Cortical cytoplasm and configurational changes of RGC-6 cells exposed to cytochalasin B

RGC-6 cells, grown to confluency in monolayer cultures, are typically fusiform. In sections of these cells, fixed in situ and examined in the electron microscope, a discontinuous zone of cortical cytoplasm (5–7 nm wide), was found subjacent to the plasma membrane of the free surface. It was composed largely of 5–7 nm microfilaments distributed in an apparently irregular nework and appeared to comprise a discrete zone separating the plasma membrane from the underlying endoplasm. Following incubation with cytochalasin B (5 μg/ml), the configuration of the cells changed markedly. The perikaryal cytoplasm appeared to swell, while the cellular processes became attenuated and often terminated in flower-like tips. Although more discontinuous and somewhat less dense, the fine structure of the cortical cytoplasm appeared little affected by incubation with cytochalasin B for one hour. By 48 hours no zone of cortical cytoplasm was found. Subplasmalemmal microfilaments were no longer identifiable and endoplasmic organelles were found in direct contact with the plasma membrane of the free surface, which appeared to have collapsed onto the underlying endoplasm. The change in cell configuration resulting from exposure to cytochalasin B occurred at 37°C but not at 4°C, suggesting a dependency on metabolic activity. It did not, however, appeared related directly to protein synthesis. Whereas incorporation of ³H-leucine was decreased to 46.9% and 62.9% of control values after one hour or two hours exposure to cytochalasin B, respectively, no change was found after 24 or 48 hours. It is suggested that the effect of cytochalasin B on cell form, which appeared not to be a direct effect on either protein synthesis or subplasmalemmal microfilaments, may represent a primary response of the cortical cytoplasmic matrix.     

Effects of colchicine and cytochalasin B on vasopressin- and cyclic adenosine monophosphate-induced changes in toad urinary bladder

Coincident with an increase in water permeability, the ridge-like surface structures of toad bladder granular cells transform to individual microvilli after stimulation with vasopressin (VP) or cyclic adenosine monophosphate (cAMP) by a mechanism that is yet to be defined. To explore the possible role of microtubules and microfilaments in this cell response, colchicine and cytochalasin B were employed to determine whether interference with the function of these components of the cytoskeletal system would prevent the VP- and cAMP-induced conversion of ridges to microvilli. Incubation of toad urinary bladders in 10(-4) M colchicine for 4 hours or 10(-5) M cytochalasin B for 90 minutes before stimulation with 20 mU. per ml. of VP markedly inhibited osmotic water flow. However, neither agent prevented the striking conversion of ridges to surface microvilli induced by VP and cAMP as seen with scanning electron microscopy. In addition, the ridges characteristic of granular cells were maintained in control bladders incubated with colchicine or cytochalasin B, but left unstimulated. Under the conditions of these experiments, these findings suggest that microtubules and microfilaments are not essential for maintenance of normal surface configuration in granular cells of toad urinary bladder, and that they are not involved in the mechanism responsible for VP- and cAMP-induced surface changes that occur in association with increased water permeability of this epithelium.     

Effects of 1,2-propanediol on the cytoskeletal organization of the mouse oocyte.

The effects of 1,2-propanediol, a cryoprotectant used for the freezing of embryos, were tested on the organization of microtubules and microfilaments in mouse oocytes arrested in metaphase II. At low doses (less than or equal to 1.0 M), 1,2-propanediol induced disorganization of the meiotic spindle but at 1.5 M and higher, it stabilized the spindle. Cytoplasmic asters were observed at all doses tested. An extensive network of free microtubules was observed at 1.0 M and 2.0 M 1,2-propanediol, the former having the stronger disruptive effect on the spindle. Higher doses of 1,2-propanediol (greater than or equal to 1.5 M) caused the oocytes to form cytoplasmic blebs. These blebs lacked detectable cortical microfilaments. In contrast, the microfilament-rich area of the cell cortex overlying the meiotic spindle was not modified.     

