Bioluminescence as a tool of measuring ATP turnover during actin polymerization and during its interaction with cytochalasin B

1984 ◽  
Vol 317 (6) ◽  
pp. 695-696
Author(s):  
K. Ritter ◽  
S. Fischer ◽  
P. Dancker
Keyword(s):  
Actin Polymerization ◽  
Cytochalasin B ◽  
Atp Turnover

scholarly journals Activation of human neutrophils by mastoparan. Reorganization of the cytoskeleton, formation of phosphatidylinositol 3,4,5-trisphosphate, secretion up-regulation of complement receptor type 3 and superoxide anion production are stimulated by mastoparan

1992 ◽  
Vol 282 (2) ◽  
pp. 393-397 ◽  
Author(s):  
J Norgauer ◽  
M Eberle ◽  
H D Lemke ◽  
K Aktories
Keyword(s):  
Pertussis Toxin ◽  
Actin Polymerization ◽  
Cytochalasin B ◽  
Superoxide Radical ◽  
Human Neutrophils ◽  
Superoxide Anion Production ◽  
Radical Production ◽  
Rapid Formation ◽  
Primary Granules ◽  
Type 3

In human neutrophils, mastoparan induced rapid F-actin polymerization which was followed by a slow and sustained depolymerization to below the initial F-actin content. Incubation of neutrophils with pertussis toxin inhibited mastoparan-stimulated actin polymerization; however it did not prevent sustained depolymerization of F-actin. Analyses of phospholipids performed in parallel revealed that mastoparan stimulated rapid formation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and consumption of phosphatidylinositol 4,5-bisphosphate (PIP2). Pertussis toxin treatment blocked mastoparan-induced formation of PIP3. Furthermore, mastoparan stimulated the release of N-acetylglucosaminidase from primary granules. Cytochalasin B enhanced mastoparan-stimulated secretion. Mastoparan triggered superoxide radical production in a cytochalasin B-sensitive manner and induced complement type 3 receptor (CR3) up-regulation.

scholarly journals Cytochalasins inhibit nuclei-induced actin polymerization by blocking filament elongation.

1980 ◽  
Vol 84 (2) ◽  
pp. 455-460 ◽  
Author(s):  
D C Lin ◽  
K D Tobin ◽  
M Grumet ◽  
S Lin
Keyword(s):  
Binding Sites ◽  
Actin Polymerization ◽  
Cytochalasin B ◽  
Molecular Size ◽  
Muscle Actin ◽  
Competitive Displacement ◽  
Low Concentrations ◽  
Relative Affinity ◽  
Cytochalasin E ◽  
Low Ionic Strength

Polylysine was found to induce polymerization of muscle actin in a low ionic strength buffer containing 0.4 mM MgCl2. The rate of induced polymerization was dependent on the amount and on the molecular size of the polylysine added. A similar effect was obtained by adding actin nuclei (containing about 2-4 actin subunits) cross-linked by p-N,N'-phenylenebismaleimide to G-actin under the same conditions, suggesting that the effect of polylysine is due to promotion of the formation of actin nuclei. Polymerization induced by polylysine and by cross-linked actin nuclei was inhibited by low concentrations (10(-8)-10(-6)M) of cytochalasins. Binding experiments showed that actin filaments, but not actin monomers, contained high-affinity binding sites for [3H]cytochalasin B (one site per 600 actin monomers). The relative affinity of several cytochalasins for these sites (determined by competitive displacement of [3H]dihydrocytochalasin B) was: cytochalasin D greater than cytochalasin E approximately equal to dihydrocytochalasin B. The results of this study suggest that cytochalasins inhibit nuclei-induced actin polymerization by binding to highly specific sites at the point of monomer addition, i.e., the elongation site, in actin nuclei and filaments.

scholarly journals Direct measurement of actin polymerization rate constants by electron microscopy of actin filaments nucleated by isolated microvillus cores.

