Xenbase Image ID: 122918

Figure 1. CENP-E is a slow, processive motor that maintains microtubule attachment for long periods. (A) Purified CE473-GFP. The arrow points to the purified protein band. (B) Experimental setup for imaging CENP-E single molecules moving along microtubules with TIRF microscopy. X-rhodamine–labeled GMPCPP microtubules were immobilized on a coverslip using antitubulin antibody, and a low concentration of CE473-GFP (0.5–1 nM) was flowed into a flow chamber. (C) Frames from time-lapse imaging of CENP-E motor–GFP moving along microtubules. A fluorescent speckle on the microtubule is indicated with a red line. Two CENP-E molecules moving at different speeds are indicated with arrows. Green, CE473-GFP; red, X-rhodamine–labeled GMPCPP microtubules. (D) Kymographs showing processive movements of the CENP-E motor domain. X-rhodamine speckles on stable microtubules produced vertical red lines. Single molecules of CE473-GFP are shown in green. Yellow arrowheads indicate starts and stops of processive movements. The actual durations of kymographs 1, 2, and 3 were 1,205 s, 1,190 s, and 1,205 s, respectively. (E) Velocity distribution of single CENP-E motor molecules. The median velocity is 8 nm/s (equal to 0.48 μm/min; red line; n = 320; four independent preparations). (F) MSD (ρ(t)) of CENP-E fitted with ρ(t) = v2t2 + 2Dt (n = 90). Error bars indicate SEM. (G) The run time of CENP-E motor was distributed exponentially. The mean run time was determined by fitting the data into a cumulative distribution function. The inset shows the one-cumulative probability of CENP-E run time plotted on a log scale. The mean run time is 195 ± 13 s (mean ± SEM; n = 320). (H) The run length of CENP-E motor was determined by fitting the data into a cumulative distribution function. The inset shows the one-cumulative probability of CENP-E run length plotted on a log scale. The mean run length is 1.5 ± 0.1 μm (mean ± SEM; n = 320). (G and H) Red lines are the exponential fits to the bar graphs. Bars, 2 μm. Image published in: Kim Y et al. (2008) © 2008 Kim et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View

Return to previous page