Figure 2.
Preferential destabilization of dynamic microtubule minus ends by KIF2A. (A) Snapshot of dynamic microtubules polymerizing from brightly labeled GMPCPP seeds (AlexaFluor647 tubulin, magenta) in the presence of mScarlet-KIF2A (cyan). Below, mScarlet-KIF2A channel alone. (B) The sequence of kymographs for representative microtubules polymerizing from bright seeds (AlexaFluor647 tubulin, magenta) in the presence of increasing concentrations of mScarlet-KIF2A (cyan). mScarlet-KIF2A channels were additionally background subtracted and contrast was independently adjusted for each condition to show minus-end binding at a similar brightness. (C) Dynamics of microtubule plus-ends growing in the presence of increasing concentrations of mScarlet-KIF2A (left) or mCherry-MCAK (right), with a tubulin concentration of 12.5 µM. Both proteins show no major impact on growth speed (top) and a similar increase in catastrophe rate (bottom) with increasing concentrations. Number of growth speeds measured per condition: mScarlet-KIF2A: 0 nM, n = 502; 0.5 nM, n = 379; 2 nM, n = 508; 5 nM, n = 405; 20 nM, n = 572; 100 nM, n = 259; mCherry-MCAK: 0 nM, n = 275; 5 nM, n = 275; 10 nM, n = 406; 20 nM, n = 317; 40 nM, n = 164. Number of catastrophe rates measured per condition: mScarlet-KIF2A: 0 nM, n = 127; 0.5 nM, n = 90; 2 nM, n = 104; 5 nM, n = 71; 20 nM, n = 64; 100 nM, n = 26; mCherry-MCAK: 0 nM, n = 57; 5 nM, n = 60; 10 nM, n = 61; 20 nM, n = 41; 40 nM, n = 14. Error bars are SD for growth speed and SEM for catastrophe frequency. (D) Fraction of time in the growth phase for minus and plus ends of dynamic microtubules in the presence of increasing concentrations of mScarlet-KIF2A or mCherry-MCAK and 12.5 µM tubulin. Lines are drawn as a guide to the eye. Number of minus-end traces measured per condition: mScarlet-KIF2A: 0 nM, n = 127; 0.5 nM, n = 99; 2 nM, n = 60; 5 nM, n = 63; mCherry-MCAK: 0 nM, n = 59; 5 nM, n = 21; 10 nM, n = 33; 20 nM, n = 35. Number of plus end traces measured per condition: mScarlet-KIF2A: 0 nM, n = 131; 0.5 nM, n = 93; 2 nM, n = 95; 5 nM, n = 70; 20 nM, n = 67; 100 nM, n = 32; mCherry-MCAK: 0 nM, n = 58; 5 nM, n = 65; 10 nM, n = 67; 20 nM, n = 49; 40 nM, n = 34. Measurements of 0% were inferred by manual inspection of conditions in which seed ends only depolymerized. Error bars are SEM. (E) Snapshot of microtubules (magenta) growing from brightly labeled seeds in the presence of mScarlet-KIF2A (top, cyan) or mCherry-MCAK (bottom, gray) in the presence of ADP and absence of ATP. Yellow arrowheads indicate mScarlet-KIF2A accumulated at the slower-growing minus ends; mCherry-MCAK did not significantly decorate either end of the growing microtubule in the presence of ADP. Images are averages of two frames taken 5 s apart. (F) Representative kymographs showing minus end tracking of ADP-bound mScarlet-KIF2A (cyan) and partially suppressed minus-end growth of microtubules growing from brightly labeled seeds (magenta). (G) Microtubule minus-end growth speeds in the presence of increasing concentrations of mScarlet-KIF2A (left) or 100 nM mCherry-MCAK (right) in ADP. Number of growth speeds measured per condition: mScarlet-KIF2A: 0, n = 404; 5, n = 303; 10, n = 330; 20, n = 336; 40, n = 397; mCherry-MCAK: 100, n = 323. Error bars are SD.

