bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cytoplasmic dynein is required to oppose the force that moves nuclei towards the hyphal tip in the filamentous ascomycete Ashbya gossypii
Authors: Alberti-Segui Christine , Dietrich Fred , Altmann-Jöhl Regula , Hoepfner Dominic , Philippsen Peter 
Published: April 2021

ABSTRACT
We have followed the migration of GFP-labelled nuclei in multinucleate hyphae of Ashbya gossypii. For the first time we could demonstrate that the mode of long range nuclear migration consists of oscillatory movements of nuclei with, on average, higher amplitudes in the direction of the growing tip. We could also show that mitotic division proceeds at a constant rate of 0. 64 μm/minute which differs from the biphasic kinetics described for the yeast Saccharomyces cerevisiae. Furthermore we were able to identify the microtubule-based motor dynein as a key element in the control of long range nuclear migration. For other filamentous fungi it had already been demonstrated that inactivating mutations in dynein led to severe problems in nuclear migration, i.e. generation of long nuclei-free hyphal tips and clusters of nuclei throughout the hyphae. This phenotype supported the view that dynein is important for the movement of nuclei towards the tip. In A. gossypii the opposite seems to be the case. A complete deletion of the dynein heavy chain gene leads to nuclear clusters exclusively at the hyphal tips and to an essentially nucleus-free network of hyphal tubes and branches. Anucleate hyphae and branches in the vicinity of nuclear clusters show actin cables and polarized actin patches, as well as microtubules. The slow growth of this dynein null mutant could be completely reverted to wild-type-like growth in the presence of benomyl, which can be explained by the observed redistribution of nuclei in the hyphal network.
Movies available on-line: http://www.biologists.com/JCS/movies/jcs1921.html
Inward rectifier potassium channel Kir2.2 is associated with Synapse-Associated Protein SAP97
Authors: Leonoudakis Dmitri , Mailliard William S. , Wingerd Kevin L. , Clegg Dennis O. , Vandenberg Carol A. 
Published: April 2021

ABSTRACT
The strong inwardly rectifying potassium channels Kir2.x are involved in maintenance and control of cell excitability. Recent studies reveal that the function and localization of ion channels are regulated by interactions with members of the membrane-associated guanylate kinase (MAGUK) protein family. To identify novel interacting MAGUK family members, we constructed GST-fusion proteins with the C termini of Kir2.1, Kir2.2 and Kir2.3. GST affinity-pulldown assays from solubilized rat cerebellum and heart membrane proteins revealed an interaction between all three Kir2.x C-terminal fusion proteins and the MAGUK protein synapse-associated protein 97 (SAP97). A truncated form of the C-terminal GST-Kir2.2 fusion protein indicated that the last three amino acids (S-E-I) are essential for association with SAP97. Affinity interactions using GST-fusion proteins containing the modular domains of SAP97 demonstrate that the second PSD-95/Dlg/ZO-1 (PDZ) domain is sufficient for interaction with Kir2.2. Coimmunoprecipitations demonstrated that endogenous Kir2.2 associates with SAP97 in rat cerebellum and heart. Additionally, phosphorylation of the Kir2.2 C terminus by protein kinase A inhibited the association with SAP97. In rat cardiac ventricular myocytes, Kir2.2 and SAP97 colocalized in striated bands corresponding to T-tubules. In rat cerebellum, Kir2.2 was present in a punctate pattern along SAP97-positive processes of Bergmann glia in the molecular layer, and colocalized with astrocytes and granule cells in the granule cell layer. These results identify a direct association of Kir2.1, Kir2.2 and Kir2.3 with the MAGUK family member SAP97 that may form part of a macromolecular signaling complex in many different tissues.
Modulation of MHC class II transport and lysosome distribution by macrophage-colony stimulating factor in human dendritic cells derived from monocytes
Authors: Baron Carole L. , Raposo Graça , Scholl Suzy M. , Bausinger Huguette , Tenza Danielle , Bohbot Alain , Pouillart Pierre , Goud Bruno , Hanau Daniel , Salamero Jean 
Published: April 2021

