bluuebunny/crossref_metadata_2025_split huggingface dataset:
The membrane skeleton of acetylcholine receptor domains in rat myotubes contains antiparallel homodimers of β-spectrin in filaments quantitatively resembling those of erythrocytes
Authors: Pumplin David W. 
Published: April 2021

ABSTRACT
I used immunogold labeling and quick-freeze, deep-etch, rotary replication to characterize the membrane skeleton at regions with high concentrations of acetylcholine receptor domains in receptor clusters of cultured rat muscle cells. This membrane skeleton consists of a network of filaments closely applied to the cytoplasmic membrane surface. The filaments are specifically decorated by immunogold labeling with a monoclonal antibody, VIIF7, that recognizes an isoform of β-spectrin colocalizing with acetylcholine receptors. The filaments are 32±11 nm in length and three to four filaments (average 3.1-3.3) join at each intersection to form the network. These parameters are nearly identical to those reported previously for the membrane skeleton of erythrocytes. Depending on the amount of platinum coating, filament diameters range from 9 to 11 nm in diameter, and are 1.4 nm larger on average than spectrin filaments of erythrocytes replicated at the same time. Filaments are decorated with gold particles close to one end, consistent with the location of the epitope recognized by the monoclonal antibody. Computer modeling shows that all filament intersections in the membrane skeletal network are equally capable of being labeled by the monoclonal antibody. This pattern of labeling is consistent with a network containing antiparallel homodimers of β-spectrin.
N-cadherin in adult rat cardiomyocytes in culture II. Spatio-temporal appearance of proteins involved in cell-cell contact and communication. Formation of two distinct N-cadherin/catenin complexes
Authors: Hertig Cecilia M. , Butz Stefan , Koch Sabine , Eppenberger-Eberhardt Monika , Kemler Rolf , Eppenberger Hans M. 
Published: April 2021

ABSTRACT
The spatio-temporal appearance and distribution of proteins forming the intercalated disc were investigated in adult rat cardiomyocytes (ARC). The ‘redifferentiation model’ of ARC involves extensive remodelling of the plasma membrane and of the myofibrillar apparatus. It represents a valuable system to elucidate the formation of cell-cell contact between cardiomyocytes and to assess the mechanism by which different proteins involved in the cell-cell adhesion process are sorted in a precise manner to the sites of function. Appearance of N-cadherin, the catenins and connexin43 within newly formed adherens and gap junctions was studied.
Here first evidence is provided for a formation of two distinct and separable N-cadherin/catenin complexes in cardiomyocytes. Both complexes are composed of N-cadherin and α-catenin which bind to either β-catenin or plakoglobin in a mutually exclusive manner. The two N-cadherin/catenin complexes are assumed to be functionally involved in the formation of cell-cell contacts in ARC; however, the differential appearance and localization of the two types of complexes may also point to a specific role during ARC differentiation. The newly synthesized β-catenin containing complex is more abundant during the first stages in culture after ARC isolation, while the newly synthesized plakoglobin containing complex progressively accumulates during the morphological changes of ARC.
ARC formed a tissue-like pattern in culture whereby the new cell-cell contacts could be dissolved through Ca2+ depletion. Presence of cAMP and replenishment of Ca2+ content in the culture medium not only allowed reformation of cell-cell contacts but also affected the relative protein ratio between the two N-cadherin/catenin complexes, increasing the relative amount of newly synthesized β-catenin over plakoglobin at a particular stage of ARC differentiation.
The clustered N-cadherin/catenin complexes at the plasma membrane appear to be a prerequisite for the following gap junction formation; a temporal sequence of the appearance of adherens junction proteins and of gap junctions forming connexin43 is suggested.
ATP is required in platelet serotonin exocytosis for protein phosphorylation and priming of secretory vesicles docked on the plasma membrane
Authors: Morimoto Takako , Ogihara Satoshi 
Published: April 2021

