bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mutation of the predicted p34cdc2 phosphorylation sites in numa impair the assembly of the mitotic spindle and block mitosis
Authors: Compton Duane A. , Luo Chenghua 
Published: April 2021

ABSTRACT
NuMA is a 236 kDa intranuclear protein that is distributed into each daughter cell during mitosis through association with the pericentrosomal region of the mitotic spindle. NuMA’s interaction with the microtubules of the mitotic spindle is mediated through its 45 kDa carboxyl-terminal globular tail, and there is indirect evidence suggesting that NuMA’s interaction with the mitotic spindle is controlled in a mitosis-specific manner. Consistent with this evidence is the fact that all four of the predicted p34cdc2 consensus phosphorylation sites in the NuMA protein are located in the carboxyl-terminal globular domain, and we demonstrate here that NuMA is phosphorylated in a mitosis-specific fashion in vivo. To test if the predicted p34cdc2 phosphorylation sites are necessary for NuMA’s mitosis-specific interaction with the mitotic spindle, we have introduced mutations into the human NuMA cDNA that convert these predicted p34cdc2 phosphorylation sites from threonine or serine residues into alanine residues, and subsequently determined the cell cycle-dependent localization of these altered NuMA proteins following their expression in tissue culture cells. While none of these specific mutations in the NuMA sequence alters the faithful targeting of the protein into the interphase nucleus, mutation of threonine residue 2040 alone or in combination with mutations in other potential p34cdc2 phosphorylation sites abolishes NuMA’s ability to associate normally with the microtubules of the mitotic spindle. Instead of binding to the mitotic spindle these mutant forms of NuMA concentrate at the plasma membrane of the mitotic cell. Cells expressing these mutant forms of NuMA have disorganized mitotic spindles, fail to complete cytokinesis normally, and assemble micronuclei in the subsequent interphase. These data suggest that NuMA’s interaction with the microtubules of the mitotic spindle is controlled by cell cycle-dependent phosphorylation in addition to differential subcellular compartmentalization, and the characteristics of the dominant negative phenotype induced by these mutant forms of NuMA support a role for NuMA in the organization of the mitotic spindle apparatus.
Centriolar cycle of fused cells
Authors: Manandhar G. , Onishchenko Galina E. 
Published: April 2021

ABSTRACT
Ultrastructure of centrioles of fused cells containing heterophasic interphase nuclei, premature chromosome condensation, and a telophase-like nucleus were studied. The study indicated that the heterophasic cellular environments contributed by the different cell partners exert a mutually opposite effect on the structure of centrioles. The G1-cell partner suppresses replication of S-centrioles. Asynchronous replications of G1 and S-stage centrioles were observed in some G1-S, G1-G2 and S-G2 fused cells. In interphase-mitotic fused cells, centrioles of interphase stages underwent mitotic activation when their nuclei were induced to premature chromosome condensation. Daughter centrioles of G1-, S-, and G2-stages were also capable of mitotic activation independently if they were separated from their mother centriole. Inactive centrioles were observed in some cells containing G1-premature chromosome condensation. When mitotic nuclei were induced to telophase-like nucleus formation, their centrioles were also inactivated. Concomitant events of induced nuclear and centriolar changes suggest that they might have been controlled by the heterophasic cytoplasmic factors through similar pathways.
Coupling between gamma irradiation, p53 induction and the apoptotic response depends upon cell type in vivo
Authors: Midgley Carol A. , Owens Bronwyn , Briscoe Cecilie V. , Thomas David Brynmor , Lane David P. , Hall Peter A. 
Published: April 2021

ABSTRACT
The accumulation of p53 protein following whole body irradiation of adult mice was studied using a new polyclonal antibody to mouse p53. While dramatic accumulation of the protein was apparent in splenocytes, thymocytes and osteocytes no p53 protein accumulation was detected in the hepatocytes of the irradiated mouse. Thus the upstream initiating signals that control the induction of p53 are controlled in a tissue specific manner. While massive apoptosis accompanies p53 induction in thymocytes and splenocytes it is not seen in the osteocytes. Thus the downstream consequences of p53 induction are also tightly controlled. These results have profound significance for an understanding of the role of the p53 tumour suppression pathway in different tissues.
The mechanism of nuclear transport of natural or artificial transport substrates in digitonin-permeabilized cells
Authors: Cserpán Imre , Udvardy Andor 
Published: April 2021

