bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyclin A-cdc2 kinase does not trigger but delays cyclin degradation in interphase extracts of amphibian eggs
Authors: Lorca Thierry , Labbé Jean-Claude , Devault Alain , Fesquet Didier , Strausfeld Ulrich , Nilsson Joakin , Nygren Per-Ake , Uhlen Mathias , Cavadore Jean-Claude , DorÉE Marcel
Published: April 2021

Purified cyclin B-cdc2 kinase has been shown previously to trigger cyclin degradation in interphase frog extracts by initiating a cascade of reactions that includes cyclin ubiquitinylation and ends with proteolysis. However, cyclin A-cdc2 kinase was not assayed in these early experiments. Here we have shown that full-length recombinant human cyclin A failed to induce cyclin degradation when it was added to frog extracts free of cyclin B, although it formed an active kinase complex with Xenopus cdc2.

A highly purified kinase complex containing a truncated human cyclin A and starfish cdc2 also failed to switch on the cyclin degradation pathway. In contrast, both recombinant cyclin B and highly purified cyclin B-cdc2 kinase readily triggered degradation of both cyclins B and A in frog extracts. Whilst free cyclin A had no inhibitory effect, cyclin A-cdc2 kinase delayed degradation of both cyclins A and B induced by cyclin B-cdc2 kinase. The finding that cyclin A-cdc2 kinase cannot turn on, and even delays, cyclin destruction may be essential to prevent premature inactivation of MPF (maturation-promoting factor) before complete condensation of chromosomes and formation of the metaphase spindle.
Ability to organize microtubules in taxol-treated mitotic PtK2 cells goes with the SPN antigen and not with the centrosome
Authors: Kallajoki Markku , Weber Klaus , Osborn Mary
Published: April 2021

The SPN antigen plays an essential role in mitosis, since microinjection of antibodies causes mitotic arrest. Here we show, by examination of the relative locations of SPN antigen, the centrosomal 5051 antigen and tubulin in normal mitotic, and in taxol-treated mitotic cells, that the SPN antigen is involved in organizing the microtubules of the spindle. The 210 kDa protein defined as SPN antigen relocates from the nuclear matrix to the centrosome at prophase, remains associated with the poles at metaphase and anaphase, and dissociates from the centrosomes in telophase. In taxol-treated mitotic cells, SPN staining shows a striking redistribution while 5051 antigen remains associated with centrosomes. SPN antigen is seen at the plasma membrane end of the rearranged microtubules. SPN antigen is always at the center of the multiple microtubule asters (5 to 20 per cell) induced by taxol, whereas 5051 again remains associated with the centrosomal complex (1 to 2 foci per cell). Microtubule nucleation is associated with the SPN antigen rather than with the 5051 antigen. Microinjection of SPN-3 antibody into taxol-treated mitotic PtK2 cells causes disruption of the asters as judged by tubulin staining of the same cells. Finally, SPN antigen extracted in soluble form from synchronized mitotic HeLa cells binds to, and sediments with, pig brain microtubules stabilized by taxol. This association of SPN antigen with microtubules is partially dissociated by 0.5 M NaCl but not by 5 mM ATP. Thus SPN antigen binds to microtubules in vitro and seems to act as a microtubular minus-end organizer in mitotic cells in vivo.
Overexpression of the csa cell adhesion molecule under its own camp-regulated promoter impairs morphogenesis in dictyostelium
Authors: Faix Jan , Gerisch Günther , Noegel Angelika A.
Published: April 2021

The contact site A (csA) glycoprotein is a strictly developmentally regulated plasma membrane component responsible for the EDTA-stable (Ca2+-indepen-dent) form of intercellular adhesion in Dictyostelium discoideum. Using inverse polymerase chain reaction and a terminator vector we have isolated a 1.6 kb genomic fragment carrying a 1.1 kb upstream region of the csA gene. This fragment had promoter activity in D. discoideum cells, giving rise to a 3′-truncated csA RNA that was regulated like the mRNA of the endogenous gene. Cyclic AMP pulses strongly enhanced transcription from the cloned csA promoter. These findings provide evidence that the cloned region of the csA gene comprises the complete promoter. It contains a G/C-rich octamer motif similar to other cAMP-regulated D. discoideum promoters. When the csA protein was strongly overexpressed under the developmental control of the csA promoter, morphogenesis was substantially altered. Aggregation was delayed, and secondary centres were formed along aggregation streams that led to fragmentation of the aggregates and multiple slug formation. At high cell density a substantial portion of aggregated cells was left behind on the substratum when slugs and fruiting bodies were built. The transformation vector was also employed to rescue a csA-negative mutant, HG1287, from its cell adhesion defect.
A cell surface-associated centrosomal layer of microtubule-organizing material in the inner pillar cell of the mouse cochlea
Authors: Tucker John B. , Paton Calum C. , Richardson Guy P. , Mogensen Mette M. , Russell IAN J.
Published: April 2021

