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. 2014 Dec 22;107(12):2872–2880. doi: 10.1016/j.bpj.2014.10.061

Figure 1.

Figure 1

Increasing acetylation increases axonemal dynein speed on porcine microtubules. (A) Schematic of tubulin acetylation and deacetylation by αTAT and SIRT2, respectively. The orange and red circles represent α- and β-tubulins, respectively. The yellow circle represents the acetylated form of K40. (B) Western blots of porcine microtubules treated with αTAT showing that αTAT treatment significantly increased the fraction of acetylated tubulin whereas the CTT-associated PTMs were unaffected. Tubulin loading was assayed by visualization in the gel using the TGX Stain-Free Precast Gel protocol before transfer to the nitrocellulose membrane. (C) Western blot of microtubules treated with αTAT showing that increased αTAT treatment time increases the fraction of acetylated tubulin. (D) Densitometry of the Western blot from panel C. (E) Example raw gliding assay data. (top) Typical kymograph of a microtubule (length = 2.6 μm) gliding on axonemal dynein that shows the characteristic unsteadiness of axonemal dynein gliding assays. (bottom) Plot of the microtubule’s distance traveled as a function of time. This is an example of typical gliding data after tracking. (F) Typical plot of mean microtubule gliding velocity as a function of microtubule length. This example is for the case of untreated microtubules. The data points were calculated by pooling microtubules in 2 μm bins and averaging their displacement-weighted mean velocities. The error bars are the standard error of the mean for each bin. The line is a least squares fit to Eq. 1 calculated before pooling and weighted by total distance traveled. This fit was used to calculate vmax and L0, shown with dashed lines, and it is typical of all cases. (G) The calculated vmax for each αTAT treatment condition plotted as a function of acetylation time. (H) The calculated L0 for each αTAT treatment condition plotted as a function of acetylation time. The data points for panels G and H were calculated by pooling the microtubule tracks from at least three movies per condition. A total of 213, 140, 280, 257, 1034, 94, 474, and 438 microtubules were analyzed for the αTAT treatment times of 0, 1, 2, 5, 10, 20, 30, and 90 min, respectively. The error bars in G and H are the standard errors for each parameter. To see this figure in color, go online.