bluuebunny/crossref_metadata_2025 huggingface dataset:
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

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

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

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

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.
Suppression of apoptosis by v-ABL protein tyrosine kinase is associated with nuclear translocation and activation of protein kinase C in an interleukin-3-dependent haemopoietic cell line
Authors: Evans Caroline A. , Lord Janet M. , Owen-Lynch P. Jane , Johnson Gerald , Dive Caroline , Whetton Anthony D.
Published: April 2021

We previously demonstrated that activation of v-ABL protein tyrosine kinase resulted in suppression of apoptosis following interleukin-3 removal using an interleukin-3-dependent haemopoietic cell line transfected with a temperature-sensitive mutant of the v-abl oncoprotein (IC.DP). Cellular signalling events associated with the activation of v-ABL included increased levels of sn-1,2-diacylglycerol, an activator of protein kinase C. Calphostin C, a PKC inhibitor, restored apoptosis to interleukin-3-deprived IC.DP cells expressing active v-ABL. However, chronic exposure to the phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate to downregulate protein kinase C did not attenuate the survival of IC.DP cells expressing active v-ABL. Translocation of a classical protein kinase C isozyme(s) to the nuclear fraction was observed 6 hours after activation of v-ABL, when nuclear protein kinase C activity was increased approximately 2-fold. The protien kinase C isozyme responsible, which was only partially downregulated by 12-O-tetradecanoyl phorbol 13-acetate, was identified as protein kinase C βII. This translocation of protein kinase C βII to the nucleus was inhibited by calphostin C. Taken together, these results suggest that nuclear translocation and activation of PKCβII may play a role in v-ABL-mediated suppression of apoptosis.
Delayed cyclin A and B1 degradation in non-transformed mammalian cells
Authors: Girard Franck , Fernandez Anne , Lamb Ned
Published: April 2021

Cyclins A and B are known to exhibit significant differences in their function, cellular distribution and timing of degradation at mitosis. On the basis of observations in marine invertebrates and Xenopus, it was proposed that cyclin destruction triggers cdc2 kinase inactivation and anaphase onset. However, this model has recently been questioned, both in Xenopus and in budding yeast.

In this report, we present evidence for delayed degradation of both cyclins A and B1 in non-transformed mammalian cells. Indeed, by means of indirect immunofluorescence and confocal microscopy, we show that cyclins A and B1 are present up to anaphase in REF52, Hs68, human primary fibroblasts and NRK epithelial cells. In marked contrast, cyclin A is shown to be degraded within metaphase and cyclin B just at the transition to anaphase in HeLa and two transformed cell lines, derivatives of normal NRK and REF52. These results further support the notion that cyclin destruction might be not correlated with anaphase onset in normal cells and highlight a significant difference in the fate of mitotic cyclins between transformed and non-transformed cells.
Gap junctions and cell polarity: Connexin32 and connexin43 expressed in polarized thyroid epithelial cells assemble into separate gap junctions, which are located in distinct regions of the lateral plasma membrane domain
Authors: Guerrier Annie , Fonlupt Pierre , Morand Isabelle , Rabilloud Rachida , Audebet Christine , Krutovskikh Vladimir , Gros Daniel , Rousset Bernard , Munari-Silem Yvonne
Published: April 2021

Epithelial cells of the thyroid gland present an uncommon connexin expression pattern, they coexpress connexin32 and connexin43. In the present work, we have analyzed the membrane distribution of these two connexins to determine: (i) whether they co-assemble in the same gap junctions or form separate gap junctions; and (ii) whether their location is somehow related to the thyroid cell polarity. Immunofluorescence analyses of the localization of the two connexins in thyroid tissue sections revealed that connexin32 and connexin43 are located in different regions of the plasma membrane. We further analyzed the location of each of the two connexins with regard to that of the tight junction-associated protein, ZO1. Laser scanning confocal microscope observations of connexin32 or connexin43 and ZO1 double-immunolabelled thyroid cells, gave evidence for a separate localization of gap junctions made of each of these two connexins. Connexin32 gap junctions appeared as fluorescent spots scattered over the lateral membrane domain, while connexin43 gap junctions formed a meshed network superimposable with that of tight junctions in the subapical region of the cells. Western blot analyses of the distribution of connexins in thyroid plasma membrane subfractions obtained by ultracentrifugation on a sucrose gradient led to the identification of membrane sub-populations enriched in either connexin32 gap junctions or connexin43 gap junctions. Connexin32 gap junctions and connexin43 gap junctions were found to differ in their resistance to solubilization by N-lauroylsarcosine. Increasing concentrations of this detergent from 0.12% to 0.42% caused a progressive solubilization of connexin43 while connexin32 remained membrane-bound. These data demonstrate that connexin32 and connexin43, co-expressed by polarized thyroid cells, form separate gap junctions with distinct membrane location, gap junctions made up of connexin43 being precisely located within tight junctions. We report here, for the first time, a relationship between connexin distribution and cell polarity.
Inhibition of regulated catecholamine secretion from PC12 cells by the Ca2+/Calmodulin kinase II inhibitor KN-62
Authors: Schweitzer Erik S. , Sanderson Michael J. , Wasterlain C. G.
Published: April 2021