This investigation provides evidence that pericentriolar material is divorced from the immediate vicinities of centrioles and becomes functionally associated with the plasmalemma during the differentiation of a mammalian cell type. Such events occur prior to the assembly of large transcellular microtubule bundles in columnar epithelial cells called inner pillar cells in the mouse organ of Corti. The microtubules do not radiate from a typical centrosome and its centrioles. They elongate from a microtubule-organizing centre (MTOC), which is deployed as a subapical cell surface-associated layer in each cell. Most of the dense material of this layer, and the tops of most of the microtubules, are initially concentrated around the sides of a cell about 1 μm below its apical surface. In addition, a pair of centrioles is located above the layer, which acts as if it is a pericellular concentration of the pericentriolar material of a modified centrosome. Although microtubule nucleation takes place in a centrosome-like region, 13 protofilament fidelity is not exercised. Most of the microtubules have 15 protofilaments.

Microtubule assembly progresses in these cells after the organ of Corti has been isolated for in vitro culture. However, large numbers of microtubules elongate from pericentriolar material juxtaposed against the centrioles. Hence, there is some reversion by the centrosomes of cultured cells to the operational configuration regarded as typical for animal tissue cells in general.
The flagellar beat of rat sperm is organized by the interaction of two functionally distinct populations of dynein bridges with a stable central axonemal partition
Authors: Lindemann Charles B. , Orlando Angela , Kanous Kathleen S.
Published: April 2021

Two distinct patterns of microtubular sliding were observed in rat sperm flagellar axonemes. The particular pattern of sliding was determined by the extraction conditions used to prepare the sperm for axoneme disintegration. Sperm prepared by incubating concentrated suspensions of Triton X-100-extracted sperm at pH 9.0 disintegrated by extruding the doublets and outer dense fibers numbered 4 through 7 in response to Mg-ATP. Sperm prepared by incubating motile Triton X-100-extracted models at 37°C for 1 to 3 hours extruded doublets and outer dense fibers 9, 1 and 2. Axonemes disintegrated by both regimens tended to have doublets 3 and 8 (with their corresponding outer dense fibers), as well as the central pair, in place. In numerous instances, the 3-central-8 complex with outer dense fibers 3 and 8 could be found isolated in midpiece sections prepared from both methods. The 3-central-8 partition was also sometimes seen in isolation in cross-sections of the principal piece where it remained attached to the fibrous sheath. The flagellar remnant produced by extrusion of fibers 4 through 7 under high pH conditions was generally straight or randomly curved. In contrast, the flagellar remnant produced by extrusion of the 9–1–2 bundle of fibers was most often curved into a hook in the midpiece region. While the hook-like configuration was not Ca2+-dependent, it may be based on a related mechanism. The sliding of the 9–1–2 group of fibers is a consequence of dynein-tubulin sliding between the 2 and 3 doublets. This sliding pattern appears to be preferentially activated in the motile sperm models in EGTA, but seldom if ever produced sliding in the high-pH-extracted models. We conclude that the 3-central pair-8 complex and associated outer dense fibers form an I-beam-like partition that does not participate in sliding, but acts as a structural foundation for organizing a planar beat. In addition, it is clear that preferential activation of certain dynein arms can be evoked, depending on the treatment regimen employed. This shows definitively that the types of microtubule sliding in the two bend directions are not identical.
Immunological evidence that plants use both hdel and kdel for targeting proteins to the endoplasmic reticulum
Authors: Napier Richard M. , Fowke Larry C. , Hawes Chris , Lewis Mike , Pelham Hugh R. B.
Published: April 2021

