bluuebunny/crossref_metadata_2025_split huggingface dataset:
Maize meiotic spindles assemble around chromatin and do not require paired chromosomes
Authors: Chan Annette , Cande W. Zacheus 
Published: April 2021

ABSTRACT
To understand how the meiotic spindle is formed and maintained in higher plants, we studied the organization of microtubule arrays in wild-type maize meiocytes and three maize meiotic mutants, desynaptic1 (dsy1), desynaptic2 (dsy2), and absence of first division (afd). All three meiotic mutations have abnormal chromosome pairing and produce univalents by diakinesis. Using these three mutants, we investigated how the absence of paired homologous chromosomes affects the assembly and maintenance of the meiotic spindle. Before nuclear envelope breakdown, in wild-type meiocytes, there were no bipolar microtubule arrays. Instead, these structures formed after nuclear envelope breakdown and were associated with the chromosomes. The presence of univalent chromosomes in dsy1, dsy2, and afd meiocytes and of unpaired sister chromatids in the afd meiocytes did not affect the formation of bipolar spindles. However, alignment of chromosomes on the metaphase plate and subsequent anaphase chromosome segregation were perturbed. We propose a model for spindle formation in maize meiocytes in which microtubules initially appear around the chromosomes during prometaphase and then the microtubules self-organize. However, this process does not require paired kinetochores to establish spindle bipolarity.
Deficient epithelial-fibroblast heterocellular gap junction communication can be overcome by co-culture with an intermediate cell type but not by E-cadherin transgene expression
Authors: Woodward T. L. , Sia M. A. , Blaschuk O. W. , Turner J. D. , Laird D. W. 
Published: April 2021

ABSTRACT
Epithelial, fibroblast and intermediate cell lines were employed to examine the mechanism(s) essential for heterocellular gap junction intercellular communication in vitro. These cell lines were characterized extensively for cell type based on morphology, intermediate cytoskeletal proteins, cell adhesion molecules and their associated proteins, tight junction proteins as well as functional differentiation. All cell types expressed connexin43 and were dye-coupled in homocellular culture. Epithelial and intermediate cells or fibroblasts and intermediate cells readily assembled heterocellular connexin43-positive gap junction plaques when co-cultured, while gap junction plaques in mixed cultures of epithelial cells and fibroblasts were rare. Dye microinjection studies were used to show that there was little gap junction intercellular communication between epithelial cells and fibroblasts. However, intermediate cells were able to communicate with epithelial cells and, to a lesser extent, fibroblasts and could transfer dye to both epithelial cells and fibroblasts when all three cell types were cultured together. Fibroblasts that were stably transfected with a cDNA encoding E-cadherin had a greater tendency to aggregate and exhibited a more epithelial-like phenotype but heterocellular gap junction intercellular communication with epithelial cells, which endogenously express E-cadherin, was not enhanced. These results suggest that mutual expression of E-cadherin is insufficient to stimulate gap junction formation between epithelial cells and fibroblasts. Moreover, our results also demonstrate that communication gaps between epithelial cells and fibroblasts can be bridged by intermediate cells, a process that may be important in mammary gland development, growth, differentiation and cancer.
Expression and potential role of Fsrg1, a murine bromodomain-containing homologue of the Drosophila gene female sterile homeotic
Authors: Rhee Kunsoo , Brunori Michele , Besset Valérie , Trousdale Rhonda , Wolgemuth Debra J. 
Published: April 2021

