bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nucleolar localization of murine nuclear DNA helicase II (RNA helicase A)
Authors: Zhang Suisheng , Herrmann Christine , Grosse Frank 
Published: April 2021

ABSTRACT
Nuclear DNA helicase II (NDH II) is a highly conserved member of the DEXH superfamily of eukaryotic helicases, whose physiological role is still unclear. To explore the function of NDH II, we studied the intracellular distribution of NDH II of different mammalian species by immunofluorescence and compared these findings with the known role of the Drosophila homologue MLE that is involved in sex-specific gene dosage compensation. NDH II displayed an apparent nucleolar localization in murine cells, whereas in cells from all other mammalian species examined so far the protein was confined to the nucleoplasm and apparently excluded from the nucleoli. The nucleolar localization of mouse NDH II strongly suggests a role in ribosomal RNA biosynthesis. Immunoelectron microscopic studies revealed that the mouse NDH II was found at the dense fibrillar components of the nucleoli, and a significant percentage of NDH II molecules colocalized with the RNA polymerase I (Pol I) transcription factor UBF (upstream binding factor). Additionally, the nucleolar localization of NDH II coincided with a preferential immunolabeling pattern of nascent transcripts with bromouridine (BrUMP). Furthermore, mouse NDH II redistributed in mitosis in a manner highly correlated with Pol I activity. Conditions leading to the inhibition of Pol I activity in the interphase decreased the amount of NDH II in the nucleoli that diffused into the nucleoplasm and the cytosol. Contrary to the effect of inhibiting rRNA synthesis, treatment of mouse cells with the translation inhibitor cycloheximide did not compromise the nucleolar localization of murine NDH II.
mAKAP: an A-kinase anchoring protein targeted to the nuclear membrane of differentiated myocytes
Authors: Kapiloff Michael S. , Schillace Robynn V. , Westphal Ann M. , Scott John D. 
Published: April 2021

ABSTRACT
The compartmentalization of second messenger-activated protein kinases contributes to the fidelity of hormone-mediated signal transduction events. For example, the cAMP-dependent protein kinase is tethered at specific intracellular locations through association with A-kinase anchoring proteins (AKAPs). We now report the cloning of mAKAP, an anchoring protein found predominantly in heart, skeletal muscle and brain, and whose expression is induced in neonatal ventriculocytes by treatment with hypertrophic stimuli. mAKAP is targeted to the nuclear membrane of differentiated myocytes. Analysis of mAKAP-green fluorescent protein (GFP) fusion constructs revealed that nuclear membrane targeting is conferred by two regions of the protein, between residues 772-915 and 915-1065, which contain spectrin-like repeat sequences. Heterologous expression of the mAKAP targeting sequences displaced the endogenous anchoring protein from the nuclear membrane, demonstrating that mAKAP targeting is saturable. Collectively, these data suggest that a domain containing spectrin-like repeats mediates targeting of the anchoring protein mAKAP and the cAMP-dependent protein kinase holoenzyme to the nuclear membrane in response to differentiation signals.
Protozoan paradigms for cell biology
Authors: Vickerman Keith , Coombs Graham H. 
Published: April 2021

ABSTRACT
This article introduces a miniseries of three commentaries on parasite cell biology. The reviews were written as a tribute to Keith Vickerman FRS on his retirement as Regius Professor of Zoology at the University of Glasgow and are based on presentations given at a symposium held to honour his pioneering work in the field. On page 2799 of this issue, Michael Ferguson reviews the structure and function of GPI anchors, and the contributions that studies of trypanosomes have made. In subsequent issues, James Alexander, Abhay Satoskar and David Russell discuss Leishmania species as models of intracellular parasitism, and Michael Turner presents a holistic view of antigenic variation in Trypanosoma brucei infections.
The structure, biosynthesis and functions of glycosylphosphatidylinositol anchors, and the contributions of trypanosome research
Authors: Ferguson Michael A. J. 
Published: April 2021

