Figure 1.
Tubulin acetylation and deacetylation.
(a) Schematic representation of the acetylation and deacetylation of microtubules by αTAT and HDAC6, respectively. (b) Western blots of non-treated, acetylated, and deacetylated tubulin with anti-b-tubulin (SAP4G5, Abnova), anti-AcK40 (6–11B-1, Thermo Scientific), anti-detyrosinated-tubulin (AB3201, Millipore), anti-Δ2-tubulin (pab0202, covalab), and anti-polyglutamylated-tubulin (GT335, Enzo). αTAT-treated tubulin is highly acetylated, whereas HDAC6-treated tubulin is deacetylated. The levels of detyrosination, decarboxylation, and polyglutamylation are not affected by αTAT and HDAC6.
Figure 2.
Tubulin acetylation does not affect microtubule velocity in kinesin-1 gliding assays.
(a) Schematic representation of the experimental setup for multi-motor gliding assays. We measured the velocities of short rhodamine-labeled acetylated and deacetylated microtubules propelled by surface-bound motors in presence of 1 mM ATP. The mean gliding velocities of 568 deacetylated microtubules (b) and 532 acetylated microtubules (c) did not differ significantly. The errors represent the SD of the Gaussian distributions.
Figure 3.
Tubulin acetylation does not affect the kinetics of kinesin-1 stepping on microtubules.
(a) Schematic representation of the experimental setup for single-motor stepping assays. Single rKin430-EGFP motors walk on a microtubule attached to the surface via anti-tubulin antibodies. By tracking the positions of the motors we determined the mean velocities and mean run lengths for deacetylated and acetylated microtubules. The mean velocities of (b) 1977 motors on deacetylated and (c) 1283 motors on acetylated microtubules did not differ significantly. The errors represent the SD of the Gaussian distributions. Mean run lengths of (d) 1949 single motors on deacetylated and (e) 1270 motors on acetylated microtubules were determined by fitting the cumulative probability distributions. The run lengths on deacetylated and acetylated microtubules did not differ significantly. The errors were estimated with the bootstrap technique.