Intrahepatic cholestasis as a canalicular motility disorder. Evidence using cytochalasin

A rich network of actin-containing microfilaments are associated with the plasma membrane of the liver cells. These filaments are especially numerous in the pericanalicular region. Recently, active contractions of bile canaliculi have been observed in normal coupled isolated hepatocytes. In this report, we document that this motility behavior is abolished by cytochalasins B and D. Other cytoplasmic contractile movements are also reduced or lost after a brief initial period of enhanced surface activity with the formation of zeiotic blebs. The lack of contractile activity of bile canaliculi is accompanied by the gradual dilation of canalicular lumina. Since there is overwhelming evidence that the functional effects of the cytochalasins on cell motility are due to their effects on actin filaments, we propose that the altered canalicular contractility observed is due to the effects of cytochalasin on pericanalicular actin filaments. If bile canalicular contractility is a requirement for normal bile flow, then interference with this mechanism may be a factor in the pathogenesis of some types of intrahepatic cholestasis.     

Changes in the organization and antigenic determinants of intermediate filaments of rat hepatocytes after infusion of cytochalasin B in vivo.

The changes in cytokeratin intermedial filaments (IFs) after cytochalasin B (CB) infusion of rat liver in vivo were studied by light and electron microscopy, immunofluorescent staining (IMF), and immunoelectron microscopy (IEM). The CB treatment caused a change in the IFs at the cell border associated with a change in the distribution of microfilaments. The IFs at the cell border were partially disrupted. Actin aggregates were localized at points where IFs had condensed together. The pericanalicular sheath was intact but very dilated. These results indicated that the CB treatment caused an irregular distribution of the microfilaments at the cell periphery but spared the actin at the bile canaliculus. Cytokeratin staining by IMF was markedly decreased or absent; however, IEM clearly showed the presence of nonstaining IFs after CB treatment. These results indicated that the antigenic determinant of normal cytokeratin IFs became masked after CB treatment. The results indicate that F-actin disassembly induced by CB affects both the organization and conformation of cytokeratins associated with loss of integrity of the plasma membrane and vesicular uptake of plasma proteins by hepatocytes.     

Ultrastructure of the choroid plexus epithelium of pigeons treated with drugs. II. Effect of cytochalasin D and colchicine

A remarkable projection of bleblike protrusions, the expulsion of organelles into the protrusions formed on the apical surface, and the separation into the ventricular lumen of these protrusions was the general cellular response of choroidal epithelial cells to intravenous injection of cytochalasin D (CD). The compact microfilament mass and agglomeration of microtubules at the base of the cluster of protrusions reflect the results of cell contraction and displacement of microfilaments induced by CD. In earlier stages after intravenous injections of colchicine, an obvious increase in the number of varioussized vesicles, vacuoles, and lysosomes in the Golgi region was detected. In the later stages, these organelles were seen to accumulate in the basal portion of the epithelial cells. These changes were accompanied by an increase in vacuoles and the disorganization and displacement of the Golgi complex, and they coincided with a decrease in the number of microtubules in apical and basal cytoplasm. These findings suggest that the action of colchicine results in destruction of the three-dimensional architecture between cytoskeletal network and cell organelles. The present results suggest that the cytoskeletal network plays a role in the spatial coordination of the three-dimensional architecture of cell organelles. The study also indicates that the structural differences in the ventricles of the choroid plexus in drug-treated pigeons are manifestations of regional functional specialization in different parts of the ventricular system.     

Effects of cytotoxins on intracellular Na/K balance in mouse embryonic cell

Cytoplasmic Na/K imbalance induced by cytotoxic substances (ethylene glycol and cytochalasin B) was studied. Incubation in Dulbecco’s medium with these cytotoxins caused changes in K and Na concentrations in the mouse two-cell embryo blastomer. The effects of both substances resulted in a drop of Na content in the embryonic cell. Washing from cytochalasin B restored the initial Na/K balance in the cytoplasm. Possible adaptive mechanisms involved in the regulation of intracellular ionic homeostasis are discussed. __________ Translated from Kletochnye Tehnologii v Biologii i Meditsine, No. 2, pp. 102–105, April, 2008     

Aflatoxin B1 and acetaminophen induce different cytoskeletal responses during prelethal hepatocyte injury