ABSTRACT
The discovery of glycosylphosphatidylinositol (GPI) membrane anchors has had a significant impact on several areas of eukaryote cell biology. Studies of the African trypanosome, which expresses a dense surface coat of GPI-anchored variant surface glycoprotein, have played important roles in establishing the general structure of GPI membrane anchors and in delineating the pathway of GPI biosynthesis. The major cell-surface molecules of related parasites are also rich in GPI-anchored glycoproteins and/or GPI-related glycophospholipids, and differences in substrate specificity between enzymes of trypanosomal and mammalian GPI biosynthesis may have potential for the development of anti-parasite therapies. Apart from providing stable membrane anchorage, GPI anchors have been implicated in the sequestration of GPI-anchored proteins into specialised membrane microdomains, known as lipid rafts, and in signal transduction events.
Subcellular localization of GFP-myosin-V in live mouse melanocytes
Authors: Tsakraklides Vasiliki , Krogh Karin , Wang Lillian , Bizario João C. S. , Larson Roy E. , Espreafico Enilza M. , Wolenski Joseph S. 
Published: April 2021

ABSTRACT
Class-V myosins are two-headed actin-based mechanoenzymes that function in the transport and subcellular localization of organelles and possibly in the outgrowth of cellular processes. To determine which domains of myosin-V are involved in intracellular localization of this motor protein, we have expressed fusions of the green fluorescent protein with segments from two distinct myosin-V heavy chains. The expression patterns of constructs encoding four different domains of chick brain myosin-Va were compared to a single construct encoding the globular tail region of mouse myosin-Vb. In transfected mouse melanocytes, expression of the NH2-terminal head (catalytic domain) of chick brain myosin-Va codistributed with actin filaments throughout the cytoplasm. A similar construct encoding the myosin–Va head with the associated neck (light chain binding sites), also codistributed with actin filaments. The GFP-head-neck peptide was also highly concentrated in the tips of filopodia in B16 melanocytes wild type for myosin-Va (MYO5a gene), but was concentrated throughout the entire filopodia of S91-6 melanocytes derived from dilute mice with mutations in the MYO5a gene. Evidence is also presented that the globular tail of myosin-Va, but not myosin-Vb, targets this motor molecule to the centrosome as confirmed by colocalization in cells stained with antibodies to γ-tubulin. Expression of the GFP-myosin-Va globular tail causes displacement of endogenous myosin-V from centrosomes as visualized by immunolabeling with antibodies to the head domain of myosin-V. Treatment with the microtubule-disrupting drug nocodazole markedly reduces myosin-V staining at the centrosome. In contrast, there was no detectable diminution of myosin-V staining at the centrosome in cells treated with the actin filament-disrupting drug cytochalasin D. Thus, while localization of the myosin-V motor domain to actin-rich regions is consistent with conventional models of actomyosin-based motility, localization to the centrosome occurs in the complete absence of the myosin-V motor domain and is dependent on intact microtubules.
In vivo satellite cell activation via Myf5 and MyoD in regenerating mouse skeletal muscle
Authors: Cooper R. N. , Tajbakhsh S. , Mouly V. , Cossu G. , Buckingham M. , Butler-Browne G. S. 
Published: April 2021

ABSTRACT
Regeneration of adult skeletal muscle is an asynchronous process requiring the activation, proliferation and fusion of satellite cells, to form new muscle fibres. This study was designed to determine the pattern of expression in vivo of the two myogenic regulatory factors, Myf5 and MyoD during this process. Cardiotoxin was used to induce regeneration in the gastrocnemius and soleus muscles of heterozygous Myf5-nlacZ mice, and the muscles were assayed for the presence of β-galactosidase (Myf5) and MyoD. Adult satellite cells identified by M-cadherin labelling, when activated, initially express either MyoD or Myf5 or both myogenic factors. Subsequently all proliferating myoblasts express MyoD and part of the population is β-galactosidase (Myf5) positive. Furthermore, we demonstrate that activated satellite cells, which express either Myf5 or MyoD, do not accumulate selectively on fast or slow muscle fibres.
Src induces morphological changes in A431 cells that resemble epidermal differentiation through an SH3- and Ras-independent pathway
Authors: Jin Fang , Reynolds Albert B. , Hines Michelle D. , Jensen Pamela J. , Johnson Keith R. , Wheelock Margaret J. 
Published: April 2021