1981 ◽  
Vol 88 (3) ◽  
pp. 654-659 ◽  
Author(s):  
T D Pollard ◽  
M S Mooseker
Keyword(s):  
Electron Microscopy ◽  
Growth Rates ◽  
Rate Constants ◽  
Actin Polymerization ◽  
Cytochalasin B ◽  
Monomer Concentration ◽  
Dissociation Rate ◽  
Filament Bundles ◽  
Dissociation Rate Constants ◽  
Pointed End

We used actin filament bundles isolated from intestinal brush-border microvilli to nucleate the polymerization of pure muscle actin monomers into filaments. Growth rates were determined by electron microscopy by measuring the change in the length of the filaments as a function of time. The linear dependence of the growth rates on the actin monomer concentration provided the rate constants for monomer association and dissociation at the two ends of the growing filament. The rapidly growing ("barbed") end has higher association and dissociation rate constants than the slowly growing ("pointed") end. The values of these rate constants differ in 20 mM KCl compared with 75 mM KCl, 5 mM MgSO4. 2 microM cytochalasin B blocks growth entirely at the barbed end, apparently by reducing both association and dissociation rate constants to near zero, but inhibits growth at the pointed end to only a small extent.

Complex Influence of Cytochalasin B on Actin Polymerization

1979 ◽  
Vol 34 (7-8) ◽  
pp. 555-557 ◽  
Author(s):  
P. Dancker ◽  
I. Low
Keyword(s):  
Light Scattering ◽  
Actin Polymerization ◽  
Time Course ◽  
Cytochalasin B ◽  
Degree Of Polymerization ◽  
Viscosity Measurements ◽  
Low Concentrations ◽  
Scattering Measurements ◽  
Final Length ◽  
Complex Influence

Abstract In the presence of very low concentrations (about 2 x 10-7 ᴍ) of cytochalasin B (CB) the time course of actin polymerization is much more sigmoidal when followed by viscosity measurements than when followed by light scattering measurements. This suggests that under these conditions actin polymers do not immediately reach their final length but only via short ”bent“ polymers which can be detected only by light scattering but not by viscosity measurements. At higher CB concentrations (about equimolar to those of actin) CB reduces the average degree of polymerization and favors the nucleation step necessary for polymerization.

Polymerization of Actin in the Absence and Presence of Cytochalasin B: Problems of Determining "Critical Concentration"

1986 ◽  
Vol 41 (7-8) ◽  
pp. 781-786 ◽  
Author(s):  
Bernd Fussmann ◽  
Peter Dancker
Keyword(s):  
Fluorescence Intensity ◽  
Actin Polymerization ◽  
Cytochalasin B ◽  
Critical Concentration ◽  
Degree Of Polymerization ◽  
Specific Fluorescence ◽  
Highly Nonlinear ◽  
Final Degree ◽  
Actin Concentration

Abstract Various concentrations of actin (0.3 or 1 mᴍ MgCl2, 1 mᴍ ATP , 1 mᴍ EGTA) reached their final degree of polymerization (measured by a pyrene dye attached to actin) earlier in the presence of cytochalasin B than in its absence. The curves relating concentrations of polymeric F-actin to total actin concentration were under these conditions highly nonlinear making an unam biguous extrapolation to zero F-actin concentration (to deduce the “critical concentration” of actin poly­ merization) im possible. The concentration of actin, above which polymerization occurred, was unaltered by cytochalasin B (although for reasons not yet understood the specific fluorescence intensity of polymerized actin was lower in the presence of cytochalasin B than in its absence). The results show that a distinct “critical concentration” of actin polymerization must not always be well defined.

scholarly journals Effects of cytochalasin B on DNA methylation and histone modification in parthenogenetically activated porcine embryos

Reproduction ◽  
2016 ◽  
Vol 152 (5) ◽  
pp. 519-527 ◽  
Author(s):  
Xiaoxiao Hou ◽  
Jun Liu ◽  
Zhiren Zhang ◽  
Yanhui Zhai ◽  
Yutian Wang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Embryonic Development ◽  
Histone Modification ◽  
Actin Polymerization ◽  
Cytochalasin B ◽  
Epigenetic Modification ◽  
Cell Activity ◽  
Cleavage Rate ◽  
Expression Levels ◽  
Mammalian Embryos

