bluuebunny/crossref_metadata_2025_split huggingface dataset:
Gap junction regulation in the uterus and ovaries of immature rats by estrogen and progesterone
Authors: Risek Boris , Klier F. George , Phillips A. , Hahn D. W. , Gilula N. B. 
Published: April 2021

ABSTRACT
The effects of estrogen (E2) and progesterone (P) were examined on the expression levels of multiple gap junction (GJ) gene products (α1=Cx43, β1=Cx32, β2=Cx26) in the uterus and ovaries of immature rats by immunohisto- chemistry, electron microscopy and northern blot analysis. E2 induced the expression of α1 connexin in the uterus (specifically in the myometrium and in endometrial stroma proximal to luminal epithelium) and ovaries. The E2- induced α1 expression was completely suppressed by P in the uterus, but only partly in ovaries. Steroid hormones also modulated the quantity, size, and distribution of β1 and β2 containing junctional plaques along lateral cell borders in polarized luminal and glandular uterine epithelia. Small GJs were detected at basolateral regions in proliferative luminal epithelium following administration of E2. In contrast, large GJs were localized at subapical- lateral cell borders of the secretory epithelium following P- treatment. The co-administration of E2+P had a synergis- tic effect on β1 and β2 expression in the luminal epithelium, but an inhibitory effect on β2 expression in glandular epithelium. Myometrial GJs were detected in freeze- fracture replicas as aggregates containing regularly arranged particles with particle free zones. In contrast, GJs in secretory epithelium contained particles which were arranged in a non-crystalline fashion. These GJs contained domains of mixed and segregated β1 and β2 antigens within a single plaque as revealed by laser scanning confocal microscopy analysis of immuno-double-labeled secretory epithelium. The demonstration of segregated antigens within a single GJ plaque indicates the possibility of multiple channel populations formed by homo-oligomeric connexons. These results suggest that different connexins can be differentially regulated by steroid hormones in different cell types, and that the same steroid hormone can have different effects on the same connexin in different cell types.
Mapping of adherens junction components using microscopic resonance energy transfer imaging
Authors: Kam Zvi , Volberg Tova , Geiger Benjamin 
Published: April 2021

ABSTRACT
Quantitative microscopic imaging of resonance energy transfer (RET) was applied for immunological high resolution proximity mapping of several cytoskeletal components of cell adhesions. To conduct this analysis, a micro-scopic system was developed, consisting of a highly stable field illuminator, computer-controlled filter wheels for rapid multiple-color imaging and a sensitive, high resolu-tion CCD camera, enabling quantitative data recording and processing. Using this system, we have investigated the spatial inter-relationships and organization of four adhesion-associated proteins, namely vinculin, talin, α-actinin and actin. Cultured chick lens cells were double labeled for each of the junctional molecules, using fluores-cein- and rhodamine-conjugated antibodies or phalloidin. RET images were acquired with fluorescein excitation and rhodamine emission filter setting, corrected for fluorescein and rhodamine fluorescence, and normalized to the fluo-rescein image. The results pointed to high local densities of vinculin, talin and F-actin in focal adhesions, manifested by mean RET values of 15%, 12% and 10%, respectively. On the other hand, relatively low values (less than 1%) were observed following double immunofluorescence labeling of the same cells for α-actinin. Double indirect labeling for pairs of these four proteins (using fluorophore-conjugated antibodies or phalloidin) resulted in RET values of 5% or lower, except for the pair α-actinin and actin, which yielded significantly higher values (13-15%). These results suggest that despite their overlapping staining patterns, at the level of resolution of the light microscope, the plaque proteins vinculin and talin are not homogeneously interspersed at the molecular level but form segregated clusters. α-Actinin, on the other hand, does not appear to form such clusters but, rather, closely interacts with actin. We discuss here the conceptual and applicative aspects of RET measurements and the implications of the results on the subcellular molecular organization of adherens-type junctions.
Identification of intrinsic dimer and overexpressed monomeric forms of γ-tubulin in Sf9 cells infected with baculovirus containing the Chlamydomonas γ-tubulin sequence
Authors: Vassilev Alexei , Kimble Mary , Silflow Carolyn D. , LaVoie Matthew , Kuriyama Ryoko 
Published: April 2021