ABSTRACT
Characterization of nuclear protein transport in digitonin-permeabilized cells revealed that the number of the nuclear localization signal sequences (NLS) within the transport substrate basically influences the mechanism of the transport reaction.
Phycoerythrine-NLS transport substrate carrying a maximum of 4-5 conjugated NLSs/subunit, or Bsp methyl-transferase-NLS fusion protein were efficiently transported into the nuclei of digitonin-permeabilized cultured cells without any exogenously added cytosolic protein. All the characteristic properties of in vivo nuclear transport are faithfully reproduced with these transport substrates: (i) the transport requires a functional NLS in the transported protein, a transport-incompetent mutant NLS being ineffective; (ii) the transport is energy dependent; (iii) the wild type nuclear localization peptide efficiently competes for transport, while the transport-incompetent mutant peptide does not; and (iv) wheat germ agglutinin inhibits this transport reaction. Nuclear transport observed with these substrates was not due to any damage of the nuclear membrane or inefficient extraction of the cytosolic proteins during the permeabilization of the cells. The nuclear transport was proportional to the number of conjugated NLSs.
Nuclear transport of phycoerythrine carrying 7-8 conjugated NLSs/subunit required the addition of exogenous cytosolic proteins. This transport also fulfilled all the characteristic properties of an authentic nuclear transport.
Nuclear transport with different combinations of transport substrates further supported the assumption that distinct transport mechanisms operate for different substrates. From a mixture of PE-NLS7-8 and Bsp methyltransferase-NLS, the highly conjugated substrate was completely retained in the cytoplasm in the absence of exogenous cytosol, while Bsp methyltransferase-NLS was efficiently transported. Exogenous cytosol promoted the nuclear transport of the highly conjugated substrate.
Colocalization of a high molecular mass phosphoprotein of the nuclear matrix (p255) with spliceosomes
Authors: Bisotto Sandra , Lauriault Pascal , Duval Marie , Vincent Michel 
Published: April 2021

ABSTRACT
It was previously demonstrated that monoclonal antibody CC-3 binds to a phosphorylation dependent epitope present on a 255 kDa nuclear protein (p255). We show here that in interphase cells, p255 distributes to typical nuclear speckles that correspond to the localization of spliceosome components as revealed by antibodies to the m3G cap of snRNAs or to the non-snRNP splicing factor SC-35. Immunofluorescence and immunoblot studies indicated that p255 is resistant to extraction with non-ionic detergents, nucleases and high ionic strength buffers and may thus be defined biochemically as a nuclear matrix phosphoprotein. To determine the nature of the association of p255 with the nuclear structure, its distribution was studied at different stages of the cell cycle and after the cells were treated with nucleases or heat shocked. We found that the antigen diffused into the cytoplasm during metaphase but was reorganized into cytoplasmic speckles during anaphase-telophase transition, where it colocalized with SC-35. Nuclear matrix preparations that were digested with DNases and RNases showed that interphasic p255 still localized to nuclear speckles even though snRNA and snRNP antigens were removed. Heat-shocked cells labelled with monoclonal antibody CC-3 exhibited more rounded and less interconnected speckles, identical to those decorated by anti-SC-35 antibody under such conditions. These results indicate that p255 and SC-35 are present in the same nuclear structures, to which they are more tightly bound than the snRNP antigens. They further suggest that both proteins are implicated in spliceosome assembly or attachment.
Cyclic AMP modulates the rate of ‘constitutive’ exocytosis of apical membrane proteins in Madin-Darby canine kidney cells
Authors: Brignoni Mirtha , Pignataro Omar P. , Rodriguez Marcelo L. , Alvarez Adriana , Vega-Salas Dora E. , Rodriguez-Boulan Enrique , Salas Pedro J. I. 
Published: April 2021