ABSTRACT
We have isolated a cDNA which is a murine homologue of the Drosophila gene female sterile homeotic (fsh). This homologue, which we have designated Fsrg1*, contains two bromodomains and an ET motif characteristic of the Fsh sub-class of bromodomain-containing proteins. Northern blot hybridization analysis of adult tissues revealed that Fsrg1 was expressed at low levels rather ubiquitously, but most abundantly in the testis and ovary. Polyclonal antibodies raised against an Fsrg1 fusion protein were used to characterize the Fsrg1 gene product in tissues. Constructs were also generated in which the Fsrg1 cDNA was tagged with epitopes for hemaglutinin and used in transfection experiments. Immunoblot analysis revealed that the Fsrg1 protein migrates with a relative molecular mass of ˜110 kDa, although the cDNA sequence would predict a protein of ˜88 kDa. The migration at ˜110 kDa was observed for both in vivo protein and protein produced in cultured cells. The Fsrg1 protein was localized to the nucleus when expressed in cultured cells, consistent with the presence of a nuclear localization signal motif in the Fsrg1 sequence. No kinase activity was detected for this nuclear protein as assessed in either autokinase or specific substrate assays. In situ hybridization analysis revealed strikingly high expression of Fsrg1 in granulosa cells of growing follicles in the adult ovary and suggested its possible involvement in folliculogenesis. Additional clues to its potential function were provided by the demonstration of its high level of expression in epithelia of tissues which undergo hormonally-modulated remodeling.
Localization and regulation of the cdk-activating kinase (Cak1p) from budding yeast
Authors: Kaldis Philipp , Pitluk Zachary W. , Bany I. Amy , Enke Deborah A. , Wagner Marisa , Winter Edward , Solomon Mark J. 
Published: April 2021

ABSTRACT
Eukaryotic cell cycles are controlled by the activities of cyclin-dependent kinases (cdks). The major cdk in budding yeast, Saccharomyces cerevisiae, is Cdc28p. Activation of Cdc28p requires phosphorylation on threonine 169 and binding to a cyclin. Thr-169 is phosphorylated by the cdkactivating kinase (CAK), Cak1p, which was recently identified as the physiological CAK in budding yeast. Here we present our further characterization of yeast Cak1p. We have found that Cak1p is dispersed throughout the cell as shown by immunofluorescence; biochemical subcellular fractionation confirmed that most of the Cak1p is found in the cytoplasm. Cak1p is a monomeric enzyme in crude yeast lysates. Mutagenesis of potential sites of activating phosphorylation had little effect on the activity of Cak1p in vitro or in vivo. Furthermore, Cak1p contains no posttranslational modifications detectable by two-dimensional isoelectric focusing. We found that Cak1p is a stable protein during exponential growth but that its expression decreases considerably when cells enter stationary phase. In contrast, Cak1p levels oscillate dramatically during meiosis, reflecting regulation at both the transcriptional and post-translational level. The localization and regulation of Cak1p are in contrast to those of the known vertebrate CAK, p40MO15.
Nucleoskeleton and initiation of DNA replication in metazoan cells
Authors: Ortega J. Miguel , DePamphilis Melvin L. 
Published: April 2021

ABSTRACT
To determine whether or not initiation sites for DNA replication in mammalian cells are defined by association with nuclear structure, attachments between the nucleoskeleton and the hamster DHFR gene initiation zone were examined. Nucleoskeletons were prepared by encapsulating cells in agarose and then extracting them with a nonionic detergent in a physiological buffer. The fraction of DNA that remained following endonuclease digestion was resistant to salt, sensitive to Sarkosyl, and essentially unchanged by glutaraldehyde crosslinking. Although newly replicated DNA was preferentially attached to the nucleoskeleton, no specific sequence was preferentially attached within a 65 kb locus containing the DHFR gene, two origins of bi-directional replication and at least one nuclear matrix attachment region. Instead, the entire region went from preferentially unattached to preferentially attached as cells progressed from G1 to late S-phase. Thus, initiation sites in mammalian chromosomes are not defined by attachments to the nucleoskeleton. To further assess the relationship between the nucleoskeleton and DNA replication, plasmid DNA containing the DHFR initiation region was replicated in a Xenopus egg extract. All of the DNA associated with the nucleoskeleton prior to S-phase without preference for a particular sequence and was released upon mitosis. However, about half of this DNA was trapped rather than bound to the nucleoskeleton. Thus, attachments to the nucleoskeleton can form in the absence of either DNA replication or transcription, but if they are required for replication, they are not maintained once replication is completed.
Chromatin binding and polymerization of the endogenous Xenopus egg lamins: The opposing effects of glycogen and ATP
Authors: Lourim David , Krohne Georg 
Published: April 2021