When stimulated by the cholinergic agonist carbachol, PC12 cells rapidly secrete a large fraction of the intracellular catecholamines by exocytotic release from the large dense-core secretory vesicles in a Ca2+-dependent manner. To investigate whether Ca2+/calmodulin kinase II plays a role in the regulated secretion of catecholamines, we examined the effect of the specific Ca2+/calmodulin kinase II inhibitor KN-62 on the carbachol-induced release of norepinephrine from PC12 cells. Approximately 50% of the regulated release of norepinephrine, stimulated either by carbachol or direct depolarization, was inhibited by pretreatment with KN-62, while the remaining 50% was resistant to KN-62 and therefore independent of Ca2+/calmodulin kinase II. In contrast, H7, an inhibitor of protein kinase C, had no effect on any of the stimulated release. FURA 2 imaging experiments demonstrated that KN-62 does not act by blocking the stimulation-induced increase in intracellular [Ca2+]. The most likely model consistent with these data is that all the dense-core vesicles fuse with the plasma membrane in a Ca2+-dependent process, but that approximately 50% of the vesicles require an additional step that is dependent on the action of Ca2+/calmodulin kinase II. This step occurs between the influx of Ca2+ and the fusion of vesicle membranes with the plasma membrane, and may be analogous to the Ca2+/calmodulin kinase II phosphorylation of synapsin which mobilizes small, clear synaptic vesicles for exocytosis at the synapse.
Transcriptional activation of endothelial cell integrin αv by protein kinase C activator 12(S)-HETE
Authors: Tang Dean G. , Diglio Clement A. , Bazaz Rajesh , Honn Kenneth V.
Published: April 2021

Previous work demonstrated that 12(S)-HETE [12(S)hydroxyeicosatetraenic acid], a lipoxygenase metabolite of arachidonic acid, stimulates the surface expression of integrin αvβ3 on mouse lung vascular endothelial cells (CD clone 3) in a post-transcriptional and protein kinase C (PKC)-dependent fashion. In this study we examined the effect of 12(S)-HETE on the expression of integrin receptors αvβ3 and α5β1 in a different clone of a mouse endothelial cell population derived from lung microvasculature (designated CD clone 4). The results indicated that 12(S)-HETE transcriptionally activates the gene expression of integrin αv as assessed by quantitative reverse transcription/polymerase chain reaction/Southern hybridization, RNase protection assay, solution hybridization, and northern blotting. The induction of αv mRNA occurred within 1 hour, peaked at ∼4 hours (2-to 4-fold increase), persisted for up to 16 hours, and thereafter gradually declined. The PKC activator phorbol 12-myristate 13-acetate (PMA) induced the αv mRNA, in a similar way. 12(S)-HETE treatment did not, in contrast, alter the mRNA levels of integrin subunit α5 or β1. The induction of αv mRNA appeared to be protein synthesis-independent, since cycloheximide did not alter the 12(S)-HETE effect. 12(S)-HETE also did not appear to alter the mRNA halflife of αv. On the other hand, 12(S)-HETE-induced increase in αv mRNA levels was PKC-dependent, since pretreatment of CD clone 4 cells with calphostin C significantly inhibited 12(S)-HETE-increased αv mRNA. Nuclear runoff experiments revealed that the increase in αv mRNA results from an enhanced gene transcription. Facilitated αv gene transcription resulted in an increased surface expression of αvβ3 protein, which resulted in an increased cell adhesion to vitronectin. The above observations, in conjunction with our previous experimental data, suggest that 12(S)-HETE may employ diverse mechanisms to stimulate the integrin αvβ3 expression in vascular endothelial cells, which could play important roles in tumor cell adhesion, angiogenesis, hemostasis, and many other vascular events.
Interactions of macromolecules with the mammalian cell surface
Authors: Wall Jonathan , Ayoub Fayad , O”Shea Paul
Published: April 2021