ABSTRACT
Madin-Darby canine kidney and other epithelial cell lines (e.g. Caco-2, MCF-10A and MCF-7) develop intracellular vacuoles composed of apical membrane displaying microvilli (VACs) when impaired from forming normal cell-to-cell contacts. In a previous publication, we showed that VACs are rapidly exocytosed upon treatment with 8-Br-3’,5’-cyclic adenosine monophosphate (8-Br-cAMP), a membrane-permeable analog of cAMP, and that this exocytosis correlates with variations in the cellular cAMP concentration in response to the cell-cell contacts. In the present work, we tested the hypothesis that cAMP may be a positive modulator of the ‘constitutive’ exocytic pathway. To mimic conditions in cells with incomplete intercellular contacts, the intracellular levels of cAMP were decreased by means of two independent approaches: (i) pores were induced in the plasma membrane with the polypeptidic antibiotic subtilin, thus allowing small molecules (including cAMP) to permeate and move out of the cytoplasm; and (ii) adenylate cyclase and protein kinase A were blocked with specific inhibitors. In all cases, the intracellular levels of cAMP were measured and, in porated cells, equilibrated to simulate the corresponding physiological intracellular concentrations. The decrease in cAMP within the physiological range resulted in a decreased rate of transport of an apical marker of the constitutive pathway (influenza virus hemagglutinin) from the trans -Golgi network to the apical plasma membrane. Likewise, the delivery of a number of cellular apical proteins to the plasma membrane was retarded at low cAMP concentrations. The inhibitors of adenylate cyclase failed to block basolateral delivery of vesicular stomatitis virus G protein. This differential modulatory effect may represent a differentiation-dependent control of the insertion of apical membrane in epithelial cells.
Expression of cartilage-specific molecules is retained on long-term culture of human articular chondrocytes
Authors: Kolettas Evangelos , Buluwela Lakjaya , Bayliss Michael T. , Muir Helen I. 
Published: April 2021

ABSTRACT
Normal human adult articular chondrocytes were used to determine how the chondrocyte phenotype is modulated by culture conditions following long-term culture. We report here for the first time that human articular chondrocytes have a lifespan in the range of 34-37 population doublings. While chondrocytes cultured as monolayers displayed a fibroblastoid morphology and grew faster, those cultured as suspensions over agarose adopted a round morphology and formed clusters of cells reminiscent of chondrocyte differentiation in intact cartilage, with little or no dNa synthesis. These morphologies were independent of the age of the culture. Despite, these morphological differences, however, chondrocytes expressed markers at mRNA and protein levels characteristic of cartilage: namely, types II and IX collagens and the large aggregating proteoglycans, aggrecan, versican and link protein, but not syndecan, under both culture conditions. However, they also expressed type I collagen α1(I) and α2(I) chains. It has been suggested that expression of collagen α1(I) by chondrocytes cultured as monolayers is a marker of the loss of the chondrocyte phenotype. However, we show here, using reverse transcriptase/polymerase chain reaction, that normal fresh intact human articular cartilage expresses collagen α1(I). The data show that following long-term culture human articular chondrocytes retain their differentiated characteristics and that cell shape does not correlate with the expression of the chondrocyte phenotype. It is proposed that loss of the chondrocyte phenotype is marked by the loss of one or more cartilage-specific molecules rather than by the appearance of non-cartilage-specific molecules.
Multiple mRNAs encode the avian lysosomal membrane protein LAMP-2, resulting in alternative transmembrane and cytoplasmic domains
Authors: Hatem Christine L. , Gough Nancy R. , Fambrough Douglas M. 
Published: April 2021

