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The Journal of Neuroscience, December 13, 2006, 26(50):13054-13066; doi:10.1523/JNEUROSCI.4330-06.2006

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Cellular/Molecular
Molecular Dynamics of a Presynaptic Active Zone Protein Studied in Munc13-1–Enhanced Yellow Fluorescent Protein Knock-In Mutant Mice

Stefan Kalla,¹ Michal Stern,³ Jayeeta Basu,⁴ Frederique Varoqueaux,¹ Kerstin Reim,¹ Christian Rosenmund,⁴ Noam E. Ziv,³ and Nils Brose^1,2

¹Department of Molecular Neurobiology, ²Deutsche Forschungsgemeinschaft Research Center for Molecular Physiology of the Brain, Max-Planck-Institute of Experimental Medicine, D-37075 Göttingen, Germany, ³Department of Physiology and Rappaport Family Institute for Research in the Medical Sciences, Technion Faculty of Medicine, Haifa 31096, Israel, and ⁴Department of Molecular and Human Genetics and Department of Neuroscience, Baylor College of Medicine, Houston, Texas 77030

Correspondence should be addressed to either of the following: Nils Brose, Department of Molecular Neurobiology, Max-Planck-Institute of Experimental Medicine, Hermann-Rein-Strasse 3, D-37075 Göttingen, Germany, Email: brose{at}em.mpg.de; or Noam Ziv, Department of Physiology and Rappaport Family Institute for Research in the Medical Sciences, Technion Faculty of Medicine, P.O. Box 9649 Bat Galim, Haifa 31096, Israel, Email: noamz{at}netvision.net.il

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 (₁ 3 min; ₂ 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.

Key words: CAZ; live imaging; priming; synapse; protein turnover; protein degradation

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Received Oct. 4, 2006; revised Nov. 9, 2006; accepted Nov. 9, 2006.

Correspondence should be addressed to either of the following: Nils Brose, Department of Molecular Neurobiology, Max-Planck-Institute of Experimental Medicine, Hermann-Rein-Strasse 3, D-37075 Göttingen, Germany, Email: brose{at}em.mpg.de; or Noam Ziv, Department of Physiology and Rappaport Family Institute for Research in the Medical Sciences, Technion Faculty of Medicine, P.O. Box 9649 Bat Galim, Haifa 31096, Israel, Email: noamz{at}netvision.net.il

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