Tubulin post-translational modifications (PTMs) occur spatiotemporally throughout cells and are suggested to be involved in a wide range of cellular activities. However, the complexity and dynamic distribution of tubulin PTMs within cells have hindered the understanding of their physiological roles in specific subcellular compartments. Here, we develop a method to rapidly deplete tubulin glutamylation inside the primary cilia, a microtubule-based sensory organelle protruding on the cell surface, by targeting an engineered deglutamylase to the cilia in minutes. This rapid deglutamylation quickly leads to altered ciliary functions such as kinesin-2-mediated anterograde intraflagellar transport and Hedgehog signaling, along with no apparent crosstalk to other PTMs such as acetylation and detyrosination. Our study offers a feasible approach to spatiotemporally manipulate tubulin PTMs in living cells. Future expansion of the repertoire of actuators that regulate PTMs may facilitate a comprehensive understanding of how diverse tubulin PTMs encode ciliary as well as cellular functions.

We developed an ultrasound-chemical hybrid tool to precisely manipulate cellular activities. A focused ultrasound coupled with gas-filled microbubbles was used to rapidly trigger the influx of membrane-impermeable chemical dimerizers into living cells to regulate protein dimerization and location without inducing noticeable toxicity. With this system, we demonstrated the successful modulation of phospholipid metabolism triggered by a short pulse of ultrasound exposure. Our technique offers a powerful and versatile tool for using ultrasound to spatiotemporally manipulate the cellular physiology in living cells.