The characterisation of fluoresceinphosphatidylethanol amine (FPE) as a real-time indicator of the electrostatic nature of the cell membrane surface is described. The con ditions appropriate for the labelling of cell membranes and the implementation of FPE as a tool to monitor the inter actions of various proteins and peptides with membranes are outlined. Some complications attributed to the ery throcyte glycocalyx are examined. In addition it is shown using neuraminidase as an example, that some types of enzyme-catalysed reactions on the cell surface may be monitored in real time. It is also shown that information concerning the binding of several proteins such as serum albumin and monoclonal antibodies are accessible with this technique. The albumin in particular is shown to exhibit a saturation of binding, the analysis of which indicates that the dissociation constant for erythrocytes was determined to be 8 µM and for lymphocytes to be almost 3 µM. On the basis of this comparison together with artificial membranes, the membrane protein components of the lym phocyte surface are implicated in the binding of albumin or the erythrocyte membrane proteins reduce the affinity of the cell surface for albumin.
Expression and glycosylation of the filamentous brush border glycocalyx (FBBG) during rabbit enterocyte differentiation along the crypt-villus axis
Authors: Maury J. , Bernadac A. , Rigal A. , Maroux S.
Published: April 2021

The filamentous brush border glycocalyx forming the “enteric surface coat” of the intestinal epithelium is composed in rabbits of a 400 kDa mucin-type glycoprotein, which was purified using the 3A4 monoclonal antibody. This monoclonal antibody recognizes a filamentous brush border glycocalyx-specific glycosidic structure containing an O-acetylated sialic acid, which is absent from all the other glycoproteins in the epithelium, with the exception of certain goblet cell mucins. Here we establish that only 50% of the rabbits tested synthesized this glycosidic structure. Upon immunolabeling surface epithelia and sections of jejunum from these rabbits, the carbohydrate epitope rec ognized by the 3A4 mAb was found to be present on the fil amentous brush border glycocalyx of a variable number of enterocytes, which were patchily distributed over all the villi. This heterogeneous expression of 3A4 antigenicity, which was also observed in the crypts, suggests the existence of differences between the patterns of differen tiation of enterocytes, which results in the expression of different pools of glycosyltransferases and/or acetyl trans ferases. In mature enterocytes, the 3A4 determinants were present only on the filamentous brush border glycocalyx, which is anchored solely to the membrane microdomain at the tip of brush border microvilli. However, expression of 3A4 antigenicity begins in the median third of crypts, in enterocytes with a short, thin brush border devoid of apical filamentous brush border glycocalyx. Here the 3A4 epitopes were present over the whole brush border membrane. A few cells higher up, the hyperpolarized expression of filamentous brush border glycocalyx, i.e. its segregation at the tip of the microvilli, began and both the apical filamentous brush border glycocalyx and the whole brush border membrane were labeled with the 3A4 mAb. The labeling of the lateral membrane of microvilli gradually decreased and then disappeared during the migration of the cells to the upper part of the crypts. The 3A4 structure was also detected in some of the granules of some rare goblet cells in the vicinity of the most deeply located labeled enterocytes, whereas no labeled goblet cells were ever observed at higher cell locations.
Mitotic disassembly of the Golgi apparatus in vivo
Authors: Misteli Tom , Warren Graham
Published: April 2021

Populations enriched in prophase cells were obtained either by using a cell line with a temperature-sensitive mutation in the mitotic kinase, p34cdc2, or by treating cells with olomoucine, an inhibitor of this kinase. Both methods resulted in efficient and reversible block of the cells at the G2/M boundary. After cells were released from the cell cycle block, the morphological changes to the Golgi apparatus were characterised using both quantitative con ventional electron microscopy and immuno-gold microscopy. The early mitotic phases were divided into six stages (G2 to pro-metaphase) based on the morphology of the nucleus. During prophase the cross-sectional length of Golgi stacks decreased prior to unstacking. At the same time, small vesicular profiles, typically 50-70 nm in diameter, accumulated in the vicinity of the stacks. The dis appearance of Golgi stacks was accompanied by the transient appearance of tubular networks. By the time cells entered prometaphase, the stacks had completely disas sembled and only clusters consisting of Golgi vesicles and short tubular elements were left. When cells were released from the G2/M boundary and pulsed briefly with [AlF4]- to prevent uncoating of transport vesicles, vesicular profiles with a morphology reminiscent of COP-coated vesicles appeared. These vesicular profiles were either associated with Golgi stacks or, at later stages, with clusters, but were formed at all stages of disassembly. Together these results provide further support for our model that continued budding of vesicles from the rims of Golgi cisternae is at least partly responsible for the disassembly of the Golgi apparatus.
Induction and regulation of connexin26 by glucagon in primary cultures of adult rat hepatocytes
Authors: Kojima Takashi , Mitaka Toshihiro , Shibata Yosaburo , Mochizuki Yohichi
Published: April 2021

