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Study of a lysis medium stabilizing microfilaments and microtubules in vitro and in vivo
Author(s) -
Foucault G.,
Raymond M. N.,
Coffe G.,
Pudles J.
Publication year - 1985
Publication title -
biology of the cell
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.543
H-Index - 85
eISSN - 1768-322X
pISSN - 0248-4900
DOI - 10.1111/j.1768-322x.1985.tb00324.x
Subject(s) - microfilament , tubulin , microtubule , cytoskeleton , heavy meromyosin , biology , lysis , actin , egta , in vivo , biochemistry , biophysics , polymerization , colchicine , in vitro , microbiology and biotechnology , cell , chemistry , calcium , genetics , organic chemistry , polymer
Determination of experimental conditions which allow the evaluation of the variations in the ratio of non polymerized and polymerized forms of actin and tubulin during the reorganization of the cytoskeletal cell system is of most valuable importance. In order to prepare cell homogenates which would reflect the in vivo situation, we tested in vitro a lysis medium which stabilized both microfilaments and microtubules, which were determined by DNase inhibition assays and colchicine binding assays respectively. This lysis medium containing 10 mM potassium phosphate, 1mM magnesium chloride, 5 mM EGTA, 1 M hexylene glycol, 1% Triton X‐100, pH 6.4, used at 4 degrees C a) diffused rapidly into the cells; b) did not denature actin and tubulin; c) did not displace the equilibrium between non polymerized and polymerized forms of actin and tubulin, allowing biochemical assays on cell homogenates; d) blocked the evolution of the cytoskeletal system and permitted structural studies; e) and allowed the decoration of microfilaments by heavy meromyosin.