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PloS One 2013TFIIB (transcription factor IIB) is a transcription factor that provides a bridge between promoter-bound TFIID and RNA polymerase II, and it is a target of various...
TFIIB (transcription factor IIB) is a transcription factor that provides a bridge between promoter-bound TFIID and RNA polymerase II, and it is a target of various transcriptional activator proteins that stimulate the pre-initiation complex assembly. The localization and/or attachment matrix of TFIIB in the cytoplast is not well understood. This study focuses on the function of TFIIB and its interrelationship with α-tubulins in a mouse model. During oocyte maturation TFIIB distributes throughout the entire nucleus of the germinal vesicle (GV). After progression to GV breakdown (GVBD), TFIIB and α-tubulin co-localize and accumulate in the vicinity of the condensed chromosomes. During the MII stage, the TFIIB signals are more concentrated at the equatorial plate and the kinetochores. Colcemid treatment of oocytes disrupts the microtubule (MT) system, although the TFIIB signals are still present with the altered MT state. Injection of oocytes with TFIIB antibodies and siRNAs causes abnormal spindle formation and irregular chromosome alignment. These findings suggest that TFIIB dissociates from the condensed chromatids and then tightly binds to microtubules from GVBD to the MII phase. The assembly and disassembly of TFIIB may very well be associated with and driven by microtubules. TFIIB maintains its contact with the α-tubulins and its co-localization forms a unique distribution pattern. Depletion of Tf2b in oocytes results in a significant decrease in TFIIB expression, although polar body extrusion does not appear to be affected. Knockdown of Tf2b dramatically affects subsequent embryo development with more than 85% of the embryos arrested at the 2-cell stage. These arrested embryos still maintain apparently normal morphology for at least 96h without any obvious degeneration. Analysis of the effects of TFIIB in somatic cells by co-transfection of BiFC plasmids pHA-Tf2b and pFlag-Tuba1α further confirms a direct interaction between TFIIB and α-tubulins.
Topics: Animals; Antibodies; Antineoplastic Agents; Chromatids; Demecolcine; Embryo, Mammalian; Embryonic Development; Female; Gene Expression Regulation, Developmental; Kinetochores; Meiosis; Mice; Microinjections; Microtubules; Oocytes; Oogenesis; Plasmids; RNA, Small Interfering; Signal Transduction; Spindle Apparatus; Transcription Factor TFIIB; Tubulin
PubMed: 24244602
DOI: 10.1371/journal.pone.0080039 -
Gut Feb 2004Enteric glia protect the integrity of the gut, as loss of enteric glial fibrillary acidic protein (GFAP) positive (+) glia leads to a haemorrhagic jejunoileitis. Crohn's...
BACKGROUND
Enteric glia protect the integrity of the gut, as loss of enteric glial fibrillary acidic protein (GFAP) positive (+) glia leads to a haemorrhagic jejunoileitis. Crohn's disease (CD) and necrotising enterocolitis (NEC) show pathological changes in enteric glia. Therefore, factors controlling GFAP+ enteric glia are of great interest. The aim of the present study was to characterise enteric glia and determine the effect of interleukin 1beta (IL-1beta), interleukin 4 (IL-4), tumour necrosis factor alpha (TNF-alpha), and lipopolysaccharides (LPS) on cultured enteric glia.
METHODS
Dissected rat colon and cultured enteric glia cells were double labelled with anti-GFAP and anti-S-100 antibodies. For regulatory studies, enteric glia cells were treated with cytokines and LPS. Proliferation was assayed using bromodeoxyuridine (BrdU) and mitosis of enteric glia was blocked by demecolcine.
RESULTS
We were able to distinguish GFAP negative (-) from GFAP+ glia subtypes in situ and in primary cultures. Incubation of cells with IL-1beta, TNF-alpha, and LPS led to a significant increase in GFAP+ enteric glia while IL-4 had no effect on GFAP expression. After incubation with IL-1beta, total intracellular GFAP of enteric glia cells was increased. Upregulation of GFAP+ enteric glia could also be observed after stimulation with IL-1beta on blocking mitosis. BrdU uptake in stimulated enteric glia showed no increased proliferation rate.