DNA methylation and histone modification play important roles in the development of mammalian embryos. Cytochalasin B (CB) is an actin polymerization inhibitor that can significantly affect cell activity and is often used in studies concerning cytology. In recent years, CB is also commonly being used inin vitroexperiments on mammalian embryos, but few studies have addressed the effect of CB on the epigenetic modification of embryonic development, and the mechanism underlying this process is also unknown. This study was conducted to investigate the effects of CB on DNA methylation and histone modification in the development of parthenogenetically activated porcine embryos. Treatment with 5 μg/mL CB for 4 h significantly increased the cleavage rate, blastocyst rate and total cell number of blastocysts. However, the percentage of apoptotic cells and the expression levels of the apoptosis-related genesBCL-XL,BAXandCASP3were significantly decreased. Treatment with CB significantly decreased the expression levels ofDNMT1,DNMT3a,DNMT3b,HAT1andHDAC1at the pronuclear stage and promoted the conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). After CB treatment, the level of AcH3K9 was upregulated and the level of H3K9me3 was downregulated. When combined with Scriptaid and 5-Aza-Cdr, CB further improved the embryonic development competence and decreased the expression ofBCL-XL,BAXandCASP3. In conclusion, these results suggest that CB could improve embryonic development and the quality of the blastocyst by improving the epigenetic modification during the development of parthenogenetically activated embryos.

Evidence that Cytochalasins Reduce the Size of Polymerization Nuclei during Actin Polymerization

1990 ◽  
Vol 45 (3-4) ◽  
pp. 300-302 ◽  
Author(s):  
Hanns Wendel ◽  
Peter Dancker
Keyword(s):  
Actin Polymerization ◽  
Cytochalasin B ◽  
Monomer Concentration ◽  
Fungal Metabolites ◽  
Double Logarithmic Plot ◽  
Logarithmic Plot

Abstract In a double-logarithmic plot of the rate of actin polymerization against monomer concentration, the slope has a lower value in the presence of the fungal metabolites cytochalasin B and D than in their absence. This suggests that actin oligomers which serve as polymerization nuclei are smaller in the presence of cytochalasins than in their absence.

Altered actin polymerization dynamics in various malignant cell types: evidence for differential sensitivity to cytochalasin B

1996 ◽  
Vol 52 (9) ◽  
pp. 1339-1346 ◽  
Author(s):  
Christos Stournaras ◽  
Eftichia Stiakaki ◽  
Sevasti B. Koukouritaki ◽  
Panayotis A. Theodoropoulos ◽  
Maria Kalmanti ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Cytochalasin B ◽  
Cell Types ◽  
Malignant Cell ◽  
Differential Sensitivity

scholarly journals Membrane glycoproteins and platelet cytoskeleton in immune complex- induced platelet activation

Blood ◽  
1993 ◽  
Vol 81 (6) ◽  
pp. 1505-1512 ◽  
Author(s):  
J Kang ◽  
C Cabral ◽  
L Kushner ◽  
EW Salzman
Keyword(s):  
Platelet Activation ◽  
Immune Complexes ◽  
Actin Polymerization ◽  
Cytochalasin B ◽  
Specific Marker ◽  
Platelet Response ◽  
Membrane Glycoproteins ◽  
Filamentous Actin ◽  
Serotonin Secretion ◽  
Platelet Binding

To clarify the mechanism of platelet activation by immune complexes and the possible involvement of surface glycoproteins (GPs), we studied platelet activation induced by heat-aggregated IgG (HAG). We examined the effects of monoclonal antibodies (MoAbs) against GPIb, GPIIb/IIIa, and the Fc receptor on resting platelets and on platelets stimulated by HAG. HAG increased the cytosolic ionized calcium concentration ([Ca2+]i) and stimulated protein (P47 and P20) phosphorylation, phosphatidic acid (PA) synthesis, serotonin secretion, and platelet aggregation. IV.3, an anti-Fc gamma RII receptor MoAb, inhibited HAG binding to platelets and all subsequent platelet responses. Like IV.3, MoAbs against GPIIb/IIIa (Tab, 10E5, AP-3) or GPIb (AP-1, 6D1) strongly inhibited platelet activation by HAG. However, while anti-GPIIb/IIIa MoAbs inhibited binding of IV.3 and HAG to platelets, anti-GPIb MoAbs had little effect on platelet binding of IV.3 or HAG. These observations suggest a close topographical and functional association of GPIIb/IIIa with Fc gamma RII in the platelet response to HAG. Cytochalasin B, an inhibitor of actin polymerization, also inhibited platelet activation but not HAG or IV.3 binding. Measurement of the fluorescence of 7-nitrobenz-2-oxa-1,3-(NBD)-phallacidin, a specific marker for filamentous actin (F-actin), showed that both cytochalasin B and AP-1 blocked the increase of F-actin induced by HAG. The common effects of anti-GPIb MoAbs and of cytochalasin B suggest that unlike the activity of GPIIb/IIIa, the ability of anti-GPIb to inhibit the activation of platelets by immune complexes is associated with perturbation of the cytoskeleton.

