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Figure 1. Katanin activity (250 nM) on microtubules results in polymer loss of taxolâ€stabilized microtubules with or without the CTT. (a) Example timeseries (i) for control microtubules without katanin (time between frames 50 s), (ii) for control microtubules with katanin (time between frames 10 s), (iii) for â€CTT microtubules without katanin (time between frames 50 s), and (iv) for â€CTT microtubules with katanin (time between frames 40 s). Scale bars 5 μm. All microtubules were taxol stabilized, and a single preparation of katanin was used. (b) Long time data: Total loss of polymer over 400 s for (i) control microtubules without katanin (red squares, N = 43 microtubules in six chambers) and with katanin (blue circles, N = 35 microtubules in seven chambers), (ii) for â€CTT microtubules without katanin (magenta squares, N = 33 microtubules in four chambers) and with katanin (green circles, N = 32 microtubules in six chambers). Data fit (lines through data sets) to Equation (1). Error bars represent standard error of the mean for average over N microtubules. (c) The percent of polymer lost from microtubules plotted for control microtubules without katanin (red bar), and control microtubules with katanin (blue bar), â€CTT microtubules without katanin (magenta bar), and â€CTT microtubules with katanin (green bar). Error bars represent the minimum uncertainty of the measurement, 7%. (d) Short time data: Total loss of polymer over the first 100 s for (i) control microtubules without katanin (red squares, N = 43, six chambers) and with katanin (blue circles, N = 35 microtubules in seven chambers), and (ii) â€CTT microtubules without katanin (magenta squares, N = 33 microtubules in four chambers) and with katanin (green circles, N = 32 microtubules in six chambers). Data fit (lines through data sets) to Equation (2). Error bars represent standard error of the mean over N microtubules. (e) The rate of polymer loss from Equation (2) fits plotted for control microtubules without katanin (red bar), and control microtubules with katanin (blue bar), â€CTT microtubules without katanin (magenta bar), and â€CTT microtubules with katanin (green bar). Error bars represent the error in the rate parameter from Equation (2). All fit parameters are reported in Tables S1–S4. CTT, Câ€terminal tail [Color figure can be viewed at wileyonlinelibrary.com]
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Figure 2. Filament depolymerization depends on the presence of katanin (250 nM). (a) Example kymographs (i) for control microtubules without katanin, (ii) for control microtubules with katanin, (iii) for â€CTT microtubules without katanin, and (iv) for â€CTT microtubules with katanin. All microtubules were taxol stabilized. Vertical scale bars 5 min. Horizontal scale bars 5 μm. (b) Example depolymerization rate measurement method using kymograph. The linear loss of polymer from the microtubule end is measured in the xâ€direction displacement, Δx. The amount of time it takes to lose the polymer is measured in the yâ€direction displacement, Δt. The depolymerization rate is given by v = Δx/Δt. Vertical scale bar 5 min. Horizontal scale bar 5 μm. (c) Cumulative distribution plots of the depolymerization rates for control microtubules without katanin (red line, N = 91 microtubule ends, 43 microtubules, six chambers), control microtubules with katanin (blue line, N = 161 microtubule ends, 32 microtubules, seven chambers), â€CTT microtubules without katanin (magenta line, N = 58 microtubule ends, 33 microtubules, four chambers), and â€CTT microtubules with katanin (green line, N = 69 microtubules ends, 43 microtubules, six chambers). (d) Boxâ€whisker plots for control microtubules without katanin (red box), control microtubules with katanin (blue box), â€CTT microtubules without katanin (magenta box), and â€CTT microtubules with katanin (green box). On box plots, middle lines of the boxes represent the median and the top and the bottom represent the third and first quartiles, respectively. Open circles represent outlier data. (e)The percentage of filaments that displayed a nonâ€zero depolymerization rate for control microtubules without katanin (red bar), control microtubules with katanin (blue bar), â€CTT microtubules without katanin (magenta bar), and â€CTT microtubules with katanin (green bar). Error bars represent the standard error of proportion. CTT, Câ€terminal tail [Color figure can be viewed at wileyonlinelibrary.com]
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Figure 3. Depolymerization of taxolâ€stabilized, â€CTT microtubules using katanin lacking enzymatic activity. (a) Depolymerization rates plotted as cumulative distributions for â€CTT microtubules without katanin and with ATP (magenta lines, N = 58 microtubule ends, 33 microtubules, four chambers), with katanin and ATP (green lines, N = 69 microtubule ends, 43 microtubules, six chambers), with katanin and ADP (orange line, N = 45 microtubule ends, 21 microtubules, two chambers), and with Walker B E306Q mutant katanin with ATP (purple line, N = 53 microtubule ends, 13 microtubules, three chambers). (b) Boxâ€whisker plots of the depolymerization rates for â€CTT microtubules without katanin (magenta box), with wildtype katanin and ATP (green box), with wildtype katanin with ADP (orange box), and with Walker B mutant katanin and ATP (purple box). On box plots, middle lines of the boxes represent the median and the top and the bottom represent the third and first quartiles, respectively. Open circles represent outlier data. All data taken at 250 nM katanin on taxolâ€stabilized â€CTT microtubules. CTT, Câ€terminal tail [Color figure can be viewed at wileyonlinelibrary.com]