ABSTRACT
Calcium-evoked secretion generally requires the presence of millimolar concentrations of Mg-ATP. We investigated the role of Mg-ATP in the secretion of serotonin from electropermeabilized bovine platelets. The secretion of serotonin was lost within 5 minutes when the Mg-ATP concentration was diluted to less than 0.1 mM, but was maintained when ATP-γS (adenosine 5′-O-3-thiotriphosphate) was used instead of ATP. Okadaic acid, a potent inhibitor of protein phosphatase, could also maintain the exocytotic activity even when ATP was diluted. Decrease in the secretory activity was paralleled by a decrease in phosphorylation level of four proteins after dilution of ATP, but the activity was maintained when the thiophosphorylation level of these proteins was maintained. Two of these proteins were digested by a protease, calpain, which has been shown to lead to a loss in the exocytotic activity. Electron microscopic studies showed that calcium did not induce the formation of distinct bridge-like structures between the granule membrane and the plasma membrane in Mg-ATP-diluted cells, previously shown as the structure transiently formed prior to fusion of the two membranes. Anchorage of the secretory dense granules to the plasma membrane and the presence of the amorphous structures between the granules and the plasma membrane were unchanged by dilution of ATP. These results indicate that ATP is not required for the anchorage itself, but is required to prime anchored granules for calcium-triggered secretion. Maintenance of the phosphorylated state of proteins by ATP enables the calcium trigger to form the bridge-like structures preceding membrane fusion events.
Role of γ-tubulin in mitosis-specific microtubule nucleation from the schizosaccharomyces pombe spindle pole body
Authors: Masuda Hirohisa , Shibata Takehiko 
Published: April 2021

ABSTRACT
The ability of the Schizosacchromyces pombe spindle pole body to nucleate microtubules is activated at the onset of mitosis for forming a mitotic spindle, but it is inactivated during interphase. We have previously developed an in vitro assay for studying the molecular mechanism of spindle pole body activation using permeabilized interphase S. pombe cells and Xenopus mitotic extracts. We have shown that the interphase spindle pole body is activated indirectly by p34cdc2 protein kinase in Xenopus mitotic extracts. In this study we examined the role of γ-tubulin, a component of both interphase and mitotic spindle pole body, in formation of the microtubule nucleating complex at the mitotic spindle pole body. A polyclonal antibody specific to S. pombe γ-tubulin inhibited both activation of the interphase spindle pole body and microtubule nucleation from the mitotic spindle pole body. Addition of bacterially expressed S. pombe γ-tubulin or its aminoterminal fragments to Xenopus mitotic extracts inhibited spindle pole body activation. Affinity chromatography of partially fractionated Xenopus mitotic extracts with the amino-terminal fragment of S. pombe γ-tubulin showed that fractions bound to the fragment supported the activation. The fractions did not contain Xenopus γ-tubulin, showing that activation of the spindle pole body is not due to recruitment of Xenopus γ-tubulin to the spindle pole body. The spindle pole body activation occurred in extracts depleted of p34cdc2 protein kinase or MAP kinase. The activity of the fractions bound to the fragment was inhibited by a protein kinase inhibitor, staurosporine. These results suggest that S. pombe γ-tubulin is a component of the microtubule nucleating complex, and that the function of proteins that interact with γ-tubulin is required for activation of the spindle pole body. We present possible models for the activation that convert the immature microtubule nucleating complex at interphase into the mature microtubule nucleating complex at mitosis.
Expression of the gap junction proteins connexin31 and connexin43 correlates with communication compartments in extraembryonic tissues and in the gastrulating mouse embryo, respectively
Authors: Dahl Edgar , Winterhager Elke , Reuß Bernhard , Traub Otto , Butterweck Anette , Willecke Klaus 
Published: April 2021

ABSTRACT
We have characterized the pattern of connexin expression in embryonic and extraembryonic tissues during early mouse development. In the preimplantation blastocyst, at 3.5 days post coitum (dpc), immunofluorescent signals specific for connexin31 and connexin43 proteins were present in both the inner cell mass and the trophectoderm, as shown by confocal laser scan microscopy. Immediately after implantation at 6.5 dpc, however, we find complete compartmentation of these two connexins: connexin31 mRNA and protein are expressed exclusively in cells derived from the trophectoderm lineage, whereas connexin43 mRNA and protein are detected in cells derived from the inner cell mass. This expression pattern of connexin31 and connexin43 is maintained at 7.5 dpc when the axial polarity of the mouse embryo is established. It correlates with the communication compartments in extraembryonic tissues and the gastrulating mouse embryo, respectively. The communication boundary between those compartments may be due to incompatibility of connexin31 and connexin43 hemichannels, which do not communicate with each other in cell culture.
Hyperosmotic stress leads to reversible dissociation of the proton pump-bearing tubules from the contractile vacuole complex in paramecium
Authors: Ishida Masaki , Fok Agnes K. , Aihara Marilynn S. , Allen Richard D. 
Published: April 2021