176 CYTOCHALASIN PREVENTS SPERM HEAD INCORPORATION INTO FERTILIZED PIG OOCYTES

2012 ◽  
Vol 24 (1) ◽  
pp. 200
Author(s):  
Z. Machaty ◽  
C. Wang ◽  
K. Lee
Keyword(s):  
Embryo Development ◽  
Actin Polymerization ◽  
Cytochalasin B ◽  
Fluorescent Protein ◽  
Pcr Analysis ◽  
Control Group ◽  
Wild Type ◽  
Sry Gene ◽  
Cell Stage ◽  
Developmental Potential

At fertilization, an acrosome-reacted sperm fuses to the plasma membrane of the oocyte. It is then internalized into the ooplasm and its head forms the male pronucleus that moves toward the female pronucleus. Experiments using cytochalasin, an inhibitor of actin polymerization, suggest that filamentous actin is necessary for spermatozoa incorporation deep into the oocyte cytoplasm. However, the exact role of actin in the process is not entirely clear and the consequences of inhibiting actin polymerization on embryo development are not known. In the present study, we investigated the effect of cytochalasin B on fertilization and subsequent embryo development in the porcine model. In vitro-matured oocytes were rinsed in fertilization medium, a modified Tris-buffered medium supplemented with 0.1% BSA and 1 mM caffeine. Groups of 30 oocytes were placed into 50-μL droplets of the medium covered with mineral oil. Fresh semen was collected from both a wild-type boar and a transgenic boar heterozygous for the green fluorescent protein (GFP), washed 3 times in Dulbecco's PBS and added at a final concentration of 5 × 105 cells mL–1 to each droplet containing the oocytes. The gametes were co-incubated for 5 h at 39°C under 5% CO2 in air. In the treatment group, 10 μg mL–1 of cytochalasin B was supplemented into the fertilization droplets; control groups received an equivalent amount of dimethyl sulfoxide, the solvent used to dissolve the inhibitor. After gamete co-incubation, potential zygotes were transferred to PZM-3 medium for culture. In experiment 1, mature oocytes were fertilized with the wild-type boar sperm. Embryos at the 6- or 8-cell stage were then collected, the blastomeres were separated and DNA from each blastomere was isolated for PCR analysis to monitor the presence of the male-specific SRY gene. In experiment 2, the GFP transgenic sperm was used for fertilization. Fertilized oocytes were cultured for 7 days and the resultant embryos were examined for GFP expression using an epifluorescence microscope. The developmental stages of the embryos were also determined by staining their nuclei with Hoechst 33342. We found that after cytochalasin B treatment, only 19.1% (21/110) of the blastomeres were positive for the SRY gene, whereas in the control group, SRY was detected in 54.5% (61/112) of the cases. In addition, in the cytochalasin B-treated group, 1.9% (7/376) of the embryos expressed GFP, as indicated by green fluorescence; this percentage was 35.4% (146/412) in the control embryos. The frequency of 2-cell and blastocyst-stage embryos was similar between the control and cytochalasin B-treated groups (84.1 vs 81.0%; and 11.6 vs 12.5%, respectively). The results indicate that the presence of cytochalasin B during fertilization effectively blocks the incorporation of the male nucleus into the newly formed zygote without affecting the developmental potential of the pre-implantation embryo.

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