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.

Cilia, which either generate coordinated motion or sense environmental cues and transmit corresponding signals to the cell body, are highly conserved hair-like structures that protrude from the cell surface among diverse species. Disruption of ciliary functions leads to numerous human disorders, collectively referred to as ciliopathies. Cilia are mechanically supported by axonemes, which are composed of microtubule doublets. It has been recognized for several decades that tubulins in axonemes undergo glutamylation, a post-translational polymodification, that conjugates glutamic acid chains onto the C-terminal tail of tubulins. However, the physiological roles of axonemal glutamylation were not uncovered until recently. This review will focus on how cells modulate glutamylation on ciliary axonemes and how axonemal glutamylation regulates cilia architecture and functions, as well as its physiological importance in human health. We will also discuss the conventional and emerging new strategies used to manipulate glutamylation in cilia.

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Neovascularization is important for bone repair, vascularization, and ossification during bone repair. Ginsenoside Rg1 (G-Rg1), which is the main extract of ginseng, has been shown to promote therapeutic angiogenesis. It has been studied in the field of biomaterials, but there is no relevant report in the field of bone substitute materials. In this study, we successfully prepared the bone substitute material combining calcium sulphate (Sr-CaS) with G-Rg1 on the basis of previous research work. In vitro experiments were carried out to verify the ossification of composites by using mouse bone marrow mesenchymal stem cells (BMMSCs) and the ossification was quantified by western blot. The related proteins in the key signaling pathways for the different concentrations of G-Rg1/Sr-CaS composite extract were studied to determine whether there was receptor competition and to find the optimal ratio parameters. The vascularization of the composite was verified in the human umbilical vein endothelial cells (HUVECs) model, and finally the coordination of pro-vascularization and ossification was evaluated in the mouse critical bone defect model. The results indicated that G-Rg1/Sr-CaS composites contributed to ossification in the mouse BMMSC model and vascularization in the HUVEC model. The G-Rg1/Sr-CaS composites resulted in significantly greater bone mineral densities and bone volume/total volume of the defect group compared to the control group. Histological analysis showed that the G-Rg1/SrCaS was resorbable with satisfactory biocompatibility. The doped strontium ions enhanced the bone repair performance of G-Rg1/Sr-CaS in the mouse model and the new substitute demonstrated promising results for clinical use.