The epitopes of two monoclonal antibodies raised to a putative auxin receptor have been mapped. Carboxypeptidase A digestion of the antigen, auxin-binding protein (ABP) purified from maize, completely abolished binding of antibody MAC 256 and impaired binding of MAC 259, suggesting that they both recognise C-terminal epitopes. Published sequences of ABP showed that the C terminus was KDEL, a tetrapeptide used for targeting proteins to the ER in animal cells. We have used this short homology to confirm that the two monoclonals recognise C-terminal KDEL, showing that animal KDEL proteins and synthetic KDEL peptides are recognised and that animal cell ER is stained strongly and specifically. Sucrose density gradient fractionation of maize microsomal membranes showed that plant KDEL proteins, including ABP, fractionated with markers for the endoplasmic reticulum. However, few proteins are stained by anti-KDEL monoclonals in plants. For comparison, a monoclonal antibody raised to a synthetic HDEL peptide was also used and found to stain a set of proteins in all plant species tested. The anti-HDEL and anti-KDEL monoclonals were sequence specific, staining different proteins. On density gradient fractionation HDEL proteins also banded with ER marker activities. However, the intracellular distribution of HDEL and KDEL proteins determined by immunofluorescence was different. Whereas HDEL proteins showed a distribution characteristic of plant ER, and this localisation was confirmed by immunogold labelling of ultrathin sections and electron microscopy, KDEL proteins showed strong fluorescence in discrete parts of the cell cortex. These observations are discussed in terms of the potential these monoclonal antibodies have as markers for ER and of the role ABP plays in plant cell signalling.
Mitochondrial gene expression in the human gastrointestinal tract
Authors: Macpherson Andrew J. S. , Mayall Timothy P. , Chester Kerry A. , Abbasi Atta , Forgacs Ian , Malcolm Alan D. B. , Peters Timothy J.
Published: April 2021

In the human gastrointestinal epithelium, in situ hybridisation demonstrates that 12 S and 16 S mitochondrial ribosomal RNAs show maximal steady-state levels on the surface epithelial cells of the normal small intestine and colon. The mitochondrial mRNAs, cytochrome b and NADH dehydrogenase (IV) have a uniform distribution throughout the crypt and surface (villus) epithelial cells of the small intestine and colon. Histochemical stains for the activity of the mitochondrial respiratory chain enzymes succinate dehydrogenase and cytochrome oxidase also show almost uniform activities throughout the crypt-surface epithelial cell axis in the small and large intestines. In sections of normal human oesophagus the levels of mitochondrial ribosomal RNAs, mitochondrial mRNAs and the activities of mitochondrial respiratory chain enzymes are maximal over the basal cells of the stratified squamous epithelium. These results show a relative increase in mitochondrial ribosomal RNA expression compared with mitochondrial mRNAs in surface cells of simple intestinal epithelia.
Evolution and the meaning of metaphase*
Authors: Nicklas R. Bruce , Arana P.
Published: April 2021

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

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

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

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

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

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

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 < 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

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

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

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

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

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.
Kinetochore formation and behaviour following premature chromosome condensation
Authors: Rattner J. B. , Wang T.
Published: April 2021

The potential for interphase centromeres to support kinetochore formation following premature chromosome condensation (PCC) has been investigated. We show that the centromere remains competent to initiate kinetochore formation throughout the cell cycle. PCC-kinetochores display a typical trilaminar morphology, associate with microtubules and show movement towards the centrosome. Indirect immunofluorescence studies illustrate that the centromere/kinetochore region of prematurely condensed chromosomes associates with proteins that are normally found within this region in both a cell cycle-dependent and an independent manner.
Recycling pathways of glucosylceramide in BHK cells: distinct involvement of early and late endosomes
Authors: Kok Jan Willem , Hoekstra Karin , Eskelinen Sinikka , Hoekstra Dick
Published: April 2021

Recycling pathways of the sphingolipid glucosylceramide were studied by employing a fluorescent analog of glucosylceramide,6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosylsphingosine (C6-NBD-glucosylceramide). Direct recycling of the glycolipid from early endosomes to the plasma membrane occurs, as could be shown after treating the cells with the microtubule-disrupting agent nocodazole, which causes inhibition of the glycolipid’s trafficking from peripheral early endosomes to centrally located late endosomes.

When the microtubuli are intact, at least part of the glucosylceramide is transported from early to late endosomes together with ricin. Interestingly, also N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), a membrane marker of the fluid-phase endocytic pathway, is transported to this endosomal compartment. However, in contrast to both ricin and N-Rh-PE, the glucosylceramide can escape from this organelle and recycle to the plasma membrane. Monensin and brefeldin A have little effect on this recycling pathway, which would exclude extensive involvement of early Golgi compartments in recycling. Hence, the small fraction of the glycolipid that colocalizes with transferrin (Tf) in the Golgi area might directly recycle via the trans-Golgi network. When the intracellular pH was lowered to 5.5, recycling was drastically reduced, in accordance with the impeding effect of low intracellular pH on vesicular transport during endocytosis and in the biosynthetic pathway.

