Tubulin Acetylation Alone Does Not Affect Kinesin-1 Velocity and Run Length In Vitro

Kinesin-1 plays a major role in anterograde transport of intracellular cargo along microtubules. Currently, there is an ongoing debate of whether α-tubulin K40 acetylation directly enhances the velocity of kinesin-1 and its affinity to the microtubule track. We compared motor motility on microtubules reconstituted from acetylated and deacetylated tubulin. For both, single- and multi-motor in vitro motility assays, we demonstrate that tubulin acetylation alone does not affect kinesin-1 velocity and run length.


Introduction
Kinesin-1 is a microtubule-based motor protein that converts the chemical energy derived from ATP hydrolysis into mechanical work to translocate processively towards the plus end of a microtubule. One of the multiple functions of kinesin-1 -and the first one that was discovered -is the transport of vesicles within neurons [1]. Thereby, kinesin-1 shows a preference for axonal microtubules over dendritic microtubules [2,3] and experiments with truncated motor constructs have shown that the motor domain itself is sufficient to distinguish between the two kinds of microtubules [2]. This selectivity can be abolished by a mutation within the microtubule-binding surface of the kinesin-1 motor domain, indicating that track selection is an inherent property of the motor [4].
Acetylation of a-tubulin K40 is a well-known marker for highly posttranslationally modified, so-called 'stable', microtubules that account for the majority of the axonal microtubules [5]. Previous studies analyzed whether tubulin acetylation facilitates selective translocation of kinesin-1 in vivo. Cells were treated with trichostatin A (TSA) -an inhibitor of the histone deacetylase (HDAC) family -which subsequently caused an increase in overall tubulin acetylation [4,6]. This led to an enhanced binding of kinesin-1 to the microtubules, a higher velocity, and a loss of the preference for axonal microtubules. Moreover, the addition of TSA to cells with impaired huntingtin protein -which causes a significant reduction of vesicle velocity and increases the frequency of waiting periods [7,8] -restored velocity and frequency of vesicles back to wt levels [9]. More recently, however, two in vivo studies indicated that acetylation alone might not be sufficient to explain the preferential binding of kinesin-1 to axonal microtubules [10,11].
The inconsistent results of the mentioned studies demonstrate the limitations of in vivo experiments as the complexity of the cellular environment often does not allow for definite conclusions. Especially the interpretation of results derived from experiments with chemical inhibitors requires caution, as other proteins besides tubulin might be affected. In order to analyze whether acetylation of the K40 residue alone is sufficient to modify kinesin-1 motility, we performed in vitro multi-motor gliding and single-motor stepping assays with microtubules reconstituted from acetylated and deacetylated porcine tubulin.