ABSTRACT
Lysosomal membranes are enriched in extensively glycosylated transmembrane proteins, LAMP-1 and LAMP-2. LAMP-1 proteins have been characterized from several mammalian species and from chickens, but no nonmammalian homologues of LAMP-2 have been described, and no splice variants of either protein have been reported. Here we report the characterization of three cDNA clones encoding chicken LAMP-2. The nucleotide sequences of the cDNAs diverge at their 3’ ends within the open reading frame, resulting in sequences that code for three different transmembrane and cytoplasmic domains. Southern analysis suggests that a single gene encodes the common region of chicken LAMP-2. The position of the divergence and the identity of the common sequence are consistent with alternative splicing of 3’ exons. Analysis of the mRNAs present in adult chicken tissues suggests tissue-specific expression of the three chicken LAMP-2 variants, with LAMP-2b expressed primarily in the brain. The cytoplasmic domain of LAMP-type proteins contains the targeting signal for directing these molecules to the lysosome. Using chimeras consisting of the lumenal domain of chicken LEP100 (a LAMP-1) and the transmembrane and cytoplasmic domains of the LAMP-2 variants, we demonstrate in transfected mouse L cells that all three LAMP-2 carboxyl-terminal regions are capable of targeting the chimeric proteins to lysosomes. Levels of expression, subcellular distribution, and glycosylation of the LAMP proteins have all been shown to change with differentiation in mammalian cells and to be correlated with metastatic potential in certain tumor cell lines. Alternative splicing of the LAMP-2 transcript may play a role in these changes.
Kinetochore motility after severing between sister centromeres using laser microsurgery: Evidence that kinetochore directional instability and position is regulated by tension
Authors: Skibbens Robert V. , Rieder Conly L. , Salmon E. D. 
Published: April 2021

ABSTRACT
During mitosis in vertebrate somatic cells, the single attached kinetochore on a mono-oriented chromosome exhibits directional instability: abruptly and independently switching between constant velocity poleward and away from the pole motility states. When the non-attached sister becomes attached to the spindle (chromosome bi-orientation), the motility of the sister kinetochores becomes highly coordinated, one moving poleward while the other moves away from the pole, allowing chromosomes to congress to the spindle equator. In our kinetochore-tensiometer model, we hypothesized that this coordinated behavior is regulated by tension across the centromere produced by kinetochore movement relative to the sister kinetochore and bulk of the chromosome arms. To test this model, we severed or severely weakened the centromeric chromatin between sister kinetochores on bi-oriented newt lung cell chromosomes with a laser microbeam. This procedure converted a pair of tightly linked sister kinetochores into two monooriented single kinetochore-chromatin fragments that were tethered to their chromosome arms by thin compliant chromatin strands. These single kinetochore-chromatin fragments moved substantial distances off the metaphase plate, stretching their chromatin strands, before the durations of poleward and away from the pole movement again became similar. In contrast, the severed arms remained at or moved closer to the spindle equator. The poleward and away from the pole velocities of single kinetochore-chromatin fragments in prometaphase were typical of velocities exhibited by sister kinetochores on intact chromosomes from prometaphase through midanaphase A. However, severing the chromatin between sister kinetochores uncoupled the normally coordinated motility of sister kinetochores. Laser ablation also uncoupled the motilities of the single kinetochore fragments from the bulk of the arms. These results reveal that kinetochore directional instability is a fundamental property of the kinetochore and that the motilities of sister kinetochores are coordinated during congression by a stiff centromere linkage. We conclude that kinetochores act as tensiometers that sense centromere tension generated by differential movement of sister kinetochores and their chromosome arms to control switching between constant velocity P and AP motility states.
Identification and localization of three classes of myosins in pollen tubes of Lilium longiflorum and Nicotiana alata
Authors: Miller Deborah D. , Scordilis Stylianos P. , Hepler Peter K. 
Published: April 2021

ABSTRACT
The presence and localization of actin and myosin have been examined in pollen tubes of Lilium longiflorum and Nicotiana alata. Immunoblot analysis of pollen tube extracts with antibodies to actin, myosins IA and IB, myosin II, and myosin V reveals the presence of these contractile proteins. Immunofluorescence microscopy using various methods to preserve the pollen tubes; chemical fixation, rapid freeze fixation and freeze substitution (RF-FS) followed by rehydration or by embeddment in a methacrylate mixture, was performed to optimize preservation. Immunocytochemistry reaffirmed that actin is localized longitudinally in the active streaming lanes and near the cortical surface of the pollen tube. Myosin I was localized to the plasma membrane, larger organelles, the surface of the generative cell and the vegetative nucleus, whereas, myosin V was found in the vegetative cytoplasm in a punctate fashion representing smaller organelles. Myosin II subfragment 1 and light meromyosin were localized in a punctate fashion on the larger organelles throughout the vegetative cytoplasm. In addition, isolated generative cells and vegetative nuclei labeled only with the myosin I antibody. Competition studies indicated the specificity of the heterologous antibodies utilized in this study suggesting the presence of three classes of myosins in pollen. These results lead to the following hypothesis: Myosin I may move the generative cell and vegetative nucleus unidirectionally through the pollen tube to the tip, while myosin V moves the smaller organelles and myosins I and II move the larger organelles (bidirectionally) that are involved in growth.
αv and α3 integrin subunits are associated with myofibrils during myofibrillogenesis
Authors: McDonald K. A. , Lakonishok M. , Horwitz A. F. 
Published: April 2021