ABSTRACT
To study the effect of hyperosmotic stress on the structure and function of the contractile vacuole complex of Para- mecium multimicronucleatum, we employed two different monoclonal antibody markers: one to a decorated spongiome antigen (A4) and a second to an antigen found on all other membranes of the contractile vacuole complex (G4). A hyperosmotic condition was produced by adding sorbitol to the axenic culture medium which induced both dose- and time-dependent decreases in the vacuole’s expulsion rate. The addition of 150 mM sorbitol to the medium (making a final osmolarity of 230 mOsmol) was sufficient to completely stop the expulsion of the contractile vacuole. Immunofluorescence demonstrated that the blocking of fluid output was accompanied by the disappearance of most fluorescence labeling from the decorated spongiome (the A4 antigen). Electron microscopy revealed that the disappearance of the labeling was accompanied by the disappearance of the decorated tubules from around the collecting canals. These tubules vesiculate. The other membranes of the contractile vacuole complex remained unaffected which was demonstrated by both electron microscopy and indirect immunolabeling using the mAb against the G4 antigen. These results show that the decorated spongiome is formed from a distinct membrane pool separate from that of the smooth spongiome, collecting canals and the contractile vacuole. Recovery of the decorated spongiome rapidly followed the return of the cell to an isotonic environment and was completed within 3 hours. Decorated tubule recovery paralleled the recovery of the function of the contractile vacuole. Recovery was also observed during continuous hyperosmotic treatment with the reappearance of the contractile vacuole activity starting at 3 hours and stabilizing at around 10 hours of incubation. Functional recovery under these conditions was accompanied by a reappearance of the decorated tubules but the total fluid output was always lower than for cells in an isotonic environment. Thus, cells were shown to be capable of adapting to high hyperosmotic conditions. We conclude that the dissociation and reassociation of the decorated spongiome is an important regulatory feature controlling the activity of the contractile vacuole complex and of intra- cellular osmoregulation in Paramecium.
MAP kinase does not inactivate, but rather prevents the cyclin degradation pathway from being turned on in Xenopus egg extracts
Authors: Abrieu Ariane , Lorca Thierry , Labbé Jean-Claude , Morin Nathalie , Keyse Stephen , Dorée Marcel 
Published: April 2021

ABSTRACT
Unfertilized frog eggs arrest at the second meiotic metaphase, due to cytostatic activity of the c-mos proto-oncogene (CSF). MAP kinase has been proposed to mediate CSF activity in suppressing cyclin degradation. Using an in vitro assay to generate CSF activity, and recombinant CL 100 phosphatase to inactivate MAP kinase, we confirm that the c-mos proto-oncogene blocks cyclin degradation through MAP kinase activation. We further show that for MAP kinase to suppress cyclin degradation, it must be activated before cyclin B-cdc2 kinase has effectively promoted cyclin degradation. Thus MAP kinase does not inactivate, but rather prevents the cyclin degradation pathway from being turned on. Using a constitutively active mutant of Ca2+/calmodulin dependent protein kinase II, which mediates the effects of Ca2+ at fertilization, we further show that the kinase can activate cyclin degradation in the presence of both MPF and the c-mos proto-oncogene without inactivating MAP kinase.
Nuclear pH gradient in mammalian cells revealed by laser microspectrofluorimetry
Authors: Seksek Olivier , Bolard Jacques 
Published: April 2021

ABSTRACT
Intracellular pH has been measured by laser microspectrofluorimetry, using the pH-sensitive dyes SNARF-1, SNARF-calcein and SNARF-1-dextran. By this technique it was possible to accurately determine pH in volumes as small as 0.5×0.5×1 µm3. The probes were loaded into the cells either by diffusion of their acetoxymethylester derivatives (SNARF-1-AM, SNARF-calcein-AM) or by microinjection (SNARF-1-dextran). When the five types of cells were studied in RPMI medium, the nuclear pH was consistently found to be 0.3 to 0.5 units above that of the cytosol. Although the presence of pores in the nuclear membrane has been taken as evidence that free diffusion of ions and small molecules can occur in and out the nucleus, we conclude that the nuclear membrane of these cells presents a permeability barrier to H+. The pH gradient was not observed in cells suspended in PBS.
pS2 transfection of murine adenocarcinoma cell line 410.4 enhances dispersed growth pattern in a 3-D collagen gel
Authors: Williams R. , Stamp G. W. H. , Gilbert C. , Pignatelli M. , Lalani E.-N. 
Published: April 2021