Results and Discussion
We prepared acetylated tubulin using mouse a-tubulin acetyltransferase (aTAT), which recently was discovered by two independent research groups to specifically acetylate a-tubulin K40 [12,13] (Fig. 1a). Tubulin K40 deacetylation was performed by incubating tubulin with recombinant human histone transacetylase-like enzyme HDAC6, the role of which has been known for several years [14]. The success of acetylation and deacetylation was proven in a Western blot with antibodies specific for a-tubulin acetyl-K40 [15] (Fig. 1b). In order to rule out effects of HDAC6 or aTAT on other posttranslational tubulin modifications we additionally performed Western blots with antibodies against detyrosinated, decarboxylated (D2), and polyglutamylated tubulin. Neither of those modifications was affected (Fig. 1b).
In multi-motor gliding assays [16] the velocities of rhodaminelabeled acetylated and deacetylated microtubules propelled by surface-bound, truncated rat kinesin-1 labeled by EGFP (rKin430-EGFP) [17] were determined in the presence of 1 mM ATP (Fig. 2a). We determined gliding velocities of 868+/230 nm?s-1 (mean +/2 SD, n = 568 microtubules) and 874+/225 nm?s-1 (n = 532) for deacetylated and acetylated tubulin, respectively ( Fig. 2b and c). The 0.7% difference in the mean velocities is statistically significant (p = 0.0003) due to the high number of observes microtubules. However, on the other hand this difference is equivalent to the effect of an increase in temperature by DT = 0.1 K (see Methods). As we are experimentally able to control the temperature only within a range of +/20.5 K, we consider the observed velocity difference to be not significant.
In both, multi-motor gliding and single-motor stepping assays with microtubules reconstituted from acetylated and deacetylated tubulin we did not observe a direct effect of K40 acetylation on kinesin-1 velocity or run length. We thus hypothesize that the differences in motor protein motility previously observed in vivo [4,6,9] might simply correlate with -but not be induced bymicrotubule acetylation. On one hand, acetylation often coexists with other posttranslational modifications. in vivo experiments indicate that detyrosination of a-tubulin in axonal microtubules might be responsible for the selective translocation of kinesin-1 [4]. Also, chemically inhibited tubulin acetylation in vivo may have effected the acetylation of additional putative acetylation sites in the tubulin dimer [19] or the acetylation of other proteins but tubulin. On the other hand, posttranslational modifications are not the only microtubule features that potentially interfere with molecular motors. Within the cell microtubules display a range of accessory proteins, so called microtubule-associated proteins (MAPs). Although the K40 acetylation site is located in the inner lumen of the microtubule [20], rendering a direct electrostatic effect on MAPs interacting with the outer surface of the microtubule unlikely, small conformational changes induced by K40 acetylation may allosterically propagate to the respective MAP binding sites. Previous results might thus be secondary effects provoked by the interaction of kinesin-1 with MAPs that potentially recognize tubulin acetylation. In particular, the protein tau, which has been described to interfere with kinesin-1 stepping [21] and tubulin acetylation [22], might play a significant role.
In summary, by performing single-and multi-motor in vitro motility assays, we found that tubulin acetylation alone does not affect kinesin-1 velocity and run length. Rather than the acetylation state of microtubules as such, its combination with additional posttranslational modifications or MAPs, may thus be responsible for guiding kinesin-1 towards axonal transport in vivo. Further experiments -including in vitro measurements with controlled populations of posttranslationally modified microtubules and additional binding partners -will be required to fully understand the selective translocation of kinesin-1.

Tubulin Acetylation and Deacetylation
Murine aTAT was recombinantly expressed in the E. coli strain BL21 as a GST fusion protein and purified as described previously [13]. The aTAT expression plasmid was a generous gift by Prof.

Gliding and Stepping Motility Assays
All experiments were performed with truncated, EGFP-labeled kinesin-1 constructs (rKin430-EGFP), which contained the first 430 aa of kinesin-1 fused to a EGFP and a His tag at the tail domain [15]. Multi-motor gliding motility assays and imaging were performed at room temperature (22-23uC) as described previously [23] except for the final assay solution (35 mM PIPES, 1 mM MgCl 2 , 1 mM EGTA, 20 mM KCl, 1 mM ATP, 0.2 mg/ ml catalase, 0.1 mg/ml glucose oxidase, 40 mM D-glucose, 10 mM DTT, 10 mM taxol, pH 7.4). Single-motor stepping assays and imaging at modest laser illumination intensity were performed as described previously [24] under the same buffer conditions as the gliding assays.

Data Analysis
Velocities of microtubules and single motors, as well as the run lengths of single motors were obtained using FIESTA tracking software [25]. Mean velocities were determined by fitting the velocity histograms to Gaussian functions. In the analysis of the stepping assays only motor molecules moving over longer distances than 200 nm along the microtubule axis were considered. The significance of velocity differences was tested using t-test statistics. The temperature change DT = 0.1 K required to increase the mean velocity from v 1 = 868 nm?s 21 to v 2 = 868 nm?s 21 was estimated assuming a two-fold velocity increase every 10 K [26] using: DT = 10 K ? log(v 2 /v 1 )/log(2) derived from: v 2 = v 1 ?2ˆ(DT/ 10 K).
To obtain bin-size independent values of the mean run lengths we analyzed the cumulative probability distributions [27]. These distributions were fitted with functions 1 -exp[(x 0 2x)/t], where the only fitted parameter t is the mean run length of the distribution and x 0 the lower limit for runs included into the analysis. As an error estimate for the mean run length, we considered 200 bootstrap samples [28] and fitted them as described above. Standard deviations of the sampled sets were used as error estimates. The significance of the differences in the run lengths was tested using Kolmogorov-Smirnov statistics [29]. Repeating the run lengths measurements at 40% and 80% increased laser intensities allowed us to extrapolate to run lengths free of photobleaching effects [30]. For both, deacetylated and acetylated microtubules, we found that these photobleach-free run lengths (though approximately 8% longer than the values given in the main text) did not differ significantly. All fitting was performed in MATLAB (MathWorks, USA).