Reconstituting what we see
A long-term goal of our research is to reconstitute key cell division processes with purified components. We have taken an important first step in this direction.
Fig. 3
Microtubule fibers move relative to each other in the cell division apparatus. Tubulin dynamics observed by FSM (fluorescent speckle microscopy) for the region highlighted in middle of the mitotic spindle are shown in the movie. Tubulin speckles move apart at ~ 4 microns/min (2V). Please click on the image to view a movie.
Fig. 4
Loss of Eg5 function results in monopolar spindles. A mammalian cell treated with monastrol, a cell-permeable small molecule inhibitor, is shown.
Motor protein micromechanics: anti-parallel microtubule sliding
(in collaboration with Christoph Schmidt’s laboratory at the Vrije University, Amsterdam)The movement of two microtubules relative to each other is believed to be important for many aspects of intracellular transport, including the separation of centrosomes and establishing the bipolar shape of the mitotic spindle. Similar to the muscle, where myosins slide filaments to generate forces, kinesins and dyneins are believed to generate forces pushing and pulling spindle microtubules.
In the mitotic spindle, FSM shows microtubules sliding in opposite directions (see Fig. 3). A widely conserved mitotic kinesin Eg5 (kinesin-5) has been suggested to be the motor protein responsible for this activity for a number of reasons, including: (1) Eg5’s homotetrameric bipolar structure with two motor domains at each end of a central stalk (2) Loss of Eg5 function in all eukaryotic cells results in monopolar spindle formation. These structures lack microtubules that have anti-parallel overlap (see Fig. 4).
Using recombinant full length homo-tetrameric Eg5 and microtubules, optical traps for micromanipulation and optimized surface chemistry, we have recently shown that Eg5 can walk on each microtubule it crosslinks, sliding apart anti-parallel microtubules. See Movie 4.
The assay system we have developed is being used to examine force production by Eg5 molecules. When combined with other mitotic kinesins, it will allow us to examine how different molecules can work together or in opposition to organize microtubules. These experiments are laying the groundwork for assembling a ‘minimal spindle’ with pure proteins.
For more information, please see (Kapitein L. et al. Nature 2005).
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