bluuebunny/crossref_metadata_2025 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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.
Two regulated secretory pathways for newly synthesized parotid salivary proteins are distinguished by doses of secretagogues
Authors: Castle J. David , Castle Anna M.
Published: April 2021

Low doses of the muscarinic agonist pilocarpine (0.1-1 µM) and the β-adrenergic agonist isoproterenol (0.5-4 nM) stimulate a minor regulated secretory pathway for salivary proteins in rat parotid lobules. Newly synthesized proteins (labeled biosynthetically) are selectively discharged, and they are secreted in the same relative proportions as observed in constitutive-like unstimulated secretion but different from the proportions of older proteins that are discharged by granule exocytosis in response to higher doses of secretagogue. The response to low doses of agonists is transient and involves output of no more than 1-2% of tissue-associated amylase. The same increase in output of pulse-labeled proteins is observed when agonist is added at various chase times (1.5-6 hours), implying that release occurs from a post-Golgi storage pool. Stimulation for 40 minutes significantly depletes the storage pool as a second stimulation elicits smaller output. Stimulation also partially depletes labeled proteins from subsequent constitutive-like secretion after the agonist is removed implying that the constitutive-like and low dose agonist mediated pathways draw on the same pool of secretory proteins. While these results indicate that acinar cells have a second regulated secretory pathway, this new pathway is unlikely to contribute uniquely to the protein composition of parotid secretion. Rather it may serve a different role in secretion at the apical cell surface.
PTL-1, a microtubule-associated protein with tau-like repeats from the nematode Caenorhabditis elegans
Authors: Goedert M. , Baur C. P. , Ahringer J. , Jakes R. , Hasegawa M. , Spillantini M. G. , Smith M. J. , Hill F.
Published: April 2021

Tau, MAP2 and MAP4 are structural microtubule-associated proteins (MAPs) that promote the assembly and stability of microtubules. They share three or four imperfect tandem repeats of an amino acid motif, which is involved in the binding to microtubules. All sequences to date containing this motif are of mammalian origin. We report here the cloning and functional characterisation of a new member of this family of proteins from the nematode Caenorhabditis elegans. This protein exists as two isoforms of 413 and 453 amino acids with four or five tandem repeats that are 50% identical to the tau/MAP2/MAP4 repeats. Both isoforms bind to microtubules and promote microtubule assembly, with the five-repeat isoform being more effective at promoting assembly than the four-repeat isoform. When expressed in COS cells, the five-repeat isoform co-localises with microtubules and induces the formation of microtubule bundles, whereas its expression in Sf9 cells leads to the extension of long unipolar processes. In view of its length, amino acid sequence and functional characteristics, we have named this invertebrate structural MAP ‘Protein with Tau-Like Repeats’ (PTL-1). In C. elegans PTL-1 is expressed in two places known to require microtubule function. It is first seen in the embryonic epidermis, when circumferentially oriented microtubules help to distribute forces generated during elongation. Later, it is found in mechanosensory neurons which contain unusual 15 protofilament microtubules required for the response to touch. These findings indicate that MAPs of the tau/MAP2/MAP4 family are found throughout much of the animal kingdom, where they may play a role in specialised processes requiring microtubules.
Structure and dynamic organization of centromeres/prekinetochores in the nucleus of mammalian cells
Authors: He D. , Brinkley B. R.
Published: April 2021

Although considerable research has been focused on understanding the structure and molecular organization of the centromere-kinetochore complex of mitotic chromosomes, few reports have dealt with the centromere (prekinetochore) in the interphase nucleus. In the present study, we utilized anti-centromere antibodies from the serum of patients with the autoimmune disease, scleroderma CREST (calcinosis, Raynaud’s phenomenon, esophageal dismotility, sclerodactyly, telangiectasia), as probes to investigate the structure and morphogenesis of the centromere in interphase nuclei of three cell lines using laser scanning confocal microscopy and immunoelectron microscopy. Of particular interest were the chromosomes of the Indian muntjac (2n=6 in females and 2n=7 in males), whose large centromeres are thought to have evolved through the tandem fusion of smaller centromeres of a Chinese muntjac-like progenitor species (2n=46). The various forms and patterns of centromeres observed in the nucleus correlated with stages in the cell cycle as determined by bromodeoxyuridine labeling and apparently represent stages in prereplication, replication and maturation. Immunoelectron microscopic studies using CREST antisera indicated that the high order structure of chromatin associated with each prekinetochore undergoes a regular unfolding-refolding cycle, displaying small beadlike subunits tandemly arranged along a linear thread of centromeric DNA, much like that reported for mitotic chromosomes. Individual centromeres/prekinetochores form a stable association with the 9-13 nm core filaments of the nucleoskeletal network in the nucleus that later become the chromosome scaffold of mitotic chromosomes. Our findings provide morphological support for the hypothesis that the spatial arrangements of individual centromeres within the nucleus may have influenced centromeric translocations and fusions during chromosome evolution. Therefore, the centromere-kinetochore complex, best known for its essential role in partitioning chromosomes in mitosis and meiosis, may also function in chromosome movements and associations in interphase.
Cloning and characterization of a new armadillo family member, p0071, associated with the junctional plaque: evidence for a subfamily of closely related proteins
Authors: Hatzfeld Mechthild , Nachtsheim Christina
Published: April 2021

