Figure 1.
Figure 1. GFP-XCTK2 accumulates between microtubule pairs in contrast to Eg5-GFP. (A, left) Schematic representations of the molecular architectures of tetrameric plus end–directed motor Eg5 and dimeric minus end–directed motor XCTK2. Large round and small rectangular symbols indicate motor domains and nonmotor microtubule binding domains, respectively. (middle) Scheme of a microtubule pair in the sliding assay. Long, dimly fluorescently labeled, biotinylated microtubules are attached to a biotin-PEG–coated (gray) glass surface via neutravidin links. Short, brightly fluorescently labeled, unbiotinylated microtubules (top) are bound to the immobilized microtubules via cross-linking motor proteins. The arrows on the bottom indicate the incidence angle of the excitation light, which is totally internally reflected and gives rise to an evanescent field (light blue gradient). (right) TIRF microscopy images of Cy5-labeled microtubule (MT) pairs cross-linked by GFP-XCTK2 present in solution at a concentration of 50 nM, illustrating accumulation of GFP-XCTK2 in microtubule overlaps. (B) Quantification of GFP-XCTK2 in Cy5-microtubule pairs in assay buffer containing 120 mM KCl. (left) Binding curves showing averaged TIRF intensities of GFP-XCTK2 along microtubule overlaps and single microtubules at varying concentrations of GFP-XCTK2. The error bars are standard deviations, indicating the width of the fluorescence intensity distribution. The Kd for the binding of GFP-XCTK2 to the microtubule overlap derived from the fit is 69 ± 15 nM (error is standard error of the mean). (middle) Dual-color kymograph (see also the scheme on top) of XCTK2-driven microtubule pair sliding showing the GFP-XCTK2 signal in green and the Cy5-microtubule signal in red (separate channels are shown individually in bottom panels). The length of the overlap between the two microtubules is decreasing over time. A bright microtubule moving to the right is pushed over the end of an immobilized, dim microtubule; XCTK2 accumulation is confined to the overlap region. (right) Averaged speeds of microtubule sliding at varying concentrations of GFP-XCTK2. Error bars are standard errors of means. (C) Quantification of Eg5-GFP in Cy5-microtubule pairs in assay buffer containing 50 mM KCl. (left) Binding curves showing averaged TIRF intensities of Eg5-GFP along microtubule overlaps and single microtubules at varying concentrations of Eg5-GFP. Error bars are standard deviations. Absolute intensity values are not comparable with B (left) because the excitation intensity was increased. (middle) Dual-color kymograph of Eg5-driven microtubule pair sliding showing the Eg5-GFP signal in green and the Cy5-microtubule signal in red. (right) Averaged speeds of microtubule sliding at varying concentrations of Eg5-GFP. Error bars are standard errors of the mean. A.U., arbitrary unit. Bars: (A, B [horizontal], and C [horizontal]) 5 µm; (B and C, vertical) 120 s.

GFP-XCTK2 accumulates between microtubule pairs in contrast to Eg5-GFP. (A, left) Schematic representations of the molecular architectures of tetrameric plus end–directed motor Eg5 and dimeric minus end–directed motor XCTK2. Large round and small rectangular symbols indicate motor domains and nonmotor microtubule binding domains, respectively. (middle) Scheme of a microtubule pair in the sliding assay. Long, dimly fluorescently labeled, biotinylated microtubules are attached to a biotin-PEG–coated (gray) glass surface via neutravidin links. Short, brightly fluorescently labeled, unbiotinylated microtubules (top) are bound to the immobilized microtubules via cross-linking motor proteins. The arrows on the bottom indicate the incidence angle of the excitation light, which is totally internally reflected and gives rise to an evanescent field (light blue gradient). (right) TIRF microscopy images of Cy5-labeled microtubule (MT) pairs cross-linked by GFP-XCTK2 present in solution at a concentration of 50 nM, illustrating accumulation of GFP-XCTK2 in microtubule overlaps. (B) Quantification of GFP-XCTK2 in Cy5-microtubule pairs in assay buffer containing 120 mM KCl. (left) Binding curves showing averaged TIRF intensities of GFP-XCTK2 along microtubule overlaps and single microtubules at varying concentrations of GFP-XCTK2. The error bars are standard deviations, indicating the width of the fluorescence intensity distribution. The Kd for the binding of GFP-XCTK2 to the microtubule overlap derived from the fit is 69 ± 15 nM (error is standard error of the mean). (middle) Dual-color kymograph (see also the scheme on top) of XCTK2-driven microtubule pair sliding showing the GFP-XCTK2 signal in green and the Cy5-microtubule signal in red (separate channels are shown individually in bottom panels). The length of the overlap between the two microtubules is decreasing over time. A bright microtubule moving to the right is pushed over the end of an immobilized, dim microtubule; XCTK2 accumulation is confined to the overlap region. (right) Averaged speeds of microtubule sliding at varying concentrations of GFP-XCTK2. Error bars are standard errors of means. (C) Quantification of Eg5-GFP in Cy5-microtubule pairs in assay buffer containing 50 mM KCl. (left) Binding curves showing averaged TIRF intensities of Eg5-GFP along microtubule overlaps and single microtubules at varying concentrations of Eg5-GFP. Error bars are standard deviations. Absolute intensity values are not comparable with B (left) because the excitation intensity was increased. (middle) Dual-color kymograph of Eg5-driven microtubule pair sliding showing the Eg5-GFP signal in green and the Cy5-microtubule signal in red. (right) Averaged speeds of microtubule sliding at varying concentrations of Eg5-GFP. Error bars are standard errors of the mean. A.U., arbitrary unit. Bars: (A, B [horizontal], and C [horizontal]) 5 µm; (B and C, vertical) 120 s.

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