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First published online 7 April 2009
doi: 10.1242/jcs.044248

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Motile microtubule crosslinkers require distinct dynamic properties for correct functioning during spindle organization in Xenopus egg extract

European Molecular Biology Laboratory, Cell Biology and Biophysics Unit, Meyerhofstr. 1, 69117 Heidelberg, Germany

View larger version (33K): [in this window] [in a new window]   Fig. 1. Characterization of chimeric Kid-Eg5-GFP and Kin1-Eg5-GFP in vitro. (A) Aligned sequences of the regions of the motor-to-stalk transition of the chimeric constructs Kid-Eg5-GFP and Kin1-Eg5-GFP. The original sequences of Kid and Kinesin-1 (Kin1) are in green and blue respectively, and the Eg5 sequence is in black. (B) Purified Kid-Eg5-GFP and Kin1-Eg5-GFP on a Coomassie-stained gel. (C) Velocity distributions of microtubules propelled by Eg5 (black), Kid-Eg5-GFP (green), Kin1-Eg5-GFP (blue) and the average velocities in a bar graph. Error bars indicate s.d. Insets are representative kymographs of moving Alexa-Fluor-568-labeled microtubules during a period of 2 minutes. Scale bars: 2 µm for Eg5 and Kid-Eg5-GFP, 10 µm for Kin1-Eg5-GFP. (D) Table (top) summarizing the size-exclusion chromatography results. Elution volumes of three constructs in two buffers containing KCl concentrations as indicated and, for comparison, a previously published value are presented (Kwok et al., 2006). Fluorescence images (bottom) of Alexa-Fluor-568-labeled microtubules bundled by Eg5, Kid-Eg5-GFP or Kin1-Eg5-GFP at KCl concentrations as indicated. Scale bar: 10 µm.

View larger version (38K): [in this window] [in a new window]   Fig. 2. Kid-Eg5-GFP and Kin1-Eg5-GFP form bundles instead of monoasters in Eg5-depleted Xenopus egg extract. (A) Scheme of the experimental procedure. Eg5-depleted mitotic extract, in which monoasters usually form, is supplemented with either Kid-Eg5-GFP or Kin1-Eg5-GFP. (B) Western blot showing the amount of Eg5 in mock-depleted and Eg5-depleted extract, and of Kid-Eg5-GFP or Kin1-Eg5-GFP in Eg5-depleted extracts after their addition close to endogenous levels. (C) Fluorescence images (top) showing examples of structures observed in the extract 45 minutes after addition of chimeric motors. (Middle) Percentages of observed structures in extract. The number of the structures counted were: 115 and 86 in mock-treated and Eg5-depleted extract, respectively, and 46 and 133 in Eg5-depleted extract supplemented with Kid-Eg5-GFP and Kin1-Eg5-GFP, respectively. Error bars indicate s.d. (Bottom) Localization of Kid-Eg5-GFP, Kin1-Eg5-GFP and wild-type Eg5-GFP in representative predominating structures as observed by fluorescence microscopy. In the merged images, Eg5-GFP constructs are shown in green, Hoechst-stained DNA in blue, TAMRA-labeled-microtubules in red. Scale bars: 10 µm. (D) Percentages of the different observed structures formed in Eg5-depleted extract after addition of Kid-Eg5-GFP (top) and Kin1-Eg5-GFP (bottom) as a function of different concentrations of added chimeric constructs. Error bars indicate s.d.

View larger version (31K): [in this window] [in a new window]   Fig. 3. Addition of Kid-Eg5-GFP or Kin1-Eg5-GFP led to a collapse of preformed spindles in Xenopus egg extract. (A) Scheme of the experimental procedure. Mitotic extract with preassembled spindles were supplemented with either Kid-Eg5-GFP or Kin1-Eg5-GFP. (B) Western blot showing the amount of endogenous Eg5 and of added Kid-Eg5-GFP or Kin1-Eg5-GFP in extract. (C; top panels) Fluorescence images of representative structures observed during spindle collapse. DNA was stained with Hoechst (blue) and microtubules were labeled with TAMRA (red). Scale bars: 10 µm. (Bottom) Percentages of these structures were plotted as a function of the time after addition of either Kid-Eg5-GFP (left) or Kin-Eg5-GFP (right). Error bars indicate s.d.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Kid-Eg5-GFP preferentially localizes to the midzone of preformed spindles in extract, where it enhances apparently outward forces leading to spindle elongation. (A) Spindle length as a function of the time passed after addition of either Kid-Eg5-GFP (green) or control buffer (grey). Error bars indicate s.d. (B) Fluorescence images showing the localization of wild-type Eg5-GFP (top left) and of Kid-Eg5-GFP (bottom left) 5-7 minutes after they were added to pre-assembled spindles. Eg5 constructs are shown in green; TAMRA-labeled microtubules in red. Average distributions of wild-type Eg5-GFP and Kid-Eg5-GFP (right) along the axis of four and six spindles, respectively. Scale bars: 10 µm. (C) Schematic model illustrating the pathway to single bundle structures either from preformed spindles after subsequent addition of chimeric Eg5 (left) or from chromatin nucleating microtubules in Eg5-depleted extract supplemented with chimeric Eg5 (right).

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