Reconstruction Of Presynaptic Active Zone Via Liquid-liquid Phase Separation System

Synaptic transmission between neurons depends on the process of synaptic vesicle secretion that occurs in the specific region attached to the presynaptic membrane,termed presynaptic active zone.The ogranization of presynaptic active zone relies on the densed network scaffolds formed by proteins as RIM1??RBP2?ELKS1 and Liprin-?3.Studies have shown that as the core components of presynaptic active zone,RIM1? and RBP2 could undergo phase separation,which then fulfills the recruitment of vesicles and calcium channels,associating active zone with liquid-liquid pahse separation.ELKS1 and Liprin-?3,as another two scaffold proteins,could interact with RIM1?.But whether they could induce and participate in the phase separation formed by RIM1? and RBP2 remain further investigation.In this respect,the present study adopts the research methods integrating cell biology,biochemistry,and biophysics to first explore in detail the interactions of the main scaffold proteins of the presynaptic membrane,and finds that ELKS1 and RBP2 do have mutual effects and could induce LLPS.Second,the ability of LLPS of these main scaffold proteins were investigated,with the cell-and-in vitro phase separation experiments.Third,this study fulfills the reconstruction of ELKS1/Liprin-?3/RIM1?/RBP2 liquid-liquid phase separation system in vitro and in HEK293 T cells.Lastly,it is confirmed that the complexes system could recruit the key synaptic secretion protein,i.e.,Munc13-1,in HEK293 T cells.To summarize,this study focuses on the interactions among the presynaptic active region scaffold proteins,including ELKS1,Liprin-?3,RIM1?,and RBP2,explores their ability regarding phase separation inducing in vitro,and constructs ELKS1/Liprin-?3/RIM1?/RBP2 liquid-liquid phase separation system.Results and findings in this study not only provide a fine-grained elaboration of the relationship within the scaffold proteins in the presynaptic active zone,but also lay a solid foundaton for further research pertaining to the reshaping of the presynaptic active zone in vitro.