In the adult rat hepatocyte, the gap junction proteins consist of a major component, connexin32 (Cx32) and a minor component, connexin26 (Cx26). Although we recently reported our success in inducing and maintaining Cx32 in adult rat hepatocytes cultured in serum-free L-15 medium supplemented with epidermal growth factor and 2% dimethyl sulfoxide, it was very difficult to induce Cx26 in the primary hepatocytes. In the present study, we found that the addition of 10−7 M glucagon into the culture medium could dramatically induce Cx26 mRNA and protein. Although the expression of Cx32 mRNA was also influenced by glucagon, the increase of the expression was small. Immunocytochemically, Cx26-positive spots were observed between most adjacent cells and were co-localized with the Cx32-positive spots. We also examined whether 0.5 mM dibutyl cyclic AMP could induce expression of Cx26 in the cells. The effect of dexamethasone on the expression of Cx26 mRNA compared to that of Cx32 mRNA was examined. For the induction and maintenance of Cx26 mRNA, more than 10−7 M dexamethasone was necessary in this culture. These results suggest that expression of Cx26 in hepatocytes may be regulated by the concentrations of glucagon and glucocorticoid hormones.
Protein translation components are colocalized in granules in oligodendrocytes
Authors: Barbarese Elisa , Koppel Dennis E. , Deutscher Murray P. , Smith Candra L. , Ainger Kevin , Morgan Frank , Carson John H.
Published: April 2021

The intracellular distribution of various components of the protein translational machinery was visualized in mouse oligodendrocytes in culture using high resolution fluorescence in situ hybridization and immunofluorescence in conjunction with dual channel confocal laser scanning microscopy. Arginyl-tRNA synthetase, elongation factor 1a, ribosomal RNA, and myelin basic protein mRNA were all co-localized in granules in the processes, veins and membrane sheets of the cell. Colocalization was evaluated by dual channel cross correlation analysis to determine the correlation index (% colocalization) and correlation distance (granule radius), and by single granule ratiometric analysis to determine the distribution of the different components in individual granules. Most granules contained synthetase, elongation factor, ribosomal RNA and myelin basic protein mRNA. These results indicate that several different components of the protein synthetic machinery, including aminoacyl-tRNA synthetases, elongation factors, ribosomes and mRNAs, are colocalized in granules in oligodendrocytes. We propose that these granules are supramolecular complexes containing all of the necessary macromolecular components for protein translation and that they represent a heretofore undescribed subcellular organization of the protein synthetic machinery. This spatial organization may increase the efficiency of protein synthesis and may also provide a vehicle for transport and localization of specific mRNAs within the cell.
An anti-αv-integrin antibody that blocks integrin function inhibits the development of a human melanoma in nude mice
Authors: Mitjans Francesc , Sander Diane , Adán Jaime , Sutter Arne , Martinez Josep M. , Jäggle Carlota-S. , Moyano Josep M. , Kreysch Hans-Georg , Piulats Jaime , Goodman Simon L.
Published: April 2021

A series of murine monoclonal antibodies were raised against purified human αvβ3 integrin and against M21 human melanoma cells. Five notable hybridomas were identified by ELISA on purified integrins, and the isolated antibodies bound the αv-chain. These antibodies, 17E6, 20A9, 23G5, 14D9.F8 and 10G2, recognised the extracellular domains of the integrin, and were shown to be reactive in FACS, immunoprecipitation, ELISA, and ELISA on fixed cells with M21, M21-L4, and UCLA-P3, but not with the αv-deficient M21-L or M21-L-IIb (M21-L transfected with GpIIb integrin).

