bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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 (&lt;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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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.