Comparative effects of cryosolvents on tubulin association, thermal stability, and binding of microtubule-associated proteins.
Organic cryosolvents essential for cryopreservation of living cells have a colligative effect on water properties, but also affect cellular structures such as the membrane, actin, or tubulin cytoskeleton. The effects of cryosolvents on actin and its binding proteins are starting to be well investigated. In parallel, tubulin assembly characteristics were investigated comparatively, with 0-30% 1,2-propanediol, dimethyl sulfoxide, or glycerol, and with or without microtubule-associated proteins, at 37 or 4 degrees C. Tubulin association was monitored by spectrometry and sedimentation, providing the concentration in free protein, cold-depolymerizable microtubules, and cold-resistant associations. At 37 degrees C, 1,2-propanediol and dimethyl sulfoxide induce a similar association level and cold stability of the assemblies. Glycerol yields a lower level of tubulin association. Cold stability of the assemblies requires the presence of solvent, the amount of which is modulated by microtubule-associated proteins (MAPs): 15% 1,2-propanediol or dimethyl sulfoxide, decreasing down to 10% with MAPs, or 10% glycerol with MAPs only. At 4 degrees C, some cold-stable association is promoted by 1,2-propanediol or dimethyl sulfoxide above 10-15%, in the presence or absence of MAPs, but not with glycerol. In addition, protein content of the various fractions obtained with MAPs and 30% solvent was examined by densitometry of electrophoresis gels. Cold-labile associations obtained at 37 degrees C with 1,2-propanediol or dimethyl sulfoxide are lacking in tubulin and enriched in tau proteins relative to control or glycerol. Associations formed at 37 degrees C and stable to subsequent cold treatment, or at 4 degrees C, regardless of the solvent, present a large tubulin content, as well as few tau proteins and high-molecular-weight MAPs.
Pajot-Augy
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Unité de Recherche en Développement Concerté INRA-INSERM (U.310), Institut de Biologie Physico-chimique, Paris, France.
D000818:Animals; D001921:Brain / Q000378:metabolism; D015925:Cryopreservation / Q000379:methods; D003451:Cryoprotective Agents / Q000494:pharmacology*; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D009391:Nephelometry and Turbidimetry; D012997:Solvents; D013552:Swine; D013816:Thermodynamics; D014404:Tubulin / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*
D003451:Cryoprotective Agents; D008869:Microtubule-Associated Proteins; D012997:Solvents; D014404:Tubulin