ABSTRACT
The role of Src family tyrosine kinases in cellular proliferation is well established; however, their role in cellular differentiation is less well understood. In this study we have investigated the role played by Src in the differentiation of squamous epithelial cells. Transfection of activated Src into A431 cells resulted in morphological changes that resembled epidermal differentiation. When we used Src mutants to characterize the observed phenotypic changes, we found that protein tyrosine kinase activity, correct membrane localization and the activity of the SH2 domain were required, but the SH3 domain was not. Furthermore, downstream activity of Ras was not required for the Src-mediated changes in A431 cells.
A staufen-like RNA-binding protein in translocation channels linking nurse cells to oocytes in Notonecta shows nucleotide-dependent attachment to microtubules
Authors: Hurst Suzanne , Talbot Nicholas J. , Stebbings Howard 
Published: April 2021

ABSTRACT
In Drosophila melanogaster the staufen gene encodes an RNA-binding protein that is essential for the correct localization of certain nurse cell-derived transcripts in oocytes. Although the mechanism underlying mRNA localization is unknown, mRNA-staufen complexes have been shown to move in a microtubule-dependent manner, and it has been suggested that staufen associates with a motor protein which generates the movement. We have investigated this possibility using Notonecta glauca in which nurse cells also supply the oocytes with mRNA, but via greatly extended nutritive tubes comprised of large aggregates of parallel microtubules. Using a staufen peptide antibody and RNA probes we have identified a staufen-like protein, which specifically binds double-stranded RNA, in the nutritive tubes of Notonecta. We show that while the staufen-like protein does not co-purify with microtubules from ovaries using standard procedures it does so under conditions of motor-entrapment, specifically in the presence of AMP-PNP. We also show that the staufen-like protein is subsequently removed by ATP and GTP, but not ADP. Nucleotide-dependent binding to microtubules is typical of a motor-mediated interaction and the pattern of attachment and detachment of the staufen-like protein correlates with that of a kinesin protein within the ovaries. Our findings indicate that the staufen-like RNA-binding protein attaches to, and is transported along, Notonecta ovarian microtubules by a kinesin motor.
A mouse model of intestinal stem cell function and regeneration
Authors: Slorach Euan M. , Campbell F. Charles , Dorin Julia R. 
Published: April 2021

ABSTRACT
We present here an in vivo mouse model for intestinal stem cell function and differentiation that uses postnatal intestinal epithelial cell aggregates to generate a differentiated murine small intestinal mucosa with full crypt-villus architecture. The process of neomucosal formation is highly similar to that of intestinal regeneration. Both in vivo grafting and primary culture of these cells reveal two different epithelial cell populations, which display properties consistent with intestinal epithelial transit amplifying and stem cell populations.
Using this model system with a mixture of wild-type and transgene marked cells, we have shown that neomucosae originally develop from single aggregates, but that over time the mucosae fuse to form chimaeric mucosae. Despite fusion, the chimaeric mucosae maintain crypt clonality and villus polyclonality, demonstrating that clonal segregation persists during intestinal epithelial regeneration.
The S. pombe zfs1 gene is required to prevent septation if mitotic progression is inhibited
Authors: Beltraminelli Nicola , Murone Maximilien , Simanis Viesturs 
Published: April 2021

ABSTRACT
Schizosaccharomyces pombe cdc16p is required to limit the cell to forming a single division septum per cell cycle; the heat-sensitive loss-of-function mutant cdc16-116 completes mitosis, and then undergoes multiple rounds of septum formation without cell cleavage. cdc16p is a homologue of Saccharomyces cerevisiae BUB2p, and has also been implicated in the spindle assembly checkpoint function in S. pombe. To identify other proteins involved in regulating septum formation, we have screened for multicopy suppressors of the cdc16-116 mutation. In this paper, we describe one of these suppressors, zfs1. The null allele (zfs1-D1) is viable. However, at low temperatures it divides at a reduced size, while at higher temperatures, it partially suppresses heat sensitive mutants in genes signalling the onset of septum formation. Zfs1-D1 cells show an increased rate of chromosome loss during exponential growth. Moreover, if assembly of the spindle is prevented, zfs1-D1 cells do not arrest normally, but the activity of cdc2p kinase decays, and cells form a division septum without completing a normal mitosis. We conclude that zfs1 function is required to prevent septum formation and exit from mitosis if the mitotic spindle is not assembled. The suppression of cdc16-116 by zfs1 is independent of dma1 function and the spindle assembly checkpoint genes mad2 and mph1. The genetic interactions of zfs1 with genes regulating septum formation suggest that it may be a modulator of the signal transduction network controlling the onset of septum formation and exit from mitosis.
Mechanical loading regulates tenascin-C expression in the osteotendinous junction
Authors: Järvinen Tero A. H. , Jozsa Laszlo , Kannus Pekka , Järvinen Teppo L. N. , Kvist Martti , Hurme Timo , Isola Jorma , Kalimo Hannu , Järvinen Markku 
Published: April 2021

