Figure 3.
Microtubules grown from GDP-tubulin are highly stable. (A) Rescue events at the minus-end of microtubules polymerized first with GDP-tubulin (210 µM, green) then GTP-tubulin (10 µM, magenta). A representative kymograph shows the behavior of a microtubule recorded during the second step as shown above (gray square). White arrows indicate rescue events occurring at the junction between lattices grown from GDP-tubulin and GTP-tubulin. S, seed. Histogram shows the distance of rescue events from the junction (dashed vertical line, position 0) between lattices grown from GDP-tubulin (green) and GTP-tubulin (magenta). n = 122 rescue events from at least three independent experiments. Most rescue events occur at the junction between lattices grown from GDP-tubulin and GTP-tubulin. (B) Control experiment: rescue events at the minus-end of microtubules polymerized with GTP-tubulin (12 µM, green) then GTP-tubulin (10 µM, magenta). Representative kymograph and histogram as in A. The white arrowhead indicates a microtubule that depolymerizes across the junction. n = 170 rescue events from at least three independent experiments. (C) Percentages of depolymerizing microtubules that cross the junction or stop at the junction. n, the total number of events from at least three independent experiments. Triangles represent the percentage of depolymerizing microtubules that stop at the junction for each experiment. ****P < 0.0001 (two-sided Fisher’s test). (D) To assess spastin severing on microtubules constituted by segments of GDP- and GTP-tubulin-grown lattices, GDP-tubulin (210 µM, light pink) is polymerized from seed (S), then GTP-tubulin is polymerized (15 µM, magenta), before perfusing GFP-spastin. The time series shows hybrid microtubules severed by spastin, which binds along the whole lattice. Time in min:sec. (E) Fragmentation of GDP- vs. GTP-tubulin-grown microtubules. Fragmentation time is the time at which fragmentation starts. Boxes and whiskers show 25–75 and 10–90 percentiles, respectively; the solid line represents the median. n, total number of microtubules from at least three independent experiments. Triangles represent the mean fragmentation time for each experiment. ***P < 0.001 (non-parametric Mann–Whitney test). (F) Procedure to monitor the depolymerizing activity of MCAK at the end of GTP- (15 µM) or GDP- (210 µM) tubulin-grown microtubules with a time series of microtubules assembled in the presence of MCAK. Arrows, microtubule ends. Time, min:sec. (G) Distribution of GDP- versus GTP-tubulin-assembled-microtubule lengths after MCAK perfusion at two timepoints. n, total number of microtubules from at least three independent experiments.

Microtubules grown from GDP-tubulin are highly stable. (A) Rescue events at the minus-end of microtubules polymerized first with GDP-tubulin (210 µM, green) then GTP-tubulin (10 µM, magenta). A representative kymograph shows the behavior of a microtubule recorded during the second step as shown above (gray square). White arrows indicate rescue events occurring at the junction between lattices grown from GDP-tubulin and GTP-tubulin. S, seed. Histogram shows the distance of rescue events from the junction (dashed vertical line, position 0) between lattices grown from GDP-tubulin (green) and GTP-tubulin (magenta). n = 122 rescue events from at least three independent experiments. Most rescue events occur at the junction between lattices grown from GDP-tubulin and GTP-tubulin. (B) Control experiment: rescue events at the minus-end of microtubules polymerized with GTP-tubulin (12 µM, green) then GTP-tubulin (10 µM, magenta). Representative kymograph and histogram as in A. The white arrowhead indicates a microtubule that depolymerizes across the junction. n = 170 rescue events from at least three independent experiments. (C) Percentages of depolymerizing microtubules that cross the junction or stop at the junction. n, the total number of events from at least three independent experiments. Triangles represent the percentage of depolymerizing microtubules that stop at the junction for each experiment. ****P < 0.0001 (two-sided Fisher’s test). (D) To assess spastin severing on microtubules constituted by segments of GDP- and GTP-tubulin-grown lattices, GDP-tubulin (210 µM, light pink) is polymerized from seed (S), then GTP-tubulin is polymerized (15 µM, magenta), before perfusing GFP-spastin. The time series shows hybrid microtubules severed by spastin, which binds along the whole lattice. Time in min:sec. (E) Fragmentation of GDP- vs. GTP-tubulin-grown microtubules. Fragmentation time is the time at which fragmentation starts. Boxes and whiskers show 25–75 and 10–90 percentiles, respectively; the solid line represents the median. n, total number of microtubules from at least three independent experiments. Triangles represent the mean fragmentation time for each experiment. ***P < 0.001 (non-parametric Mann–Whitney test). (F) Procedure to monitor the depolymerizing activity of MCAK at the end of GTP- (15 µM) or GDP- (210 µM) tubulin-grown microtubules with a time series of microtubules assembled in the presence of MCAK. Arrows, microtubule ends. Time, min:sec. (G) Distribution of GDP- versus GTP-tubulin-assembled-microtubule lengths after MCAK perfusion at two timepoints. n, total number of microtubules from at least three independent experiments.

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