ABSTRACT
A new member of the tubulin superfamily, γ-tubulin, is localized at microtubule-organizing centers (MTOCs) in a variety of organisms. Chlamydomonas cDNA coding for the full-length sequence of γ-tubulin was expressed in insect ovarian Sf9 cells using the baculovirus expression system. Approximately half of the induced 52 kDa γ-tubulin was recovered in the supernatant after centrifugation of Sf9 cell lysates at 18,000 g for 15 minutes. When the cell super-natant was analyzed by FPLC on a Superdex 200 sizing column, Chlamydomonas γ-tubulin separated into two major peaks. The lagging peak contained a monomeric form of γ-tubulin with a sedimentation coefficient of 2.5 S, which interacted with the Superdex column in a salt-dependent manner. The leading peak, with an apparent molecular mass of 900 kDa, corresponded to a molecular chaperonin complex, and TCP1 chaperonin released folded γ-tubulin polypeptide from the complex in the presence of MgATP. The released γ-tubulin monomers were capable of binding to microtubules in vitro and biochemical quanti-ties of active monomers were further purified using a com-bination of size-exclusion and ion-exchange column chro-matography. The endogenous Sf9 cell γ-tubulin migrated faster than Chlamydomonas γ-tubulin with an apparent molecular mass of 49 kDa on gels. Analyses on gel filtration and sucrose density gradient centrifugation showed that, while overexpressed Chlamydomonas γ-tubulin was present in a monomeric form, endogenous γ-tubulin from Sf9 and HeLa cells exists as a dimer. These results may suggest the possibility that γ-tubulin could form a heterodimer with hitherto unknown molecule(s).
Mutants lacking myosin II cannot resist forces generated during multicellular morphogenesis
Authors: Shelden Eric , Knecht David A. 
Published: April 2021

ABSTRACT
We have used fluorescent labeling, confocal microscopy and computer-assisted motion analysis to observe and quantify individual wild-type and myosin II mutant cell behavior during early multicellular development in Dic-tyostelium discoideum. When cultured with an excess of unlabeled wild-type cells, labeled control cells are randomly distributed within aggregation streams, while myosin II mutant cells are found primarily at the lateral edges of streams. Wild-type cells move at average rates of 8.5±4.9 μm/min within aggregation streams and can exhibit regular periodic movement at 3.5 minute intervals; half as long as the 7 minute period reported previously for isolated cells. Myosin II mutants under the same conditions move at 5.0±4.8 μm/min, twice as fast as reported previously for isolated myosin II mutant cells, and fail to display regular periodic movement. When removed from aggregation streams myosin II mutant cells move at only 2.5±2.0 μm/min, while wild-type cells under these conditions move at 5.9±4.5 μm/min. Analysis of cell morphology further reveals that myosin II mutant cells are grossly and dynamically deformed within wild-type aggregation streams but not when removed from streams and examined in isolation. These data reveal that the loss of myosin II has dramatic consequences for cells undergoing multicellular develop-ment. The segregation of mutant cells to aggregation stream edges demonstrates that myosin II mutants are unable to penetrate a multicellular mass of wild-type cells, while the observed distortion of myosin II mutant cells suggests that the cortex of such cells is too flacid to resist forces generated during movement. The increased rate of mutant cell movement and distortion of mutant cell mor-phology seen within wild-type aggregation streams further argues both that movement of wild-type cells within a mul-ticellular mass can generate traction forces on neighboring cells and that mutant cell morphology and behavior can be altered by these forces. In addition, the distortion of myosin II mutant cells within wild-type aggregation streams indicates that myosin is not required for the formation of cell-cell contacts. Finally, the consequences of the loss of myosin II for cells during multicellular development are much more severe than has been previously revealed for isolated cells. The techniques used here to analyze the behavior of individual cells within multicellular aggregates provide a more sensitive assay of mutant cell phenotype than has been previously available and will be generally applicable to the study of motility and cytoskeletal mutants in Dictyostelium.
Stimulation of tyrosine phosphorylation of distinct proteins in response to antibody-mediated ligation and clustering of α3 and α6 integrins
Authors: Jewell Kevin , Kapron-Bras Carolyn , Jeevaratnam Premala , Dedhar Shoukat 
Published: April 2021

