Recovery of tubulin functions after freeze-drying in the presence of trehalose.
Microtubules represent cytoplasmic structures that are indispensable for the maintenance of cell morphology and motility generation. Due to their regular structural organization, microtubules have become of great interest for preparation of in vitro nanotransport systems. However, tubulin, the major building protein of microtubules, is a thermolabile protein and is usually stored at -80 degrees C to preserve its conformation and polymerization properties. Here we describe a novel method for freeze-drying of assembly-competent tubulin in the presence of a nonreducing sugar trehalose. Even after prolonged storage at ambient temperature, rehydrated tubulin is capable of binding antimitotic drugs and assembling to microtubules that bind microtubule-associated proteins in the usual way. Electron microscopy confirmed that rehydrated tubulin assembles into normal microtubules that are able to generate motility by interaction with the motor protein kinesin in a cell-free environment. Freeze-drying also preserved preformed microtubules. Rehydrated tubulin and microtubules can be used for preparation of diverse in vitro and in vivo assays as well as for preparation of bionanodevices.
Dráberová
Eduarda
E
Laboratory of Biology of Cytoskeleton, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, CZ-142 20 Prague 4, Czech Republic.
Sulimenko
Vadym
V
Sulimenko
Tetyana
T
Böhm
Konrad J
KJ
Dráber
Pavel
P
D003078:Colchicine / Q000737:chemistry / Q000378:metabolism; D005612:Freeze Drying / Q000379:methods*; D016547:Kinesins / Q000378:metabolism; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism; D055550:Protein Stability; D013696:Temperature; D014199:Trehalose / Q000737:chemistry*; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure
D014404:Tubulin; D014199:Trehalose; D016547:Kinesins; D003078:Colchicine