A FRET-based Biosensor For Visualizing The Tension In Active Zone Of Neural Synapse

Objective: The tension changes in active zone of neural synapse have close association with the formation of neural synapses,endocytosis and exocytosis of synaptic vesicles.It helps to understand the mechanism of synaptic transmission by exploring tension changes in active zone during the release of synaptic vesicles.Methods: Three tension probes based on fluorescence resonance energy transfer(FRET)are designed and constructed in current experiment,named BKTS,RKTS,and CKTS,respectively.They all consist of a tension sensor and two anchoring proteins which are located at two terminals.The tension sensor comprises an elastic spider silk protein inserted between two fluorescence proteins,ECFP and YPet.With this sensor,tension changes can be transformed into FRET efficiency changes of these two fluorescence proteins.The two specific anchoring proteins can bind with active zone.The N-terminal of BKTS binds with SH3-?domain of Rab3-interacting molecules-binding protein(RIM-BP)preferentially,while C-terminal binds with cell membrane within active zone.RKTS has the same binding site in C-terminal but binds with SH3-?or SH3-? domain of RIM-BP in N-terminal.Both terminals of CKTS bind with cell membrane within active zone through C1 domain and KRas,respectively.High potassium solution is applied to induce synaptic vesicle release after the probes are transfected into neural cells.The fluorescence pictures of ECFP and YPet are taken at the same time under laser scanning confocal microscope.Their FRET ratio pictures,calculated as YPet/ECFP,are analyzed by Matlab programs,combined with Kmeans method.Results: Under high potassium solution stimuli,FRET ratio values of BKTS,RKTS,and CKTS change significantly,while the ratio values of their control probes remain unchanged.During the release of synaptic vesicles,the FRET ratio values of BKTS and CKTS are continuously decreased in nerve endings,while the ratio of RKTS keeps increasing.Results show a more significant tension change tendency in active zone after clustering ratio pictures with Kmeans method.These findings indicate that all three probes can react to the release of synaptic vesicles with good sensitivity,and these probes are capable of detecting tension changes in different parts of active zone.Kmeans method is able to extract tension changes in active zone from the complex tension changes in nerve endings.This method plays a role of signal amplification in describing tension changes more accurately in active zone.Conclusion: Three FRET biosensors are designed and constructed successfully in current project to visually detect tension changes in different parts of synaptic active zone.They reflect tension changes in active zone from different aspects and reveal the effects of different proteins on the release of synaptic vesicles.These probes provide effective and visual tools for exploring synaptic transmission mechanism more deeply.