Influence Of Cytochalasin Research Articles

Cell spreading as a hydrodynamic process.

Many cell types have the ability to move themselves by crawling on extra-cellular matrices. Although cell motility is governed by actin and myosin filament assembly, the pattern of the movement follows the physical properties of the network ensemble average. The first step of motility, cell spreading on matrix substrates, involves a transition from round cells in suspension to polarized cells on substrates. Here we show that the spreading dynamics on 2D surfaces can be described as a hydrodynamic process. In particular, we show that the transition from isotropic spreading at early time to anisotropic spreading is reminiscent of the fingering instability observed in many spreading fluids. During cell spreading, the main driving force is the polymerization of actin filaments that push the membrane forward. From the equilibrium between the membrane force and the cytoskeleton, we derive a first order expression of the polymerization stress that reproduces the observed behavior. Our model also allows an interpretation of the effects of pharmacological agents altering the polymerization of actin. In particular we describe the influence of Cytochalasin D on the nucleation of the fingering instability.

Cariopathologic changes in human binuclear and mononuclear T-cells under the influence of cytochalasin B and lead chloride <i>in vitro</i>

The influence of PbCl2 on PHA-stimulated culture of human¢s T-cells was analyzed in the investigation. The number of mitotic cells was increased in cultures with PbCl2. Increased number of cells with morphological characteristics of apoptosis was also observed in these cultures. Addition of cytochalasin B lead to intensification of cell¢s death under the influence of lead chloride.

A novel method to quantify mechanical tension in cell monolayers.

A new technology to analyze mechanical properties of adherent cell monolayers grown on elastic silicon membranes is introduced. Measurements were performed using 3T3 (NIH) fibroblasts under the influence of Cytochalasin D and Thrombin. The stress-strain relation of the cell monolayer-silicon-composite was monitored. The drum-like construct of the culture chamber opens new roads for studying the mechanics of cell monolayers and of ultra flat tissue constructs. Steady state as well dynamic mechanical studies can be performed. Defined mechanical boundary conditions together with the known number and orientation of the cells allow precise information on the average tension exerted by a single cell within the monolayer.

Vitrificação de oócitos e embriões bovinos produzidos in vitro e expostos à citocalasina B

In this study, the influence of cytochalasin B on the survival and development after vitrification of oocytes and in vitro produced (IVP) bovine blastocysts was evaluated. In the Experiment I, 956 oocytes were matured for 22h and were immediately vitrified (Vitri treatment) or exposed for 15-20 minutes, to 7.5µg/ml (CB7.5Vitri treatment) or 45µg/ml (CB45Vitri treatment) cytochalasin B solutions, before vitrification. After 30 seconds of exposure to SV1 [400µl TCM-Hepes with 10% fetal serum (SF), 50µl ethylene glycol (EG) and 50µl DMSO], and 20 seconds to SV2 (300µl trehalose 1,0M + 20% SF, 100µl EG and 100µl DMSO) solutions, the oocytes were vitrified in Open Pulled Straws (OPS). The rewarming was performed at 37-38oC in two steps of 5 minutes each, into 0.3 and 0.15M trehalose solutions, respectively. There was no difference (P>0.05) in the cleavage and embryo rates between Vitri, Cito7.5Vitri and Cito45Vitri treatments, which were inferior to control group (P 0.05), which were lower than those observed in the Control group (P<0.05). The results show that, independently of dose studied (7.5 or 45mg/ml), cytochalasin B has not a beneficial effect to the vitrification of oocytes and IVP bovine blastocysts.