Cell contacts of the adherens type are organized around transmembrane proteins of the cadherin family. Whereas the extracellular domains mediate homophilic interactions between cadherins of neighbouring cells the cytoplasmic domains organize a set of proteins into the junctional plaque. Among these junctional plaque proteins are members of the armadillo gene family, β-catenin, plakoglobin (γ-catenin), B6P/plakophilin and p120. These proteins are assumed to play a key role in cell cell signalling through intercellular junctions.

Here we report cloning of a cDNA encoding a new armadillo family member, p0071, closely related to p120 and B6P/plakophilin and more distantly related to armadillo, plakoglobin, β-catenin and other members of the gene family. The deduced amino acid sequence encodes a basic protein of 1,211 amino acids with a central armadillo repeat region which is conserved in sequence and organization of its ten individual motifs between p120, B6P/plakophilin and p0071. In contrast the end domains of the three proteins are variable in size and sequence. The RNA coding for p0071 is expressed in all tissues examined. Using antibodies generated against the armadillo repeat region of the protein we show that p0071 is localized at cellcell borders and is expressed in the desmosomal plaque of some cultured epithelial cells. The protein seems to be an accessory component of the desmosomal plaque as well as of other adhesion plaques and might be involved in regulating junctional plaque organization and cadherin function.

Our data provide evidence for a subfamily of armadillo related proteins that share not only structural features but also have in common their localisation in the junctional plaque. We therefore suggest that family members exert similar functions and might be involved in cell signalling through cell contacts.
Oscillating mitotic newt lung cell kinetochores are, on average, under tension and rarely push
Authors: Waters Jennifer C. , Skibbens Robert V. , Salmon E. D.
Published: April 2021

Experimentally introduced tension on kinetochores and their centromeres has been shown to stabilize kinetochore attachment to microtubules, modify kinetochore directional instability, and regulate cell-cycle progression into anaphase. In mitosis, kinetochore tension and the stretch of centromere chromatin are produced by the movement of sister kinetochores toward opposite poles and astral ejection forces on the chromosome arms. However, newt lung cell kinetochores oscillate between poleward and away from the pole motility states throughout mitosis, indicating kinetochores are not under constant tension. To test whether kinetochores are under net tension while they are oscillating, and how often they are under compression and pushing into the chromosome, we measured the distance between sister kinetochores in newt lung cells using both video-enhanced differential interference contrast microscopy (VE-DIC) and immunofluorescence microscopy. We found that for chromosomes in which sister kinetochores are attached to opposite spindle poles, centromeres are, on average, stretched (2.2 μm in living cells and 1.8 μm in fixed cells) with respect to the inter-kinetochore ‘rest’ length (1.1 μm in living and fixed cells). For chromosomes in which only one kinetochore is attached to the spindle, the centromere chromatin associated with the tethered kinetochore is, on average, stretched to approximately half of the average inter-kinetochore distance measured for chromosomes in which both kinetochores are attached. We conclude that while newt lung cell kinetochores oscillate between states of P and AP movement, they are under tension approximately 90% of the time and under compression less than 6% of the time.
Integrin-dependent induction of early growth response genes in capillary endothelial cells
Authors: Dike Laura E. , Ingber Donald E.
Published: April 2021