ABSTRACT
We have previously identified and quantitated three B-type lamin isoforms present in the nuclei of mature Xenopus laevis oocytes, and in cell-free egg extracts. As Xenopus egg extracts are frequently used to analyze nuclear envelope assembly and lamina functions, we felt it was imperative that the polymerization and chromatin-binding properties of the endogenous B-type egg lamins be investigated. While we have demonstrated that soluble B-type lamins bind to chromatin, we have also observed that the polymerization of egg lamins does not require membranes or chromatin. Lamin assembly is enhanced by the addition of glycogen/glucose, or by the depletion of ATP from the extract. Moreover, the polymerization of egg cytosol lamins and their binding to demembranated sperm or chromatin assembled from naked λ-DNA is inhibited by an ATP regeneration system. These ATP-dependent inhibitory activities can be overcome by the coaddition of glycogen to egg cytosol. We have observed that glycogen does not alter ATP levels during cytosol incubation, but rather, as glycogen-enhanced lamin polymerization is inhibited by okadaic acid, we conclude that glycogen activates protein phosphatases. Because protein phosphatase 1 (PP1) is the only phosphatase known to be specifically regulated by glycogen our data indicate that PP1 is involved in lamin polymerization. Our results show that ATP and glycogen effect lamin polymerization and chromatin binding by separate and opposing mechanisms.
A role for microtubule dynamics in phagosome movement
Authors: Blocker Ariel , Griffiths Gareth , Olivo Jean-Christophe , Hyman Anthony A. , Severin Fedor F. 
Published: April 2021

ABSTRACT
We have shown previously that intracellular phagosome movement requires microtubules. Here we provide evidence that within cells phagosomes display two different kinds of microtubule-based movements in approximately equal proportions. The first type occurs predominantly in the cell periphery, often shortly after the phagosome is formed, and at speeds below 0.1 µm/second. The second is faster (0.2-1.5 µm/second) and occurs mainly after phagosomes have reached the cell interior. Treating cells with nanomolar concentrations of taxol or nocodazole alters microtubule dynamics without affecting either total polymer mass or microtubule organisation. Such treatments slow the accumulation of phagosomes in the perinuclear region and reduce the number of slow movements by up to 50% without affecting the frequency of fast movements. This suggests that a proportion of slow movements are mediated by microtubule dynamics while fast movements are powered by microtubule motors. In macrophages, interphase microtubules radiate from the microtubule organising centre with their plus-end towards the cell periphery. To understand the behaviour of ‘early’ phagosomes at the cell periphery we investigated their ability to bind microtubule plus-ends in vitro. We show that early phagosomes have a strong preference for microtubule plus-ends, whereas ‘late’ phagosomes do not, and that plus-end affinity requires the presence of microtubule-associated proteins within cytosol. We suggest that phagosomes can bind to the plus-ends of dynamic microtubules and move by following their shrinkage or growth.
Long-range and selective autoregulation of cell-cell or cell-matrix adhesions by cadherin or integrin ligands
Authors: Levenberg Shulamit , Katz Ben-Zion , Yamada Kenneth M. , Geiger Benjamin 
Published: April 2021

ABSTRACT
In this study we demonstrate that local stimulation of cell surface cadherins or integrins induces a selective enhancement of adherens junction or focal contact assembly, respectively, throughout the cell. N-cadherin transfected CHO cells (CHO-Ncad) were incubated with different ligands including N-cadherin extracellular domain (NEC), anti-N-cadherin antibodies, fibronectin and concanavalin A (ConA), conjugated to synthetic beads. Electron microscopic examination indicated that both cadherin- and integrin-reactive beads bound tightly to the cell surface and were apparently endocytosed after several hours of incubation. The ConA-beads remained largely at the cell surface. Immunofluorescence labeling of the cells with antibodies to different adhesion-associated molecules indicated that both NEC- and anti-N-cadherin-conjugated beads induced a major increase in the level of junction-associated cadherin and β-catenin labeling and a modest increase in junctional vinculin labeling, compared to untreated cells or cells bound to ConA-beads. FN-conjugated beads, on the other hand, significantly enhanced vinculin labeling at focal contacts and suppressed cadherin and β-catenin staining in cell-cell junctions. The cadherin-reactive beads specifically stimulated tyrosine phosphorylation at cell-cell junctions, while the FN-beads increased the levels of focal contact-associated phosphotyrosine, as shown by immunofluorescence labeling of the cells for phosphotyrosine. Inhibition of this phosphorylation by genistein resulted in a complete suppression of the effects of both types of beads. These findings indicate that specific cadherin- and integrin-mediated surface interactions can trigger positively cooperative long-range signaling events which lead to the selective assembly of cell-cell or cell-matrix adhesions, and that these signals involve tyrosine phosphorylation.
The spa2-related protein, sph1p, is important for polarized growth in yeast
Authors: Roemer Terry , Vallier Laura , Sheu Yi-Jun , Snyder Michael 
Published: April 2021

