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. 2014 Jan 1;25(1):95–106. doi: 10.1091/mbc.E13-07-0376

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© 2014 Mazel et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 3: — Colocalization of dynein complexes with short microtubules. (A) Two-color TIRF microscopic analysis of the EGFP-labeled dynein heavy chain (EGFP-Dync1h1) and mCherry-labeled MAP2c–decorated microtubules in COS7 cells. A subpopulation of dynein speckles (green) overlaps with MAP2c-induced microtubules (red) at the cell cortex. (B) Determination of the percentage of speckles that overlap with MAP2c-decorated short microtubules reveals their preferential association: About 28% of speckles are colocalized with short microtubules. By shifting the dynein channel by >1 pixel relative to the microtubule channel, this apparent colocalization is reduced to the expected value for random overlap, which was calculated to be ∼23%. The observed colocalization was consistently higher than the expected random overlap (4.3 ± 0.6%, p < 0.0001; Student's paired t test; n = 9 cells). See Supplemental Figure S7A for analysis of additional dynein components. (C) Left, five different dynamic behaviors can be distinguished by automated speckle and short-microtubule tracking. The relative fractions for observation of these cases are shown (based on 10,478 three-frame subtrajectories from four cells). Measurements are shown as percentages with SEM. The two most common cases are underlined. Right, examples of observations belonging to these cases are shown above the schematics of their interpretation. The first example illustrates case 2, in which a dynein speckle moves toward the trailing end of a moving short microtubule (white arrowheads). The example for the more frequently observed case 3 shows a short microtubule, which slides along a stationary dynein speckle. In the rarely observed case 5, dynein speckles usually accumulate at the trailing end of motile microtubules and translocate together with them in the same direction (large white arrows; see also Supplemental Figure S7C). For the example of case 2, first the dynein speckle moves leftward (white arrowheads) while the microtubule is stationary, corresponding to case 2, followed by a short rightward movement (white small arrows) of the microtubule and a relatively stationary dynein speckle characteristic of case 3, suggesting that microtubules and dynein speckles can rapidly switch their motile behavior. See also Supplemental Movies S5–S8. MT: microtubule.