Abstract
Kinesins play a role in organizing the mitotic spindle through the crosslinking of microtubules (MTs), made possible through binding sites at opposite ends of the holoenzyme. Here, we developed a method to test kinesin MT crosslinking action under physiological conditions.
Keywords: in vitro bundling assay, Fluorescence microtubules, Kinesin motors, DEAE-Dextran coated glass chamber, Physiological [ATP]
Background
Microtubule-based motor proteins are important as they use the chemical energy from ATP to generate force to translocate microtubules (MTs) vectorially. Of these MT based motor proteins, the superfamily known as kinesins, are responsible for directional transport and movement along microtubules. Some kinesins have MT-binding sites at both ends of the holoenzyme, so they can crosslink MTs into bundles under physiological ATP conditions (Tao et al., 2006). Due to this bundling activity, they have important roles in organizing and maintaining the mitotic spindle, whose action depends upon the polarity patterns of its microtubules (van den Wildenberg et al., 2008). Previous bundling assays didn’t include ATP, or used a non-hydrolysable ATP analog, which could generate artificial results. Here we developed a method using physiological ATP conditions. By purifying these full-length motor proteins, it has allowed us to determine their crosslinking activity under physiological conditions.
Materials and Reagents
Equipment
Procedure
Data analysis
The microtubule (MT) bundling assay was repeated three times, with three separate batches of reagents. 15 images were collected for each experiment. To ensure consistency, it is recommended that the assay be repeated three times, with 15 images collected for each. Figure 2 shows a kinesin holoenzyme crosslinking MTs into bundles. Figure 2A shows a robust bundle due to crosslinking activity of the kinesin motor. However, in Figure 2B the kinesin-1 head dimers do not bundle MTs, suggesting that the binding sites at opposite ends of the motor are required for crosslinking, rather than action of two heads at one end. Figure 2. Example of bundling assay. Fluorescence microscopy of purified kinesin holoenzyme bundling MTs (A) while kinesin-1 head dimers unable to bundle MTs under same condition (B). Scale bar = 10 µm. (Tao et al., 2016)
Notes
Recipes
*Note: Listed concentrations are final concentrations, buffer is prepared in ddH2O.
Acknowledgments
This MT bundling assay was inspired by ‘Tum/RacGAP functions as a switch activating the Pav/kinesin-6 motor’ (Tao et al., 2016) and ‘Purification and assay of mitotic motors’ (Tao and Scholey, 2010). This work was funded by the NIH grant GM046409 to W.S. and NIH grant GM 55507 to J.M.S.
References
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