Video shows the dynamics of microtubules (MTs) using a GFP fusion construct of EB3 (end binding protein 3) that transiently binds to growing MT plus ends, generating a punctate pattern of EB3-GFP comets throughout the cell. RPE-1 cells were infected with a retroviral vector to express EB3-GFP. Cells on round coverslips were transferred to a homemade holding device, and 600 µl of the appropriate medium was added. The video was acquired with a microscope (IX70 Delta Vision Spectris; Olympus), temperature-controlled at 37C, using a 60× NA 1.4 differential interference contrast (DIC) oil Plan-Apochromat objective, ex 470 em 520, and a camera (CoolSNAP HQ; Roper Industries) with an exposure time of 100 ms and a frame rate of .5 s. Acquisition software used was SoftWoRx version 3.3.4 (Applied Precision). This is the raw data file for Video 1 and a frame from this is shown in Fig 1E of J Cell Biol. 2010. 190: 991-1003.