![Figure 6. Effect of a nonmotor microtubule cross-linking protein on microtubule sliding and self-organization. (A) Schematic representations of XCTK2 and the DoubleTail construct. Numbers indicate amino acid position of the XCTK2 coding sequence. (B) GFP-DoubleTail cross-links microtubules (MTs). (left) Merged microtubule channels and individual color channels of microtubule pairs formed with 0.8 nM XCTK2 GFP-DoubleTail. (right) Fraction of microtubule pairs with parallel and antiparallel orientation. (C) Addition of XCTK2 GFP-DoubleTail to an antiparallel microtubule sliding assay with 1 nM mCherry-XCTK2 motor. (left) Binding of GFP-DoubleTail to single microtubules and to microtubule overlaps. (middle) GFP-DoubleTail competes with mCherry-XCTK2 for binding to microtubules. Black lines are global fits to the DoubleTail binding and competitive inhibition curves for microtubule overlaps with the shared fit parameter Kd for the DoubleTail binding to microtubule pairs, yielding a Kd of 10.4 ± 3.0 nM (error is standard error of the mean). (right) Increasing concentrations of GFP-DoubleTail decreases the sliding velocity. The black line is a fit assuming protein friction exerted by DoubleTail (see Materials and methods). A.U., arbitrary unit. (D, left) Typical kymographs of microtubule pairs in a motor competition experiment with 0.66 nM Eg5 and 0.5 nM XCTK2 in the absence or presence of 16 nM GFP-DoubleTail. (right) Quantification of the mean absolute sliding velocity of antiparallel microtubules under these conditions. (E) Self-organization of microtubules and antagonistic kinesins (100 nM mCherry-XCTK2 and 66 nM Eg5-GFP) in the absence or presence of 1 µM Z-GFP-DoubleTail. Merged images of microtubules (red) and mCherry-XCTK2 (green) after 36 min. Entire recorded time course is shown in Video 10. Error bars are standard errors of the mean (B, C [right], and D) or standard deviations (C, left and middle). Bars: (B) 10 µm; (D, horizontal) 5 µm; (D, vertical) 120 s; (E) 50 µm.](https://cdn.rupress.org/rup/content_public/journal/jcb/189/3/10.1083_jcb.200910125/6/jcb_200910125r_rgb_fig6.jpeg?Expires=1771613784&Signature=R8TkM8R10iWVyigU3UFOHgJtPrVp~xJolUQh8E3IZgkeRINSpwh7bSzZ4MjescVajmOW~ZWAOuM1al3cYEFi5DaDg6QRS701iJ0oNOnwmC7Jacd5w5vQkVoeC3YB7Livi6nzIS0UDtVyMkIPwQMkSg5qYntRUl9B8i8PtrbhU1cUkAYMGzHZV76xeINST-BAhFcF-0Dlwq6Iat4r67iXK6LGFcM7GGiPVDz3SH6WhClSOEKO-DaHoI-g2ArbeOlTaYIKZeZTibcJi5Wl2WO7iSTkKRS6rd-eiI3Cmm~uZqEV7m2qCxtPVY6PtPSsIKQM5zoDvYfpkOkKn0p0v5MQ6w__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Effect of a nonmotor microtubule cross-linking protein on microtubule sliding and self-organization. (A) Schematic representations of XCTK2 and the DoubleTail construct. Numbers indicate amino acid position of the XCTK2 coding sequence. (B) GFP-DoubleTail cross-links microtubules (MTs). (left) Merged microtubule channels and individual color channels of microtubule pairs formed with 0.8 nM XCTK2 GFP-DoubleTail. (right) Fraction of microtubule pairs with parallel and antiparallel orientation. (C) Addition of XCTK2 GFP-DoubleTail to an antiparallel microtubule sliding assay with 1 nM mCherry-XCTK2 motor. (left) Binding of GFP-DoubleTail to single microtubules and to microtubule overlaps. (middle) GFP-DoubleTail competes with mCherry-XCTK2 for binding to microtubules. Black lines are global fits to the DoubleTail binding and competitive inhibition curves for microtubule overlaps with the shared fit parameter Kd for the DoubleTail binding to microtubule pairs, yielding a Kd of 10.4 ± 3.0 nM (error is standard error of the mean). (right) Increasing concentrations of GFP-DoubleTail decreases the sliding velocity. The black line is a fit assuming protein friction exerted by DoubleTail (see Materials and methods). A.U., arbitrary unit. (D, left) Typical kymographs of microtubule pairs in a motor competition experiment with 0.66 nM Eg5 and 0.5 nM XCTK2 in the absence or presence of 16 nM GFP-DoubleTail. (right) Quantification of the mean absolute sliding velocity of antiparallel microtubules under these conditions. (E) Self-organization of microtubules and antagonistic kinesins (100 nM mCherry-XCTK2 and 66 nM Eg5-GFP) in the absence or presence of 1 µM Z-GFP-DoubleTail. Merged images of microtubules (red) and mCherry-XCTK2 (green) after 36 min. Entire recorded time course is shown in Video 10. Error bars are standard errors of the mean (B, C [right], and D) or standard deviations (C, left and middle). Bars: (B) 10 µm; (D, horizontal) 5 µm; (D, vertical) 120 s; (E) 50 µm.
