Figure S4.
Severing of stabilized microtubule seeds by the severase spastin and mGFP-spastin. (A) Time sequence of TIRF microscopy images of immobilized GMPCPP microtubules (AlexaFluor647 5%, magenta) in the absence or presence of 50 nM of spastin. (B) Time sequence of TIRF microscopy images of immobilized GMPCPP microtubules (Atto647 5%, magenta) and mGFP-spastin (yellow) in the absence or presence of 20 nM of mScarlet-KIF2A (cyan). Time in B and C is min:s. (C) Time course of fluorescence intensities of GMPCCP microtubules (magenta), mGFP-spastin (yellow), and mScarlet-KIF2A (cyan) measured along the initial contour of microtubules in the presence of 50 nM mGFP-spastin (squares) or 50 nM mGFP–spastin and 20 nM KIF2A (circles). Number of microtubules analyzed per condition: 50 nM mGFP-spastin, n = 81; 50 nM mGFP-spastin and 20 nM KIF2A, n = 87. Data for plots were pooled from at least two independent experiments. Error bars are SEM. For symbols without visible error bars, error bars are smaller than the symbol size. (D) TIRF microscopy images of 100 nM mGFP-spastin in the absence or presence of immobilized γTuRC. γTuRC concentrations were used for immobilization as indicated. The fluorescence intensities of mGFP-spastin (bottom, right; see Materials and methods) represent the background intensity and are independent of the γTuRC density on the surface, demonstrating the absence of interaction between γTuRC and spastin. Moreover, no colocalization is observed, also indicating absence of interaction. Data for plots were pooled from at least two independent experiments. Error bars are SEM. For symbols without visible error bars, error bars are smaller than the symbol size.

Severing of stabilized microtubule seeds by the severase spastin and mGFP-spastin. (A) Time sequence of TIRF microscopy images of immobilized GMPCPP microtubules (AlexaFluor647 5%, magenta) in the absence or presence of 50 nM of spastin. (B) Time sequence of TIRF microscopy images of immobilized GMPCPP microtubules (Atto647 5%, magenta) and mGFP-spastin (yellow) in the absence or presence of 20 nM of mScarlet-KIF2A (cyan). Time in B and C is min:s. (C) Time course of fluorescence intensities of GMPCCP microtubules (magenta), mGFP-spastin (yellow), and mScarlet-KIF2A (cyan) measured along the initial contour of microtubules in the presence of 50 nM mGFP-spastin (squares) or 50 nM mGFP–spastin and 20 nM KIF2A (circles). Number of microtubules analyzed per condition: 50 nM mGFP-spastin, n = 81; 50 nM mGFP-spastin and 20 nM KIF2A, n = 87. Data for plots were pooled from at least two independent experiments. Error bars are SEM. For symbols without visible error bars, error bars are smaller than the symbol size. (D) TIRF microscopy images of 100 nM mGFP-spastin in the absence or presence of immobilized γTuRC. γTuRC concentrations were used for immobilization as indicated. The fluorescence intensities of mGFP-spastin (bottom, right; see Materials and methods) represent the background intensity and are independent of the γTuRC density on the surface, demonstrating the absence of interaction between γTuRC and spastin. Moreover, no colocalization is observed, also indicating absence of interaction. Data for plots were pooled from at least two independent experiments. Error bars are SEM. For symbols without visible error bars, error bars are smaller than the symbol size.

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