Journal: Molecular cell

Article Title: A tethering mechanism controls the processivity and kinetochore-microtubule plus-end enrichment of the kinesin-8 Kif18A

doi: 10.1016/j.molcel.2011.07.022

Figure Lengend Snippet: A) Photobleaching of Kif18A-GFP in a metaphase HeLa cell. Irradiation was targeted to the indicated region (dashed yellow line). Enlarged images show K-MT plus-end fluorescence before and after photobleaching. Time is indicated in seconds and is relative to irradiation. Scale bars, 10 µm and 2 µm (enlarged images). B) Kinetics of Kif18A-GFP fluorescence recovery at a K-MT plus-end. A representative plot of normalized Kif18A-GFP fluorescence at a single kinetochore against time is shown. The recovery of Kif18A-GFP at K-MT plus-ends fit a single exponential (black line). C) Taxol causes equatorial enrichment of Kif18A-GFP. Still images from a video of a HeLa cell stably expressing Kif18A-GFP treated with 10 µM taxol. Time is indicated in min and is relative to taxol addition. Scale bar, 10 µm. D) Kif18A is enriched at kinetochores in taxol-treated HeLa cells. The localizations of endogenous Kif18A (red) and kinetochores (green) in a control HeLa cell or in a cell treated with 10 µM taxol for 15 min are shown. Insets are higher magnification views of the boxed regions. Scale bars, 10 µm and 1 µm (enlarged images). E) Photobleaching of Kif18A-GFP in a taxol-treated metaphase HeLa cell. Time is indicated in seconds and is relative to irradiation. Scale bars, 10 µm and 2 µm (enlarged images). F) Kinetics of Kif18A-GFP fluorescence recovery at a K-MT plus-end in a cell treated with 10 µM taxol. A representative plot of normalized Kif18A-GFP fluorescence at a single kinetochore against time is shown.

Article Snippet: Graphs of fluorescence intensities versus time were generated in SigmaPlot (Systat Software), and the resulting data fit to a single exponential, F t =F 0 +F inf *(1-e −kt ), essentially as described ( Howell et al., 2000 ).

Techniques: Irradiation, Fluorescence, Stable Transfection, Expressing, Control

Journal: Molecular cell

Article Title: A tethering mechanism controls the processivity and kinetochore-microtubule plus-end enrichment of the kinesin-8 Kif18A

doi: 10.1016/j.molcel.2011.07.022

Figure Lengend Snippet: A) Metaphase localization of Kif18A truncation mutants. The localizations of full-length GFP-Kif18A, GFP-Kif18A-N406, Kif18A-N480-GFP, and GFP-Kif18A-C307 in cells co-stained for tubulin (red) and Hec1 (blue) are shown. Scale bar, 5 µm. B) The C-terminal tail domain of Kif18A is required for the motor to accumulate at K-MT plus-ends. Representative linescans showing the distribution of Kif18A (green) along metaphase K-MTs (red) near the kinetochore (Hec1 peak, blue). C) The tail domain of Kif18A increases the dwell time of the motor on spindle MTs. Still images from photoconversion runs of tdEOS-Kif18A-FL and Kif18A-N480-tdEOS are shown. An image of fluorescence from the GFP channel is shown at moment of photoconversion (t=0). Regions that were photoconverted and subjected to analysis are outlined. Time is indicated in sec and is relative to the time of photoconversion. Scale bar, 10 µm. D) Decay kinetics of tdEOS-Kif18A-FL (blue) and Kif18A-N480-tdEOS (pink) fluorescence from the mitotic spindle. Normalized mean fluorescence of photoconverted tdEOS-Kif18A-FL (n=8) and Kif18A-N480-tdEOS (n=11) versus time in sec are shown. Asterisks denote time points corresponding to the final images shown in Figure 2C. Black lines represent fits of the data to single exponentials. Error bars represent SEM.

Article Snippet: Graphs of fluorescence intensities versus time were generated in SigmaPlot (Systat Software), and the resulting data fit to a single exponential, F t =F 0 +F inf *(1-e −kt ), essentially as described ( Howell et al., 2000 ).

Techniques: Staining, Fluorescence