Figure 3.
Treadmilling of dynamic microtubules driven by KIF2A. (A) Time sequence snapshots of microtubules growing from brightly labeled seeds (magenta, Atto647) in the presence of 20 nM untagged KIF2A. Circles track the depolymerizing minus ends of selected microtubules that undergo treadmilling. A solid circle indicates the microtubule is still attached to the brightly labeled GMPCPP seed, whereas a dashed circle indicates the seed has completely depolymerized and the microtubule (including the minus end) is composed of only GTP/GDP tubulin. For better distinction and identification of the selected microtubules, both solid and dashed circles are color coded. Arrows indicate the direction of “travel” across the glass surface. The asterisk indicates that the microtubule was lost from view after this timepoint following a plus-end catastrophe. (B) Representative kymographs of microtubules (magenta) that are released from the stabilized GMPCPP seed after depolymerization by mScarlet-KIF2A (cyan), indicated by the black arrowhead. Minus-end depolymerization was heterogeneous: asterisks indicate instances of increased depolymerization speed. Insets: Enlarged regions of kymographs showing mScarlet-KIF2A localization at minus ends undergoing periods of “slow” (10 nM KIF2A inset) and “fast” (20 nM KIF2A inset) depolymerization. Inset scale bars: horizontal, 1 µm; vertical, 30 s. (C) Average depolymerization rates of minus ends of treadmilling microtubules, measured from microtubules that are no longer attached to seeds, in the presence of various concentrations of mScarlet-KIF2A. Each measurement is plotted as a semi-transparent dot (with random x-jitter to improve visualization) and the mean is overlaid as a black bar. Inset: The same data with a 0 nM KIF2A timepoint included (where each measurement is made from each instance of depolymerization after catastrophe) and an extended y axis. Number of depolymerization speeds measured per condition: mScarlet-KIF2A: 0 nM, n = 264; 10 nM, n = 74; 20 nM, n = 76; 40 nM, n = 62; 60 nM, n = 68.

Treadmilling of dynamic microtubules driven by KIF2A. (A) Time sequence snapshots of microtubules growing from brightly labeled seeds (magenta, Atto647) in the presence of 20 nM untagged KIF2A. Circles track the depolymerizing minus ends of selected microtubules that undergo treadmilling. A solid circle indicates the microtubule is still attached to the brightly labeled GMPCPP seed, whereas a dashed circle indicates the seed has completely depolymerized and the microtubule (including the minus end) is composed of only GTP/GDP tubulin. For better distinction and identification of the selected microtubules, both solid and dashed circles are color coded. Arrows indicate the direction of “travel” across the glass surface. The asterisk indicates that the microtubule was lost from view after this timepoint following a plus-end catastrophe. (B) Representative kymographs of microtubules (magenta) that are released from the stabilized GMPCPP seed after depolymerization by mScarlet-KIF2A (cyan), indicated by the black arrowhead. Minus-end depolymerization was heterogeneous: asterisks indicate instances of increased depolymerization speed. Insets: Enlarged regions of kymographs showing mScarlet-KIF2A localization at minus ends undergoing periods of “slow” (10 nM KIF2A inset) and “fast” (20 nM KIF2A inset) depolymerization. Inset scale bars: horizontal, 1 µm; vertical, 30 s. (C) Average depolymerization rates of minus ends of treadmilling microtubules, measured from microtubules that are no longer attached to seeds, in the presence of various concentrations of mScarlet-KIF2A. Each measurement is plotted as a semi-transparent dot (with random x-jitter to improve visualization) and the mean is overlaid as a black bar. Inset: The same data with a 0 nM KIF2A timepoint included (where each measurement is made from each instance of depolymerization after catastrophe) and an extended y axis. Number of depolymerization speeds measured per condition: mScarlet-KIF2A: 0 nM, n = 264; 10 nM, n = 74; 20 nM, n = 76; 40 nM, n = 62; 60 nM, n = 68.

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