ABSTRACT
The Saccharomyces cerevisiae protein Sph1p is both structurally and functionally related to the polarity protein, Spa2p. Sph1p and Spa2p are predicted to share three 100-amino acid domains each exceeding 30% sequence identity, and the amino-terminal domain of each protein contains a direct repeat common to Homo sapiens and Caenorhabditis elegans protein sequences. sph1- and spa2-deleted cells possess defects in mating projection morphology and pseudohyphal growth. sph1Δ spa2Δ double mutants also exhibit a strong haploid invasive growth defect and an exacerbated mating projection defect relative to either sph1Δ or spa2Δ single mutants. Consistent with a role in polarized growth, Sph1p localizes to growth sites in a cell cycle-dependent manner: Sph1p concentrates as a cortical patch at the presumptive bud site in unbudded cells, at the tip of small, medium and large buds, and at the bud neck prior to cytokinesis. In pheromone-treated cells, Sph1p localizes to the tip of the mating projection. Proper localization of Sph1p to sites of active growth during budding and mating requires Spa2p. Sph1p interacts in the two-hybrid system with three mitogen-activated protein (MAP) kinase kinases (MAPKKs): Mkk1p and Mkk2p, which function in the cell wall integrity/cell polarization MAP kinase pathway, and Ste7p, which operates in the pheromone and pseudohyphal signaling response pathways. Sph1p also interacts weakly with STE11, the MAPKKK known to activate STE7. Moreover, two-hybrid interactions between SPH1 and STE7 and STE11 occur independently of STE5, a proposed scaffolding protein which interacts with several members of this MAP kinase module. We speculate that Spa2p and Sph1p may function during pseudohyphal and haploid invasive growth to help tether this MAP kinase module to sites of polarized growth. Our results indicate that Spa2p and Sph1p comprise two related proteins important for the control of cell morphogenesis in yeast.
Coexpression of both types of desmosomal cadherin and plakoglobin confers strong intercellular adhesion
Authors: Marcozzi Cristiana , Burdett Ian D. J. , Buxton Roger S. , Magee Anthony I. 
Published: April 2021

ABSTRACT
Desmosomes are unique intercellular junctions in that they invariably contain two types of transmembrane cadherin molecule, desmocollins and desmogleins. In addition they possess a distinct cytoplasmic plaque structure containing a few major proteins including desmoplakins and the armadillo family member plakoglobin. Desmosomal cadherins are putative cell-cell adhesion molecules and we have tested their adhesive capacity using a transfection approach in mouse L cells. We find that L cells expressing either one or both of the desmosomal cadherins desmocollin 2a or desmoglein 1 display weak cell-cell adhesion activity that is Ca2+-dependent. Both homophilic and heterophilic adhesion could be detected. However, co-expression of plakoglobin with both desmosomal cadherins, but not with desmoglein 1 alone, resulted in a dramatic potentiation of cell-cell aggregation and the accumulation of detergent-insoluble desmosomal proteins at points of cell-cell contact. The effect of plakoglobin seems to be due directly to its interaction with the desmosomal cadherins rather than to its signalling function. The data suggest that the desmosome may obligatorily contain two cadherins and is consistent with a model in which desmocollins and desmogleins may form side by side heterodimers in contrast to the classical cadherins that are homodimeric. Plakoglobin may function by potentiating dimer formation, accretion of dimers to cell-cell contact sites or desmosomal cadherin stability.
Tight junctions
Authors: Balda Maria S. , Matter Karl 
Published: April 2021

