Like a flexible Lego railwaytrack: How stable microtubules form within cells
). For GTP-bound tubulin, polymerization/depolymerization was repeated for three cycles. In the final cycle, tubulin was depolymerized in PEM supplemented with 0.1 mM GTP, snap-frozen, and stored in liquid nitrogen. To prepare GMPCPP-bound tubulin, two cycles were conducted in the same way as GTP-bound tubulin, and purified tubulin was subsequently subjected to two polymerization/depolymerization cycles using 0.1 mM GMPCPP instead of 1 mM GTP.
GMPCPP-bound tubulin labeled with Alexa Fluor 488 and biotin was incubated at 27°C for 1 h to promote nucleation, followed by 1 h of polymerization at 37°C. Polymerized microtubule samples were purified by centrifugation at 20,000The glass chamber was treated as follows: first, 1 mg/ml NeutrAvidin was loaded into the chamber and incubated for 2 min to let it bind to biotin molecules covalently connected to the glass surface.
Because the binding activity of D2 was lost quickly after being thawed , we prepared new samples every 30 min.During this experiment, 1 μM D2-mCherry in imaging buffer was used for the D2-mCherry samples, and variable concentrations of KIF5C in BRB30 with 25% glycerol were used for the KIF5C pretreatment. A microtubule-immobilized glass chamber was first incubated with D2-mCherry for 5 min.
For the compaction experiment, pretreatment with 2.0 μM KIF5C was repeated as above except the buffer used for the KIF5C dissociation was ADP buffer and not a high-salt buffer.
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