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Liprin-α1/SYD-2 Determines Size of Electron-dense Projections in Presynaptic Active Zones in C. elegans


Liprin-α1Figure Caption: 3D structure of NMJ synapses by serial reconstruction of 40-nm-thin HPF EM sections and EM tomography. (A) Six consecutive 40-nm sections (1–6) of a presynaptic cholinergic active zone with a schematic drawing of the electron dense projection and surrounding synaptic vesicles below each section highlighting the tethers projecting from the electron dense projection to contact docked synaptic vesicles. (B) 3D reconstruction from serial sections of a wild-type cholinergic neuromuscular junction synapse. The contacting postsynaptic muscle arm is shown in gray. (C) 3D model of the active zone reconstructed from serial sections 1–6. (D) 3D model of the electron dense projection only displayed as trace slabs to highlight the branch point. (E) Volume segmentation of a tomogram from a 250-nm-thick high pressure frozen/freeze substitution plastic section showing a cholinergic neuromuscular junction. Higher magnifications of the electron dense projection and close surrounding structures are shown below. Color codes are axon membrane (gray), the dense projection with emanating tethers (red), synaptic vesicles (blue), dense core vesicle (black), endoplasmic reticulum (orange), microtubules (yellow), and mitochondria (green).

January, 2014 La Jolla -- Fast synaptic neurotransmission relies on triggered release of neurotransmitters from synaptic vesicles after fusion with the plasma membrane. The release of synaptic vesicles is a highly regulated process of sequential events: First synaptic vesicles are recruited to the presynaptic active zone, then docked to the plasma membrane in a release-competent state, which guarantees their rapid release after the influx of calcium into the presynaptic terminal.

A network of proteins that form the presynaptic active zones spatially and temporally regulates the release of synaptic vesicles. Most active zones manifest as electron-dense projections (DPs) surrounded by synaptic vesicles. Despite the importance of DPs to triggering synaptic vesicle release, there have been few high-resolution analyses of DP structures.

The absence of this kind of data caused an international research team to apply electron microscopy (EM) at the National Center for Microscopy and Imaging Research at UCSD to the structures. Their work revealed the existence of the electron-dense protein matrix, the DP, at the center of active zones. Furthermore, their work showed that DPs at C. elegans neuromuscular junctions (NMJs) were highly structured, composed of regular building units forming bays in which synaptic vesicles are docked to the active zones membrane. Furthermore, larger ribbon-like DPs -- multimers of the basic building unit -- were found at synapses between inter- and motoneurons. More specifically, the ultrastructure of active zones DPs was resolved using high-pressure freezing cryofixation and serial EM reconstruction and EM tomography. A subsequent analysis revealed a previously unknown basic ultrastructural unit for DPs.

While the precise 3D ultrastructure of these DPs and their building principle remain to be determined, the team reported that DPs in C. elegans cholinergic and GABAergic motoneurons follow a previously unknown building principle, with the smallest 3D ultrastructural unit resembling a three-pointed triadic structure. From a central branch point, three short branches extend planar along the presynaptic plasma membrane. Adjacent branches form bay-like structures in which synaptic vesicles are frequently found in contact with the subjacent plasma membrane.

As a result, NCMIR scientists and collaborators believes that these bays to be putative release sites within the active zones. They also demonstrated that DP size is determined by the activity of the active zones protein SYD-2/Liprin-α1. Whereas the loss of SYD-2 function led to smaller DPs, SYD-2 gain-of-function mutants displayed larger ribbon-like DPs through increased recruitment of ELKS-1/ELKS. Therefore, the study’s data suggest that a main role of SYD-2/Liprin-α1 in synaptogenesis is to regulate the polymerization of DPs.

The team wondered: Is the regulation of DP size by SYD-2 a form of synaptic plasticity? Their work showed that even DPs at NMJ active zones, which normally contain small DPs, can be elongated by increasing SYD-2 activity. This suggests that all active zones have the ability to form longer DPs dependent on SYD-2 activity.

This international team of scientists also provided evidence that SYD-2 activity levels control formation of fine structures essential for proper recruitment o f synaptic vesicles to the active zones membrane, indicating that SYD-2 plays a role in DP higher-order assembly and/or stabilization. This study might serve as a first step toward unifying the molecular roles of SYD-2/Liprin-α1 as a scaffold required to assemble a molecular network controlling the productive polymerization of elongated structures in different systems and cell types. In that case, Liprin-α1 activity would be required to shift a dynamic equilibrium to assemble and recruit scaffold components needed for polymerization. Thus, the activity status of SYD-2/Liprin-α1 might be used to transiently recruit scaffolds such that low-affinity interactions are stabilized to be productive.

This study was performed by researchers from the European Neuroscience Institute, the Schwann-Schleiden-Centre for Molecular Cell Biology, Center for Molecular Physiology of the Brain (all of Göttingen, Germany), Hannover Medical School (Germany), UCSD, Hokkaido University (Japan), the University of Illinois at Chicago, and the University of Freiburg (Germany).

Citation: Maike Kittelmann, Jan Hegermann, Alexandr Goncharov, Hidenori Taru, Mark H. Ellisman, Janet E. Richmond, Yishi Jin, and Stefan Eimer, Liprin-α1/SYD-2 Determines the Size of Dense Projections in Presynaptic Active Zones in C. elegans, The Journal of Cell Biology, 2013, 203:849-863.

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