One antibody, 17E6, strongly perturbed cell attachment mediated by αv integrins, reacting at least with αvβ3, αvβ5, and αvβ1, and strongly inhibiting cell attachment to αv-ligands vitronectin and fibronectin with an IC50 of ∼0.1 µg ml-1. Furthermore, 17E6 at this concentration could induce cell retraction from the substrate, while LM609 (anti-αvβ3) and control antibody 14E2 (anti-200 kDa melanoma surface protein) at 1,000-fold higher concentrations had minimal effects on cell morphology. The action of 17E6 was reversible and was not due to toxic effects: in vitro 17E6 at 0.1 mg ml-1 did not affect either cell proliferation or DNA synthesis.

In two nude-mouse tumour models, subcutaneous tumour development and a lung colonisation (‘experimental metastasis’) assay, injection of 17E6 strongly inhibited tumour development, while isotype-matched controls had no effect. There was no obvious mechanism of cell or of complement-mediated tumour cytotoxicity; the antibody did not mediate ADCC or AECDC, or complement fixation. The data strongly support previous studies which have indicated the importance of αv-integrins, and especially αvβ3, in the tumour progression of human melanoma.
Short- and long-term mechanisms of tau regulation in PC12 cells
Authors: Sadot Einat , Barg Jacob , Rasouly David , Lazarovici Phillip , Ginzburg Irith
Published: April 2021

Induction by nerve growth factor of neurite outgrowth in PC12 cells is transcription-dependent and is associated with the accumulation of tau protein. It was recently shown that short-term treatment with staurosporine, a protein kinase alkaloid inhibitor, induced an elevation of tau protein levels and outgrowth of stable neurites. In this study, we analyzed the mechanism(s) by which nerve growth factor and staurosporine exert their effects on tau levels. We demonstrate that nerve growth factor affects tau mRNA stability, thus contributing to the observed increase in tau mRNA levels. On the other hand, tau mRNA levels were not affected by the treatment with staurosporine. We also demonstrate that the phosphorylation of tau protein was reduced after treatment of PC12 cells with nerve growth factor or staurosporine, as shown by immunoblot analysis using specific antibodies and alkaline phosphatase treatment. Thus, regulation of tau levels by nerve growth factor appears to be mediated by transcriptional, post-transcriptional and posttranslational steps, whereas the effect of staurosporine on tau levels may be attributed to its effect on the state of phosphorylation of the protein.
Protocadherins and diversity of the cadherin superfamily
Authors: Suzuki Shintaro T.
Published: April 2021

Recent cadherin studies have revealed that many cadherins and cadherin-related proteins are expressed in various tissues of different multicellular organisms. These proteins are characterized by the multiple repeats of the cadherin motif in their extracellular domains. The members of the cadherin superfamily are divided into two groups: classical cadherin type and protocadherin type. The current cadherins appear to have evolved from a protocadherin type. Recent studies have proved the cell adhesion role of classical cadherins in embryogenesis. In contrast, the bio-logical role of protocadherins is elusive. Circumstantial evidence, however, suggests that protocadherins are involved in a variety of cell-cell interactions. Since protocadherins, and many other new cadherins as well, have unique properties, studies of these cadherins may provide insight into the structure and biological role of the cadherin superfamily.
Mutations in the fission yeast silencing factors clr4
+ and rik1
+ disrupt the localisation of the chromo domain protein Swi6p and impair centromere function
Authors: Ekwall Karl , Nimmo Elaine R. , Javerzat Jean-Paul , Borgstrøm Britta , Egel Richard , Cranston Gwen , Allshire Robin
Published: April 2021

Transcriptional silencing is known to occur at centromeres, telomeres and the mating type region in the nucleus of fission yeast, Schizosaccharomyces pombe. Mating-type silencing factors have previously been shown also to affect transcriptional repression within centromeres and to some extent at telomeres. Mutations in the clr4+, rik1+ and swi6+ genes dramatically reduce silencing at certain centromeric regions and cause elevated chromosome loss rates. Recently, Swi6p was found to co-localise with the three silent chromosomal regions. Here the involvement of clr4+, rik1+ and swi6+ in centromere function is investigated in further detail. Fluorescence in situ hybridisation (FISH) was used to show that, as in swi6 mutant cells, centromeres lag on late anaphase spindles in clr4 and rik1 mutant cells. This phenotype is consistent with a role for these three gene products in fission yeast centromere function. The Swi6 protein was found to be delocalised from all three silent chromosomal regions, and dispersed within the nucleus, in both clr4 and rik1 mutant cells. The phenotypic similarity observed in all three mutants is consistent with the products of both the clr4+ and rik1+ genes being required to recruit Swi6p to the centromere and other silent regions. Mutations in clr4, rik1 and swi6 also result in elevated sensitivity to reagents which destabilise microtubules and show a synergistic interaction with a mutation in the β-tubulin gene (nda3). These observations suggest that clr4+ and rik1+ must play a role in the assembly of Swi6p into a transcriptionally silent, inaccessible chromatin structure at fission yeast centromeres which is required to facilitate interactions with spindle microtubules and to ensure normal chromosome segregation.
Cell surface expression of a functional rubella virus E1 glycoprotein by addition of a GPI anchor
Authors: Bernasconi Eric , Fasel Nicolas , Wittek Riccardo
Published: April 2021

