Fig. 4. Phosphorylation regulates the binding of AtMAP65-1 to microtubules. (A) Cosedimentation assays of taxol-stabilised microtubules with the recombinant wild-type AtMAP65-1 (WT) or the mutants, AtMAP65-1^2D (2D), AtMAP65-1^4D(4D), AtMAP65-1^7D(7D) and AtMAP65-1^9D(9D). The amount of AtMAP65-1 proteins in the supernatant and pellet was measured on the Coomassie-stained SDS-PAGE gels using densitometry and the percentage of the total recombinant AtMAP65-1 protein used in the reaction that was recovered in the pellet was calculated as described in the Materials and Methods. (B) Turbidimetric analysis of a 14 µM tubulin solution and a tubulin solution mixed in an equimolar ratio with AtMAP65-1 or AtMAP65-1^9D. (C) Localisation of GFP:AtMAP65-1^4D(4D), GFP:AtMAP65-1^7D(7D), GFP:AtMAP65-1^9D(9D) fusion proteins in interphase and anaphase cells. (D) The ratio between the midzone and the pole signals of GFP:AtMAP65-1 (WT), GFP:AtMAP65-1^4D(4D), GFP:AtMAP65-1^7D(7D) and GFP:AtMAP65-1^9D(9D) fusion proteins. Ten anaphase cells were analysed for each transgenic cell line.