Our results thus demonstrate the existence of at least two recycling pathways for glucosylceramide and indicate the relevance of early endosomes in recycling of both proteins and lipids.
Effects of the expression of mammalian annexins in yeast secretory mutants
Authors: Creutz Carl E. , Kambouris Nicholas G. , Snyder Sandra L. , Hamman Helen C. , Nelson Michael R. , Liu Wei , Rock Philip
Published: April 2021

The hypothesis that calcium-dependent membrane-binding proteins of the annexin family can influence intracellular membrane trafficking was tested by expressing five mammalian annexins in wild-type yeast cells (Saccharomyces cerevisiae) and in 13 yeast secretory (sec) mutants. Expression of human synexin (annexin VII) inhibited the growth of sec2, sec4 and sec15 mutants at a semi-permissive temperature. These three sec mutants are defective in the final step in the secretory pathway, the process of exocytosis. The inhibition of growth correlated with reduced viability and increased accumulation of internal invertase in these mutants when expressing synexin. Bovine endonexin (annexin IV) partially suppressed the growth defect of a sec2 mutant incubated at a semi-permissive temperature. Human synexin, human lipocortin (annexin I), and murine p68 (annexin VI) reduced the lag time associated with adaptation of sec2 mutants to galactose-containing medium. These interactions suggest that the annexins may influence specific steps in membrane trafficking associated with cell growth, secretion and plasma membrane remodelling.
Contact stimulation of cell migration
Authors: Thomas Linda A. , Yamada Kenneth M.
Published: April 2021

Mass migrations of dense cell populations occur periodically during embryonic development. It is known that extracellular matrices, through which the cells migrate, facilitate locomotion. However, this does not explain how cells, such as neural crest, can migrate as a dense cohort of cells in essentially continuous contact with one another. We report here that unique behavioral characteristics of the migrating cells may contribute to cohesive migration. We used time-lapse video microscopy to analyze the migration of quail neural crest cells and of two crest derivatives, human melanoma cells and melanocytes. These cells migrated poorly, if at all, when isolated, but could be stimulated up to 200-fold to travel following contact with migrating cells. This phenomenon, which we have termed “contact-stimulated migration,” appeared to activate and sustain migration of the mass of cells. Cells that became dissociated from the others ceased directional migration, thereby limiting aberrant cell dispersion. Fibroblasts were minimally responsive to this novel phenomenon, which may be crucial for major, mass cell migrations.
Molecular characterization of the secondary constriction region (qh) of human chromosome 9 with pericentric inversion
Authors: Luke Sunny , Verma Ram S. , Mathews Robert A. Contethomas
Published: April 2021

Pericentric inversion of the secondary constriction region (qh) of human chromosome 9 is a frequent occurrence. This structural alteration is regarded as a normal familial variant, termed heteromorphism, and is inherited in a Mendelian fashion without any apparent phenotypic consequences. We characterized the qh region of chromosome 9 from five individuals using a series of molecular cytogenetic techniques. Four out of the five individuals have an additional area composed of alphoid DNA sequences on the inverted chromosome 9 while one case was found to have an apparently intact alphoid DNA sequence. Although the direct function(s) of alphoid DNA sequences remain unclear, the centromeric breakage involving these sequences in inverted chromsome 9 raises a series of questions pertaining to the monocentric, dicentric and pseudodicentric nature of pericentric inversions. Nevertheless, these findings have prompted us to suggest that the structural organization of alphoid DNA sequences of the centromeric region of chromosome 9 are apparently “breakage prone” and may be associated with a higher incidence of pericentric inversions. Furthermore, the hierarchical organization of various satellite DNA families (α-satellite, β-satellite and satellite III) within the primary and secondary constriction regions of chromosomes 9 are elucidated here.
Characterization of the human involucrin promoter using a transient β-galactosidase assay
Authors: Carroll Joseph M. , Taichman Lorne B.
Published: April 2021

Involucrin, a component of the cornified cell envelope, is expressed specifically in differentiating keratinocytes of stratified squamous epithelia. To explore the regulation of involucrin expression, 3.7 kb of upstream sequences of the human involucrin gene was cloned into a plasmid containing a -galactosidase reporter gene and transfected into early passage keratinocytes and a variety of human cell types. The full-length construct gave maximal and tissue-specific expression. Deletion analysis showed that sequences between 900 and 2500 bp upstream of the transcriptional start site and the intron located between the transcriptional and translational start sites were required for maximal expression. Further analysis of the intron indicated that its effects on expression were independent of it being present in nascent RNA and suggested that sequences within the intron have regulatory activity. These results suggest that the involucrin intron operates in vivo to regulate expression in the epidermis.