Rubella virus (RV) envelope glycoproteins E1 and E2 are targeted to the Golgi as heterodimers. While E2 contains a transmembrane Golgi retention signal, E1 is arrested in a pre-Golgi compartment in the absence of E2, and appears to require heterodimerization in order to reach the Golgi. Various forms of E1 with deletions in the ectodomain or lacking the cytoplasmic (CT) and transmembrane (TM) domains, as well as the 29 C-terminal amino acid residues of the ectodomain were also retained intracellularly. We therefore investigated the possibility of targetting E1 to the plasma membrane by addition of a glycosylphosphatidylinositol (GPI) anchor. We found that E1GPI was transported to the cell surface where it retained the hem-adsorption activity characteristic of the wild-type E1/E2 heterodimer. Furthermore, coexpression of a mammalian GPI-specific phospholipase D (GPI-PLD) resulted in the release of E1GPI and in constitutive expression of a soluble form of E1. This study thus demonstrates that the GPI anchor has a dominant effect over the E1 pre-Golgi retention signal and that E1 is sufficient for hemadsorption.
The osteoclast generation: an in vitro and in vivo study with a genetically labelled avian monocytic cell line
Authors: Solari F. , Flamant F. , Cherel Y. , Wyers M. , Jurdic P.
Published: April 2021

Osteoclasts are multinucleate giant cells responsible for bone resorption. Osteoclast precursors are hematopoietic mononucleate cells, which give rise to osteoclasts after fusion. Nevertheless, the precise stage of differentiation where osteoclast precursors diverge from other hematopoi-etic lineages is still debated. We describe here both in vitro and in vivo approaches to the study of the osteoclast differ-entiation pathway.

We used cells of the BM2 avian monocytic cell line, which are able to differentiate into macrophages both in vitro and in vivo. In order to follow the progeny of BM2 monocytes, we have derived a BM2 cell clone expressing the nlslacZ gene (BM2nlslacZ) which has still retained the main features of the parental cell line.

In vitro, when BM2nlslacZ cells were triggered toward macrophages, they participated in the formation of multi-nucleate osteoclast-like cells as seen by their blue nuclei. Furthermore, when BM2nlslacZ cells were injected into the blood stream of chicken embryos, they could give rise to blue nucleate macrophages in the bone marrow, as well as to osteoclasts with blue nuclei in bone.

Finally, we have shown that fusion of tagged mononucleate precursor cells not only occurs with other mononucleate precursor cells but also with mature multinucleate osteoclasts.

This work shows that cells already engaged in the late stages of the monocytic differentiation pathway are able to differentiate into osteoclasts and that osteoclast divergence takes place after the monocyte stage.
Localisation of the Ki-67 antigen within the nucleolus: Evidence for a fibrillarin-deficient region of the dense fibrillar component
Authors: Kill Ian R.
Published: April 2021

The Ki-67 antigen is detected in proliferating cells in all phases of the cell division cycle. Throughout most of inter-phase, the Ki-67 antigen is localised within the nucleolus. To learn more about the relationship between the Ki-67 antigen and the nucleolus, we have compared the distribution of Ki-67 antibodies with that of a panel of antibodies reacting with nucleolar components by confocal laser scanning microscopy of normal human dermal fibroblasts in interphase stained in a double indirect immunofluor-escence assay. During early G1, the Ki-67 antigen is detected at a large number of discrete foci throughout the nucleoplasm, extending to the nuclear envelope. During S-phase and G2, the antigen is located in the nucleolus. Double indirect immunofluorescence studies have revealed that during early to mid G1 the Ki-67 antigen is associated with reforming nucleoli within discrete domains which are distinct from domains containing two of the major nucleolar antigens fibrillarin and RNA polymerase I.

