Fig. 3. Selective visualization of growing MT ends with CLIP-170. CHO cells were transfected by nuclear microinjection of GFP-CLIP-170 DNA, allowed to express GFP-CLIP protein, then microinjected with Cy3-tubulin and, finally, enucleated to generate cytoplasts. Time-lapse sequences were obtained to show dynamics of MTs and CLIP-170. (A) Low magnification of MTs, CLIP-170 and merged image (green, Cy3-MTs; red, CLIP-170). (B) Time-lapse sequence of region boxed in panel A. Two dynamic MTs are indicated by arrowheads. CLIP-170 is present at their plus ends during growth phases but disappears within 5 seconds after transition from growth to pause or shortening phase. Numbers in top-left corners indicate time in seconds. Interval between acquisitions of images in alternating channels was 2.5 seconds, which gave an interval between successive images in either the GFP or Cy3 channel of 5 seconds. Scale bar, 5 µm. (C) Life history plots of MTs (green) and CLIP-170 tracks (red) indicated by arrowheads in panel B. CLIP-170 data points were time-shifted by 2.5 seconds to compensate for the delay between acquisition of CLIP and MT images. Absence of red data points from segments of the plots indicates when CLIP-170 disappeared from the MT end. (D) Example plots of nascent MTs (green) growing off the centrosome and corresponding CLIP-170 tracks (red) after time-shifting of 2.5 s. Plots were arbitrarily staggered along the time axis for clarity. Scales of axes indicated in upper right corner of graph. For some MTs either near the centrosome or in areas of high MT density, the MT end could not be clearly visualized. Nevertheless, CLIP-170 movement could be seen. This is represented in the plots as red points without corresponding green ones. In all cases, clear growing MTs had CLIP-170 at their ends.