Cellular Uptake of Clostridium botulinum C2 Toxin Requires Oligomerization and Acidification

The actin-ADP-ribosylating binary Clostridium botulinum C2 toxin consists of two individual proteins, the binding/translocation component C2II and the enzyme component C2I. To elicit its cytotoxic action, C2II binds to a receptor on the cell surface and mediates cell entry of C2I via receptor-mediated endocytosis. Here we report that binding of C2II to the surface of target cells requires cleavage of C2II by trypsin. Trypsin cleavage causes oligomerization of the activated C2II (C2IIa) to give SDS-stable heptameric structures, which exhibit a characteristic annular or horseshoe shape and form channels in lipid bilayer membranes. Cytosolic delivery of the enzyme component C2I is blocked by bafilomycin but not by brefeldin A or nocodazole, indicating uptake from an endosomal compartment and requirement of endosomal acidification for cell entry. In the presence of C2IIa and C2I, short term acidification of the extracellular medium (pH 5.4) allows C2I to enter the cytosol directly. Our data indicate that entry of C2 toxin into cells involves (i) activation of C2II by trypsin-cleavage, (ii) oligomerization of cleaved C2IIa to heptamers, (iii) binding of the C2IIa oligomers to the carbohydrate receptor on the cell surface and assembly with C2I, (iv) receptor-mediated endocytosis of both C2 components into endosomes, and finally (v) translocation and release of C2I into the cytosol after acidification of the endosomal compartment.

SEPTUM FORMATION IN THE DESMID XANTHIDIUM (CHLOROPHYTA): EFFECTS OF CYTOCHALASIN D AND LATRUNCULIN B SUGGEST THE INVOLVEMENT OF ACTIN MICROFILAMENTS

Septum formation and mitosis were investigated by light and electron microscopical techniques as well as through the completion of inhibitor experiments in the unicellular desmid Xanthidium armatum (Bréb.) Rabenh. In untreated cells, numerous endoplasmic reticulum (ER) cisternae permeated the mitotic apparatus and secretory vesicles, and ER formed a band in front of the linearly growing septum, indicating the predetermined direction of septum growth. Under the influence of cytochalasin D (CD), the vesicle/ER band lost its proper orientation, which led to a malformed septum wall; moreover, abnormal septum branches could potentially have developed. Whereas the septum of an untreated cell only grew at its edge, the CD‐induced branches (also with a vesicle/ER band in front) represented additional growing zones. These observations indicated that actin filaments were involved in establishing, maintaining, and orienting the “preforming” vesicle/ER band and, thus, the later septum. Latrunculin B (LB) had more severe effects on septum formation than did CD. Only small accumulations of septum material were found at the septum edge, and no aberrant growth of the septum occurred in LB‐treated as in CD‐treated cells. This could be explained by the more rapid disturbance of all actin‐driven processes after LB treatment, which was assumed, because even low concentrations of the drug rapidly inhibited cytoplasmic streaming.

Effect of cytochalasin D on articular cartilage cell phenotype and shape in long-term organ culture.

It has been documented, on the basis of cell culture experiments, that cytochalasin treatment promotes a round cell shape in chondroblast cultures by altering the cytoskeleton, and that it simultaneously alters the balance between production of type I and type II collagens. The aim in this study was to monitor the deposition of pro-type-I and type II collagens, and possible changes in articular cartilage layers in the mandibular condyle of the mouse under the influence of Cytochalasin D (CD) when total craniomandibular joints of 5-day-old mice were cultured in one block. The experimental group comprised 20 Balb/c mice of both sexes. Twenty in vitro controls were cultured without the administration of cytochalasin. The mice in the third group were used as in vivo controls. The cells in the prechondroblast layer responded with a rapid change in shape when treated with CD and assumed a rounded morphology. The total thickness of the cell layer was reduced at 7 days. Immunostaining against pro-type-I collagen was intense in the narrow fibrous and prechondroblast layers in the CD-treated group, whereas the stained area was wider and the staining gradually reduced in the deeper cartilage layers in the in vitro controls. Staining against type II collagen became weaker at the end of the culturing period of the CD-treated group, whereas in the in vitro controls the staining against type II collagen was clearly visible at all observation times. These phenomena can be explained by changes in differentiation and the altered cell cycle of the chondroblasts in organ culture under the influence of CD.

Bioelectrorheological Model of the Cell. 7. Cellular Deformation in the Presence of Cytochalasin B.