Within mature nucleoli the Ki-67 antigen is absent from regions containing RNA polymerase I and displays only partial co-localisation within domains containing either fib-rillarin or B23/nucleophosmin. Following disruption of nucleolar structure, induced by treatment of cells with the drug 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole or with actinomycin D, the Ki-67 antigen translocates to nucleoplasmic foci which are associated with neither fib-rillarin nor RNA polymerase I. However, in treated cells the Ki-67 Ag remains associated with, but not co-localised to, regions containing B23/nucleophosmin. Our observa-tions suggest that the Ki-67 antigen associates with a fib-rillarin-deficient region of the dense fibrillar component of the nucleolus. Integrity of this region is lost following either nucleolar dispersal or nucleolar segregation.
Mutations which block the binding of calmodulin to Spc110p cause multiple mitotic defects
Authors: Stirling Douglas A. , Rayner Timothy F. , Prescott Alan R. , Stark Michael J. R.
Published: April 2021

We have generated three temperature-sensitive alleles of SPC110, which encodes the 110 kDa component of the yeast spindle pole body (SPB). Each of these alleles carries point mutations within the calmodulin (CaM) binding site of Spc110p which affect CaM binding in vitro; two of the mutant proteins fail to bind CaM detectably (spc110-111, spc110-118) while binding to the third (spc110-124) is tem-perature-sensitive. All three alleles are suppressed to a greater or lesser extent by elevated dosage of the CaM gene (CMD1), suggesting that disruption of CaM binding is the primary defect in each instance. To determine the consequences on Spc110p function of loss of effective CaM binding, we have therefore examined in detail the progression of synchronous cultures through the cell division cycle at the restrictive temperature. In each case, cells replicate their DNA but then lose viability. In spc110-124, most cells duplicate and partially separate the SPBs but fail to generate a functional mitotic spindle, a phenotype which we term ‘abnormal metaphase’. Conversely, spc110-111 cells initially produce nuclear microtubules which appear well-organised but on entry into mitosis accumulate cells with ‘broken spindles’, where one SPB has become completely detached from the nuclear DNA. In both cases, the bulk of the cells suffer a lethal failure to segregate the DNA.
Fibronectin regulates calvarial osteoblast differentiation
Authors: Moursi Amr M. , Damsky Caroline H. , Lull Jonathan , Zimmerman Deborah , Doty Stephen B. , Aota Shin-ichi , Globus Ruth K.
Published: April 2021

The secretion of fibronectin by differentiating osteoblasts and its accumulation at sites of osteogenesis suggest that fibronectin participates in bone formation. To test this directly, we determined whether fibronectin-cell interactions regulate progressive differentiation of cultured fetal rat calvarial osteoblasts. Spatial distributions of α5 integrin subunit, fibronectin, osteopontin (bone sialoprotein I) and osteocalcin (bone Gla-protein) were similar in fetal rat calvaria and mineralized, bone-like nodules formed by cultured osteoblasts. Addition of anti-fibronectin antibodies to cultures at confluence reduced subsequent formation of nodules to less than 10% of control values, showing that fibronectin is required for normal nodule morphogenesis. Anti-fibronectin antibodies selectively inhibited steady-state expression of mRNA for genes associated with osteoblast differentiation; mRNA levels for alkaline phosphatase and osteocalcin were suppressed, whereas fibronectin, type I collagen and osteopontin were unaffected. To identify functionally relevant domains of fibronectin, we treated cells with soluble fibronectin fragments and peptides. Cell-binding fibronectin fragments (type III repeats 6-10) containing the Arg-Gly-Asp (RGD) sequence blocked both nodule initiation and maturation, whether or not they contained a functional synergy site. In contrast, addition of the RGD-containing peptide GRGDSPK alone did not inhibit nodule initiation, although it did block nodule maturation. Thus, in addition to the RGD sequence, other features of the large cell-binding fragments contribute to the full osteogenic effects of fibronectin. Nodule formation and osteoblast differentiation resumed after anti-fibronectin antibodies or GRGDSPK peptides were omitted from the media, showing that the inhibition was reversible and the treatments were not cytotoxic. Outside the central cell-binding domain, peptides from the IIICS region and antibodies to the N terminus did not inhibit nodule formation. We conclude that osteoblasts interact with the central cell-binding domain of endogenously produced fibronectin during early stages of differentiation, and that these interactions regulate both normal morphogenesis and gene expression.
Differential expression and cell envelope incorporation of small proline-rich protein 1 in different cornified epithelia
Authors: Jarnik Michal , Kartasova Tonja , Steinert Peter M. , Lichti Ulrike , Steven Alasdair C.
Published: April 2021