CONCLUSIONS
Two different types of enteric glia based on GFAP expression exist in the gut. Proinflammatory cytokines and LPS cause a dramatic increase in GFAP+ enteric glia. This suggests that cytokines play an important role in controlling GFAP+ enteric glia which might in turn be involved in modulating the integrity of the bowel during inflammation.
Topics: Animals; Blotting, Western; Cell Division; Cells, Cultured; Colitis; Colon; Cytokines; Dose-Response Relationship, Drug; Enteric Nervous System; Fluorescent Antibody Technique, Indirect; Glial Fibrillary Acidic Protein; Interleukin-1; Interleukin-4; Lipopolysaccharides; Neuroglia; Rats; Rats, Wistar; Tumor Necrosis Factor-alpha
PubMed: 14724154
DOI: 10.1136/gut.2003.012625 -
The Journal of Cell Biology Jun 1983We studied the characteristics of cytoplasmic microtubule reassembly from endogenous tubulin pools in situ using a Brij 58-lysed 3T3 cell system. Cells that were...
We studied the characteristics of cytoplasmic microtubule reassembly from endogenous tubulin pools in situ using a Brij 58-lysed 3T3 cell system. Cells that were pretreated in vivo with colcemid retain endogenous tubulin in the depolymerized state after lysis. When lysed cells were removed from colcemid block and incubated in GTP-PIPES reassembly buffer at pH 6.9, microtubules repolymerized randomly throughout the cytoplasm, appeared to be free-ended and were generally not associated with the centrosomes. However, tubulin could be induced to polymerize in an organized manner from the centrosomes by increasing the pH to 7.6 in the presence of ATP and cAMP. Microtubules polymerized in ATP had significantly longer lengths than those assembled in GTP or UTP. When cells not treated with colcemid were lysed, the integrity of the cytoplasmic microtubule complex (CMTC) was maintained during subsequent incubation in reassembly buffer. However, in contrast to unlysed, living cells, microtubules of lysed cells were stable to colchicine. A significant fraction of the CMTC was stable to cold-induced disassembly whereas microtubules reassembled after lysis were extremely cold-sensitive. When cells not treated with colcemid were lysed and incubated in millimolar Ca++, microtubules depolymerized from their distal ends and a much reduced CMTC was observed. Ca++ reversal with EGTA rapidly resulted in a reformation of the CMTC apparently by elongation of Ca++ resistant microtubules.
Topics: Adenosine Triphosphate; Animals; Calcium; Cell Membrane; Cetomacrogol; Cyclic AMP; Demecolcine; Egtazic Acid; Fibroblasts; Guanosine Triphosphate; Mice; Microscopy, Electron, Scanning; Microtubules; Polyethylene Glycols; Tubulin; Uridine Triphosphate
PubMed: 6304113
DOI: 10.1083/jcb.96.6.1631 -
The Journal of Experimental Medicine Apr 2008Diapedesis of leukocytes across endothelial cells is a crucial step in both the innate and adaptive immune responses. Surface molecules on leukocytes and endothelial...