ABSTRACT
We describe the first model system employing human pS2 gene transfer and expression in a non-pS2-expressing cell line, mouse mammary adenocarcinoma 410.4, in order to analyse the potential effect of human trefoil peptide pS2 in glandular epithelium. Two selected clones, AA4 and AD4, were established and shown to have incorporated the pS2 cDNA sequence into the genome, express pS2 containing transcript and produce the pS2 peptide. When grown in 3-D collagen gels both transfectants show striking morphological changes compared to the vector control clone (VA5). VA5 forms large cohesive spherical aggregates with rare coarse spicular outgrowths, accompanied by prominent hyalinised extracellular matrix deposition. pS2 transfectants form poorly cohesive, stellate colonies with very little or no matrix deposition, radiating long cords composed of single elongated cells, an effect previously observed in other cell lines with hepatocyte growth factor. pS2 transfection had no demonstrable effect on proliferation and this is not a morphogenetic phenomenon, as tubulogenesis is not seen. Motility assays suggest that the pS2 ‘dispersant’ effect in collagen gels is due to an increase in cell motility. There were no measurable alterations in either E-cadherin expression or E-cadherin-dependent cell-cell aggregation. pS2 may play a role in maintenance and restitution of mucosal integrity by accelerating migration/dispersion.
Isolation of the Schizosaccharomyces pombe RAD54 homologue, rhp54
+, a gene involved in the repair of radiation damage and replication fidelity
Authors: Muris Daan F. R. , Vreeken Kees , Carr Antony M. , Murray Johanne M. , Smit Claudia , Lohman Paul H. M. , Pastink Albert 
Published: April 2021

ABSTRACT
The RAD54 gene of Saccharomyces cerevisiae encodes a putative helicase, which is involved in the recombinational repair of DNA damage. The RAD54 homologue of the fission yeast Schizosaccharomyces pombe, rhp54+, was isolated by using the RAD54 gene as a heterologous probe. The gene is predicted to encode a protein of 852 amino acids. The overall homology between the mutual proteins of the two species is 67% with 51% identical amino acids and 16% similar amino acids. A rhp54 deletion mutant is very sensitive to both ionizing radiation and UV. Fluorescence microscopy of the rhp54 mutant cells revealed that a large portion of the cells are elongated and occasionally contain aberrant nuclei. In addition, FACS analysis showed an increased DNA content in comparison with wild-type cells. Through a minichromosome-loss assay it was shown that the rhp54 deletion mutant has a very high level of chromosome loss. Furthermore, the rhp54 mutation in either a rad17 or a cdc2.3w mutant background (where the S-phase/mitosis checkpoint is absent) shows a significant reduction in viability. It is hypothesized that the rhp54+ gene is involved in the recombinational repair of UV and X-ray damage and plays a role in the processing of replication-specific lesions.
Ran, a GTPase involved in nuclear processes: its regulators and effectors
Authors: Avis Johanna M. , Clarke Paul R. 
Published: April 2021

ABSTRACT
Ran is a small GTPase that has been implicated in a variety of nuclear processes, including the maintainance of nuclear structure, protein import, mRNA processing and export, and cell cycle regulation. There has been significant progress in determining the role of Ran in nuclear protein import. However, it has been unclear whether this role is sufficient to account for the diverse effects of disrupting Ran functions. Recently, several proteins have been identified that bind specifically to Ran and are, therefore, possible effectors. Other experiments using dominant mutants of Ran that block its GTP/GDP cycle have suggested that Ran may have multiple roles. Here, these results are summarised and discussed with respect to the action of Ran.
Transmembrane phospholipid distribution revealed by freeze-fracture replica labeling
Authors: Fujimoto Kazushi , Umeda Masato , Fujimoto Toyoshi 
Published: April 2021

