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G-protein-coupled receptors (GPCRs) play important roles in cellular functions. However, their intracellular organization is largely unknown. Through investigation of the cannabinoid receptor 1 (CB1), we discovered periodically repeating clusters of CB1 hotspots within the axons of neurons. We observed these CB1 hotspots interact with the membrane-associated periodic skeleton (MPS) forming a complex crucial in the regulation of CB1 signaling. Furthermore, we found that CB1 hotspot periodicity increased upon CB1 agonist application, and these activated CB1 displayed less dynamic movement compared to non-activated CB1. Our results suggest that CB1 forms periodic hotspots organized by the MPS as a mechanism to increase signaling efficacy upon activation., Despite the importance of G-protein-coupled receptors in many cellular functions, their intracellular organisation is largely unknown. The authors identified periodically repeating clusters of cannabinoid receptor 1 hotspots within neuronal axons that are dynamically regulated by CB1 agonists.

Prophylactic vaccines against SARS-CoV-2 have been extensively developed globally to overcome the COVID-19 pandemic. However, recently emerging SARS-CoV-2 variants B.1.1.7 and B.1.351 limit the vaccine protection effects and successfully escape antibody cocktail treatment. Herein, based on our previously engineered adeno-associated viral (AAV) vector, AAV-ie, and systematic immunogen screening, we developed an AAV-ie-S1 vaccine with thermostability, high efficiency, safety, and single-dose vaccination advantage. Importantly, the AAV-ie-S1 immune sera efficiently neutralize B.1.1.7 and B.1.351, indicating a potential to circumvent the spreading of SARS-CoV-2.

G-protein-coupled receptors (GPCRs) play important roles in cellular functions. However, their intracellular organization is largely unknown. Through investigation of the cannabinoid receptor 1 (CB1), we discovered periodically repeating clusters of CB1 hotspots within the axons of neurons. We observed these CB1 hotspots interact with the membrane-associated periodic skeleton (MPS) forming a complex crucial in the regulation of CB1 signaling. Furthermore, we found that CB1 hotspot periodicity increased upon CB1 agonist application, and these activated CB1 displayed less dynamic movement compared to non-activated CB1. Our results suggest that CB1 forms periodic hotspots organized by the MPS as a mechanism to increase signaling efficacy when being activated.

Hearing loss is the most common sensory disorder. While gene therapy has emerged as a promising treatment of inherited diseases like hearing loss, it is dependent on the identification of gene delivery vectors. Adeno-associated virus (AAV) vector-mediated gene therapy has been approved in the US for treating a rare inherited eye disease but no safe and efficient vectors have been identified that can target the diverse types of inner ear cells. Here, we identify an AAV variant, AAV-inner ear (AAV-ie), for gene delivery in mouse inner ear. Our results show that AAV-ie transduces the cochlear supporting cells (SCs) with high efficiency, representing a vast improvement over conventional AAV serotypes. Furthermore, after AAV-ie-mediated transfer of the Atoh1 gene, we find that many SCs trans-differentiated into new HCs. Our results suggest that AAV-ie is a useful tool for the cochlear gene therapy and for investigating the mechanism of HC regeneration., There are currently few AAV vectors that can effectively target the diverse cell types of the inner ear. Here the authors design AAV-ie for gene delivery to the mouse cochlea, targeting cochlear supporting cells.

Clinical therapies of pluripotent stem cells (PSCs)-based transplantation have been hindered by frequent development of teratomas or tumors in animal models and clinical patients. Therefore, clarifying the mechanism of carcinogenesis in stem cell therapy is of great importance for reducing the risk of tumorigenicity. Here we differentiate Oct4-GFP mouse embryonic stem cells (mESCs) into neural progenitor cells (NPCs) and find that a minority of Oct4+ cells are continuously sustained at Oct4+ state. These cells can be enriched and proliferated in a standard ESC medium. Interestingly, the differentiation potential of these enriched cells is tightly restricted with much higher tumorigenic activity, which are thus defined as differentiation-resistant ESCs (DR-ESCs). Transcriptomic and epigenomic analyses show that DR-ESCs are characterized by primordial germ cell-like gene signatures (Dazl, Rec8, Stra8, Blimp1, etc.) and specific epigenetic patterns distinct from mESCs. Moreover, the DR-ESCs possess germ cell potential to generate Sycp3+ haploid cells and are able to reside in sperm-free spermaduct induced by busulfan. Finally, we find that TGFβ signaling is overactivated in DR-ESCs, and inhibition of TGFβ signaling eliminates the tumorigenicity of mESC-derived NPCs by inducing the full differentiation of DR-ESCs. These data demonstrate that these TGFβ-hyperactivated germ cell-like DR-ESCs are the main contributor for the tumorigenicity of ESCs-derived target cell therapy and that inhibition of TGFβ signaling in ESC-derived NPC transplantation could drastically reduce the risk of tumor development.