Diapedesis of leukocytes across endothelial cells is a crucial step in both the innate and adaptive immune responses. Surface molecules on leukocytes and endothelial cells critical for diapedesis have been identified, but the mechanisms underlying this process are not understood. Homophilic interaction between platelet/endothelial cell adhesion molecule (PECAM) on leukocytes and PECAM at the endothelial border triggers targeted recycling of membrane from a reticulum localized close to the endothelial cell lateral border. This membrane surrounds the transmigrating leukocyte (Mamdouh, Z., X. Chen, L.M. Pierini, F.R. Maxfield, and W.A. Muller. 2003. Nature. 421:748-753). How this process occurs and whether it is required for diapedesis independent of PECAM are not known. We now report that targeted recycling from this lateral border recycling compartment (LBRC) is required for diapedesis, is mediated by kinesin family molecular motors, and requires normally functioning endothelial microtubules. Selective disruption of microtubules or inhibition of kinesin motor domain blocked targeted recycling and diapedesis of monocytes. Furthermore, targeted recycling of membrane from the LBRC was required for transmigration of lymphocytes, which migrate independently of PECAM. Thus, trafficking of membrane from the LBRC to surround leukocytes may be a general requirement for migration of leukocytes across the endothelial cell border. Furthermore, these data provide the first demonstration of a role for endothelial microtubules and kinesins in promoting diapedesis, and a mechanism to explain targeted recycling.
Topics: Antigens, CD; Cadherins; Cell Compartmentation; Cell Membrane; Cell Movement; Cells, Cultured; Demecolcine; Endothelial Cells; Humans; Intercellular Adhesion Molecule-1; Intercellular Junctions; Kinesins; Leukocytes; Lymphocytes; Microtubules; Molecular Motor Proteins; Monocytes; Platelet Endothelial Cell Adhesion Molecule-1; Protein Structure, Tertiary; Protein Transport
PubMed: 18378793
DOI: 10.1084/jem.20072328 -
Proceedings of the National Academy of... May 1987Human Daudi lymphoblastoid cells, which are highly sensitive to the antiproliferative action of human leukocyte alpha-interferon (IFN-alpha), and IFN-resistant and...
Human Daudi lymphoblastoid cells, which are highly sensitive to the antiproliferative action of human leukocyte alpha-interferon (IFN-alpha), and IFN-resistant and IFN-sensitive Daudi subclones (Cl2 and Cl1, respectively), contain 2300 (Kd = 20 X 10(-12) M), 3000 (Kd = 45 X 10(-12) M), and 3700 (Kd = 52 X 10(-12) M) IFN-alpha binding sites per cell, respectively. Thus, these IFN-sensitive and IFN-resistant cells have similar numbers of high-affinity IFN-alpha receptors. IFN-receptor complexes that are insoluble in Triton X-100 accumulate in IFN-sensitive but not in IFN-resistant cells. The ligand-induced accumulation of Triton-insoluble complexes in IFN-sensitive cells was inhibited by cytochalasin B. This suggests that the solubility change of IFN-receptor complexes results from their interaction with the cytoskeletal matrix. The dissociation of IFN-alpha from IFN-sensitive and IFN-resistant cells can be resolved into fast and slow components. IFN-alpha dissociates more slowly from IFN-sensitive cells than from IFN-resistant cells. Very slow dissociation of IFN-alpha from Triton-insoluble complexes correlates with this difference. These observations suggest that IFN-receptor complexes become coupled to the cytoskeletal matrix in IFN-sensitive but not in IFN-resistant cells, and that such interaction is an important element in the mechanism of the antiproliferative action of IFN-alpha on Daudi cells.
Topics: Burkitt Lymphoma; Cell Line; Cell Membrane; Cytochalasin B; Cytoskeleton; Demecolcine; Humans; Interferon Type I; Kinetics; Receptors, Immunologic; Receptors, Interferon
PubMed: 2953024
DOI: 10.1073/pnas.84.10.3249 -
Kidney International Aug 1996In an in vitro model of monocyte adhesion to glomerular cells, U-937 myelomonocytic leukemia cells irreversibly bind to human mesangial cell monolayers. Adhesion is...