ABSTRACT
The interaction of cells with components of the extracellular matrix through their integrin receptors results in the stimulation of tyrosine phosphorylation of several proteins, suggesting that these receptors play a key role in signal transduction. Here we report that antibody-mediated ligation and clustering of α3β1 and α6β1/α6β4 integrins resulted in the stimulation of tyrosine phosphorylation of proteins that are specific for each heterodimer. Thus, ligation and clustering of the α3β1 integrin on human prostate carcinoma cells (PC-3) and human umbilical vein endothelial cells (HUVEC) with anti-α3 antibodies resulted in the stimulation of tyrosine phosphorylation of a 55 kDa protein. In contrast, ligation and clustering of the α6β1 integrin on these cells with anti-α6 antibody resulted in the dramatic stimulation of tyrosine phosphorylation of a 90 kDa protein in addition to a 52 kDa protein, and ligation and clustering of α5β1 on HUVEC did not result in the apparent stimulation of tyrosine phosphorylation of any proteins. Clustering with anti-β1 antibodies triggered the tyrosine phosphorylation of all of these proteins, whereas ligation and clustering of PC-3 cells with an anti-β4 antibody resulted in the tyrosine phosphorylation of a distinct 62 kDa protein. Since the PC-3 cells express both α6β1 and α6β4, these data suggest that these two receptors can transduce distinct signals. All of the phosphorylations could be inhibited by treating the cells with Genistein, a tyrosine kinase inhibitor. Antibody-mediated ligation and clustering of integrins on the two types of cells did not result in the stimulation of tyrosine phosphorylation of pp125 focal adhesion kinase, although this was observed upon cell attachment and spreading on fibronectin, laminin and anti-α3 monoclonal antibody. Collectively, these data demonstrate that cross-linking of different integrin het-erodimers can stimulate tyrosine kinase activities, leading to the phosphorylation of distinct proteins, which are also different from those observed when cells are allowed to spread on a matrix.
The state of actin assembly regulates actin and vinculin expression by a feedback loop
Authors: Bershadsky Alexander D. , Glück Ursula , Denisenko Oleg N. , Sklyarova Tatyana V. , Spector Ilan , Ben-Ze’ev Avri 
Published: April 2021

ABSTRACT
Actin filaments are major determinants of cell shape, motility and adhesion, which control important biological processes including embryonic development and wound healing. These processes are associated with changes in actin assembly, which is regulated by controlling the balance between polymerized and non-polymerized actin. To maintain a significant pool of non-polymerized actin, mechanism(s) linking actin synthesis to its state of poly-merization were proposed. We have studied this relation-ship between actin synthesis and organization by modulat-ing actin assembly using different drugs. Unassembled actin was increased in 3T3 cells using either the Clostrid-ium botulinum C2 toxin, which ADP-ribosylates actin, or by latrunculin A, a Red Sea sponge product, which binds monomeric actin. The synthesis of actin was dramatically reduced in these cells owing to a concomitant decrease in actin RNA level. Similar results were obtained with HeLa cells grown in both monolayer and in suspension, suggest-ing that cell shape changes associated with drug treatment are not the primary cause for the effect on actin synthesis. In contrast, the scrape-loading of 3T3 cells with phalloidin, a stabilizer of polymerized actin that increased the level of assembled actin, resulted in elevated actin synthesis and RNA content. The expression of vinculin, a major component of adhesion plaques and cell-cell junctions, which is involved in actin-membrane associations, was altered in parallel with that of actin in cells treated with these drugs. The decrease in actin RNA resulted from destabilization of actin mRNA in cells where unassembled actin level was elevated. This is suggested by the unchanged transcription of actin in isolated nuclei from drug-treated cells, and by demonstrating that actin mRNA was degraded faster in cells after C2 toxin treatment than in control cells. This feedback control mechanism is mainly confined to the cytoplasm, as it remained active in enucleated cells. The results suggest the existence of an autoregulatory pathway for the expression of actin and other microfilament-associ-ated proteins which is linked to the state of actin polymer-ization in the cell.
The levels of ubiquitinated histone H2A are highly upregulated in transformed human cells: Partial colocalization of uH2A clusters and PCNA/cyclin foci in a fraction of cells in S-phase
Authors: Vassilev Alex P. , Rasmussen Hanne H. , Christensen Erik I. , Nielsen Søren , Celis Julio E. 
Published: April 2021

