Cytochalasin B: Does It Affect Actin-Like Filaments?

Forer, Arthur; Emmersem, J.; Behnke, O.

doi:10.1126/science.175.4023.774

Cited by 91 publications

(11 citation statements)

References 20 publications

(11 reference statements)

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“…rough ER, Golgi and vesicles, we inhibited secretory transport at different steps. 293T cells were treated with the following drugs: brefeldinA (BFA) which inhibits anterograde ER export to the Golgi, but allows retrograde Golgi-ER transport, resulting in a fusion of the ER and the Golgi and blocking of secretion [21]; monensin A which blocks transport within the Golgi [22]; nocodazole which causes microtubule depolymerization leading to blocking of microtubule-dependent translocation of vesicles between the ER and the Golgi [23]; and cytochalasin B which disrupts the actin cytoskeleton and thereby blocks membrane transport along actin filaments [24]. Treating cells with these drugs resulted in significant inhibition of Gluc secretion into the conditioned medium with about 90% decrease with BFA, 65% with monensin A, 75% with nocodazole, and 30% with cytochalasin B at the concentration used over 24 hrs ( Fig.…”

Section: Resultsmentioning

confidence: 99%

A Highly Sensitive Assay for Monitoring the Secretory Pathway and ER Stress

et al. 2007

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BackgroundThe secretory pathway is a critical index of the capacity of cells to incorporate proteins into cellular membranes and secrete proteins into the extracellular space. Importantly it is disrupted in response to stress to the endoplasmic reticulum that can be induced by a variety of factors, including expression of mutant proteins and physiologic stress. Activation of the ER stress response is critical in the etiology of a number of diseases, such as diabetes and neurodegeneration, as well as cancer. We have developed a highly sensitive assay to monitor processing of proteins through the secretory pathway and endoplasmic reticulum (ER) stress in real-time based on the naturally secreted Gaussia luciferase (Gluc).Methodology/Principle FindingsAn expression cassette for Gluc was delivered to cells, and its secretion was monitored by measuring luciferase activity in the conditioned medium. Gluc secretion was decreased down to 90% when these cells were treated with drugs that interfere with the secretory pathway at different steps. Fusing Gluc to a fluorescent protein allowed quantitation and visualization of the secretory pathway in real-time. Expression of this reporter protein did not itself elicit an ER stress response in cells; however, Gluc proved very sensitive at sensing this type of stress, which is associated with a temporary decrease in processing of proteins through the secretory pathway. The Gluc secretion assay was over 20,000-fold more sensitive as compared to the secreted alkaline phosphatase (SEAP), a well established assay for monitoring of protein processing and ER stress in mammalian cells.Conclusions/SignificanceThe Gluc assay provides a fast, quantitative and sensitive technique to monitor the secretory pathway and ER stress and its compatibility with high throughput screening will allow discovery of drugs for treatment of conditions in which the ER stress is generally induced.

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Section: Resultsmentioning

confidence: 99%

A Highly Sensitive Assay for Monitoring the Secretory Pathway and ER Stress

et al. 2007

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“…We harvested these cells 9 days after challenge, when they had just completed a cycle of blast transformation and several cell divisions in the draining lymph nodes (17)(18)(19); they might be expected to differ physiologically from their unstimulated precursors. Sensitized cells have been described as intensely pyroninophilic (20)(21)(22), medium-sized lymphocytes (3,5,18,23) of low to moderate density (24), possessing a uropod unlike their precursors (25,26) and ultrastructural features resembling those of a cell activated by mitogen (26). Thus, they may be regarded as already partially activated, and this would account for their decreased sensitivity to PHA or cytochalasin B.…”

Section: 5ogmentioning

confidence: 99%

Regulation of Lymphocyte Responses In Vitro: Potentiation and Inhibition of Rat Lymphocyte Responses to Antigen and Mitogens by Cytochalasin B

Yoshinaga

Waksman

1972

Proc. Natl. Acad. Sci. U.S.A.

