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Protocol no. 24

CYTOSKELETAL ALTERATIONS AS A PARAMETER FOR ASSESSMENT OF TOXICITY

Changes in the balance of cytoskeletal proteins after exposure to test compounds can be detected by indirect immunofluorescence microscopy and quantitative biochemical methods.

CONTACT

INVITTOX 34 Stoney Street Nottingham NG1 1NB UK Tel: England - 0602 584740 Fax: England - 0602 503570 N.B. Due to unforeseen circumstances, INVITTOX lost contact with the informant for this protocol when the document was already completed but not cleared. We have decided to release the protocol, but are unable to put users in touch with the informant or pass on any comments or problems.

RATIONALE

It is becoming increasingly apparent that the cytoskeleton is the target site for a number of toxins and environmental pollutants. The protein constituents may be affected in two ways: stabilisation of the polymerised form, or the breakdown of these fibres into the constitutive elements. Three methods using, respectively, the binding of colchicine, electrophoresis, and fluorescent microscopy provide an overall picture of the effect of toxins on the cytoskeleton.

BASIC PROCEDURE

Fluorescent microscopy CHO cells are grown on glass coverslips, then fixed with absolute methanol and acetone. After rehydration with PBS they are incubated with either monoclonal anti-a- and anti-ß-tubulin or monoclonal anti-vimentin, rinsed, and incubated with rhodamine-labelled anti-mouse IgG in order to stain the microtubules and intermediate filaments. The microfilaments are stained by fixing the cells with paraformaldehyde, and then permeabilising with Nonidet. Rhodamine-labelled fluorescent phalloidin is used as the stain. After a final rinse, the cells are mounted in a non-fluorescent mounting solution, observed under an epifluorescent microscope and photographed. Tubulin measurement using the colchicine method Monomeric tubulin is extracted from CHO cells using "PM2G solution" containing Nonidet P-40. The remaining cytoskeletal layers are then be treated with a solubilising solution which extracts tubulin from the microtubules. The tubulin content of both extracts is determined using a colchicine binding assay and the relative amounts of polymerised and unpolymerised tubulin in control and exposed cultures are compared. Radiolabelling of cytoskeletal proteins CHO cells are grown in the presence of radioactive methionine. The cytoplasmic and microtubular proteins are extracted as outlined above. The remaining cytoskeleton (residual fraction) is dissolved with lysis buffer. By means of 2-dimensional gel electrophoresis the distribution of actin, tubulin and vimentin in the three fractions can then be compared in control and test cultures.

CRITICAL ASSESSMENT

Gross alterations to the cytoskeleton may be visualised by means of indirect immunofluorescence microscopy. Monoclonal antibodies against tubulin may be used to stain microtubules and intermediate filaments, while the capacity of phalloidin to bind irreversibly to actin is used to stain microfilaments. The information derived from such observations may then be extended by more refined biochemical assays: The use of two-dimensional gel electrophoresis allows a more detailed analysis of changes in the distribution of cytoskeletal proteins between various fractions after exposure of cells to test compounds. The high affinity of colchicine for tubulin provides a means to assay the tubulin present in the unpolymerised intracellular pool and in the microtubules. The ratio between polymerised/unpolymerised protein is a quantitative measure of toxic effects directed at the cytoskeleton. Observation of alterations to the cytoskeleton can provide a more sensitive system for the detection of some toxic effects than does the use of a more general assay, such as the uptake of radio-labelled thymidine. An illustration of the potential of this test system can be found in the paper of Scapigliati et al., (1988) which compares the effects of cholera and pertussis toxins. Fluorescence microscopy showed the microtubular stabilisation induced by cholera toxin, which resulted in the microtubule cytoskeleton being better visualised in the cells treated with cholera toxin than in control cells or those treated with pertussis toxin. The other two cytoskeletal fibres were not affected by the cholera toxin. In contrast, cells treated with pertussis toxin were found to have lost their actin filaments, while microtubules and intermediate filaments were unaffected. The effects of cholera toxin were further investigated with the other two techniques described in this protocol. Two-dimensional gel electrophoresis showed microtubular tubulin to be more abundant than monomeric tubulin, and the colchicine-binding assay quantified this observation to show that the cholera-toxin treatment resulted in a 2-fold accumulation of tubulin into the microtubules, while in control and pertussis-treated cells the tubulin was equally distributed between the unpolymerised and the microtubular fractions.

CHEMICALS TESTED

Cholera toxin Pertussis toxin

REFERENCES

  1. Borisy, J.J. (1972) A rapid method for quantitative determination of microtubule protein using DEAE-cellulose filters. Analytical Biochemistry, 50, 373-385
  2. Bravo, R., & Celis, J.E. (1980) A search for differential polypeptide synthesis throughout the cell cycle of HeLa cells. J. Cell. Biol., 84, 795-802
  3. Bravo, R., Celis, A., Mosses, D., & Celis, J.E. (1981) Distribution of HeLa cell polypeptides in cytoplasts and karyoplasts. Cell. Biol. Int. Rep., 5, 479-489
  4. Duerr, A., Palla, D. & Solomon, F. (1981) Molecular analysis of cytoplasmic microtubules "in situ": identification of both widespread and specific proteins. Cell, 24, 203-211.
  5. Holmgren, J.L., Lindholm, L. & Lonnroth, I. (1974) Interaction of cholera toxin derivatives and lymphocytes. I. Binding properties and interference with lectin-induced cellular stimulation. J. Exp. Med., 139, 801-819.
  6. Laemmli, U,K. (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature, 227, 680-685
  7. Nicosia, A., Perugini, M., Franzini, C., Casagli, M.C., Borri, M.G., Antoni, G., Almoni, M., Neri, P., Ratti, G. & Rappuoli, R. (1986) Cloning and sequencing of the pertussis toxin genes: operon structure and gene duplication. Proceedings of the National Academy of Sciences, USA, 83: 4631-4635.
  8. Scapigliati, G., Rappuoli, R., Silvestri, S., & Pallini, V. (1988) Cytoskeletal alterations as a parameter for assessment of toxicity. Xenobiotica 18, 715-724.

IP-24 © July 1991