bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evolution and the meaning of metaphase*
Authors: Nicklas R. Bruce , Arana P. 
Published: April 2021

ABSTRACT
We used an evolutionary test to ask whether the congression of chromosomes to the spindle equator is important in itself or just a mitotic happenstance. If congression matters, then it might evolve if absent initially. Previous workers established that newly made trivalents, meiotic units of three chromosomes, generally do not congress to the spindle equator. Instead, these young trivalents lie close to the pole to which two of the three chromosomes are oriented. We studied ancient sex-chromosome trivalents that arose hundreds of thousands to several million years ago in several species of praying mantids and one grasshopper. All these old trivalents lie near the spindle equator at metaphase; some of them congress as precisely to the equator as the ordinary chromosomes in the same cells. We conclude that congression evolved independently two or three times in the materials studied. Therefore, the metaphase position of chromosomes midway between the poles appears to matter, but why? In the praying mantids, the evident answer is that metaphase is a quality-control checkpoint. Sometimes the three chromosomes are not associated in a trivalent but rather are present as a bivalent plus an unpaired chromosome, which lies near one pole. Earlier workers showed that such cells are blocked in metaphase and eventually degenerate; this prevents the formation of sperm with abnormal combinations of sex chromosomes. We suggest that the quality-control system would have trouble distinguishing an unpaired chromosome from an uncongressed, newly arisen trivalent, both of which would lie near a spindle pole. If so, the confused quality-control system would block anaphase imprudently, causing a loss of cells that would have produced normal sperm. Hence, we conclude that the congression of the trivalent to the equator probably evolved along with the metaphase quality-control checkpoint. The mechanism of congression in old trivalents is uncertain, but probably involves an interesting force-sensitive regulation of the motors associated with particular chromosomes. We also examined the congression of two newly made quadrivalents when they orient with three kinetochores to one pole and one to the other. As others have described, one of these quadrivalents does not congress, while the other quadrivalent comes closer than expected to the spindle equator. Such variation in the extent of congression may provide materials on which natural selection can act, leading to the evolution of congression. The trivalents of praying mantids are attractive materials for further studies of the mechanism of congression and of the idea that metaphase is a checkpoint for progression through the cell cycle.
Relocation of nucleolar proteins around chromosomes at mitosis A study by confocal laser scanning microscopy
Authors: Gautier Thierry , Robert-Nicoud Michel , Ö Elle Guilly Marie-N , Hernandez-Verdun Daniele 
Published: April 2021

ABSTRACT
The behaviour of nucleolar antigens known to associate with chromosomes at mitosis was investigated in mammalian cells (HeLa, HEp-2, PtK1, CHO) by immunofluorescence and confocal laser scanning microscopy. Serial optical sections through mitotic cells, from prophase to telophase, were used to generate threedimensional images of the antigen distribution. Our results indicate that, at the onset of mitosis, these antigens leave the nucleoli in a highly ordered manner to form a network extending from the nucleoli towards the nuclear envelope. The migration begins at very early prophase, when the condensation of the chromosomes is not yet visible. After completion of the migration at late prophase, the labelling is found at the chromosome periphery. The antigens remain distributed as a sheath surrounding the chromosomes from prophase to telophase. Therefore, the proteins involved in the formation of this perichromosomal layer have different behaviour than those of the prenucleolar bodies. The antigens appear to interact strongly with chromosomes, since they are not lost during chromosome isolation in hypotonic buffer. Each chromosome is entirely covered from one telomere to the other, except in the centromeric region. Thus the relocation of these nucleolar proteins does not appear to be the result of a passive accumulation at the chromosome periphery, but seems rather to be due to an active targeting to specific sites. Consequently, these proteins may have a determining function in the progression of the cells through mitosis, possibly by participating in the protection and stabilization of the chromosomes.
Independent control of locomotion and orientation during Dictyostelium discoideum chemotaxis
Authors: Duijn Bert Van , Haastert Peter J. M. Van 
Published: April 2021

