by partial activation of Cdc25C and may be prevented by tyrosine phosphatase inhibitors and by the protein kinase inhibitor staurosporine, suggesting that Cdc25C and kinases distinct from Cdc2 are required for these mitotic events. Using in-gel assays, we show that a 45-kDa protein kinase normally activated at mitosis is also activated by okadaic acid independently of Cdc2 kinase. The 45-kDa kinase can utilize GTP, is stimulated by spermine and is inhibited by heparin. These properties are characteristic of the kinase CK2, but immunoprecipitation studies indicate that it is not CK2. The data underline the importance of a tyrosine phosphatase, possibly Cdc25C, and of kinases other than Cdc2 in the structural changes the cell undergoes at mitosis, and indicate that entry into mitosis involves the activation of multiple kinases working in concert with Cdc2 kinase.
α-COP can discriminate between distinct, functional di-lysine signals in vitro and regulates access into retrograde transport
Authors: Schröder-Köhne Stephan , Letourneur Francois , Riezman Howard
Published: April 2021

Emp47p is a yeast Golgi transmembrane protein with a retrograde, Golgi to ER transport di-lysine signal in its cytoplasmic tail. Emp47p has previously been shown to recycle between the Golgi complex and the ER and to require its di-lysine signal for Golgi localization. In contrast to other proteins with di-lysine signals, the Golgi-localization of Emp47p has been shown to be preserved in ret1-1 cells expressing a mutant α-COP subunit of coatomer. Here we demonstrate by sucrose gradient fractionation and immunofluorescence analysis that recycling of Emp47p was unimpaired in ret1-1. Furthermore we have characterized three new alleles of ret1 and showed that Golgi localization of Emp47p was intact in cells with those mutant alleles. We could correlate the ongoing recycling of Emp47p in ret1-1 with preserved in vitro binding of coatomer from ret1-1 cells to immobilized GST-Emp47p-tail fusion protein. As previously reported, the di-lysine signal of Wbp1p was not recognized by ret1-1 mutant coatomer, suggesting a possible role for α-COP in the differential binding to distinct di-lysine signals. In contrast to results with α-COP mutants, we found that Emp47p was mislocalised to the vacuole in mutants affecting β’-, γ-, δ-, and ζ-COP subunits of coatomer and that the mutant coatomer bound neither to the Emp47p nor to the Wbp1p di-lysine signal in vitro. Therefore, the retrograde transport of Emp47p displayed a differential requirement for individual coatomer subunits and a special role of α-COP for a particular transport step in vivo.
Histone H3 phosphorylation is required for the initiation, but not maintenance, of mammalian chromosome condensation
Authors: Hooser Aaron Van , Goodrich David W. , Allis C. David , Brinkley B. R. , Mancini Michael A.
Published: April 2021

The temporal and spatial patterns of histone H3 phosphorylation implicate a specific role for this modification in mammalian chromosome condensation. Cells arrest in late G2 when H3 phosphorylation is competitively inhibited by microinjecting excess substrate at mid-S-phase, suggesting a requirement for activity of the kinase that phosphorylates H3 during the initiation of chromosome condensation and entry into mitosis. Basal levels of phosphorylated H3 increase primarily in late-replicating/early-condensing heterochromatin both during G2 and when premature chromosome condensation is induced. The prematurely condensed state induced by okadaic acid treatment during S-phase culminates with H3 phosphorylation throughout the chromatin, but in an absence of mitotic chromosome morphology, indicating that the phosphorylation of H3 is not sufficient for complete condensation. Mild hypotonic treatment of cells arrested in mitosis results in the dephosphorylation of H3 without a cytological loss of chromosome compaction. Hypotonic-treated cells, however, complete mitosis only when H3 is phosphorylated. These observations suggest that H3 phosphorylation is required for cell cycle progression and specifically for the changes in chromatin structure incurred during chromosome condensation.