The influence of cytochalasin B on the mechanical properties of Neurospora crassa cells subjected to a periodic electric field was investigated. Shear and extensional deformations were considered and studied separately. Conditions were found under wh

Bioelectrorheological model of the cell. 7. Cellular deformation in the presence of cytochalasin B

The influence of cytochalasin B on the mechanical properties of Neurospora crassa cells subjected to a periodic electric field was investigated. Shear and extensional deformations were considered and studied separately. Conditions were found under which shear deformations become irreversible. Rheological models helped in the interpretation of the results in terms of the different response to the shear stress of the three hypothetical supramolecular regions of the membrane-skeleton network (F, S and 0). Rheological parameters for the above regions related to the proposed models were calculated. The models were satisfactorily fitted to the experimental results. The influence of cytochalasin B on the course of extensional deformation of cells was investigated and characterized semiquantitatively.

Influence of cytochalasin B (CB) on GP Ib distribution after thrombin or TRAP and before surface activation

The receptor for von Willebrand factor (vWF) on human platelets, glycoprotein (GP) Ib/IX, has been shown in our studies to be an immobile complex when stimulated in suspension or on surfaces. Recent investigations have revealed that GP Ib/IX remains immobile on platelets activated in suspension followed by exposure to formvar surfaces that cause the cells to spread. However, since channels of the open canalicular system (OCS) are evaginated back on to the exposed surface during spreading, it was suggested that our study missed the clearance of GP Ib/IX from the exposed surface to internal membranes. The present study has added cytochalasin B after exposure of platelets to thrombin or TRAP in suspension in order to prevent spreading and movement of GP Ib/IX during subsequent exposure to surface activation on formvar grids. Results indicate that GP Ib/IX receptors remain randomly dispersed from edge to edge on platelets activated by thrombin or TRAP in suspension 10 minutes before treatment with CB followed by surface activation. Statistical analysis of the frequency of immunogold particles binding to monoclonal antibodies attached to GP Ib/IX revealed no significant reduction in frequency, translocation from cell edges or concentration of GP Ib/IX receptors in or around channels of the OCS. Results support the concept that GP Ib/IX is not cleared from exposed surfaces to the OCS of platelets activated by thrombin or TRAP and surface activation.

Localization of GPIb/IX and GPIIb/IIIa on Discoid Platelets

The organization and reorganization of mobile receptors, GPIIb/IIIa and GPIb/IX, on surface- and suspension-activated platelets have been studied in detail, but their distribution on resting, discoid platelets is uncertain. The present study has treated platelets in suspension with cytochalasin E before mounting on formvar grids or glass slide fragments in order to preserve their discoid appearance, then probed the organization of GPIIb/IIIa with fibrinogen coupled to gold particles (Fgn/Au) and GPIb/IX with bovine or ristocetin-activated human plasma detected by combined anti-vWF antibody and protein A coupled to gold particles. Multimers of vWF had the same tortuous, linear distribution from edge to edge observed previously on surface-activated platelets. However, the gold particles marking the complex of vWF-anti-vWF bound to GPIb/M were closer together on the discoid cells. Fgn/Au particles bound to GPIIb/IIIa receptors were uniformly distributed from edge to edge on many discoid platelets. On others they tended to clump or cluster in strips or patches. The latter organization of Fgn/Au-GPIIb/IIIa receptors may be due to the rugose nature of the discoid platelet surface or an influence of cytochalasin E. Definition of mobile receptor organization on discoid cells provides a useful baseline for determining their fate following surface or suspension activation.

