RT Journal Article SR Electronic T1 Molecular Dynamics of a Presynaptic Active Zone Protein Studied in Munc13-1–Enhanced Yellow Fluorescent Protein Knock-In Mutant Mice JF The Journal of Neuroscience JO J. Neurosci. FD Society for Neuroscience SP 13054 OP 13066 DO 10.1523/JNEUROSCI.4330-06.2006 VO 26 IS 50 A1 Stefan Kalla A1 Michal Stern A1 Jayeeta Basu A1 Frederique Varoqueaux A1 Kerstin Reim A1 Christian Rosenmund A1 Noam E. Ziv A1 Nils Brose YR 2006 UL http://www.jneurosci.org/content/26/50/13054.abstract AB GFP (green fluorescent protein) fusion proteins have revolutionized research on protein dynamics at synapses. However, corresponding analyses usually involve protein expression methods that override endogenous regulatory mechanisms, and therefore cause overexpression and temporal or spatial misexpression of exogenous fusion proteins, which may seriously compromise the physiological validity of such experiments. These problems can be circumvented by using knock-in mutagenesis of the endogenous genomic locus to tag the protein of interest with a fluorescent protein. We generated knock-in mice expressing a fusion protein of the presynaptic active zone protein Munc13-1 and enhanced yellow fluorescent protein (EYFP) from the Munc13-1 locus. Munc13-1–EYFP-containing nerve cells and synapses are functionally identical to those of wild-type mice. However, their presynaptic active zones are distinctly fluorescent and readily amenable for imaging. We demonstrated the usefulness of these mice by studying the molecular dynamics of Munc13-1–EYFP at individual presynaptic sites. Fluorescence recovery after photobleaching (FRAP) experiments revealed that Munc13-1–EYFP is rapidly and continuously lost from and incorporated into active zones (τ1 ∼ 3 min; τ2 ∼ 80 min). 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. These experiments, performed in a minimally perturbed system, provide evidence that presynaptic active zones of mammalian CNS synapses are highly dynamic structures. They demonstrate the usefulness of the knock-in approach in general and of Munc13-1–EYFP knock-in mice in particular for imaging synaptic protein dynamics.