In the final stages of terminal differentiation in the epidermis and other squamous epithelia, a ∼15 nm thick protein layer called the cornified cell envelope (CE) assembles on the keratinocytes’ inner surface. Its constituent proteins are covalently crosslinked by the action of transglutaminases. Recent studies have indicated that the expression of CE precursor proteins may vary in different tissues. To investigate such variations further, we have studied the CEs of two different keratinizing epithelia of mouse: epidermis and forestomach, with particular focus on their contents of loricrin and the small proline-rich proteins (SPRs). To this end, we have applied electron microscopic immunocytochemistry and estimated the CE protein compositions by mathematical modeling of their amino acid compositions. Ultrastructurally, forestomach resembles the epidermis in having well defined cornified and granular layers. Minor but significant differences are: in forestomach, striated material resembling lamellar granules is intercalated between the cornified squames; and in forestomach granular layer cells, loricrin-containing L-granules are more abundant, and filaggrin-containing F-granules less abundant than in epidermis.

In forestomach, dense labeling with anti-SPR1 antibody was observed at the margin of cornified layer cells; and in the granular layer, diffuse but positive labeling of both cytoplasm and nucleus. In contrast, epidermis was uniformly negative. Isolated forestomach CEs (but not epidermal CEs), labeled positively on the cytoplasmic side, consistent with the presence of covalently crosslinked SPR1. Our compositional analysis predicts the content of loricrin in forestomach CEs to be very high (∼65%), as in the epidermis, and accompanied by ∼18% content of total SPRs. Of these, a substantial proportion should be SPR1, according to our immunolabeling data. In contrast, epidermal CEs are calculated to have a much lower amount of SPRs or SPR-like proteins (∼8%), with a negligible content of SPR1. Thus both kinds of CEs have loricrin as their major constituent but differ in their respective complements of SPRs, which are thought to inter-connect loricrin molecules in the final phase of CE assembly. Applying a basic concept of materials science, it may be that the observed differences in their SPR contents reflect differences in the mechanical and chemical properties required for the function of the respective CEs.
Active RNA polymerases are localized within discrete transcription ‘factories’ in human nuclei
Authors: Iborra Francisco J. , Pombo Ana , Jackson Dean A. , Cook Peter R.
Published: April 2021

Nascent transcripts in permeabilized HeLa cells were elongated by ∼30-2,000 nucleotides in Br-UTP or biotin-14-CTP, before incorporation sites were immunolabelled either pre- or post-embedding, and visualized by light or electron microscopy. Analogues were concentrated in ∼2,100 (range 2,000-2,700) discrete sites attached to a nucleoskeleton and surrounded by chromatin. A typical site contained a cluster (diameter 71 nm) of at least 4, and probably about 20, engaged polymerases, plus associated transcripts that partially overlapped a zone of RNA polymerase II, ribonucleoproteins, and proteins rich in thiols and acidic groups. As each site probably contains many transcription units, these results suggest that active polymerases are confined to these sites, which we call transcription ‘factories’. Results are consistent with transcription occurring as templates slide past attached polymerases, as nascent RNA is extruded into the factories.
Dynamics of calcium regulation in Paramecium and possible morphogenetic implication
Authors: Prajer Malgorzata , Fleury Anne , Laurent Michel
Published: April 2021

This paper is the first report of the use of a fluorescent indicator (Dextran-coupled calcium green-1) for imaging of cytosolic free calcium in ciliate cells. Using this technique in Paramecium, we show that a very transient increase in the mean intracellular calcium concentration accompanied exocytosis. It has long been postulated based on indirect experimental evidence, that a calcium wave which would spread across the cortex at the time of cell division, would be the primary event that triggers morphogenesis in these species. We theoretically show that a unifying interpretation can be given for the possible occurrence of a single wave and that of multiple oscillations of cytosolic calcium: both of which correspond to two different behaviors of the same dynamic system. Experimental conditions allowing the visualization of possible calcium periodicities in the interphase Paramecium cell are much more easily fulfilled than those permitting the observation of a single wave at the time of cell division. Hence, experiments were performed on interphase cells. After microinjection of calcium indicator into a mutant strain which is defective in exocytosis, we observed Ca2+ oscillations with a period close to 2 minutes. Hence, we conclude that Paramecium possesses all the dynamic elements required to generate, at the time of cell division, a morphogenetic calcium wave.