ABSTRACT
The macrophage-colony stimulating factor (M-CSF) has been already shown to affect the function of dendritic cells (DC). Therefore, the differentiation of dendritic cells into macrophages (MΦ) might represent a pathway which could inhibit the immune response initiated by DC. Because Major Histocompatibility Complex class II molecules (MHC-II) are crucial for DC function, we asked whether M-CSF may influence the intracellular transport of MHC-II in monocyte derived DC. We found that, at early stages, M-CSF induced first a rapid redistribution of MHC-II from the MHC-II containing compartments (MIIC) to the plasma membrane and second an increase in MHC-II synthesis as observed with LPS or TNF-α. These processes were associated with the sorting of MHC-II from lysosomal membranes which underwent a drastic structural reorganization. However, in contrast to tumor necrosis factor (TNF)-α or lipopolysaccharide (LPS), M-CSF neither potentiated the allostimulatory function of DC nor allowed the stabilization of MHC-II at the cell surface, but rather increased MHC-II turnover. We conclude that the rapid modulation of MHC-II transport and distribution may participate in the inhibitory effect of M-CSF on DC function and differentiation.
Insulin signalling
Authors: Bevan Paul 
Published: April 2021

ABSTRACT
Insulin binding to its receptor results in receptor autophosphorylation on tyrosine residues and the tyrosine phosphorylation of insulin receptor substrates (IRS-1, IRS-2 and IRS-3) by the insulin receptor tyrosine kinase. This allows association of IRSs with the regulatory subunit of phosphoinositide 3-kinase (PI3K) through its SRC homology 2 (SH2) domains. Once activated, the catalytic subunit phosphorylates phosphoinositides at the 3′ position of the inositol ring or proteins at serine residues. PI3K activates PtdIns(3,4)P2 / PtdIns(3,4,5)P3-dependent kinase 1 (PDK1), which activates PKB/Akt, a serine kinase. PKB in turn deactivates glycogen synthase kinase 3 (GSK-3), leading to activation of glycogen synthase and thus glycogen synthesis. Activation of PKB also results in the translocation of GLUT-4 vesicles from their intracellular pool to the plasma membrane, where they allow uptake of glucose into the cell. PKB also leads to mTOR-mediated activation of protein synthesis by PHAS/elf4 and p70s6k.
AKAP signaling complexes at the cytoskeleton
Authors: Diviani Dario , Scott John D. 
Published: April 2021

ABSTRACT
Targeting of protein kinases and phosphatases to the cytoskeleton enhances the regulation of signal transduction events. The assembly of cytoskeletal signaling complexes facilitates the relay of messages from membrane receptors to specific sites on the actin cytoskeleton. These signals influence fundamental cell properties, such as shape, movement and division. Targeting of the cAMP-dependent kinase (PKA) to the cytoskeleton is achieved through interaction with A-kinase anchoring proteins (AKAPs). AKAPs maintain multivalent signaling complexes by binding additional enzymes, including kinases and phosphatases.
The life cycle of actin patches in mating yeast
Authors: Smith Michael G. , Swamy Sudhindra R. , Pon Liza A. 
Published: April 2021

ABSTRACT
Actin patches are core components of the yeast actin cytoskeleton that undergo redistribution during establishment of cell polarity. Using 4D imaging, we observe the life cycle of actin patches in living yeast for the first time. We observe assembly of actin patches at sites of polarized growth, and disassembly of actin patches concomitant with movement away from those sites. The total lifetime of an actin patch is 10.9±4.2 seconds. These findings indicate that actin patches are labile structures, and that the localization of actin patches during establishment of cell polarity occurs by assembly of these structures at sites of polarized cell surface growth. These findings were confirmed and extended by analysis of myosin I proteins and their receptor, verprolin, proteins implicated in actin assembly in yeast. Deletion of type I myosins or their receptor has no effect on the velocity of actin patch movement. However, these mutants show a 65% reduction in number of patch movements and a three-fold increase in patch lifetime. Finally, the actin patch resident proteins Abp1p, fimbrin, and Arp2p show normal association with actin patches in myosin I and verprolin mutants. However, cofilin accumulates in abnormal ‘bars’ of G-actin in myo3Δ,myo5Δ and vrp1Δ strains, and Las17p/Bee1p is not associated with actin patches in vrp1Δ strains. These findings imply a multi-step process for actin patch assembly. Early events in this process, including assembly of Abp1p, fimbrin and Arp2p with F-actin, can occur throughout the cell and do not require myosin I proteins or their receptor. Later events in this process are myosin I-dependent, and are required for assembly of actin patches at sites of polarized cell surface growth.
Movies available on-line: http://www.biologists.com/JCS/movies/jcs1990.html
Transcription-dependent nucleocytoplasmic distribution of hnRNP A1 protein in early mouse embryos
Authors: Vautier Dominique , Chesné Patrick , Cunha Celso , Calado Angelo , Renard Jean-Paul , Carmo-Fonseca Maria 
Published: April 2021

