|Ph.D Student||Stern Michal|
|Subject||Active Zone Maintenance and Stability Studied in|
Munc13-1 yfp/yfp Knock-in Mice
|Department||Department of Medicine||Supervisor||PROF. Noam Ziv|
|Full Thesis text - in Hebrew|
Synapses are cellular structures specialized for passing signals among neurons as well as non-neuronal targets. The Active Zone is an axonal specialization that organizes the presynaptic compartment and plays key roles in orchestrating synaptic transmission. Active Zone stability, function and dynamics have been studied intensively by live imaging microscopy, and in particular by using synaptic molecules tagged with green fluorescent protein (GFP) and its derivatives. However, the common use of artificial expression constructs for expressing such fusion proteins usually results in significant overexpression levels and decouples their expression from endogenous spatiotemporal regulatory mechanisms.
In this study, these difficulties were bypassed by using a unique Knock-In mice model expressing the Cytomatrix at the Active Zone (CAZ) protein Munc13-1 fused to enhanced yellow fluorescent protein (EYFP) from the Munc13-1 locus (Munc13-1YFP/YFP). Presynaptic active zones from these mice are distinctly fluorescent since the entire population of Munc13-1 molecules is tagged with EYFP. Indeed, we found that Munc13-1-EYFP is targeted correctly to functional presynaptic Active Zones and that expression levels of Munc13-1-EYFP are suitable for informative fluorescence imaging.
Using this model, we aimed to study cellular and molecular mechanisms that underlie the structural preservation and maintenance of the Active Zone and in particular the transport, stability and exchange of endogenous Munc13-1 and the contribution of important CAZ molecules to Active Zone integrity. We found that Munc13-1-EYFP is rapidly and continuously lost from and incorporated into Active Zones possibly from two Munc13-1-EYFP pools. Munc13-1-EYFP steady-state levels and exchange kinetics were not affected by proteasome inhibitors or acute synaptic stimulation, but exchange kinetics were reduced by chronic suppression of spontaneous activity. At longer time scales, we found that Munc13-1-EYFP fluorescence levels did not remain constant. In fact, significant, spontaneously occurring changes in Munc13-1-EYFP cluster sizes at individual synapses were observed.
By comparing Munc13-1-EYFP dynamics with synaptic vesicle dynamics we found that Munc13-1-EYFP and synaptic vesicles are recruited to nascent synapses with similar kinetics. This was also observed during active zone elimination.
Finally, by crossing these mice with mutant mouse lines we found that the genetic elimination of neither RIM1α nor Bassoon did not dramatically affect CAZ stability.
To summarize, the use of this Knock-In mice model enabled the investigation of synapse formation, maintenance and dynamics in an innovative and minimally perturbed system, and provided evidence for the highly dynamic nature of the synapse and its limited stability over time.