In an in vitro model of monocyte adhesion to glomerular cells, U-937 myelomonocytic leukemia cells irreversibly bind to human mesangial cell monolayers. Adhesion is enhanced in mesangial cells proliferating in response to fetal bovine serum, and in the presence of several cytokines and vasoactive agents. In the present study, co-culture with U-937 followed by removal of non-adherent cells time-dependently decreased viability of mesangial cells, measured either by fluorometry after dual labeling with calcein acetoxymethylester and ethidium homodimer, or by the release of lactate dehydrogenase. The cytotoxic effects of co-culture with U-937 cells were significantly reduced by a combination of free radical scavengers, indicating involvement of reactive oxygen species. U-937 cells also stimulated subsequent proliferation of mesangial cells, assessed by [3H]-TdR incorporation and direct cell counts 24 hours later (from 1,034 +/- 83 to 14,611 +/- 959 and from 2,931 +/- 201 to 19,400 +/- 2,124 cpm/well, quiescent/cycling mesangial cells, respectively, P < 0.01). Controls to rule out TdR incorporation by adherent U-937 cells included selective [3H]-TdR labeling and demecolcine pretreatment. Cell counts at 24 hours confirmed U-937-induced proliferation of quiescent HMC, from 50,575 +/- 3,596 to 143,012 +/- 10,039 cells/cm2 (P < 0.01). Agents that promote U-937 cell adhesion, such as the TxA2 mimetic, U-46619, or angiotensin II, enhanced cytotoxicity while inhibiting the proliferation of both quiescent and cycling mesangial cells, when added during co-culture and the subsequent 24 hours (+1 microM U-46619, 1,875 +/- 131 and 2,546 +/- 125 cpm/well, respectively, 79,793 +/- 5,744 cells/cm2, P < 0.01 vs. U-937 only; +1 microM Ang II, 5066 +/- 560 and 5,784 +/- 306 cpm/well, respectively, 81,068 +/- 4,671 cells/cm2, P < 0.05). Blocking antibodies against the adhesion molecule ICAM-1 and leukocyte counterreceptors (LFA-1, VLA-4) prevented the proliferative response, which could not be duplicated with the conditioned media of U-937 alone or co-cultured with mesangial cells. These findings may reflect the interactions occurring in vivo between infiltrating leukocytes and resident cells during glomerular inflammation.
Topics: Animals; Antibodies, Blocking; Cattle; Cell Adhesion; Cell Adhesion Molecules; Cell Communication; Cell Division; Cell Line; Cells, Cultured; Culture Media, Conditioned; Glomerular Mesangium; Humans; Models, Biological; Monocytes; Thymidine
PubMed: 8840268
DOI: 10.1038/ki.1996.331 -
Developmental Biology Oct 1999Completion of cytokinesis, abscission, has been studied little despite the intensive studies of the onset and contractile mechanism of the earlier phases of division. It...
Completion of cytokinesis, abscission, has been studied little despite the intensive studies of the onset and contractile mechanism of the earlier phases of division. It has been well documented that microtubule (MT) disruption before furrow stimulation prevents furrowing, while MT disruption after furrow stimulation allows division to proceed. We have confirmed those findings using the MT inhibitors, nocodazole and demecolcine. In addition, we have found that MT disruption after furrow stimulation but before completion of division prevents abscission as evidenced by the observation that prospective daughter cells in MT-disrupted eggs maintain electrical continuity. Continued observation of eggs revealed that the furrow in MT-disrupted eggs did not result in abscission, but rather held steady until the time when controls underwent second cleavage, at which point the furrows regressed. These findings extend the recent reports that MTs are required for completion of division in mammalian tissue culture cells and frog eggs, to invertebrates, suggesting a common mechanism of abscission for animal cells.
Topics: Animals; Cell Cycle; Cell Division; Electrophysiology; Female; Microtubules; Nocodazole; Oocytes; Sea Urchins
PubMed: 10491270
DOI: 10.1006/dbio.1999.9395 -
Proceedings of the National Academy of... Oct 1978Three experimental situations have been found in which cultured sensory neurons from embryonic chicken will form growth cones from positions along the length of the...