ABSTRACT
A unique feature of certain members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins is that they shuttle continuously between nucleus and cytoplasm and their accumulation in the nucleus is transcription-dependent. An extensively characterised protein of this group is hnRNP A1. To date, most studies addressing the transcription-dependent transport of hnRNP A1 have been performed on cultured cell lines treated with transcription inhibitors. Here we have analysed the nucleocytoplasmic distribution of hnRNP A1 in early mouse embryos, where the haploid pronuclei remain transcriptionally inactive for a period of several hours. Consistent with its small molecular size (36 kDa), the hnRNP A1 protein diffuses passively through the nuclear pores and equilibrates between the nucleus and the cytoplasm of transcriptionally inactive embryos. In contrast, following transcriptional activation the A1 protein becomes accumulated in the nucleus. This accumulation of the A1 protein in the nucleus is blocked by the lectin wheat germ agglutinin (WGA), which binds to nuclear pore proteins and prevents translocation of receptor-cargo complexes through the pores. This indicates that a carrier-mediated transport pathway is required for the concentration of A1 in transcriptionally active nuclei. To further analyse how transcription is coupled to nucleocytoplasmic transport, we transplanted transcriptionally inactive pronuclei into the cytoplasm of transcriptionally active embryos. The results show that the presence of newly synthesised RNAs in the cytoplasm is not sufficient to induce the accumulation of hnRNP A1 in the nucleus. Rather, the appearance of nascent transcripts in the nucleus appears to be the crucial event. Since hnRNP A1 is a shuttling protein, an increase in its steady state nuclear concentration could be the result of either faster nuclear import or slower export to the cytoplasm. We propose that binding of A1 to nascent transcripts retards its export to the cytoplasm and therefore contributes to its concentration in the nucleus.
N-WASP, WAVE and Mena play different roles in the organization of actin cytoskeleton in lamellipodia
Authors: Nakagawa Hiroyuki , Miki Hiroaki , Ito Masayo , Ohashi Kazuyo , Takenawa Tadaomi , Miyamoto Shigeaki 
Published: April 2021

ABSTRACT
WASP- and Ena/VASP-family proteins have been reported to regulate the cortical actin cytoskeleton as downstream effectors of the Rho-family small G-proteins Rac and Cdc42, but their functions are little understood. We observed the localization of the WASP family proteins, N-WASP and WAVE, and the Ena/VASP family protein, Mena, in protruding lamellipodia. Rat fibroblast cell line 3Y1 protruded lamellipodia on poly-L-lysine-coated substrate without any trophic factor. N-WASP and Cdc42 were concentrated along the actin filament bundles of microspikes but not at the tips. By immunofluorescence and immunoelectron microscopy, both WAVE and Mena were observed to localize at the lamellipodium edge. Interestingly, Mena tended to concentrate at the microspike tips but WAVE did not. At the edge of the lamellipodium, the correlation between the fluorescence from Mena and actin filaments stained with the specific antibody and rhodamine-phalloidin, respectively, was much higher than that between WAVE and actin filament. The Ena/VASP homology 2 (EVH2) domain of avian Ena, an avian homolog of Mena, was localized to the lamellipodium edge and concentrated at the tip of microspikes. The SCAR homology domain (SHD) of human WAVE was distributed along the lamellipodium edge. These results indicate that N-WASP, WAVE and Mena have different roles in the regulation of the cortical actin cytoskeleton in the protruding lamellipodium. WAVE and Mena should be recruited to the lamellipodium edge through SHD and the EVH2 domain, respectively, to regulate the actin polymerization near the cell membrane. N-WASP should regulate the formation of the actin filament bundle in addition to activating Arp2/3 complex in lamellipodium under the control of Cdc42.
N-cadherin is developmentally regulated and functionally involved in early hematopoietic cell differentiation
Authors: Puch Sabine , Armeanu Sorin , Kibler Christine , Johnson Keith R. , Müller Claudia A. , Wheelock Margaret J. , Klein Gerd 
Published: April 2021