ABSTRACT
We propose the use of membrane splitting by freezefracture for differential phospholipid analysis of protoplasmic and exoplasmic membrane leaflets (halves). Unfixed cells or tissues are quick-frozen, freeze-fractured, and platinum-carbon (Pt/C) shadowed. The Pt/C replicas are then treated with 2.5% sodium dodecyl sulfate (SDS) to solubilize unfractured membranes and to release cytoplasm or contents. While the detergent dissolves unfractured membranes, it would not extract lipids from split membranes, as their apolar domains are stabilized by their Pt/C replicas. After washing, the Pt/C replicas, along with attached protoplasmic and exoplasmic membrane halves, are processed for immunocytochemical labeling of phospholipids with antibody, followed by electron microscopic observation. Here, we present the application of the SDS-digested freeze-fracture replica labeling (SDS-FRL) technique to the transmembrane distribution of a major membrane phospholipid, phosphatidylcholine (PC), in various cell and intracellular membranes. Immunogold labeling revealed that PC is exclusively localized on the exoplasmic membrane halves of the plasma membranes, and the intracellular membranes of various organelles, e.g. nuclei, mitochondria, endoplasmic reticulum, secretory granules, and disc membranes of photoreceptor cells. One exception to this general scheme was the plasma membrane forming the myelin sheath of neurons and the Ca2+-treated erythrocyte membranes. In these cell membranes, roughly equal amounts of immunogold particles for PC were seen on each outer and inner membrane half, implying a symmetrical transmembrane distribution of PC. Initial screening suggests that the SDS-FRL technique allows in situ analysis of the transmembrane distribution of membrane lipids, and at the same time opens up the possibility of labeling membranes such as intracellular membranes not normally accessible to cytochemical labels without the distortion potentially associated with membrane isolation procedures.
Movement of Ca2+-ATPase molecules within the sarcoplasmic/endoplasmic reticulum in skeletal muscle
Authors: Kaprielian Zaven , Robinson Shawn W. , Fambrough Douglas M. , Kessler Paul D. 
Published: April 2021

ABSTRACT
The endoplasmic reticulum undergoes rapid, microscopic changes in its structure, including extension and anastomosis of tubular elements. Such dynamism is expected to manifest itself also as rapid intermixing of membrane components, at least within subdomains of the endoplasmic reticulum. Here we present evidence of a similar dynamism in the sarcoplasmic reticulum of developing skeletal muscle. The sarcoplasmic reticulum is sometimes considered a specialized type of endoplasmic reticulum, but it appears to be a rather static set of membrane-bound elements, repetitively arranged to enwrap each sarcomere of each myofibril. Both endoplasmic reticulum and sarcoplasmic reticulum contain P-type Ca2+-ATPases that transport calcium from the cytosol into their lumen. In the experiments reported here, chicken and mouse cells were fused by polyethylene glycol, natural myogenic cell fusion, or Sendai virus. The redistribution of Ca2+-ATPase molecules between chick and mouse endoplasmic reticulum/sarcoplasmic reticulum was followed by immunofluorescence microscopy in which species-specific monoclonal antibodies to chick and mouse Ca2+-ATPases were used. Redistribution was time- and temperature-dependent but independent of protein synthesis as well as the method of cell fusion. Intermixing occurred on a time scale of tens of minutes at 37°C. These results verify the dynamic nature of the sarcoplasmic reticulum and illustrate an aspect of the special relationship between endoplasmic reticulum and sarcoplasmic reticulum.
Transport of storage proteins to the vacuole is mediated by vesicles without a clathrin coat
Authors: Hohl Inge , Robinson David G. , Chrispeels Maarten J. , Hinz Giselbert 
Published: April 2021

ABSTRACT
Storage parenchyma cells of developing legume cotyledons actively transport large amounts of storage proteins to protein storage vacuoles (PSV). These proteins are synthesized on the endoplasmic reticulum and pass through the Golgi apparatus. Clathrin coated vesicles (CCV) and small electron dense vesicles found near the trans-Golgi network (TGN) have both been implicated in the Golgi-to-vacuole transport step. Recent findings that protein storage cells contain more than one type of vacuole have necessitated a re-examination of the role of both types of vesicles in vacuolar protein transport.
Immunoblots of highly purified CCV preparations and immunogold labelling with antibodies to the storage proteins vicilin and legumin, indicate that the dense vesicles, but not the CCV, are involved in storage protein transport in pea cotyledons. This result is supported by the finding that α-TIP, a protein characteristic of the PSV membrane, is absent from CCV. In addition, complex glycoproteins appear to be carried by CCV but are not detectable in the PSV. We suggest on the basis of these data that storage proteins and other vacuolar proteins such as acid hydrolases are not sorted by the same mechanism and are transported by different types of vesicles to different types of vacuoles.