Primary monolayer cultures of adult rat hepatocytes exposed to the hepatocarcinogen aflatoxin B1 (AFB1) undergo a characteristic prelethal cytomorphological change that is distinct from their response to the necrogenic noncarcinogenic hepatotoxin, acetaminophen (AAP). Since changes in cell shape are mediated, at least in part, by the F-actin cytoskeleton, we designed experiments to study early prelethal alterations in the distribution of actin microfilaments in monolayer rat hepatocytes exposed to AFB1 (100 microM) or AAP (16 mM). Using rhodamine-phalloidin fluorescence microscopy, we observed that normal hepatocytes showed a submembranous F-actin distribution with focal short microfilaments extending into filopodia along the periphery of the cell. Hepatocytes exposed to AFB1 for several hours exhibited retraction of their cytoplasm within a prominent circumferential peripheral band of F-actin microfilament bundles. Retraction of focal areas of peripheral cytoplasm was associated with an increased prominence of the radial F-actin-containing filopodia. Subsequently, there appeared peripheral blebs containing very little F-actin. Hepatocytes exposed to equivalently lethal concentrations of AAP initially remained structurally normal. After several hours, the cells exhibited a prominent polar aggregate of short microfilament bundles without the formation of blebs. Both the blebbing and the polar aggregation of F-actin bundles occurred prior to cell death as shown by lactate dehydrogenase release and trypan blue exclusion. These studies support the hypothesis that the lethal effects of these two agents may occur by different biological mechanisms that are associated with remarkably distinct prelethal cytoskeletal responses.     

Possible roles for microtubules and microfilaments in ADH action on toad urinary bladder.

Intramembranous particle aggregates in the luminal membrane of toad bladder granular cells after vasopressin stimulation have been found to correlate closely and specifically with induced alterations of water permeability. Roles for microtubules and microfilaments in mediating the latter response have been proposed on the basis of studies involving colchicine and cytochalasin B, respectively. In the present investigation the effects of these agents on both initiating and sustaining vasopressin-induced osmotic water flow and the particle aggregation phenomenon were studied. The results indicate that during initiation the aggregation and water flow responses to vasopressin are each colchicine- and cytochalasin B-sensitive and that these sensitivities can be wholly additive. However, after full vasopressin stimulation is established, the same responses demonstrate sensitivity only to cytochalasin B, not to colchicine. The findings, therefore, suggest that microtubules and microfilaments may be independently necessary for the initiation of the aggregation and water flow responses to vasopressin, and that microfilaments, but not microtubules, are required for their maintenance.     

细胞松弛素B对内皮细胞损伤修复的影响

目的观察细胞松弛素B对内皮细胞损伤修复过程中微丝骨架系统的形态结构变化,研究阻断微丝功能对内皮细胞损伤修复的影响。方法以培养单层内皮细胞损伤模型,采用免疫荧光染色和3H-TdR掺入法,研究微丝功能对创面愈合及细胞增殖的影响。结果内皮细胞在损伤修复过程中伴随微丝特殊而有序的变化。用细胞松弛素B破坏微丝,可不同程度抑制创面的愈合及细胞增殖,并呈一定的时间-剂量依赖关系。结论微丝功能在促进内皮细胞修复过程中起重要作用,可通过直接或间接效应影响DNA合成,从而影响修复过程。     

A fine structural study of human oral epithelium separated from the lamina propria by enzymatic action

Incubation of human oral mucosa in physiological solutions containing proteolytic enzymes permits separation of the preparation into its epithelial and connective tissue components. Trypsin, collagenase and elastase were utilized to effect epithelium-connective tissue separation. Elastase was the most suitable in that a reliable separation of the epithelium from the connective tissue occurred at the lamina lucida (the electron-lucent zone between the basal cell and basal lamina) with only minimal alteration of the epithelium. The most common change observed in separated epithelium was the formation of cytoplasmic protrusions or blebs on the inferior surface of the epithelial basal cell. Bleb formation was quite extensive when preparations were incubated one to two hours beyond the point where the epithelium could be separated from the connective tissue. With prolonged incubations inferior aspects of epithelial basal cells demonstrated the formation of an entirely new cytoplasmic front apparently resulting from fusion of membranes and subsequent confluence of the cytoplasm contained within the blebs. Individual hemidesmosomes or small lengths of the original inferior epithelial basal cell membrane became enclosed in membrane-bound vacuoles within the cytoplasm of the epithelial basal cell. These vacuoles were shown to have been interiorized by the absence of a ruthenium red reaction product within the vacuolar spaces. Bleb formation was shown to be strictly enzyme-induced since intact specimens demonstrated extensive basal cell blebbing following prolonged incubation. Occasional desmosomes were broken and the component halves interiorized in membrane-bound vacuoles within the cell cytoplasm. Alterations observed in epithelial basal cells as a consequence of exposure to exogenous proteolytic enzymes mimic alterations observed in many disease processes and during certain stages of development.     