Abbreviations used: ADH, alcohol dehydrogenase; β-gal, β-galactosidase; DME, Dulbecco’s modified Eagle’s medium; DDAB, dimethyldioctyldecylammonium bromide; FCS, fetal calf serum; kb, kilobase; ONPG, O-nitrophenyl β-D-galactopyranoside; PBS, phosphate buffered saline without calcium/magnesium salts; PCR, polymerase chain reaction; PtdEtn, dioleoyl-L-α-phosphatidylethanolamine; RSV, Rous sarcoma virus; SV40, simian virus 40.
Brain microtubule-associated proteins modulate microtubule dynamic instability in vitro: Real-time observations using video microscopy
Authors: Pryer Nancy K. , Walker Richard A. , Skeen Victoria Petrie , Bourns Brenda D. , Soboeiro Michael F. , Salmon Edward D.
Published: April 2021

We used video assays to study the dynamic instability behavior of individual microtubules assembled in vitro with purified tau, purified MAP2 or a preparation of unfractionated heat-stable MAPs. Axoneme-nucleated microtubules were assembled from pure tubulin at concentrations between 4 and 9 M in the presence of MAPs, and observed by video-differential interference contrast microscopy. Microtubules co-assembled with each MAP preparation exhibited the elongation and rapid shortening phases and the abrupt transitions (catastrophe and rescue) characteristic of dynamic instability. Each MAP preparation increased the microtubule elongation rate above that for purified tubulin alone by decreasing the tubulin subunit dissociation rate during elongation. The brain MAPs used in this study reduced the rate of microtubule rapid shortening, but allowed significant loss of polymer during the shortening phase.

Purified tau and MAP2 decreased the frequency of catastrophe and increased the frequency of rescue, while the heat-stable MAPs suppressed catastrophe at all but the lowest tubulin concentrations. Thus, each of these MAPs modulates, but does not abolish, dynamic instability behavior of microtubules. We propose a model to explain how MAP2 and tau bind to the microtubule lattice at sites along protofilaments so that the MAPs promote polymerization, but do not significantly block the mechanism of rapid shortening inherent in the tubulin lattice. Rapid shortening, when it occurs, proceeds primarily by the dissociation of short fragments of protofilaments, which contain the bound MAPs.
The effect of cytochalasin D on preprophase band organization in root tip cells of Allium
Authors: Eleftheriou E. P. , Palevitz B. A.
Published: April 2021

The relationship between microfilaments (Mfs) and microtubules (Mts) in the organization of the preprophase band (PPB) was investigated in Allium root tip cells subjected to treatment with cytochalasin D (CD). Mts and Mfs were visualized by indirect immunofluorescence and various parameters such as PPB width were analyzed quantitatively. In control samples, the PPB first appears as a wide Mt band that progressively narrows to an average width of 4 m in mid-prophase. Randomly oriented Mfs are present throughout the cytoplasm of most interphase control cells. Preprophase and prophase cells, however, contain cortical Mfs arranged parallel to the PPB. The Mfs initially occupy much of the cortex but in most cells they progressively become restricted to an area wider than the PPB. In the presence of CD, the PPB fails to narrow and remains at least two-fold wider than in control cells. PPB width expressed as a percentage of nuclear or cell length also increases compared to controls. Widening is concentration dependent, and the effect of 10 M CD is near maximal only 15 min after application of the drug. This rapid response suggests that a rebroadening of already condensed PPBs takes place. After as little as 15 min in CD, Mfs are replaced by many small specks and rods. Dual localizations of both Mts and Mfs show that prophase cells contain broad PPBs without Mfs. The rapid disorganization of Mfs, by CD, therefore coincides with the rebroadening of PPBs. CD-treated cells in metaphase, anaphase and telophase contain larger actin aggregates at the poles, as previously reported. The results indicate that Mfs play an important role in the narrowing of the PPB, which in turn is essential for determination of the exact position of the plane of division. They also indicate that movement of intact Mts is important in PPB organization.