ABSTRACT
Chemotaxis is cell movement in the direction of a chemical and is composed of two components: movement and directionality. The directionality of eukaryotic chemotaxis is probably derived from orientation: the detection of the spacial gradient of chemoattractant over the cell length. Chemotaxis was investigated in eukaryotic Dictyostelium discoideum cells that were permeabilized by high-voltage discharges. These permeable cells respond chemotactically to extracellular cAMP. However, locomotion is impaired if the Ca2+ concentration is clamped at submicromolar concentrations; interestingly, these non-motile cells still form pseudopodia and elongate in the direction of the cAMP gradient. These results imply that locomotion and orientation during Dictyostelium chemotaxis are independently regulated.
Chromosomes attain a metaphase position on half-spindles in the absence of an opposing spindle pole
Authors: Leslie Roger J. 
Published: April 2021

ABSTRACT
To examine the relative roles of chromosomes, spindle poles and microtubules in the formation of the metaphase spindle and metakinesis, I have experimentally placed an extra centrosome-free pronucleus close to a forming bipolar spindle in a living cell. The chromosomes from the extra nucleus induce the formation of an extra half-spindle from one pole of the otherwise normal bipolar spindle with chromosomes positioned at the putative metaphase plate. I conclude that chromosomes determine the location of half-spindles by sustaining a higher than normal density of microtubules. These results are surprising for two reasons: first, because previous in vivo experiments in tissue culture cells show that mono-oriented chromosomes with functional attachments to spindle microtubules do not support halfspindle formation but oscillate unstably or move to one spindle pole. Additionally, the generally accepted view is that chromosomes attain a metastable condition at the metaphase plate as a result of a balance between forces directed to opposite spindle poles. However, our observation that chromosomes on extra half-spindles attain a metastable position in the absence of an opposing spindle pole, suggests that Ostergren’s model does not account for metakinesis in sea urchin embryos.
Double localization of F-actin in chemoattractant-stimulated polymorphonuclear leucocytes
Authors: Lepidi Hubert , Benoliel Anne-Marie , Mege Jean-Louis , Bongrand Pierre , Capo Christian 
Published: April 2021

ABSTRACT
Uniform concentrations of chemoattractants such as formylpeptides induced a morphological polarization of human polymorphonuclear leucocytes (PMNs) and a concentration of F-actin at the cell front. They also induced a transient increase in filamentous actin (Factin) which preceded the cell shape change.
We combined fluorescence microscopy and image analysis to study the localization of F-actin, as revealed by a specific probe (bodipy™ phallacidin) in suspended PMNs stimulated by chemoattractants. F-actin exhibited remarkable concentration in focal points after a 30 s exposure to 10−8 M formylmethionyl-leucylphenylalanine (fMet-Leu-Phe), although no shape change of PMNs was detectable. A 10-min incubation with formylpeptide (10−6 to KU9 M) induced the morphological polarization of PMNs and the appearance of a principal focus of F-actin in the cell head region and a secondary focus in the cell posterior end.
The distribution of F-actin-associated fluorescence in 2D images of polarized PMNs might be due to an actual concentration of F-actin in privileged areas, to a local concentration of plasma membrane drawing filamentous actin or to variations in the cell volume. Then, we studied the distribution of a cytoplasmic marker, fluorescein diacetate and a membrane probe, TMA-DPH, in unstimulated rounded PMNs and in spherical and morphologically polarized PMNs stimulated by formylpeptide. The distribution of neither of these probes was correlated with F-actin distribution, especially in rounded PMNs stimulated 30 s with 10−8 M fMet-Leu-Phe, suggesting that F-actin was concentrated in two foci located in the cell head region and in the cell posterior end. In addition, zymosan-activated serum induced the morphological polarization of PMNs and the appearance of two foci of filamentous actin, demonstrating that binding of formylpeptide to its specific receptor was not required for F-actin reorganization. We conclude that the accumulation of F-actin probably resulted from local filament assembly and put forward the hypothesis that microfilament reorganization in two centres drives the morphological polarization of PMNs.
Radial movement of lysosomes along microtubules in permeabilized macrophages
Authors: Swanson Joel A. , Locke Albert , Ansel Patricia , Hollenbeck Peter J. 
Published: April 2021