Influence of cytochalasin a on single cell motility of melanoma cells in vitro

Influence of cytochalasin a on single cell motility of melanoma cells in vitro

Changes in actin distribution during fertilization of the mouse egg

Unfertilized mouse oocytes and eggs 1-8 h after fertilization in vitro were examined at the light microscope level for structural changes, distribution of actin (as assessed by both antiactin antibodies and NBD-phallicidin), surface binding of concanavalin A (Con A) and chromosomal distribution and condensation. The influence of cytochalasin D on these events was also assessed. Changes in actin distribution were associated with rotation of the second anaphase spindle, formation of the second polar body, the events following incorporation of the sperm nucleus, and formation and migration of the pronuclei. Cytochalasin D prevented spindle rotation, polar body formation, pronuclear migration and the restoration of Con A binding over the site of sperm entry and the site of polar body formation, but did not affect sperm fusion and entry, the loss of Con A binding at the site of sperm entry and pronuclear formation.

Membrane cycling and macrovacuolation under the influence of cytochalasin: Kinetic and morphometric studies

Fibroblasts exposed to higher doses of cytochalasin accumulate very big discrete endoplasmic vacuoles, the membrane of which is derived by internalization of plasmalemma. Morphometry confirms that the amount of surface interiorized is equal to the difference between the original cell surface area (before CD) and the reduced surface area measurable after CD-induced rounding. Correspondingly, there is a nearly two-fold increase in the activity of the ectoenzyme 5′-nucleotidase (a marker for plasma membrane) internally within the cytoplasm, after treatment with CD. Macrovacuolation increases cell volume by ~30%. Surface membrane is internalized as micropinocytotic vesicles at a rate measurable by the accumulation of HRP, a marker of fluid-phase pinocytosis. Uptake of HRP is shown to be enhanced at all times during exposure to CD, and is balanced by accelerated exocytic recycling of membrane except during a phase (~4–8 hr) in which pinocytic uptake exceeds exocytosis. Vesicular membrane accumulated intracellularly in this period is retained in the endoplasm, and by successive fusions forms vacuoles in close approximation to microfilament aggregates. Once established, this new macrovacuolar membrane compartment is in dynamic equilibrium with the cell surface, and its membrane is cycled like the plasma membrane, in a mutual exchange of pinosomes between the several vacuoles and the cell surface. In drug-free medium vacuole membrane apparently reverts to the surface by pinocytotic recycling, and the cells recover normal characteristics 4–6 hr after withdrawal of cytochalasin.

Differentiation of neuroblastoma cells under the influence of cytochalasin B

Differentiation of neuroblastoma cells under the influence of cytochalasin B

Capping of surface immunoglobulin on ‘hairy cells’ is independent of energy production

Capping, independent of metabolic energy, of surface immunoglobulin (S-Ig) on 'Hairy cells' from patients with Hairy cell leukaemia (HCL) is described. As controls leukaemic cells from a patient with a prolymphocytic leukaemia (PLL) and blood lymphocytes from healthy individuals were used. The specificity of the energy-independent capping of the HCL-cells as compared to the controls was tested by incubation of the cells at 4 degrees C in the presence of 0.1 M sodium azide with different FITC-labelled ligands. In order to find an explanation for this phenomenon, the influence of cytochalasin B, colchicine and the combination of both drugs on capping of S-Ig and concanavalin (Con-A)-receptors at 37 degrees C was investigated. Furthermore the effect of Con A on S-Ig capping and vice versa was studied. The results show that only S-Ig on HCL cells could form caps at 4 degrees C in the presence of sodium-azide. Cytochalasin B alone induced a strong inhibition of Con A capping on all 3 cell types, whereas S-Ig capping was unaffected. Colchicine alone had practically no effect. Anti-Ig inhibited subsequent patch and cap formation with Con A on both HCL cells and PLL cells, whereas Con A caps and patches were redistributed by anti-Ig on PLL cells, but not on HCL cells. Conversely, Con A could link S-Ig to other receptors, leading to inhibition of S-Ig capping at 4 degrees C on HCL cells and to co-capping of S-Ig at 37 degrees C on both cell types. In addition Con A induced redistribution of S-Ig caps. The combination of co-capping of S-Ig by Con A, followed by redistribution of the caps by FITC-anti-Ig simulated inhibition of S-Ig capping by Con A on PLL cells. The major conclusions are: in some cases inhibition of capping may actually be caused by redistribution of caps; the energy-independent capping cannot be explained by free diffusion of S-Ig in the membrane through lack of any connexion with receptor-mobility regulating systems. It is proposed that the energy requirement of capping is needed to inactivate a specific mechanism,w which restrains receptor mobility and which is non-operative in HCL cells.

