EB3-GFP comet movements in living megakaryocytes. (A-D) EB3-GFP comet movements within the tips of proplatelets. (A) First frame of a time-lapse sequence shown in Movie S3. (C) First frame of a time-lapse sequence shown in Movie S4. (B,D) Kymograph of EB3 around proplatelet tips. In the first example, EB3-GFP comets enter the proplatelet tip, circle its periphery, and then reenter the shaft of the proplatelet. Images are every 5 seconds. In the second example, EB3-GFP comets move around the periphery of the tip in both directions (arrowhead highlights clockwise movement; arrow highlights counterclockwise movement). Images are every 2 seconds. Comets also move into the tip on microtubules that end abruptly. The scale bar represents 5 μm. (E-G) Location and dynamics of microtubule plus-ends in representative preproplatelet megakaryocytes. (E-F) Anti-EB3 immunofluorescence of preproplatelet-megakaryocytes. Cells at this stage of maturation have a radial array of microtubules that emanates from the centrosome. EB3-GFP comets concentrate near the centrosome and are on the plus-ends of microtubules that radiate outward. Field width, 20 μm. (G) Dynamics of EB3-GFP comets in a megakaryocyte lacking proplatelets. This sequence of images shows EB3-GFP to concentrate on the plus-ends of microtubules as they grow from the centrosome (Movie S4). Translocations of EB3-GFP in the cell cortex, parallel to the plasma membrane, are also apparent. Elapsed time is in seconds.