ABSTRACT
The levels and distribution of ubiquitinated histone H2A (uH2A) have been studied in normal and transformed human cells using a monoclonal antibody (mAb E6C5) that reacts specifically with this ubiquitin conjugate as determined by two-dimensional gel western blotting and microsequencing. Immunoblotting experiments demonstrated that the levels of the protein are highly upregulated in SV40-transformed human fibroblasts (WI-38 SV40) and keratinocytes (K14) relative to their normal counterparts, a finding that was further confirmed by indirect immuno-fluorescence studies of formaldehyde/Triton X-100-treated cells, which showed that about 97% of the transformed cells and 26% of the normal populations reacted with the antibody to yield a fine granular nuclear staining associ-ated with the chromatin. Transformed cells contained in addition clusters of uH2A that were quite abundant and that showed variable size, shape and distribution even within a single cell line. The clusters, which were rare in normal cells, did not colocalize with other known nuclear antigens and may correspond to novel nuclear domains where ubiquitination/deubiquitination takes place. Electron microscopic immunocytochemistry of K14 cells confirmed the existence of the clusters. Double immunoflu-orescence studies of K14 keratinocytes with proliferating cell nuclear antigen (PCNA)/cyclin antibodies, which react with the nuclei of cells engaged in DNA replication, showed partial colocalization of PCNA/cyclin foci and large uH2A clusters in about 14% of the S-phase cells, and these cor-responded mainly to late S-phase cells. Inhibition of DNA replication with hydroxyurea resulted in an overall increase in the intensity of the uH2A staining as well as in a more clear colocalization of uH2A clusters and PCNA/cyclin foci. Taken together, the results support the contention that uH2A plays a role at some stage of DNA replication.
αvβ1 is a receptor for vitronectin and fibrinogen, and acts with α5β1 to mediate spreading on fibronectin
Authors: Marshall John F. , Rutherford Deborah C. , McCartney Alison C. E. , Mitjans Francesc , Goodman Simon L. , Hart Ian R. 
Published: April 2021

ABSTRACT
We have shown previously that VUP was the only line out of ten human melanoma lines that failed to express the vit-ronectin receptor αvβ3, but instead expressed αvβ1. Levels of αvβ1 expression were low on parental VUP cells so that iterative sorting by FACS, using an anti-αv antibody (13C2), was utilised to derive sublines with 8-to 10-fold higher amounts of cell surface αvβ1. There was little dif-ference between low (V−) and high (V+) αvβ1-expressing sublines with regard to adherence to collagen type I, collagen type IV or laminin substrata. However, adherence to vitronectin and fibrinogen correlated closely with αvβ1 expression (35-42% adhesion for V(+) lines versus 6-8% adhesion for V− lines on vitronectin, for example). Utilising a high αvβ1-expressing subline (V+B2) we have shown that binding to vitronectin and fibrinogen was inhibited specif-ically by function-blocking antibodies to αv (17E6 and 14D9) and β1 (A11B2). V(+) sublines spread more compared with V(−) sublines on both vitronectin and fibronectin. However, neither α5-nor αv-blocking anti-bodies had any effect on attachment or spreading of V+B2 on fibronectin whereas the combination of α5 (PID6)-and αv(17E6)-blocking antibodies abrogated binding to fibronectin almost completely. This is the first report of an αvβ1 integrin able to recognise vitronectin and fibrinogen, and also cooperate with α5β1 to mediate attachment to and spreading on fibronectin.
Dynamics of fibroblast spreading
Authors: Dunn Graham A. , Zicha Daniel 
Published: April 2021

ABSTRACT
A new technique of microinterferometry permits cellular growth and motile dynamics to be studied simultaneously in living cells. In isolated chick heart fibroblasts, we have found that the non-aqueous mass of each cell tends to increase steadily, with minor fluctuations, throughout the cell cycle. The spread area of each cell also tends to increase during interphase but fluctuates between wide limits. These limits are dependent on the cell’s mass and the upper limit is particularly sharp and directly proportional to mass. From a dynamical point of view, the spread area of a cell is determined by the balance between the rates of two antagonistic processes: protrusion of cellular material into new territory and retraction of material from previously occupied territory. The spatial asymmetry of these processes determines the translocation of the cell. We have found with the chick fibroblasts that the rates of the two processes are generally closely matched to each other and appear to be dependent on the cell’s area of spreading. Both continue incessantly in well spread cells, even when there is no net translocation of the cell, and the lower limit of each activity is directly proportional to spread area. The two processes show different behaviour, however, during changes in the spread area of the cell. Both increases and decreases in area appear to be brought about by changes in the rate of retraction, the rate of protrusion remaining relatively constant. A simple stochastic model based on a limited supply of adhesion molecules can simulate all our observations including the mass-limited spreading, the strong correlation between protrusion and retraction and the retraction-dominated changes in area. We conclude that the spread area of the cell is actively regulated, possibly by a simple automatic mechanism that adjusts the area of spreading in relation to the mass of the cell and controls the rate of protrusion to compensate rapidly for spontaneous fluctuations in retraction.
Rapid bulk replacement of acceptor membrane by donor membrane during phagosome to phagoacidosome transformation in Paramecium
Authors: Allen Richard D. , Bala Nenita P. , Ali Rose F. , Nishida Dawn M. , Aihara Marilynn S. , Ishida Masaki , Fok Agnes K. 
Published: April 2021