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Figure 4. Depolymerization speed as a function of katanin concentration. (a) Cumulative distributions of depolymerization speeds (i) for control microtubules without katanin (black lines, N = 63 filament ends) or with katanin at 250 nM (red lines, N = 40 ends), 300 nM (orange lines, N = 72 ends), 370 nM (yellow lines, N = 113 ends), 500 nM (green lines, N = 36 ends), 740 nM (blue lines, N = 36 ends) and 1.5 μM (purple lines, N = 75 ends), and (ii) for â€CTT microtubules without katanin (black lines, N = 242 filament ends) or with katanin at 250 nM (red lines, N = 56 ends), 300 nM (orange lines, N = 74 ends), 370 nM (yellow lines, N = 47 ends), 500 nM (green lines, N = 83 ends), 740 nM (blue lines, N = 84 ends) and 1.5 μM (purple lines, N = 85 ends). All microtubules were taxol stabilized and a single preparation of katanin was used for this data. (b) The relative average depolymerization rate as a function of katanin concentration. (i) (a) All data rescaled by the depolymerization rate in the absence of katanin, V0, plotted for â€CTT microtubules (filled circles) and control microtubules (filled squares). Colors match the colors from part (a). Error bars represent the rescaled standard deviation (unscaled data reported in sFigure S2) to represent the width of the distribution. Dashed lines serve as a guide to the eye. (b) Rescaled depolymerization rates for â€CTT microtubules were fit to hyperbolic function (Equation (3), black line) and hyperbolic function with cooperativity (Equation 4, dark gray line). Control microtubule data were fit to a difference between a hyperbolic function and a linear equation (Equation (5), light gray line). Fit parameters are given in Tables 5–7. Error bars represent standard error of the mean (uncertainty weighting for the fit). (ii) residuals or each fit found by taking the difference between the relative depolymerization rate, Vr, and expected depolymerization rate from each fit, Vfit, plotted as a function of katanin concentration for the hyperbolic function for â€CTT microtubule data (black line) and hyperbolic function with cooperativity for â€CTT microtubule data (dark gray line), and hyperbolic fit minus a linear relation for control microtubule data (light gray line). Dashed line represents zero. CTT, Câ€terminal tail [Color figure can be viewed at wileyonlinelibrary.com]
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Figure 5. Example of GFPâ€katanin binding to microtubules with quantification of the fluorescence intensity. (a) Representative images of control microtubules (left, red) and GFPâ€katanin (center, cyan) and color overlay (right) for katanin concentrations (i) 5 nM and (ii) 100 nM. Scale bar 5 μm. All microtubules were taxol stabilized. (b) Representative images of â€CTT microtubules (left, red) and GFPâ€katanin (center, cyan) and color overlay (right) for katanin concentrations (i) 5 nM and (ii) 100 nM. Scale bar 5 μm. All microtubules were taxol stabilized. (c) Quantification of the intensity ratio for katanin binding to microtubules rescaled by the background intensity (signal:noise). Box plots displaying all the intensity data for each measurement of (top) control microtubules and (bottom) â€CTT microtubules for 5 nM katanin (black box, gray lines), 10 nM katanin (green box, black lines), 25 nM katanin (blue box, black lines), 50 nM katanin (red box, black lines), 75 nM katanin (cyan box, black lines), and 100 nM katanin (magenta box, black lines). Number of measurements taken given in parenthesis in each plot. (d) Quantification of normalized intensity over time of katanin binding to (i) control microtubules or (ii) â€CTT microtubules. For both plots, data for 5 nM katanin (blue lines) and 100 nM katanin (red lines) are shown. CTT, Câ€terminal tail [Color figure can be viewed at wileyonlinelibrary.com]
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Figure 6. Cartoon mechanism of microtubule depolymerization of â€CTT microtubules. (left) Control microtubules (magenta) in the absence of katanin lose dimers from the ends due to normal degradation to replenish the background concentration. This loss of polymer is slow. (middle) In the presence of katanin (green) katanin can catalyze the loss of dimers from both the ends of control microtubules, called depolymerization, and from the middle of control microtubules, called severing. We observe a significant amount of mobility, association, and dissociation of katanin to and from the filaments. (right) Microtubules lacking the CTT can still bind katanin, but fewer katanins are bound and the bound katanin is less mobile. Without the CTT, katanin cannot sever microtubules but can still catalyze the loss of dimers from the ends. CTT, Câ€terminal tail [Color figure can be viewed at wileyonlinelibrary.com]
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