Figure 4.
Figure 4. Single-molecule imaging: fast turnover of CLIP-170 and EB1 at growing microtubule ends. (A, top left) Brightness distribution of the fluorescence of single H2-GFP molecules as measured when attached to a surface and bound to the growing microtubule end, as indicated. The uniformity of the distributions and their similarity indicates that the large majority of the observed H2-GFP molecules at microtubule ends are individual dimers (see also Fig. S3, available). (A, right) Individual H2-GFP molecules (added at 1 nM) binding at growing microtubule ends in the presence of an excess 74 nM of unlabeled H2, 150 nM of unlabeled EB1, and 35 μM tubulin. Images of a growing Alexa Fluor 568–labeled microtubule directly before (top) and after (bottom) a fast H2-GFP time lapse (shown as a kymograph between the microtubule images) were used to identify the growth trajectory of the microtubule end region. Binding events within the end region reaching from the distal end of the microtubule (green) to the end of the comet tail (yellow) as obtained from the averaged intensity profile (Fig. 4 A, right) were analyzed. Horizontal bar, 5 μm; vertical bar, 2 s. (A, bottom left) Histogram of the dwell times of end-associated single H2-GFP molecules. (B) Histogram of single CLIP-170–GFP molecules added at 5 nM in the presence of 70 nM of unlabeled CLIP-170, 150 nM of unlabeled EB1, and 35 μM tubulin. (C) Histogram of single EB1-GFP molecules added at 0.75 nM in the presence of 149 nM of unlabeled EB1 and 35 μM tubulin. Red lines in the histograms are single exponential fits to the data, providing a mean (single molecule) dwell time for each +TIP that is approximately two orders of magnitude lower than the mean bleaching time of the fluorescent label (Fig. S3). (D) Summary of the mean +TIP comet decoration times as determined from data shown in Fig. 3 C and the mean single molecule dwell times from histogram fits. Comet decoration times are significantly different (Mann-Whitney U test, P < 0.05), even if in a similar range. Dwell times differ from comet decoration times by more than an order of magnitude.

Single-molecule imaging: fast turnover of CLIP-170 and EB1 at growing microtubule ends. (A, top left) Brightness distribution of the fluorescence of single H2-GFP molecules as measured when attached to a surface and bound to the growing microtubule end, as indicated. The uniformity of the distributions and their similarity indicates that the large majority of the observed H2-GFP molecules at microtubule ends are individual dimers (see also Fig. S3, available). (A, right) Individual H2-GFP molecules (added at 1 nM) binding at growing microtubule ends in the presence of an excess 74 nM of unlabeled H2, 150 nM of unlabeled EB1, and 35 μM tubulin. Images of a growing Alexa Fluor 568–labeled microtubule directly before (top) and after (bottom) a fast H2-GFP time lapse (shown as a kymograph between the microtubule images) were used to identify the growth trajectory of the microtubule end region. Binding events within the end region reaching from the distal end of the microtubule (green) to the end of the comet tail (yellow) as obtained from the averaged intensity profile (Fig. 4 A, right) were analyzed. Horizontal bar, 5 μm; vertical bar, 2 s. (A, bottom left) Histogram of the dwell times of end-associated single H2-GFP molecules. (B) Histogram of single CLIP-170–GFP molecules added at 5 nM in the presence of 70 nM of unlabeled CLIP-170, 150 nM of unlabeled EB1, and 35 μM tubulin. (C) Histogram of single EB1-GFP molecules added at 0.75 nM in the presence of 149 nM of unlabeled EB1 and 35 μM tubulin. Red lines in the histograms are single exponential fits to the data, providing a mean (single molecule) dwell time for each +TIP that is approximately two orders of magnitude lower than the mean bleaching time of the fluorescent label (Fig. S3). (D) Summary of the mean +TIP comet decoration times as determined from data shown in Fig. 3 C and the mean single molecule dwell times from histogram fits. Comet decoration times are significantly different (Mann-Whitney U test, P < 0.05), even if in a similar range. Dwell times differ from comet decoration times by more than an order of magnitude.

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