ABSTRACT
The extent to which a donor membrane will be retrieved, or if it is retrieved at all after it fuses with an acceptor membrane, is usually difficult to determine. We have studied the dynamics of membrane retrieval in the phagosome system of Paramecium multimicronucleatum using six monoclonal antibody markers. Our previous freeze-fracture and transmission electron microscopic studies have indicated that extensive changes take place in the membrane of the young phagosome as it progresses through its cycle. Using immunofluorescence and immu-noelectron microscopy to determine the times of entry and exit of these individual antigens into the digestive vacuole system, we showed that two hydrophilic antigens, one located on the cytosolic and one on the lumenal side of the discoidal membrane (phagosome membrane precursor), were completely retrieved from the phagosome by tubula-tion within the first three minutes. At the same time that this membrane was retrieved, membrane from a second population of vesicles, the acidosomes, fused with the phagosome to produce the phagoacidosome. On the basis of immunogold localization on cryosections of a total of six antigens, the two specific for phagosome/discoidal vesicle membrane as well as four specific for the acidosome/phagoacidosome membrane, this replacement is total. We also showed that in the presence of the actin-active drug cytochalasin B, this replacement was essentially prevented. However, when vacuole acidification was neu-tralized by ammonium chloride, this replacement process continued unaffected after a lag. Consequently, acidifica-tion, per se, is not required to trigger the replacement of the phagosome membrane. We conclude, on the basis of these studies as well as our previous freeze-fracture studies that during phagoacidosome formation most of the acceptor membrane is retrieved and is replaced by the donor membrane. This shows that at least one cell type possesses the mechanisms needed to substantially replace the membrane of a phagosomal compartment when radical and rapid changes are needed to modulate the digestive and absorptive processes.
Characterization of mucins and proteoglycans synthesized by a mucin-secreting HT-29 cell subpopulation
Authors: Huet G. , Kim I. , Bolos C. de , Lo-Guidice J. M. , Moreau O. , Hemon B. , Richet C. , Delannoy P. , Real F. X. , Degand P. 
Published: April 2021

ABSTRACT
HT-29 cells selected by adaptation to 10−5 M methotrexate (HT-29 MTX) are a homogeneous cell population producing high amounts of mucin. Intracellular mucins and proteoglycans were isolated from these cells by ultra-centrifugation of cell lysates on a cesium bromide gradient and further separated by anion-exchange high perfomance liquid chromatography. The major mucin fraction isolated was characterized by a high hydroxy amino acid content (40%), a Thr/Ser ratio of 1.52, a high sialic acid content, and a low sulfate content. When the same procedure was applied to undifferentiated HT-29 cells, a minor mucin fraction was isolated which appeared less sialylated and more sulfated. The major proteoglycan species identified in HT-29 MTX cells showed less acidic behavior than the pro-teoglycan isolated from HT-29 cells. The effect of brefeldin A and the sugar analog GalNAc-α-O-benzyl on the synthesis and biochemical properties of mucins synthesized by HT-29 MTX cells was examined. Brefeldin A induced the synthesis of more-sulfated mucins. GalNAc-α-O-benzyl treatment resulted in mucins with an increased content of T antigen and a 13-fold lower sialic acid content. We show that GalNAc-α-O-benzyl was metabolized by the cells to Galβ1-3GalNAc-α-O-benzyl, which, in turn, was a potent competitive inhibitor of the O-glycan α-2,3-sialyltrans-ferase. These results illustrate the suitability of HT-29 MTX cells as a model to analyse mucin synthesis and sia-lylation.