ABSTRACT
The development of the myofibrillar apparatus in skeletal muscle is a process in which transmembrane linkages with adhesion molecules are implicated. Integrins are one class of transmembrane adhesion receptors which appear to mediate these interactions. Two prominent linkages are at the myotendinous junction (MTJ), which resides at the ends of the cell and connects myofibrils to the tendon, and the costameres, which encircle the girth of the cell and connect the Z-disks to the sarcolemma. In this study we report that the αv integrin subunit is a prominent component of the costamere. The αv subunit is present initially on developing myotubes in a diffuse staining pattern with some concentration along nascent myofibrils. However, it appears in a striated pattern at the costamere and inconsistently at the M-line following the striation of α-actinin and titin but before that of desmin. Its recruitment to preformed striation suggests that it is incorporated into a pre-existing structure. The presence of αv in the costamere points to a role in lateral myofibrillar anchorage. In addition, we find that the α3 subunit is transiently associated with myofibrils along portions of their lengths and at their ends during myofibrillogenesis. The α3 subunit staining shows a novel localization and junctional structure. As myofibrils become striated the α3 integrin dissociates from the localized pattern and becomes diffuse. This suggests a possible role in the stabilization of nascent myofibrils prior to striation. Antibody-induced perturbation of adhesion mediated by the integrin β1 subunit in developing myotubes inhibits assembly of the sarcomeric architecture. The observations presented here also contribute to an emerging theme, where different integrins occupy unique junctional regions on muscle.
A role for phospholipase C activity but not ryanodine receptors in the initiation and propagation of intercellular calcium waves
Authors: Hansen Michael , Boitano Scott , Dirksen Ellen R. , Sanderson Michael J. 
Published: April 2021

ABSTRACT
Mechanical stimulation of a single cell in an airway epithelial culture initiates an increase in intracellular Ca2+ concentration ([Ca2+]i) that propagates from cell to cell as an intercellular Ca2+ wave. These Ca2+ waves appear to require an increase in intracellular inositol 1,4,5-trisphosphate (IP3) concentration ([IP3]i) in the stimulated cell and are propagated between cells by the diffusion of IP3 through gap junctions. To test the hypothesis that the activation of phospholipase C (PLC) contributes to the elevation of [IP3]i and initiation of an intercellular Ca2+ wave, changes in [Ca2+]i induced by mechanical stimulation were measured by digital fluorescence microscopy in the presence of the PLC inhibitor, aminosteroid U73122. Following exposure to U73122 mechanical stimulation elevated [Ca2+]i of the stimulated cell, but did not initiate the propagation of an intercellular Ca2+ wave. By contrast, in the presence of U73343, a similar aminosteroid that does not inactivate PLC, mechanical stimulation increased the [Ca2+]i of the stimulated cell and initiated an intercellular Ca2+ wave. U73122 also blocked the elevation of [Ca2+]i of airway epithelial cells in response to ATP, a P2-receptor agonist that activates PLC to elevate [IP3]i and [Ca2+]i. In addition, the propagation of intercellular Ca2+ waves was not affected by the ryanodine-receptor agonists, caffeine or ryanodine. The hypotheses that: (1) an elevation of [IP3]i is required to initiate intercellular Ca2+ waves; (2) mechanical stimulation activates PLC; and (3) Ca2+ wave propagation in airway epithelial cells involves Ca2+ release from intracellular stores primarily via IP3 receptors are supported by these results.