Spastin's Microtubule-Binding Properties and Comparison to Katanin
Panel A shows the results of a co-sedimentation assay of microtubules and GFP-linker domain constructs. From left to right: GFP-HsSpastin wild type linker domain (approximately 34 kDa) was mixed with microtubules (50 kDa) at a stoichiometric ratio of 1∶1. The microtubule-bound fraction appears in the pellet (P) of ultracentrifugation, the unbound fraction in the supernatant (S). Two amounts were analyzed by SDS-PAGE. Lanes 7–10 show the same experiment with the triple K310KK>Q310QQ mutation. The right SDS-gel shows a co-sedimentation assay with the analogous region from D. melanogaster spastin. Weight markers (lanes 1, 6, 11 left gel, and lanes 1 and 6 right gel) are given in kDa. Panel B: Binding of the GFP-linker constructs to microtubules in a TIRF microscopy assay.The top row shows Alexa Fluor 555 microtubules in red, the middle row GFP-linker constructs in green, the bottom row an overlay of red and green channels. All images were taken at the same gain and exposure time. The images show that the GFP HsSpastin wild type linker (left column) coats all microtubules densely, while the mutant (center column) and the Drosophila (right column) constructs fail to bind to a significant degree.