ABSTRACT
In murine bone marrow-derived macrophages, lysosomes often form tubulovesicular compartments, whose extended distribution in the cytoplasm depends on the integrity of cytoplasmic microtubules. When macrophages with fluorescently labeled lysosomes were plated onto coverslips opsonized with IgG, they engaged that surface in a phagocytic response (frustrated phagocytosis). The tubular lysosomal compartment of these cells collected in a central, perinuclear region, despite the continued presence of a radiating array of cytoplasmic microtubules. Using methods developed in the study of melanophores, we permeabilized macrophages engaged in frustrated phagocytosis, then re-activated lysosome extension along microtubules. Permeabilization was selective for plasma membranes, in that high molecular weight probes such as trypan blue or IgG could enter cells, while fluorescent probes previously loaded into lysosomes via endocytosis remained contained therein. Addition of 2 niM ATP, GTP or UTP to these permeabilized cell models produced centrifugal extension of tubular lysosomes. Selective depletion of ATP, using Escherichia coli glycerol kinase, inhibited ATP-dependent extension but not that which occurred with GTP or UTP, indicating that the mechanism of radial movement can use any of these three nucleotide triphosphates. Extension was independent of pH between 6.8 and 7.4, and was inhibited by AMP-PNP and by GMP-PNP. Depolymerization of cytoplasmic microtubules with nocodazole prevented subsequent ATP-inducible lysosome extension, whereas preincubation of cells with cytochalasin D did not inhibit the response. These results are consistent with the in vitro mechanochemical properties of kinesin (Cohn et al., 1989), and support earlier evidence, obtained in living cells, that kinesin is the mechanochemical motor of lysosome extension along microtubules in macrophages.
Development and characterisation of a rat brain capillary endothelial culture: towards an in vitro blood-brain barrier
Authors: Abbott N. Joan , Hughes C. C. W. , Revest Patricia A. , Greenwood J. 
Published: April 2021

ABSTRACT
Primary culture of rat brain endothelial cells is described, based on the method of C. C. W. Hughes and P. L. Lantos. The cells have been characterised using morphological and immunocytochemical techniques, and systematic studies undertaken to determine the optimal culture medium and conditions required to grow the cells at high purity on a variety of substrata. The endothelial cells have a spindle-shaped morphology, and proliferate as plaques from small clusters of cells associated with capillary fragments in the starting material. Tight junction-like celkcell appositions are seen at the electron-microscopic level. The cells show characteristic staining for antigens recognized by antibodies against von Willebrand factor (Factor VTH-related antigen), angiotensin-converting enzyme (ACE), the transferrin receptor (Ox-26), actin and vimentin. They also show binding of the lectin from Ulex europaeus (UEA I). Potential contaminating cells include smooth muscle, fibroblasts, pericytes and meningeal cells.
Contaminants can be kept to &lt; ca. 5% by careful removal of large vessels and meninges during dissection, by brief treatment with Ca2+- and Mg2+-free saline, by growth in medium supplemented with plasma-derived serum treated for removal of platelet-derived growth factor (PDGF), and by occasional use of medium in which D-valine is substituted for L-valine. Cells attach well to collagen-coated plastic, less well to glass. Cells can be grown on transparent collagen filters (ICN, Cellagen and Costar, Transwell-Col), and on microcarrier beads (Pharmacia, Cytodex-3). The culture has proved to be a useful preparation for studies of cellular physiology, pharmacology and biochemistry of the brain endothelium, and represents a first step in producing an in vitro model of the rat blood-brain barrier.
A complex consisting of pp60c-src/pp60c-srcN and a 38 kDa protein is highly enriched in growth cones from differentiated SH-SY5Y neuroblastoma cells
Authors: Meyerson Gabrielle , Pfenninger Karl H. , Påhlman Sven 
Published: April 2021

