Faculty Bibliography

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 (tau1 approximately 3 min; tau2 approximately 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.

Presynaptic vesicle trafficking and priming are important steps in regulating synaptic transmission and plasticity. The four closely related small GTP-binding proteins Rab3A, Rab3B, Rab3C, and Rab3D are believed to be important for these steps. In mice, the complete absence of all Rab3s leads to perinatal lethality accompanied by a 30% reduction of probability of Ca2+-triggered synaptic release. This study examines the role of Rab3 during Ca2+-triggered release in more detail and identifies its impact on short-term plasticity. Using patch-clamp electrophysiology of autaptic neuronal cultures from Rab3-deficient mouse hippocampus, we show that excitatory Rab3-deficient neurons display unique time- and frequency-dependent short-term plasticity characteristics in response to spike trains. Analysis of vesicle release and repriming kinetics as well as Ca2+ sensitivity of release indicate that Rab3 acts on a subset of primed, fusion competent vesicles. They lower the amount of Ca2+ required for action potential-triggered release, which leads to a boosting of release probability, but their action also introduces a significant delay in the supply of these modified vesicles. As a result, Rab3-induced modifications to primed vesicles causes a transient increase in the transduction efficacy of synaptic action potential trains and optimizes the encoding of synaptic information at an intermediate spike frequency range.

Munc13 proteins are essential in neurotransmitter release, controlling the priming of synaptic vesicles to a release-ready state. The sequences responsible for this priming activity are unknown. Here we identify a large alpha-helical domain of mammalian Munc13-1 that is autonomously folded and is sufficient to rescue the total arrest in neurotransmitter release observed in hippocampal neurons lacking Munc13s.