Figure 7.

p150^Glued has a potent microtubule nucleation effect and EB1 has a potent microtubule elongation effect. (A) The effect of p150^Glued, EB1, both proteins, and tubulin only (control) on microtubule polymerization over time was examined using a light-scattering assay. When no microtubule seeds are present, EB1 has little effect over that of tubulin alone, but p150^Glued has a potent effect on the extent of polymerization. The combination of EB1 and p150^Glued had an even greater effect. However, the combination of EB1 and p150^Glued appeared to cause a confounding light-scattering aggregation that settled out of solution over time, similar to that seen in the pelleting experiment. The inset graph shows this effect before and after the addition of tubulin. (B) In the presence of microtubule seeds, p150^Glued had little effect on microtubule polymerization over that of the addition of tubulin alone, but the addition of EB1 had a potent effect. (C) Fluorescently labeled microtubules were pelleted onto coverslips after polymerization in the above conditions. Without seeds, very few microtubules were polymerized with tubulin alone. Long slender bundles were polymerized in the presence of EB1, and dense tangled bundles were polymerized in the presence of p150^Glued. Microtubules polymerized in the presence of both EB1 and p150^Glued had characteristics of both. (D) Microtubules polymerized much more efficiently in the presence of seeds, necessitating a larger microscopic field of view. Relatively short, simple microtubules were polymerized in the presence of tubulin alone, short, highly branched microtubule bundles were polymerized in the presence of p150^Glued, and extremely long microtubule bundles were polymerized in the presence of EB1. Again, microtubules polymerized in the presence of both EB1 and p150^Glued had characteristics of both. Scale, 10 μm.