Studies were carried out to explore how extracellular matrix molecules, such as fibronectin (FN), promote capillary endothelial (CE) cell growth. When G0-synchronized cells were plated on FN-coated dishes, expression of the immediateearly mRNAs, c-fos, c-myc and c-jun, was rapidly induced, even in the absence of serum or soluble growth factors. Moreover, plating cells on different FN densities (5-200 μg/150 mm dish), resulted in a dose-dependent increase in the steady state levels of these mRNAs. Addition of FGF potentiated gene activation and was required for maximal DNA synthesis, however, the overall steady-state level of gene induction was dictated primarily by the density of immobilized FN. Expression of junB also was induced when suspended cells bound RGD-peptide coated microbeads that promote integrin clustering, but not when the suspended cells bound beads coated with other receptor ligands (e.g. acetylated low density protein) or when they were stimulated by soluble FN or FGF in the absence of substrate adhesion. c-Jun exhibited a similar requirement for gene induction except that it also was partially induced by binding to soluble FN alone. In contrast, c-fos expression was induced by all stimuli tested. Interestingly, inhibition of Na+/H+ exchange using hexamethylene-amiloride prevented most of the FN-induced increase in c-jun expression whereas it was relatively ineffective when cells were simultaneously stimulated by both FN and FGF. These data demonstrate that cell adhesion to extracellular matrix and associated integrin binding can directly activate signaling cascades in quiescent CE cells that lead to induction of immediate-early genes associated with the G0/G1 transition and thereby, stimulate these cells to reenter the growth cycle.
The novel human protein serine/threonine phosphatase 6 is a functional homologue of budding yeast Sit4p and fission yeast ppe1, which are involved in cell cycle regulation
Authors: Bastians Holger , Ponstingl Herwig
Published: April 2021

We identified a novel human protein serine/threonine phosphatase cDNA, designated protein phosphatase 6 (PP6) by using a homology-based polymerase chain reaction. The predicted amino acid sequence indicates a 35 kDa protein showing high homology to other protein phosphatases including human PP2A (57%), human PP4 (59%), rat PPV (98%), Drosophila PPV (74%), Schizosaccharomyces pombe ppe1 (68%) and Saccharomyces cerevisiae Sit4p (61%). In human cells, three forms of PP6 mRNA were found with highest levels of expression in testis, heart and skeletal muscle. The PP6 protein was detected in lysates of human heart muscle and in bull testis. Complementation studies using a temperature sensitive mutant strain of S. cerevisiae SIT4, which is required for the G1 to S transition of the cell cycle, showed that PP6 can rescue the mutant growth arrest. In addition, a loss of function mutant of S. pombe ppel, described as a gene interacting with the pim1/spil mitotic checkpoint and involved in cell shape control, can be complemented by expression of human PP6. These data indicate that human PP6 is a functional homologue of budding yeast Sit4p and fission yeast ppel, implying a function of PP6 in cell cycle regulation.
Cell cycle-dependent phosphorylation of the 77 kDa echinoderm microtubule-associated protein (EMAP) in vivo and association with the p34cdc2 kinase
Authors: Brisch Ellen , Daggett Melissa A. F. , Suprenant Kathy A.
Published: April 2021

The most abundant microtubule-associated protein in sea urchin eggs and embryos is the 77 kDa echinoderm micro-tubule-associated protein (EMAP). EMAP localizes to the mitotic spindle as well as the interphase microtubule array and is a likely target for a cell cycle-activated kinase. To determine if EMAP is phosphorylated in vivo, sea urchin eggs and embryos were metabolically labeled with 32PO4 and a monospecific antiserum was used to immunoprecipitate EMAP from 32P-labeled eggs and embryos. In this study, we demonstrate that the 77 kDa EMAP is phosphorylated in vivo by two distinct mechanisms. In the unfertilized egg, EMAP is constitutively phosphorylated on at least five serine residues. During the first cleavage division following fertilization, EMAP is phosphorylated with a cell cycle-dependent time course. As the embryo enters mitosis, EMAP phosphorylation increases, and as the embryo exits mitosis, phosphorylation decreases. During mitosis, EMAP is phosphorylated on 10 serine residues and two-dimensional phosphopeptide mapping reveals a mitosis-specific site of phosphorylation. At all stages of the cell cycle, a 33 kDa polypeptide copurifies with the 77 kDa EMAP, regardless of phosphorylation state. Antibodies against the cdc2 kinase were used to demonstrate that the 33 kDa polypeptide is the p34cdc2 kinase. The p34cdc2 kinase copurifies with the mitotic apparatus and immunostaining indicates that the p34cdc2 kinase is concentrated at the spindle poles. Models for the interaction of the p34cdc2 kinase and the 77 kDa EMAP are presented.
Isolation and characterization of early endosomes, late endosomes and terminal lysosomes: their role in protein degradation
Authors: Tjelle Torunn Elisabeth , Brech Andreas , Juvet Lene Kristine , Griffiths Gareth , Berg Trond
Published: April 2021

Although endosomal proteolysis has been reported (e.g. for peptide hormones and lysosomal enzymes), lysosomes are believed to be the main site of degradation in the endocytic pathway.

