An Optogenetic Approach to Control Microtubule Acetylation in Living Cells

The association and conformation of proteins at the perimeter of moving cells is affected by post-translational modifications of microtubules. Acetylation of specific microtubules regulates cargo selection and trafficking to specific regions of the cell edge. Microtubule acetylation correlates with metastasis, indicating that microtubule post-translational modification is important in cancer cell biology. To study the spatio-temporal control of microtubule acetylation in live cells, we designed an analog of alpha tubulin acetyl transferase1 (αTAT1) that can be controlled with light. Unlike other tubulin modification enzymes, αTAT1 is highly specific for tubulin in its polymerized form. αTAT1 binds and transfers an acetyl moiety from acetyl coA to lysine 40 on alpha tubulin. A fragment of αTAT1 was fused to the photo-responsive LOV2 domain from Avena Sativa phototropin and to a peptide that we had engineered to bind selectively to the dark state of LOV2 (Wang et al. Nature Methods 13 755-758, 2016). Only in the dark, the LOV2 and Zdk bound to each other, occluding the microtubule binding interface. Photoactivatable αTAT1 (PA αTAT) could be fully activated in less than a second, and the half-life for return to the off state could be adjusted between 1.7 and 496 seconds by mutating residues around the LOV2 flavin. Cell assays showed extensive acetylation of microtubules upon irradiation of cells expressing PA αTAT1. Use of the analog in living cells to elucidate the role of microtubule acetylation in directed motility will be described.