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.

Biomolecules that respond to different external stimuli enable the remote control of genetically modified cells. Chemogenetics and optogenetics, two tools that can control cellular activities via synthetic chemicals or photons, respectively, have been widely used to elucidate underlying physiological processes. These methods are, however, very invasive, have poor penetrability, or low spatiotemporal precision, attributes that hinder their use in therapeutic applications. We report herein a sonogenetic approach that can manipulate target cell activities by focused ultrasound stimulation. This system requires an ultrasound-responsive protein derived from an engineered auditory-sensing protein prestin. Heterogeneous expression of mouse prestin containing two parallel amino acid substitutions, N7T and N308S, that frequently exist in prestins from echolocating species endowed transfected mammalian cells with the ability to sense ultrasound. An ultrasound pulse of low frequency and low pressure efficiently evoked cellular calcium responses after transfecting with prestin(N7T, N308S). Moreover, pulsed ultrasound can also non-invasively stimulate target neurons expressing prestin(N7T, N308S) in deep regions of mice brains. Our study delineates how an engineered auditory-sensing protein can cause mammalian cells to sense ultrasound stimulation. Moreover, owing to the great penetration of low-frequency ultrasound (~400 mm in depth), our sonogenetic tools will serve as new strategies for non-invasive therapy in deep tissues of large animals like primates.

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.