Effects of cytochalasin B on the fine structure of organized endodermal cells of the early chick embryo

Fresh pullet eggs (White Leghorn) were incubated for 36 to 48 hours. The blastoderms were exposed to cytochalasin B (CB), 10 or 40 μg/ml, for 2, 5, and 15 minutes prior to fixation by immersion in buffered chick Ringers solution containing CB, previously dissolved in dimethysulfoxide (DMSO), or by sub-blastodermic injection. Controls fixed in ovo possess relatively flat surfaces with bulges due to uptake of yolk. Numerous microappendages (blebs, microvilli and ruffles) are present, especially at cell margins. DMSO-controls present a similar cell surface except that small blebs are more prominent. The plasmalemmas of CB-treated endodermal cells possess numerous large blebs (2-10 μm in diameter), smaller blebs (0.2 μm) and microvilli. Cell dissociation occurs in selected areas resulting in rounded cells, devoid of microappendages, with peripheral processes. Transmission electron microscopic preparations of tissues similar to those used for scanning electron microscopy reveal that large blebs are filled with membranous material. Microfilaments are present but lack their normal subplasmalemmal arrangement. Microtubules and other cell organelles are apparently unaffected by CB. Evidence in this study supports the concept that cytochalasin B exerts its influence through alteration of the plasmalemma.     

Inhibition of human cutaneous anaphylaxis by cytochalasin B

The effect of cytochalasin B (which selectively inhibits the function of cytoplasmic contractile microfilaments) on IgE-mediated hypersensitivity in human skin was studied. Cytochalasin B inhibited the antigen-induced histamine release from human skin slices sensitized passively in vitro with human reaginic serum. The magnitude of inhibition of histamine release was related to the duration of preincubation of sensitized skin slices with cytochalasin B. The effect of cytochalasin B on antigen-induced histamine release from sensitized skin was reversible, indicating that the inhibition of histamine release may be due to the inhibition of contraction of cytoplasmic microfilaments in sensitized target cells by cytochalasin B.     

The effect of cytochalasin B on chick mesoderm cells as studied by scanning electron microscopy

Summary The effects of cytochalasin B (CCB) on chick mesoderm cells in vivo was examined by scanning electron microscopy (SEM). The embryos were mounted for New Culture and the mesoderm exposed by dissecting off the endoderm. Cytochalasin B was suspended in saline and the embryos flooded with the suspension. Control embryos were treated with saline alone. The embryos were reincubated for varying times at 37°C.In the treated embryos the mesoderm cells were rounded and separated from each other. Many had long branched processes and rough surfaces. These changes became more pronounced as treatment time was increased. They were also reversible on reincubating treated embryos in the absence of cytochalasin B. The morphological changes produced by CCB are thought to be due to an effect on the cytoskeleton, either a direct disruptive effect or detachment of skeletal microfilaments from the cell membrane. There may also be a direct removal of cell surface materials leading to the observed surface roughening of treated cells.     

Mechanism of T cell-mediated cytolysis: the differential impact of cytochalasins at the recognition and lethal hit stages.

We have investigated the impact of a range of cytochalasins of various classes, not only on T cell-mediated cytolysis as a whole, but also at each stage of this process using the analytical Ca⁺⁺ pulse method. We compared the results thus obtained with the effect of some of these drugs on microfilaments (assessed by electron microscopy) and on glucose metabolism. All cytochalasins blocked the recognition stage of T cell-mediated cytolysis, none blocked the target cell disintegration stage, and all cytochalasins but cytochalasin A blocked Ca⁺⁺-dependent events of the lethal hit stage. Cytochalasin A, which did not block lethal hit, clearly affected microfilaments. Cytochalasin B blocked both the lethal hit and glucose catabolism while cytochalasin A inhibited neither. The following pattern is therefore suggested. The recognition stage can be blocked by both cytochalasin A and cytochalasin B, presumably through a common effect of these drugs on microfilamental structures. The lethal hit stage can be blocked by cytochalasin B but not by cytochalasin A, presumably through the impact of the former on cell membrane permeability to glucose. These results are discussed both in terms of their practical interest relative to the use of Ca⁺⁺ and cytochalasin A to probe lytic events, and in terms of the information they provide about metabolic steps and cell structures which could be involved in the lethal hit.     

