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Fig. 7. In vitro analysis of kinesin-1 interactions with post-translationally modified MTs. (A) Dot-blot assays for different post-translational modifications of tubulin in the three types of MT used in the in vitro MT-binding and -motility assays. A range of tubulin concentrations (500-10 ng) were probed with antibodies that specifically recognise tyrosinated, detyrosinated or acetylated tubulin. Microtubules purified from pig brain were extensively modified. Microtubules purified from untreated HeLa cells [HeLa (–CPA)] were predominantly unmodified (composed of at least 95% tyrosinated tubulin). Carboxypeptidase A (CPA) treatment of HeLa cells [HeLa (+CPA)] resulted in at least 95% of tubulin being detyrosinated. (B) Tubulin from pig brain, untreated HeLa cells (–CPA) and treated HeLa cells (+CPA) resolved on SDS gel and stained with Coomassie-Blue. The two bands corresponding to 
- and β-tubulin can be seen in the modified and unmodified tubulin of HeLa cells. (C) Enhanced DIC images from in vitro motility assays for tubulin from pig brain, untreated HeLa cells (–CPA) and treated HeLa cells (+CPA). There is no discernable difference between the MTs. (D) Stills from an in vitro motility assay using unmodified tubulin from HeLa cell. Time (in seconds) is indicated in the top left hand corner. Black arrows indicate the front of the MT, asterisks show the starting position of the MT. Scale bar, 5 mm. (E) The velocity distribution of the Kif5c motor moving on the three types of MT in an in vitro motility assay. Bin sizes for the velocities are 0.02 µm second–1. Average velocities for the three types of MT were 0.55 µm second–1 (±0.05, mean ± s.d., n=207), 0.66 µm second–1 (±0.06, n=103) and 0.58 µm second–1 (±0.07, n=206) for MTs from pig brain, tyrosinated and detyrosinated MTs, respectively. Dashed curves show the calculated normal distribution for each dataset.
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