We have studied the separate roles of lysosomes and prelysosomal endocytic organelles in the degradation of ovalbumin in J774 cells. The ovalbumin was labelled with 125I-tyramine cellobiose (125I-TC-ova). The labelled degradation products formed from this probe are trapped at the site of formation.

To separate lysosomes efficiently from prelysosomal endocytic organelles we allowed the cells to endocytose a pulse of colloidal gold particles complexed with ovalbumin. By combining this density shift technique with subcellular fractionation of a postnuclear supernatant in Percoll gradients we could isolate three fractions that were sequentially involved in the endocytic pathway: a light Percoll fraction, a dense Percoll fraction and a gold fraction.

The light Percoll fraction contained early endosomes since it was transferrin positive and received endocytic markers such as ovalbumin and horseradish peroxidase (HRP) early (<5 minutes) after internalization. The dense Percoll fraction was transferrin negative, rab7 positive and received endocytic markers after 10-15 minutes of internalization. The gold-filled fraction was negative for both transferrin and rab7 but highly enriched in the lysosomal enzyme β-hexosaminidase and was therefore defined as a lysosome.

To study the role of endosomes and lysosomes in the degradation of endocytosed material we allowed the cells to take up (via the mannose receptor) 125I-TC-ova. It was found that the main degradation of 125I-TC-ova (measured as acid soluble radioactivity trapped in the organelle) took place in the late endosomes (and not in the lysosomes containing the bulk of the lysosomal enzymes). Our data therefore suggest that the late endosomes operate as an early lysosomal compartment. The terminal lysosomes may serve as storage bodies for acid hydrolases that may be called upon when needed (for instance during phagocytosis).
A novel bone marrow frozen section assay for studying hematopoietic interactions in situ: the role of stromal bone marrow macrophages in erythroblast binding
Authors: Barbé E. , Huitinga I. , Döpp E. A. , Bauer J. , Dijkstra C. D.
Published: April 2021

Bone marrow macrophages are found in intimate contact with erythroblasts. Exact mechanisms and functions of this interaction are unclear. New insights into erythroblast binding were obtained using a newly developed bone marrow frozen section assay. This modified Woodruff and Stamper assay has some important advantages compared to other adhesion assays. Erythroblasts specifically adhered to bone marrow macrophages forming clusters, as appear in vivo. Selective depletion of bone marrow macrophages by intravenous injection of dichloromethylenediphosphonate resulted in a release of immature erythroid cells to the peripheral blood. Furthermore no erythroblasts adhered to bone marrow sections without macrophages. Evaluating the binding of erythroblasts to bone marrow macrophages we found that this binding is temperature and cation dependent. The receptor for erythroblasts present on macrophages recognizes a sialyated protein as ligand on erythroblasts, since neuraminidase treatment of erythroblasts resulted in a decrease in binding. A possible candidate for the erythroblast receptor on macrophages is the ED2 antigen. ED2 is a differentiation antigen present on resident macrophages that has some biochemical features characteristic of an adhesion molecule. Erythoblast binding to bone marrow was inhibited using a monoclonal antibody directed against ED2.
The size control of fission yeast revisited
Authors: Sveiczer A. , Novak B. , Mitchison J. M.
Published: April 2021