Three experimental situations have been found in which cultured sensory neurons from embryonic chicken will form growth cones from positions along the length of the neurite. If the neurons are dissected with a remaining short axonal stump and plated into serum-free medium, they can form a morphologically normal growth cone from the stump within 15 min, even in the presence of cycloheximide or puromycin. When neurites growing in culture media with low levels of serum are cut at any point with microneedles, growth cones are produced quickly from the amputated stump, usually within 20 min. Treatment of growing neurons with low concentrations of colchicine, Colcemid, or podophyllotoxin results in the progressive appearance of lateral filopodia and regions of flattened cytoplasm that closely resemble growth cones except for their preterminal positions. These observations show that the potential to form growth cones is distributed throughout the neuron and suggest that this normally repressed in some way by the neuronal microtubules.
Topics: Axons; Cells, Cultured; Colchicine; Cycloheximide; Cytochalasin B; Demecolcine; Ganglia, Spinal; Nerve Tissue Proteins; Neurons; Podophyllotoxin; Puromycin
PubMed: 283427
DOI: 10.1073/pnas.75.10.5226 -
The Journal of Biological Chemistry Oct 2010Drugs that target microtubules are thought to inhibit cell division and cell migration by suppressing dynamic instability, a "search and capture" behavior that allows...
Drugs that target microtubules are thought to inhibit cell division and cell migration by suppressing dynamic instability, a "search and capture" behavior that allows microtubules to probe their environment. Here, we report that subtoxic drug concentrations are sufficient to inhibit plus-end microtubule dynamic instability and cell migration without affecting cell division or microtubule assembly. The higher drug concentrations needed to inhibit cell division act through a novel mechanism that generates microtubule fragments by stimulating microtubule minus-end detachment from their organizing centers. The frequency of microtubule detachment in untreated cells increases at prophase suggesting that it is a regulated cellular process important for spindle assembly and function. We conclude that drugs produce differential dose-dependent effects at microtubule plus and minus-ends to inhibit different microtubule-mediated functions.
Topics: Animals; CHO Cells; COS Cells; Cell Division; Cell Movement; Centrosome; Chlorocebus aethiops; Cricetinae; Cricetulus; Demecolcine; Dose-Response Relationship, Drug; Microtubule-Associated Proteins; Microtubules; Tubulin; Tubulin Modulators; Vinblastine
PubMed: 20696757
DOI: 10.1074/jbc.M110.160820 -
PloS One 2014Digital holographic microscopy (DHM) has emerged as a powerful non-invasive tool for cell analysis. It has the capacity to analyse multiple parameters simultaneously,...
Digital holographic microscopy (DHM) has emerged as a powerful non-invasive tool for cell analysis. It has the capacity to analyse multiple parameters simultaneously, such as cell- number, confluence and phase volume. This is done while cells are still adhered and growing in their culture flask. The aim of this study was to investigate whether DHM was able to monitor drug-induced cell cycle arrest in cultured cells and thus provide a non-disruptive alternative to flow cytometry. DHM parameters from G1 and G2/M cell cycle arrested L929 mouse fibroblast cells were collected. Cell cycle arrest was verified with flow cytometry. This study shows that DHM is able to monitor phase volume changes corresponding to either a G1 or G2/M cell cycle arrest. G1-phase arrest with staurosporine correlated with a decrease in the average cell phase volume and G2/M-phase arrest with colcemid and etoposide correlated with an increase in the average cell phase volume. Importantly, DHM analysis of average cell phase volume was of comparable accuracy to flow cytometric measurement of cell cycle phase distribution as recorded following dose-dependent treatment with etoposide. Average cell phase volume changes in response to treatment with cell cycle arresting compounds could therefore be used as a DHM marker for monitoring cell cycle arrest in cultured mammalian cells.
Topics: Animals; Cell Cycle Checkpoints; Cell Line; Demecolcine; Dose-Response Relationship, Drug; Etoposide; Holography; Mice; Microscopy; Staurosporine
PubMed: 25208094
DOI: 10.1371/journal.pone.0106546