ABSTRACT
Elastic extracellular matrix protein tenascin-C (TN) has very restricted expression in normal tissues, but is expressed in large quantities during embryogenesis and hyperplastic processes. To examine the importance of mechanical stress on the regulation of TN expression in vivo, the effects of various mechanical loading states (immobilization and three forms of subsequent remobilization) on the expression of TN were studied immunohistochemically at the bone-tendon attachment of the rat quadriceps muscle. This osteotendinous junction (OTJ) was selected as study site, since it receives its mechanical stimuli only from muscle contracting activity, which is easy to block by cast immobilization. TN was expressed abundantly in the normal OTJ. Following the removal of the mechanical stress from the junction by cast-immobilization of three weeks, the immunoreactivity of TN was almost completely absent. Normal mechanical stress in the form of free remobilization of eight weeks (free cage activity) resulted in a slight increase in TN expression, but could not restore the expression of TN to the level of the healthy contralateralleg. After the application of the increased mechanical stress (intensified remobilization of the eight weeks by low- or high-intensity treadmill running), the distribution and immunoreactivity of TN reached the level of the healthy contralateral limb in the low-intensity running group or even exceeded that in the high-intensity running group. High TN expression was seen around the chondrocytes and fibroblasts of the OTJ as well as around the collagen fibers of the tendon belly.
We conclusively show that mechanical strain regulates the expression of TN in vivo, and propose that mechanical stress is a major regulator of TN expression in fibroblasts and chondrocytes. This may be an important aspect of the regulation of TN expression during embryogenesis, tendon degeneration, wound healing, bone formation, and in the other normal or regenerative morphogenetic processes TN is postulated to take part in.
Commentary Antigenic variation in Trypanosoma brucei infections: an holistic view
Authors: Turner C. Michael R. 
Published: April 2021

ABSTRACT
Trypanosoma brucei parasites undergo clonal phenotypic (antigenic) variation to promote their transmission between mammals and tsetse-fly vectors. This process is classically considered to be a mechanism for evading humoral immune responses, but such an explanation cannot account for the high rate of switching between variable antigens or for their hierarchical (i.e. non-random) expression. I suggest that these anomalies can be explained by a new model: that antigenic variation has evolved as a bifunctional, rather than as a unifunctional, strategy that not only evades humoral immune responses but also enables competition between parasite strains in concomitantly infected hosts. This competition causes a depression of cellular responses. My proposal gives rise to a number of testable predictions. First, low numbers of trypanosomes should express some variable antigen types (VATs) in infections several weeks before these VATs are detectable. Second, as an infection progresses, the number of VATs expressed simultaneously in the population should decrease. Third, immunisation to generate a T helper 1 response against those VATs that are expressed most frequently should lower parasitaemias and reduce virulence.
Ligand-receptor interactions and trans-endocytosis of Delta, Serrate and Notch: members of the Notch signalling pathway in Drosophila
Authors: Klueg Kristin M. , Muskavitch Marc A. T. 
Published: April 2021

ABSTRACT
Molecular evidence has established that direct heterotypic interactions occur between the Drosophila receptor Notch and the ligands Delta and Serrate, and that homotypic interactions occur between Delta molecules on opposing cell surfaces. Using an aggregation assay developed for Drosophila cultured cells, we have compared the affinities of these interactions. We find that the heterotypic interactions between Notch and the ligands Delta and Serrate have higher affinities than homotypic interactions between Delta molecules. Contrary to previous suggestions, our evidence implies that the interactions between Serrate and Notch are similar in affinity to those between Delta and Notch. We find that Fringe does not detectably affect the ligand-receptor interactions of the Notch pathway in cultured cells. Furthermore, we find that Serrate, like Delta, is a transmembrane ligand that can participate in reciprocal trans-endocytosis of ligand and receptor between expressing cells. Our findings imply that qualitative differences between Delta- and Serrate-mediated Notch signalling depend on characteristics other than intrinsic ligand-receptor affinities or the ability to participate in reciprocal ligand and receptor trans-endocytosis.
A FERM domain governs apical confinement of PTP-BL in epithelial cells
Authors: Cuppen Edwin , Wijers Mietske , Schepens Jan , Fransen Jack , Wieringa Bé , Hendriks Wiljan 
Published: April 2021