An analysis was made of cell length and cycle time in time-lapse films of the fission yeast Schizosaccharomyces pombe using wild-type (WT) cells and those of various mutants. The more important conclusions about ‘size controls’ are: (1) there is a marker in G2 in WT cells provided by a rate change point (RCP) where the linear rate of length growth increases by ∼30%. The period before this RCP is dependent on size and can be called a ‘sizer’. The period after the RCP is nearly independent of size and can be called a ‘timer’. The achievement of a critical threshold size is at or near the RCP which is on average at about 0.3 of the cycle (halfway through G2). This is much earlier than was previously believed. (2) The RCP is at about the time when H1 histone kinase activity and the B type cyclin cdc13 start to rise in preparation for mitosis. The RCP is also associated with other metabolic changes. (3) In wee1 mutants, the mitotic size control is replaced by a G1/S size control which is as strong as the mitotic control. As in WT cells, there is a sizer which precedes the RCP followed by a timer but the RCP is at about the G1/S boundary and has a larger increase (∼100%) in rate. (4) cdc25 is not an essential part of the size control at mitosis or at the G1/S boundary. (5) Three further situations have been examined in which the mitotic size control has been abolished. First, induction synchronisation by block and release of cdc2 and cdc10. In the largest oversize cells which are produced, the RCP is pushed back to the beginning of the cycle. There is no sizer period but only a timer. Second, when both the antagonists wee1 and cdc25 are absent in the double mutant wee1-50 cdc25Δ. In this interesting situation there is apparently no mitotic size control and the cycle times are quantised. Third, in rum1Δ wee1-50 where the normal long G1 in wee1 is much reduced, there is probably no size control either in G1 or in G2 causing a continuous shortening of division length from cycle to cycle.
Axonal targeting of agrin in cultured rat dorsal horn neurons
Authors: Escher Gérard , Béchade Catherine , Levi Sabine , Triller Antoine
Published: April 2021

Agrin, a synaptic basal lamina protein synthesized by motoneurons is involved in the aggregation of nicotinic acetylcholine receptors (nAchRs) at the neuromuscular junction. Agrin transcripts are broadly expressed in the central nervous system (CNS) including non-cholinergic regions. This wide distribution of agrin mRNAs raises the question of its function in these areas. To approach this question, we analysed the expression and cellular distribution of agrin in primary cultures of rat embryonic dorsal horn neurons. Polymerase chain reaction analysis demonstrated that the four agrin isoform (B0, B8, B11, B19) mRNAs are expressed as early as 4 days in vitro, before the formation of functional synaptic contacts. Western blots also showed that agrin-like proteins are secreted in conditioned medium from 7 days cultures. We analysed the subcellular distribution of agrin by double immunolabeling and fluorescence microscopy. We found that agrin is synthesized by almost all neurons and was present in the somata and in the axons but not in dendrites within the sensitivity of the detection. This intra-axonal localisation of agrin could only be seen after permeabilization. Furthermore, agrin immunoreactive axons were found adjacent to gephyrin, the postsynaptic glycine receptor-associated protein. Altogether, our results suggest that, as established at the neuromuscular junction, agrin may be involved in preto postsynaptic interactions in the central nervous system.
Differentiation and integrity of cardiac muscle cells are impaired in the absence of βi integrin
Authors: Fässler Reinhard , Rohwedel Jürgen , Maltsev Victor , Bloch Wilhelm , Lentini Silvia , Guan Kaomei , Gullberg Donald , Hescheler Jürgen , Addicks Klaus , Wobus Anna M.
Published: April 2021

Cellular interactions with substrata of the microenvironment are one of the major mechanisms for differentiation and morphogenesis. Many of these interactions are mediated via the β1 integrin subfamily of cell surface receptors, which are believed to transduce signals upon cell adhesion. We have used β1 integrin-deficient embryonic stem cells to test their ability to differentiate into cardiac muscle cells. We show here by several approaches that β1 integrin is important for normal cardiogenesis. First, the in vitro differentiation of β1 integrin-deficient embryonic stem cells into cardiac muscle cells is retarded. This is demonstrated by the delayed expression of cardiac muscle-specific genes and action potentials. Second, the specification of cardiac precursor cells into pacemaker-, atrial- and ventricular-like cells is significantly impaired in β1 integrin-deficient cells. The occurrence of atrial- and ventricular-like cells is reduced and transient. Only cells exhibiting pacemaker-like action potentials of high frequency and arrhythmias survive. Third, the sarcomeric architecture is incomplete and disarranged in the absence of β1 integrin. Fourth, β1-deficient embryonic stem cells can contribute to the developing heart in chimaeric mice but many areas with β1-null cells contain cell debris. The number of β1-null cells decreases from prenatal to postnatal stages and is lost completely in 6-month-old hearts.

Thus, we conclude that interactions with the extracellular matrix via β1 integrin is necessary for differentiation and the maintenance of a specialized phenotype of cardiac muscle cells.