ABSTRACT
Nerve growth cones of primary neurons are highly enriched in the proto-oncogene product pp60c-src. In order to investigate this molecule further in growing neuronal cells, growth cone and cell body fractions were prepared from human SH-SY5Y neuroblastoma cells differentiated neuronally in vitro under the influence of phorbol ester. The fractions were characterized ultra-structurally and by biochemical criteria. The neuronal (pp60c-srcN) and the fibroblastic (pp60c-src) forms of pp60src are slightly enriched and activated in the growth cones relative to the perikarya. Immunoprecipitates of pp60src from differentiated SH-SY5Y growth cones contain at least four phosphoproteins in addition to pp60src One of these, pp38, migrates as a 100-140 kDa complex with pp60src under non-reducing conditions of gel electrophoresis. The pp38/pp60src complex is not easily detected in non-differentiated SH-SY5Y cells or perikarya of differentiated SH-SY5Y cells, but it is highly enriched in the growth cone preparation. These data suggest that growth-cone pp60src exists in a disulfide-linked oligomeric complex. The complex appears to be assembled only in the cell periphery and may be dependent upon neuronal differentiation.
The distribution of the fiber cell intrinsic membrane proteins MP20 and connexin46 in the bovine lens
Authors: Tenbroek Erica , Arneson Mark , Jarvis Lisa , Louis Charles 
Published: April 2021

ABSTRACT
MP20 is an intrinsic membrane protein previously identified in mammalian lens fiber cells. To identify a possible role for this protein in the lens, the distribution of MP20 and connexin46 has now been examined. Western immunoblotting with an anti-peptide antibody generated to the C-terminal 8 amino acids of MP20 confirmed the presence of this protein in the lens of several different mammalian species. A monoclonal antibody 5H1 was prepared that, in Western blots of bovine lens membranes, recognized the same component as an antibody to rat connexin46 (Cx46). The apparent molecular mass of this component decreased from 59 kDa to 55 kDa following treatment of lens membranes with alkaline phosphatase. A monoclonal antibody to connexin-related MP70 recognized a 70 kDa component in bovine lens membranes confirming the presence of these two different connexin proteins in bovine lens membranes. To localize MP20 and Cx46 in the bovine lens membrane, lens fiber cell bundles were immuno-fluorescently labeled with both the MP20 antibody, and the monoclonal antibody to Cx46. Cx46 was identified in large plaques on the broad faces of the lens fiber cells throughout the outer 1 mm of the lens cortex. MP20 colocalized with Cx46 only in a restricted area 0.5 mm to 1.0 mm into the lens. In other regions of the lens, MP20 appeared more diffusely distributed over the fiber cell surface, although apparently concentrated in the ball- and-socket regions at the corners of the narrow side of the inner cortical lens fiber cells. These inner cortical regions were devoid of Cx46. A difference in distribution of these two proteins was confirmed in studies of immunofluorescently labeled lens cryosections. Furthermore, immunogold electron microscopy of purified lens membranes identified MP20 in both junctional regions (with Cx46) and in single membranes. These results provide evidence for a role for MP20 in mammalian lens fiber cell junctional formation or organization.
Force contribution of the LFA-1/ICAM-1 complex to T cell adhesion
Authors: Sung K.-L. Paul , Kuhlman Peter , Maldonado Fausto , Lollo Bridget A. , chien Shu , Brian Adrienne A. 
Published: April 2021

ABSTRACT
Little is known in quantitative terms about forces between cells generated during adhesion and recognition, or about the contribution of any one set of molecular associations to the development of these forces. To determine the forces involved in adhesion dependent on lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1), we have measured the junctional avidity between single cell pairs consisting of a cloned T cell that expresses LFA-1 and a fibroblast cell that expresses MHC class n molecules and ICAM-1 after transfection. Micromanipulation was used to induce conjugation of cell pairs and to determine the force required to separate the conjugate. T cell adhesion to three related fibroblast cell lines was compared: the parent line that does not express ICAM-1 or other LFA-1 counter-receptors, and two transfectants that have high and moderate levels of surface ICAM-1 expression. The force needed to separate the conjugates varied with the fibroblast ICAM-1 expression levels. The T cell adhesion to ICAM-1-expressing fibroblasts was strong, and the critical separation stresses measured for the three cell lines were 1.4 × 103 dyn/cm2 (1 dyn=10”5 N) for the ICAM-1-negative fibroblast, 4.98 × 103 dyn/cm2 for the fibroblast with a moderate level of ICAM-1 expression, and 6.25 × 103 dyn/cm2 for the fibroblast line with the highest ICAM-1 expression. The dependence of adhesion strength on the LFA-l/lCAM-1 complex was confirmed by the use of blocking antibodies, which showed the contribution from the interaction of CD4/MHC class II to be negligible.
Integrin subunit expression by human osteoblasts and osteoclasts in situ and in culture
Authors: Clover Julie , Dodds R. A. , Gowen Maxine 
Published: April 2021

