Microtubules tightly regulate various cellular activities. Our understanding of microtubules is largely based on experiments using microtubule-targeting agents, which, however, are insufficient to dissect the dynamic mechanisms of specific microtubule populations, due to their slow effects on the entire pool of microtubules. To overcome this technological limitation, we have used chemo and optogenetics to disassemble specific microtubule subtypes, including tyrosinated microtubules, primary cilia, mitotic spindles, and intercellular bridges, by rapidly recruiting engineered microtubule-cleaving enzymes onto target microtubules in a reversible manner. Using this approach, we show that acute microtubule disassembly swiftly halts vesicular trafficking and lysosomal dynamics. It also immediately triggers Golgi and ER reorganization and slows the fusion/fission of mitochondria without affecting mitochondrial membrane potential. In addition, cell rigidity is increased after microtubule disruption owing to increased contractile stress fibers. Microtubule disruption furthermore prevents cell division, but does not cause cell death during interphase. Overall, the reported tools facilitate detailed analysis of how microtubules precisely regulate cellular architecture and functions.
24Biomolecules that respond to different external stimuli enable the remote control of genetically 25 modified cells. Chemogenetics and optogenetics, two tools that can control cellular activities 26 via synthetic chemicals or photons, respectively, have been widely used to elucidate underlying 27 physiological processes. These methods are, however, very invasive, have poor penetrability, 28 or low spatiotemporal precision, attributes that hinder their use in therapeutic applications. We 29 report herein a sonogenetic approach that can manipulate target cell activities by focused 30 ultrasound stimulation. This system requires an ultrasound-responsive protein derived from an 31 engineered auditory-sensing protein prestin. Heterogeneous expression of mouse prestin 32 containing two parallel amino acid substitutions, N7T and N308S, that frequently exist in 33 prestins from echolocating species endowed transfected mammalian cells with the ability to 34 sense ultrasound. An ultrasound pulse of low frequency and low pressure efficiently evoked 35 cellular calcium responses after transfecting with prestin(N7T, N308S). Moreover, pulsed 36 ultrasound can also non-invasively stimulate target neurons expressing prestin(N7T, N308S) 37 in deep regions of mice brains. Our study delineates how an engineered auditory-sensing 38 protein can cause mammalian cells to sense ultrasound stimulation. Moreover, owing to the 39 great penetration of low-frequency ultrasound (~400 mm in depth), our sonogenetic tools will 40 serve as new strategies for non-invasive therapy in deep tissues of large animals like primates. 41 42 43 44 45 46
Microtubules (MTs) are components of the evolutionarily conserved cytoskeleton, which tightly regulates various cellular activities. Our understanding of MTs is largely based on MT-targeting agents, which, however, are insufficient to dissect the dynamic mechanisms of specific MT populations due to their slow effects on the entire pool of MTs in cells. To address this limitation, we have used chemogenetics and optogenetics to disassemble specific MT subtypes by rapid recruitment of engineered MT-cleaving enzymes. Acute MT disassembly swiftly halted vesicular trafficking and lysosome dynamics. We also used this approach to disassemble MTs specifically modified by tyrosination and several MT-based structures including primary cilia, mitotic spindles, and intercellular bridges. These effects were rapidly reversed by inhibiting the activity or MT association of the cleaving enzymes. The disassembly of targeted MTs with spatial and temporal accuracy enables to uncover new insights of how MTs precisely regulate cellular architectures and functions.
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