Influence of MRP8 and MRP14 on tubulin polymerization. Tubulin (final concentration 1 mg/mL) was mixed with MRP8/MRP14, MRP8/phospho-MRP14 complexes (A-B), or MRP8 or MRP14 monomers (C) under either polymerizing (3.4 M glycerol in panels A and C, molar ratio of tubulin to MRP = 1:1) or nonpolymerizing conditions (panel B, 1.1 M glycerol, molar ratio of tubulin to MRP as indicated in the figure). Tubulin and MRP8/MRP14 alone were used as controls. Polymerization was induced by elevating the temperature from 1°C to 37°C within 210 seconds, and formation of tubulin filaments was monitored by measuring optical density at 340 nm for at least 1 hour. Each experiment was repeated at least 3 times. (D-H) Transmission electron micrographs of samples prepared as above (ratio of tubulin dimer to MRP8/MRP14 dimer = 10:1, 60 μM calcium). Tubulin filaments polymerized in the absence (D) or presence (E) of MRP8/MRP14; in the presence of MRP8/phospho-MRP14 (F); or in the presence of MRP8/MRP14 with localization of MRP14 by immunogold labeling (G-H). MRP8/MRP14 shows a clear colocalization with MT bundles, whereas single-stranded tubulin filaments are spared. Immunogold labeling with anti-MRP8 revealed identical patterns (data not shown). Bars represent 300 nm.