ABSTRACT
The extracellular matrix may be considered as an insoluble local mediator which plays an important role in regulating cell function. Communication between the cell and its matrix occurs via the integrins, a family of transmembrane proteins composed of non-covalently linked α and β subunits. The aim of this study was to establish which integrins are present on human bone cells in situ and in culture, using cryostat sections of undecalcified human bone, osteoclastoma tissue and cultured human osteoblasts. Integrin subunit expression was identified indirectly using alkaline phosphatase anti-alkaline phosphatase conjugates and FITC-labelled secondary antibodies. Subunits expressed by cultured human osteoblast-like cells were then quantified by FACS analysis.
Staining patterns observed in situ show that osteoblasts and osteoclasts possess different integrin subunits. Osteoblasts primarily express α1, α3 and β1 and weakly express α 2. Osteoclasts express α 2, α v, β2 and β3. Subunits α 4, α5, α6, αL, αM and β2 were not expressed by either of these cell types. Expression of β1 by all cells of the osteoblastic lineage was constitutive, but α1, and α3 subunits were expressed by osteoblasts actively synthesizing bone and some of the osteoblast lining cells. All integrin subunits identified on osteoblasts in situ were maintained on culture but there was an increased expression of α 2 and α v subunits were weakly positive. Expression of α 2, α3, α v and β1 subunits was independent of cell density but expression of α1, was much greater in confluent cultures. Integrin expression of α 2, α v, β1 and β3 by osteoclasts did not vary according to osteoclast size, location or activation state. There was no evidence of subunit polarisation towards the bone surface exhibited by either osteoblasts or osteoclasts.
These findings provide evidence to suggest that osteoblasts and osteoclasts use different integrin subunits to attach to specific ligands within the bone matrix. By transmitting signals from the environment to the interior of the cell, the type of integrin expressed can influence phenotypic behaviour and hence bone remodelling.
Cell-, age- and stage-dependent distribution of connexin43 gap junctions in testes
Authors: Risley Michael S. , Tan Ignatius P. , Roy Christine , Sáez Juan C. 
Published: April 2021

ABSTRACT
Immunocytochemical data demonstrate that the distribution of gap junction connexin43 (C×43) in rodent testes is dependent on cell type, testis maturation, and stage of the mature seminiferous epithelium. Western blotting and indirect immunofluorescence microscopy using anti-peptide antisera to C×43 revealed abundant C×43 in rat and mouse testes and mouse TM3 and TM4 cells. C×43 mRNA was detected in rat testes and mouse TM4 cells by Northern blot analysis. C×43 was localized by immunogold electron microscopy to gap junctions on Sertoli cells and Leydig cells. A punctate distribution of C×43 was observed on peritubular cell surfaces following indirect immunofluorescence of detergent-permeabilized tubule segments. In cryosections from testes of immature (to 30 days) rats, and mature rats and mice, Leydig cells showed a punctate surface distribution of C×43 following indirect immunofluorescence. A diffuse cytoplasmic fluorescence was also seen in spermatocytes and spermatogonia. C×43 staining associated with Sertoli cells was age- and stage-dependent. Over 90% of the tubules in immature testes (22-30 days) contained C×43 in the region of Sertoli-Sertoli occluding junctions and in the adluminal compartment. In mature rat testes, however, C×43 immunostaining was detected in only 60% of 1195 tubule sections where it was abundant proximal to the Sertoli cell occluding junctions. All strongly stained tubules were from stages I-VHI, while negatively stained tubules were at stages 1X-XIV. C×43 immunostaining in mature mouse testes was also stagedependent with all positive tubules at stages VI-VIII. In contrast to C ×43, C ×26 and C ×32 were detected by immunofluorescence only in the apical regions of the seminiferous epithelia in 90% of tubules from mature rats. Consistent with the observed C ×43 immunostaining, octanol-sensitive in situ dye-coupling was observed between Leydig cells, between peritubular cells and between Sertoli cells, suggesting the occurrence of functional gap junctions in these cell types. These observations provide evidence for extensive gap junction-mediated communication between a variety of testis cell types important to the support of spermatogenesis.