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Cytochalasin B, at concentrations between 0.02 and 0.2 ;Lg/ml, was slightly stimulatory to lymph-node cells from normal rats and greatly potentiated their response to phytohemagglutinin and low concentrations of concanavalin A (mitogens for thymus-derived lymphocytes); it also potentiated the response of thymocytes to phytohemagglutinin. The response of lymphnode cells to lipopolysaccharide endotoxin (a mitogen for thymus-independent lymphocytes) was also enhanced, but only at concentrations in the usual inhibitory range, possibly by inhibition of a "suppressor T-cell" response. Sensitized lymphocytes responding to antigen were not stimulated at all, except at a very high cell density, where inhibition of a "suppressor cell" response was also considered likely. At concentrations of 5-10 Mg/ml or higher, cytochalasin 1B inhibited all responses tested.Cytochalasin B is a fungal product (1) widely used as an inhibitor of cell functions that involve contraction of microfilaments (2). Its target within the cell was undetermined (3) until recently; new evidence establishes firmly its ability to interact directly with the actin moiety of actomyosin, producing a loss of viscosity and of ATPase activity (4). Cytochalasin B, in a restricted low-concentration range (<1 Mug/ml), stimulates chemotaxis (5) and phagocytosis ( §), but inhibits these functions at higher concentrations (>5 Mkg/ml). We report here its ability, in a concentration range of 0.02-0.5 ,g/ml, to stimulate lymphocyte DNA synthesis and to potentiate the responses of lymphocytes to mitogens. MATERIALS AND METHODSAnimals and Sensitization. Inbred DA rats of both sexes, 3-5 months of age, were sensitized by injection of ovalbumin (Nutritional Biochemicals Corp., Cleveland, Ohio), 100 ,g in Freund's complete adjuvant in both hind foot pads (6). Their inguinal and iliac nodes, 9 days after sensitization, were used as sources of sensitized lymph-node cells (LNC). As sources of normal LNC, cervical, axillary, inguinal, and popliteal lymph nodes were harvested from untreated rats. (Grand Island Biological Co., Grand Island, N.Y.), 100 units of penicillin per ml, and 100 ,lg of streptomycin per ml, was used throughout.Reagents. Ovalbumin, PHA (phytohemagglutinin P; Difco Laboratories, Detroit, Mich., ¶), Con A (concanavalin A, twice crystallized, Nutritional Biochemicals Corp., Cleveland, Ohio), and lipopolysaccharide B, Escherichia coli 0111:1B4 (Difco Laboratories, Detroit, Mich.) were dissolved in phosphate-buffered saline (pH 7.4) at various concentrations, and 0.1 ml of the solutions were added to appropriate cultures. Cytochalasin B (Imperial Chemical Industries Ltd., Macclesfield, Chesire, England) was dissolved at 3 mg/ml in (CH3)2SO. Just before use, the stock solution was diluted with phosphate-buffered saline to the desired concentrations, and corresponding dilutions of (CH3)2SO were prepared as controls.Estimation RESULTSEffect of Cytochalasin B on Nonstimulated Cells. Normal LNC (2 X 106/ml) were incubated with cytochalasin B at concentrati...

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“…(33) . A direct disruptive effect of cytochalasin on the microfilaments of the cell cortex (or of their insertions), on a generalized scale such as that tentatively suggested by Wessels et al .…”

Section: Contractionmentioning

confidence: 99%

“…Although the direct effect of cytochalasin B (CB) on actin fibrils in vitro is in some doubt (33,87,88), long treatment of cells with cytochalasin does not simply derange the cortical microfilaments (36) . Indeed, many such microfilaments can become organized into impressive arrays' (36) .…”

Section: Introductionmentioning

confidence: 99%

Action of Cytochalasin D on Cells of Established Lines

Miranda

Godman

Deitch

et al. 1974

The Journal of Cell Biology

216

119

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HeLa, Vero, L, HEp2, and MDBK cells respond immediately to 0.2–0.5 µg/ml cytochalasin D (CD) with sustained contraction (contracture), loss of microvilli, expression of endoplasmic contents (zeiosis), nuclear protrusion, and extension of cytoplasmic processes. The development of these changes is depicted, and the dose-response patterns in these cell lines are described. MDBK is generally most resistant and HeLa most sensitive to these effects of CD. Cells in G1 are most sensitive to CD; responsiveness decreases progressively during early S and is least in mid S through G2. CD inhibits transport of [14C]deoxyglucose in HeLa by about 45% but has no significant effect on hexose uptake in Vero and MDBK; sugar transport is thus apparently unrelated to any morphologic effect of CD. Although spreading and attachment are impeded, CD does not decrease and may even enhance the adhesiveness of established monolayers. Contraction appears to be a primary early effect of CD, upon which other visible changes follow. It is prevented by some inhibitors of energy metabolism (deoxyglucose and dinitrophenol) and does not occur in glycerinated models without ATP. The possible bases of the contractile response to CD are discussed. Although direct or indirect action of CD on some microfilaments may occur, a generalized structural disruption of contractile filaments by CD is considered unlikely.

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Cytochalasin B: Does It Affect Actin-Like Filaments?

Cited by 91 publications

References 20 publications

A Highly Sensitive Assay for Monitoring the Secretory Pathway and ER Stress

A Highly Sensitive Assay for Monitoring the Secretory Pathway and ER Stress

Regulation of Lymphocyte Responses In Vitro: Potentiation and Inhibition of Rat Lymphocyte Responses to Antigen and Mitogens by Cytochalasin B

Action of Cytochalasin D on Cells of Established Lines

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