内皮细胞微丝结构与血管内皮完整性关系的探讨

为证实内皮细胞微丝与血管内皮完整性之间的直接关系，用Ｒｈｏｄａｍｉｎｅ－ｐｈａｌｌｏｉｄｉｎ显示了体外培养的牛主动脉内皮细胞形成一微密单层细胞后的微丝形态及用细胞毒素Ｂ孵育单层内皮细胞后的细胞形态及微丝的变化。结果表明：内皮细胞内的微丝结构ＤＰＢ是维持内皮细胞之间连接的重要结构。文内还对内皮细胞微丝结构与动脉粥样硬化早期病变的关系进行了讨论。     

Differences in the Mode of Phagocytosis with Fc and C3 Receptors in Macrophages

Mouse macrophages, activated in vivo or in vitro, were made to internalize sheep erythrocytes opsonized with IgG or IgM and complement. Scanning electron microscopy and transmission electron microscopy were performed at various times after the transfer of the cultures from conditions favoring attachment to conditions favoring internalization. The receptors for Fc and C3 were distributed randomly over the macrophage surface with the exception of the extreme periphery, were Fc receptors were more abundant. The E-IgG were ingested by means of thin membrane extensions rising from the macrophage surface and enclosing the opsonized particles tightly in a cup-like structure protruding from the macrophage surface. Only afterwards were the covered particles drawn into the cell body proper. The E-IgMC were seen to sink directly into the macrophage cytoplasm without apparent involvement of membrane extensions. Experiments with cytochalasin B suggested that microfilaments were essential for the phagocytosis by Fc but much less important with the C3 receptor.     

Inhibition of endothelial cell retraction by ATP depletion.

To determine the relationship among endothelial cell (EC) retraction, cell adenosine triphosphate (ATP), and the status of cellular actin, ATP levels, F-actin content, and cytochemical redistribution in bovine pulmonary artery endothelial cells were assessed. EC monolayers 7 days after confluence were exposed to ethchlorvynol (ECV), histamine, or cytochalasin B (cyto B) for time intervals from 5-90 minutes. All 3 agents resulted in endothelial cell retraction without significant effect on cellular ATP content. Sixty-minute incubation of monolayers in glucose-free media containing antimycin A and 2-deoxyglucose depleted cellular ATP to less than 10% of control levels. ATP depleted monolayers failed to retract when incubated with ECV, histamine, or cyto B. ATP depletion resulted in loss of the prominent EC margins but only a rare gap between adjacent cells. When ATP levels were allowed to recover, the ability of EC monolayers to retract was restored. Actin filaments in control monolayers localized to a dense peripheral band of actin, a paranuclear complex, and bundles of microfilaments orientated parallel to the long axis of the cell. ECV induced complete loss of the dense peripheral band and other changes in the actin disposition. Monolayers exposed to histamine showed a retraction of the dense peripheral band of actin to a subcortical position. Cyto B caused loss of the dense peripheral band and the longitudinal microfilament bundles. Monolayers depleted of ATP lost their dense peripheral band and exhibited a disorganized, tangled web of microfilaments. Neither histamine nor ECV modified the actin distribution in ATP-depleted monolayers, whereas exposure to cyto B resulted in substantial change in actin with formation of a rim inside the cell membrane and considerable loss of actin filaments. ECV or histamine induced a small reduction in F-actin content while cyto B resulted in a 50% decline in 15 minutes. ATP depletion resulted in a 19% decrease in F-actin, with no further reduction on subsequent exposure to histamine or ECV. Cyto B treatment of ATP-depleted monolayers caused a drop in F-actin content equivalent to that observed in cells with normal ATP levels. These studies indicate that ATP is essential for changes in actin filament distribution and endothelial cell retraction produced by ECV, histamine or cyto B, and make it unlikely that any of these agents acts simply by depolymerization of actin filaments or modification of the dense peripheral band, although disruption of the dense peripheral band may facilitate retraction in the presence of adequate levels of cell ATP.     

Human T-lymphocyte rosette formation: inhibition by cytochalasin B.

The effects of cytochalasin B, colchicine and vinblastine on the rosette formation of human T lymphocytes with neuraminidase-treated human erythrocytes (nHRBC) and with sheep red blood cells (SRBC) have been studied. Pretreatment of the lymphocytes with cytochalasin B which affects microfilament action reversibly inhibits both nHRBC and SRBC rosette formation. Colchicine and vinblastine known to interact with microtubules causes no major reduction in rosette-forming cells. The results suggest that normally functioning microfilaments are necessary for nHRBC and SRBC rosetting, whereas microtubules are not essential for the blinding of the erythrocytes to the lymphocytes.