ABSTRACT
The cadherins, an important family of cell adhesion molecules, are known to play major roles during embryonic development and in the maintenance of solid tissue architecture. In the hematopoietic system, however, little is known of the role of this cell adhesion family. By RT-PCR, western blot analysis and immunofluorescence staining we show that N-cadherin, a classical type I cadherin mainly expressed on neuronal, endothelial and muscle cells, is expressed on the cell surface of resident bone marrow stromal cells. FACS analysis of bone marrow mononuclear cells revealed that N-cadherin is also expressed on a subpopulation of early hematopoietic progenitor cells. Triple-color FACS analysis defined a new CD34+ CD19+ N-cadherin+ progenitor cell population. During further differentiation, however, N-cadherin expression is lost.
Treatment of CD34+ progenitor cells with function-perturbing N-cadherin antibodies drastically diminished colony formation, indicating a direct involvement of N-cadherin in the differentiation program of early hematopoietic progenitors. N-cadherin can also mediate adhesive interactions within the bone marrow as demonstrated by inhibition of homotypic interactions of bone-marrow-derived cells with N-cadherin antibodies. Together, these data strongly suggest that N-cadherin is involved in the development and retention of early hematopoietic progenitors within the bone marrow microenvironment.
UV induces tyrosine kinase-independent internalisation and endosome arrest of the EGF receptor
Authors: Oksvold Morten P. , Huitfeldt Henrik S. , Østvold Anne Carine , Skarpen Ellen 
Published: April 2021

We have compared the activation and trafficking of epidermal growth factor receptor (EGFR) induced by UV light and EGF. Tyrosine phosphorylation of EGFR was not detected in UV-exposed cells by immunoblotting of whole cell lysates or EGFR immunoprecipitates with antibodies specific for each of the five activated autophosphorylation sites of EGFR. In addition, EGFR of UV-irradiated cells did not demonstrate increased 32P-incorporation. However, UV-exposed cells demonstrated a gel mobility shift of EGFR, which was not abolished by alkaline phosphatase treatment. UV-exposure did not induce dimerisation of EGFR. Furthermore, UV induced internalisation of EGFR without polyubiquitination or degradation. UV-exposed EGFR was transferred to early endosomes and arrested in transferrin-accessible endosomes close to the cell surface. Whereas inhibition of the EGFR tyrosine kinase effectively inhibited tyrosine phosphorylation and internalisation of EGF-activated EGFR, internalisation of UV-exposed EGFR was unaffected. UV induced neither relocalisation of Shc and Grb2 nor activation of Raf, but activation of MEK and MAPK was observed.Our work indicates that UV induces internalisation of EGFR independent of its phosphorylation or receptor tyrosine kinase activation, and altered EGFR trafficking compared with ligand-activated receptor. In addition, MAPK activation by UV does not appear to be mediated by EGFR activation.
Forskolin-mediated G1 arrest in acute lymphoblastic leukaemia cells: phosphorylated pRB sequesters E2Fs
Authors: Gützkow Kristine Bjerve , Naderi Soheil , Blomhoff Heidi K. 
Published: April 2021

Increased intracellular levels of cAMP, induced by forskolin, lead to permanent G1 arrest of Reh cells. As expected, we observed a rapid dephosphorylation of the retinoblastoma protein (pRB) within 2 hours of forskolin treatment concomitant with reduced activity of the pRB-specific kinases. Interestingly, however, the dephosphorylation of pRB, as well as the inhibition of the kinase activities, was only transient, despite the permanent arrest of cells in G1. Importantly, although the pRB-specific kinases were fully active after 48 hours, pRB became only partially rephosphorylated.
The transient dephosphorylation of pRB could be explained by the transient decrease in the activities of the pRB-specific kinases, but to understand why pRB became only partially rephosphorylated, despite fully activated kinases,we postulated that cAMP could activate a pRB-directed phosphatase. It was therefore interesting to find that the phosphatase inhibitor, tautomycin, was able to abolish the forskolin-mediated dephosphorylation of pRB, without increasing the activities of the pRB-specific kinases.
To understand how Reh cells expressing hyperphosphorylated forms of pRB can remain arrested in G1, we used three different methods to test for the ability of pRB to form functional complexes with the family of E2F transcription factors. As expected, we observed an increased complex formation between E2F-1, E2F-4 and pRB after 2 hours when pRB was in its most dephosphorylated state. Suprisingly, however, prolonged treatment with forskolin, which induced partial rephosphorylation of pRB, in fact further increased the complex formation between the E2Fs and pRB, and this also resulted in reduced E2F-promoter activity in vivo. These data imply that in Reh cells, partially phosphorylated forms of pRB retain the ability to inhibit E2F-promoter activity, and thereby prevent cells from entering into S-phase.
Delineation by lanthanum staining of filamentous elements associated with the surfaces of axonal microtubules
Authors: Burton P. R. , Fernandez H. L. 
Published: April 2021