Preferential destabilization of dynamic microtubule minus ends by KIF2A. (A) Snapshot of dynamic microtubules polymerizing from brightly labeled GMPCPP seeds (AlexaFluor647 tubulin, magenta) in the presence of mScarlet-KIF2A (cyan). Below, mScarlet-KIF2A channel alone. (B) The sequence of kymographs for representative microtubules polymerizing from bright seeds (AlexaFluor647 tubulin, magenta) in the presence of increasing concentrations of mScarlet-KIF2A (cyan). mScarlet-KIF2A channels were additionally background subtracted and contrast was independently adjusted for each condition to show minus-end binding at a similar brightness. (C) Dynamics of microtubule plus-ends growing in the presence of increasing concentrations of mScarlet-KIF2A (left) or mCherry-MCAK (right), with a tubulin concentration of 12.5 µM. Both proteins show no major impact on growth speed (top) and a similar increase in catastrophe rate (bottom) with increasing concentrations. Number of growth speeds measured per condition: mScarlet-KIF2A: 0 nM, n = 502; 0.5 nM, n = 379; 2 nM, n = 508; 5 nM, n = 405; 20 nM, n = 572; 100 nM, n = 259; mCherry-MCAK: 0 nM, n = 275; 5 nM, n = 275; 10 nM, n = 406; 20 nM, n = 317; 40 nM, n = 164. Number of catastrophe rates measured per condition: mScarlet-KIF2A: 0 nM, n = 127; 0.5 nM, n = 90; 2 nM, n = 104; 5 nM, n = 71; 20 nM, n = 64; 100 nM, n = 26; mCherry-MCAK: 0 nM, n = 57; 5 nM, n = 60; 10 nM, n = 61; 20 nM, n = 41; 40 nM, n = 14. Error bars are SD for growth speed and SEM for catastrophe frequency. (D) Fraction of time in the growth phase for minus and plus ends of dynamic microtubules in the presence of increasing concentrations of mScarlet-KIF2A or mCherry-MCAK and 12.5 µM tubulin. Lines are drawn as a guide to the eye. Number of minus-end traces measured per condition: mScarlet-KIF2A: 0 nM, n = 127; 0.5 nM, n = 99; 2 nM, n = 60; 5 nM, n = 63; mCherry-MCAK: 0 nM, n = 59; 5 nM, n = 21; 10 nM, n = 33; 20 nM, n = 35. Number of plus end traces measured per condition: mScarlet-KIF2A: 0 nM, n = 131; 0.5 nM, n = 93; 2 nM, n = 95; 5 nM, n = 70; 20 nM, n = 67; 100 nM, n = 32; mCherry-MCAK: 0 nM, n = 58; 5 nM, n = 65; 10 nM, n = 67; 20 nM, n = 49; 40 nM, n = 34. Measurements of 0% were inferred by manual inspection of conditions in which seed ends only depolymerized. Error bars are SEM. (E) Snapshot of microtubules (magenta) growing from brightly labeled seeds in the presence of mScarlet-KIF2A (top, cyan) or mCherry-MCAK (bottom, gray) in the presence of ADP and absence of ATP. Yellow arrowheads indicate mScarlet-KIF2A accumulated at the slower-growing minus ends; mCherry-MCAK did not significantly decorate either end of the growing microtubule in the presence of ADP. Images are averages of two frames taken 5 s apart. (F) Representative kymographs showing minus end tracking of ADP-bound mScarlet-KIF2A (cyan) and partially suppressed minus-end growth of microtubules growing from brightly labeled seeds (magenta). (G) Microtubule minus-end growth speeds in the presence of increasing concentrations of mScarlet-KIF2A (left) or 100 nM mCherry-MCAK (right) in ADP. Number of growth speeds measured per condition: mScarlet-KIF2A: 0, n = 404; 5, n = 303; 10, n = 330; 20, n = 336; 40, n = 397; mCherry-MCAK: 100, n = 323. Error bars are SD.

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