Within a nerve terminal, synaptic vesicles exclusively fuse at the active zone. The active zone consists of a protein scaffold that is anchored to the plasma membrane and forms release sites precisely opposed to postsynaptic receptors. Interactions between active zone proteins and Ca2+ channels have long been of central interest. Ca2+ influx through channels of the CaV2 family triggers release, and their exact positioning supports the sub-millisecond timing of synaptic transmission and determines synaptic strength. There are two competing models for roles and mechanisms of Ca2+ channels in synapse and active zone assembly. First, Ca2+ channels may be essential for synapse structure. Second, the active zone may recruit Ca2+ channels to release sites, implying that synapse structure is CaV2 independent. It has been difficult to distinguish between these models because the complexity of the Ca2+ channel gene family and their auxiliary subunits leads to extensive redundancy. Furthermore, precisely localizing Ca2+ channels has been challenging. We have overcome these hurdles by generating conditional triple knockout mice to remove all pore-forming a1 subunits of CaV2 channels, and by adapting superresolution microscopy to assess Ca2+ channel localization. Our data confirm that Ca2+ flux through these channels is essential for release triggering. Based on our preliminary data, we hypothesize that active zone assembly is independent of CaV2 channels, but instead the active zone targets CaV2 channels with nanoscale precision to release sites. Our experimental plan tests this hypothesis from three independent angles and dissects underlying mechanisms.
In aim 1, we assess the competing models by removing the pore forming a1 subunits, followed by assessment of synapse and active zone structure and function. We then propose rescue experiments to assess which sequences of CaV2 channels are required for their targeting, and we test which CaV2 sequences are sufficient to confer active zone targeting onto non-CaV2 channels.
In aim 2, we determine the precise presynaptic localization of auxiliary subunits and assess whether their presynaptic targeting depends on a1. We then test whether functional roles of these auxiliary subunits require the presence of a1.
In aim 3, we address molecular mechanisms for CaV2 targeting from the perspective of active zone scaffolds. We first determine the order of arrival of active zone and CaV2 proteins during active zone assembly, and we then determine localization and function of CaV2s and their subunits in mutants that lack specific active zone proteins. This grant will test two fundamentally different models of the relationship between Ca2+ channels and the active zone, and dissects the mechanisms that underlie Ca2+ channel anchoring at the target membrane. Precise understanding of these mechanisms is important for understanding synapses in health and disease.
Pathological alterations in neuronal signal transmission are a hallmark of many neurological disorders, including neurodegeneration, autism, schizophrenia and addiction. In the present project, we dissect molecular mechanisms that underlie the release of chemical signals from neurons. Our results will provide detailed molecular insight into neuronal communication, and will provide a framework to study how such communication fails in brain disorders.
| Wong, Man Yan; Liu, Changliang; Wang, Shan Shan H et al. (2018) Liprin-?3 controls vesicle docking and exocytosis at the active zone of hippocampal synapses. Proc Natl Acad Sci U S A 115:2234-2239 |
| de Jong, Arthur P H; Roggero, Carlos M; Ho, Meng-Ru et al. (2018) RIM C2B Domains Target Presynaptic Active Zone Functions to PIP2-Containing Membranes. Neuron 98:335-349.e7 |
| Liu, Changliang; Kershberg, Lauren; Wang, Jiexin et al. (2018) Dopamine Secretion Is Mediated by Sparse Active Zone-like Release Sites. Cell 172:706-718.e15 |
| Held, Richard G; Kaeser, Pascal S (2018) ELKS active zone proteins as multitasking scaffolds for secretion. Open Biol 8: |
| Wang, Shan Shan H; Kaeser, Pascal S (2018) A Presynaptic Liquid Phase Unlocks the Vesicle Cluster. Trends Neurosci 41:772-774 |
| Biederer, Thomas; Kaeser, Pascal S; Blanpied, Thomas A (2017) Transcellular Nanoalignment of Synaptic Function. Neuron 96:680-696 |
| Kawabe, Hiroshi; Mitkovski, Miso; Kaeser, Pascal S et al. (2017) ELKS1 localizes the synaptic vesicle priming protein bMunc13-2 to a specific subset of active zones. J Cell Biol 216:1143-1161 |
| Kaeser, Pascal S; Regehr, Wade G (2017) The readily releasable pool of synaptic vesicles. Curr Opin Neurobiol 43:63-70 |
| Wang, Yu; Woehrstein, Johannes B; Donoghue, Noah et al. (2017) Rapid Sequential in Situ Multiplexing with DNA Exchange Imaging in Neuronal Cells and Tissues. Nano Lett 17:6131-6139 |
| Held, Richard G; Liu, Changliang; Kaeser, Pascal S (2016) ELKS controls the pool of readily releasable vesicles at excitatory synapses through its N-terminal coiled-coil domains. Elife 5: |
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