EB3-GFP movements in proplatelet-producing megakaryocytes. (A) First frame from a time-lapse movie of a live megakaryocyte expressing EB3-GFP (Movie S2). The cell body is at the left of the micrograph. EB3-GFP labels growing microtubule plus-ends in a characteristic “comet” staining pattern (arrowheads) that has a bright front and dim tail. Moving comets are found along the proplatelets as well as in the megakaryocyte cell body. The scale bar is 5 μm. (B) Kymograph of the boxed region in panel A. Images are every 5 seconds. EB3-GFP comets undergo bidirectional movements in proplatelets. Some EB3-GFP comets move tipward and are highlighted in green; others that move toward the cell body are highlighted in red. (C) Comparison of the velocity distribution of comets moving in proplatelets (white bars) with those emanating from the centrosome of preproplatelet megakaryocytes (dark bars). The average rate of comet movement in the preproplatelet megakaryocytes was 10.2 ± 0.77 μm/min and the rates of movement were tightly grouped (8.9-12.3 μm/min). EB3 movements in proplatelets, however, are apparently bimodal with distinct populations moving slower and faster than those of the preproplatelet megakaryocytes. (D) EB3-GFP comets were distributed throughout the proplatelet. Proplatelets were divided into 10 segments (tip = 0, cell body = 9) and the number of GFP-EB3 comets in each was determined. The length of the evaluated proplatelet was 28 μm. Error bars indicate standard deviation