ABSTRACT
Tight junctions are the most apical intercellular junctions of epithelial and endothelial cells and create a regulatable semipermeable diffusion barrier between individual cells. On a cellular level, they form an intramembrane diffusion fence that restricts the intermixing of apical and basolateral membrane components. In addition to these well defined functions, more recent evidence suggests that tight junctions are also involved in basic cellular processes like the regulation of cell growth and differentiation.
Activation of a PP2A-like phosphatase and dephosphorylation of τ protein characterize onset of the execution phase of apoptosis
Authors: Mills Jason C. , Lee Virginia M.-Y. , Pittman Randall N. 
Published: April 2021

ABSTRACT
The execution phase is an evolutionarily conserved stage of apoptosis that occurs with remarkable temporal and morphological uniformity in most if not all cell types regardless of the condition used to induce death. Characteristic features of apoptosis such as membrane blebbing, DNA fragmentation, chromatin condensation, and cell shrinkage occur during the execution phase; therefore, there is considerable interest in defining biochemical changes and signaling events early in the execution phase. Since onset of the execution phase is asynchronous across a population with only a small fraction of cells in this stage at any given time, characterizing underlying biochemical changes is difficult. An additional complication is recent evidence suggesting that the execution phase occurs after cells commit to die; thus, agents that modulate events in the execution phase may alter the morphological progression of apoptosis but will not affect the time-course of death. In the present study, we use a single cell approach to study and temporally order biochemical and cytoskeletal events that occur specifically in the execution phase. Microtubules de-acetylate and disassemble as terminally differentiated PC12 cells enter the execution phase following removal of nerve growth factor. Using phosphorylation sensitive antibodies to τ, we show that this microtubule-stabilizing protein becomes dephosphorylated near the onset of the execution phase. Low concentrations of okadaic acid inhibit dephosphorylation suggesting a PP2A-like phosphatase is responsible. Transfecting τ into CHO cells to act as a ‘reporter’ protein shows a similar dephosphorylation of τ by a PP2A-like phosphatase during the execution phase following induction of apoptosis with UV irradiation. Therefore, activation of PP2A phosphatase occurs at the onset of the execution phase in two very different cell types following different initiators of apoptosis which is consistent with activation of PP2A phosphatase being a common feature of the execution phase of apoptosis. Experiments using either taxol to inhibit microtubule disassembly or okadaic acid to inhibit τ dephosphorylation suggest that microtubule disassembly is necessary for τ dephosphorylation to occur. Thus, we propose that an early step in the execution phase (soon after a cell commits to die) is microtubule disassembly which frees or activates PP2A to dephosphorylate τ as well as other substrates.
Retrieved constituents of large dense-cored vesicles and synaptic vesicles intermix in stimulation-induced early endosomes of noradrenergic neurons
Authors: Partoens P. , Slembrouck D. , Quatacker J. , Baudhuin P. , Courtoy P. J. , Potter W. P. De 
Published: April 2021

ABSTRACT
Two storage compartments in cultured noradrenergic neurons derived from the superior cervical ganglion from fetal pig have been defined using sucrose density gradient centrifugation and electron microscopy: (1) large dense-cored vesicles (LDV) contain noradrenaline and dopamine-β-hydroxylase (DβH); (2) small electron-lucent vesicles contain acetylcholine and p38 and represent the noradrenergic small synaptic vesicles (SSV); no small dense-cored vesicles (SDV) could be detected. Our results demonstrate that internalized LDV membrane constituents are retrieved into early endosomes, as shown by the colocalization of retrieved DβH with the endosomal markers Rab5 and HRP in sucrose density gradients and on confocal microscopical images. Recycling of the SSV membranes via an endosomal intermediate is also confirmed in noradrenergic neurons. Finally, colocalization of retrieved DβH and retrieved p38 in stimulated neurons indicates that the two sets of constituents intermix. These data provide the first experimental evidence for a common early endosome in which SSV and LDV membrane constituents are internalized after exocytosis and imply that endosomal sorting is an important process for the generation of different secretory vesicles in the noradrenergic nerve terminal.