Cytoplasmic streaming in the root cortex and its role in the delivery of potassium to the shoot

The influence of cytochalasin B (CB), a potent inhibitor of cytoplasmic streaming, on (86)Rb-labelled K(+) translocation by detopped Lycopersicon esculentum Mill., Cucumis sativus L. and Zea mays L. plants was examined by measuring the radioactivity in xylem exudate before and after the addition of CB to the medium bathing the roots. CB caused complete cessation of cytoplasmic streaming in root segments within 15 min but was without effect on either total (86)Rb uptake or exudation. Thus factors other than cytoplasmic streaming limit the movement of K(+) across the symplast of the root of higher plants.

The cytaster, a colchicine-sensitive migration organelle of cleavage nuclei in an insect egg

Time-lapse analyses of nuclear multiplication in the eggs of the gall midge Wachtliella persicariae L., documented in film D 1235 (available from the IWF, Göttingen), give evidence of a special migration organelle of cleavage nuclei. Each of these “migration cytasters” represents one greatly enlarged polar cytaster of the mitotic apparatus, which is connected to one nucleus. From the films it can be concluded that the astral rays temporarily adhere to peripheral egg structures and exert tractive forces toward the cytaster center. These forces combine and pull the accompanying daughter nucleus through the ooplasm after each mitosis. This “active” mode of migration, which is accompanied by extensive polarized transport of yolk particles toward the cytaster center, enables the energids (= cleavage nucleus and its associated island of cytoplasm) to move relative to the surrounding ooplasm. In addition, there is a “passive” mode of nuclear migration: The energids are moved by means of plasmic flows, thereby maintaining their position in relation to the surrounding ooplasm. Electron microscopic studies show solitary microtubules running radially toward the cytaster center. As a result of colchicine injection (1) the microtubules disintegrate, (2) the polarized transport of yolk particles cases, (3) the active nuclear migration stops and the nuclei are only passively moved by rhythmic ooplasmic flows. This inhibition of active nuclear migration gives further evidence that microtubules take an essential part in it. Control experiments with lumicolchicine show no effect on nuclear migration. Conversely, under the influence of cytochalasin B active nuclear migration is continued, while the ooplasmic flows are inhibited. Thus the mechanisms of active and passive nuclear migration can work independently of each other. The generation of tractive forces along the astral rays is discussed with respect to current models of spindle function.

Tumor cell locomotion--a factor in metastasis formation? Influence of cytochalasin B on a tumor dissemination pattern.

The influence of Cytochalasin B (CB) on TA3 ascites tumor cells was studied in vivo in order to assess whether CB-induced cell paralysis would affect the transplanation behavior of the cells and in particular tumor distribution after IV cell infusion ("experimental metastases"). Tumor cell pre-treatment with CB (1 mug/ml) did not alter the SC or IV transplantability of TA3 cells. Pre-treatment with 10 mug/ml CB, in contrast, consistently increased the incidence and number of extra-pulmonary tumor takes from IV transfused cells. The amount of pulmonary tumors was not significantly altered. SC transplantability was not affected by 10 mug/ml CB. The importance of cell mobility and cell surface topography for tumor cell nidation in vessels is discussed.

The influence of cytochalasin B on the response of adrenal tumor cells to acth and cyclic AMP

Cytochalasin B inhibits increase in steroid synthesis by mouse adrenal tumor cells (Y-1), produced either by ACTH or cyclic AMP. Basal levels of steroid synthesis are not decreased and the inhibitor acts by decreasing the response of the side-chain cleavage step (cholesterol → pregnenolone) to ACTH. Inhibition is reversible and is seen in medium without glucose. These observations suggest that microfilaments may play a role in the response of adrenal cells to ACTH.