ABSTRACT
The major formed elements of axons of the ventral nerve cord of the crayfish are longitudinally oriented microtubules, filamentous matrix material, and mitochondria. Lanthanum staining delineates both microtubules and filamentous material to great advantage, and it is demonstrated that filamentous elements continuous with matrix material are associated with the surfaces of microtubules. Microtubules are interconnected by filamentous elements and connexions also exist between microtubules and the plasma membrane to form a 3-dimensional latticework. This latticework is hypothesized to play a role in translocation of materials down the axon: the filamentous material probably provides the gelatinous integrity of the axoplasm and may provide the motive force for axonal transport, with the microtubules serving as attachment sites and guideways extending the length of the axon. In pieces of nerve cord treated with cytochalasin B, the filamentous material of the axoplasm is structurally unchanged. Treatment of pieces of nerve cord with hyaluronidase, however, induces the formation of large tubular elements (macrotubules), 40–50 ran in diameter, with which lateral filamentous material is also associated.
The effects of adapting human diploid cells to grow in glutamic acid media on cell morphology, growth and metabolism
Authors: Griffiths J. B. 
Published: April 2021

ABSTRACT
Two lines of human diploid cells, the W1-38 and MRC-5, were adapted to utilize glutamic acid in place of glutamine. This adaptation resulted in (a) more cells per unit culture area, (b) an alteration in cell size and protein content, (c) a morphological change of the cells from fibroblastic to epithelial-like, and (d) increased metabolic activity. Changes in the agglutin-ability of adapted cells by 3 lectins together with the results from polyacrylamide gel electrophoresis could be interpreted as a change in plasma membrane structure. Comparative studies of unadapted, adapted and transformed cells showed that adaptation to glutamic acid produced cells with a metabolism and amino acid uptake similar to those of transformed cells. These changes were reversible and were not accompanied by any apparent karyological change. The significance of these results for the study of density-dependent inhibition of growth is discussed.
Evidence for the importance of puromycyl peptides in the inhibition by ptjromycin of cell aggregation In Vitro
Authors: Dunn M. J. , Owen E. , Kemp R. B. 
Published: April 2021

ABSTRACT
Trypsin-dissociated cells from the muscle tissue of 9-day-old chick embryos were employed to investigate the effects of cycloheximide and a puromycin-cycloheximide mixture on cell aggregation, protein synthesis and respiratory metabolism.
Cycloheximide when introduced at a concentration of 10 μg/ml into a suspension of cells in Eagle’s MEM inhibited aggregation by 25 % at 24 h. At this time an inhibition of 40 % was apparent in the presence of a mixture of cycloheximide and puromycin both at a concentration of 10 μg/ml.
Both cycloheximide and the cycloheximide-puromycin mixture arrested protein synthesis of rotated cells by 90 % within 15 min of introducing the antibiotics into cell suspensions. The antibiotics retained their inhibitory effects on protein synthesis for the 24-h period of rotation.
Cycloheximide inhibited cellular oxygen uptake and carbon dioxide evolution of rotated cells by 25 % at the end of the 24-h experimental period. At this time an inhibition of 30 % was observed in the presence of the cycloheximide-puromycin mixture. The release of radioactive carbon dioxide by cycloheximide-treated cells was inhibited by 46 % at 24 h. In the presence of the antibiotic mixture, 14CO2 release was inhibited by 30 % at 4 h, but after 8 h very little further 14CO2 was evolved.
As a control, puromycin (10μg/ml) inhibited cell aggregation and respiration to an extent similar to that previously reported.
The results are discussed in terms of puromycyl peptides producing a metabolic effect on cell aggregation. It is considered that this is additional to the effect of puromycin inhibiting aggregation through the arrest of protein synthesis.