ABSTRACT
PTP-BL is a cytosolic multidomain protein tyrosine phosphatase that shares homologies with several submembranous and tumor suppressor proteins. Here we show, by transient expression of modular protein domains of PTP-BL in epithelial MDCK cells, that the presence of a FERM domain in the protein is both necessary and sufficient for its targeting to the apical side of epithelial cells. Furthermore, immuno-electron microscopy on stable expressing MDCK pools, that were obtained using an EGFP-based cell sorting protocol, revealed that FERM domain containing fusion proteins are enriched in microvilli and have a typical submembranous location at about 10-15 nm from the plasma membrane. Immunofluorescence microscopy suggested colocalization of the FERM domain moiety with the membrane-cytoskeleton linker ezrin. However, at the electron microscopy level this colocalization cannot be confirmed nor can we detect a direct interaction by immunoprecipitation assays. Fluorescence recovery after photobleaching (FRAP) experiments show that PTP-BL confinement is based on a dynamic steady state and that complete redistribution of the protein may occur within 20 minutes. Our observations suggest that relocation is mediated via a cytosolic pool, rather than by lateral movement. Finally, we show that PTP-BL phosphatase domains are involved in homotypic interactions, as demonstrated by yeast two-hybrid assays. Both the highly restricted subcellular compartmentalization and its specific associative properties may provide the appropriate conditions for regulating substrate specificity and catalytic activity of this member of the PTP family.
Differential role of mannose and glucose trimming in the ER degradation of asialoglycoprotein receptor subunits
Authors: Ayalon-Soffer Michal , Shenkman Marina , Lederkremer Gerardo Z. 
Published: April 2021

ABSTRACT
To gain insight into how sugar chain processing events modulate endoplasmic reticulum (ER)/proteasomal degradation we looked at human asialoglycoprotein receptor polypeptides H2a and H2b, variants which differ only by an extra pentapeptide (EGHRG) present in H2a. Membrane-bound H2a is a precursor of a soluble secreted form while H2b reaches the plasma membrane. Uncleaved precursor H2a molecules are completely retained in the ER and degraded as well as a portion of H2b. Inhibition of N-linked sugar chain mannose trimming stabilized both variants. In contrast, inhibition of glucose trimming with castanospermine greatly enhanced the degradation rate of H2a but not that of H2b. We studied a possible involvement of the ER chaperone calnexin, as inhibitors of glucose trimming are known to prevent calnexin binding. Incubation of cells with low concentrations of castanospermine (30 µg/ml) did not interfere with calnexin binding to H2a while causing the same accelerated degradation as high concentrations (&gt;100 µg/ml) which did inhibit the association. Castanospermine treatment after calnexin binding blocked the dissociation of the chaperone but still caused accelerated degradation. The increased degradation could be blocked by a specific proteasome inhibitor, ZL3VS. Our results suggest that extensive mannose trimming or retention of glucose residues due to lack of glucose trimming are signals for ER/proteasomal degradation independent of interaction with calnexin.
The Drosophila GMII gene encodes a Golgi α-mannosidase II
Authors: Rabouille Catherine , Kuntz Douglas A. , Lockyer Anne , Watson Rose , Signorelli Tara , Rose David R. , Heuvel Marcel van den , Roberts David B. 
Published: April 2021

ABSTRACT
In this paper we show the organisation of the Drosophila gene encoding a Golgi α-mannosidase II. We demonstrate that it encodes a functional homologue of the mouse Golgi α-mannosidase II. The Drosophila and mouse cDNA sequences translate into amino acid sequences which show 41% identity and 61% similarity. Expression of the Drosophila GMII sequence in CHOP cells produces an enzyme which has mannosidase activity and is inhibited by swainsonine and by CuSO4. In cultured Drosophila cells and in Drosophila embryos, antibodies raised against a C-terminal peptide localise this product mainly to the Golgi apparatus as identified by cryo-immuno electron microscopy studies and by antibodies raised against known mammalian Golgi proteins. We discuss these results in terms of the possible use of dGMII as a Drosophila Golgi marker.