Video 9.
Colocalization of microtubule subpopulations in differently coiled MBs. Related to Fig. 4 (D–F). Shown are 360° rotations of 3D reconstructions of confocal z-stack images of platelets triple immunostained with antibodies against acetylated α-tubulin (green), tyrosinated α-tubulin (red), and total α-tubulin (magenta). Individual stainings as well as merges are shown in the top images. Bars, 2 µm. The bottom images are generated from the different stainings after thresholding and skeletonizing of the MB. Images were acquired with a laser-scanning confocal microscope (LSM 710). (D) Platelet after 5 µM ADP activation for 10 s (strongly coiled MB, stage 5; the coiled MB is 13.3 µm, and the smaller ring is 10.4 µm). (E) Platelet after 5 µM ADP activation for 10 s (strongly coiled MB, stage 5; the coiled MB is 13.6 µm, and the smaller ring is 9 µm). (F) Platelet after 5 µM ADP activation for 10 s (strongly coiled MB, stage 5; the coiled MB is 13.4 µm, and the smaller ring is 7.8 µm). Please note that there may be two reasons for the absence or faint staining of microtubule subpopulations by the general anti–α-tubulin antibody. First, antibodies directed against acetylated and tyrosinated tubulin have a very high affinity for the individual modification and will give a strong signal even when only very few microtubules are present, whereas a higher number of microtubules may be necessary to obtain a strong signal with the general tubulin antibody. Second, there might also be an effect of steric hindrance between the mouse monoclonal anti–acetylated tubulin and the rabbit monoclonal anti–α-tubulin antibody because their epitopes are in relative close proximity.