Synaptic Ultrastructure and Regulation of Synaptic Transmission in Caenorhabditis elegans

The triggered release of synaptic vesicles (SVs) is tightly regulated in time and space by an elaborate network of proteins forming the active zone (AZ). When visualized by electron microscopy (EM), a morphological hallmark of the presynaptic AZ is an electron dense projection (DP) situated at the center of the AZ and extending into the cytoplasm. DPs are surrounded by a cluster of clear core SVs and have been proposed to play an important role in organizing and regulating SV release. However, our knowledge about its molecular composition, assembly mechanisms, and high-resolution 3D structure are limited. The neuromuscular junction (NMJ) of the nematode Caenorhabditis elegans provides an easily accessible model system to study AZ assembly, structure and function in regulated synaptic transmission. By using high-pressure-freeze (HPF) and freeze substitution (FS) EM in combination with tomography, I could show that C. elegans DPs are highly structured. DPs at cholinergic and GABAergic NMJs are branched, forming bay-like slots in which SV can usually be found docked to the AZ membrane. These bays thus may correspond to SV release sites. Large neuron-neuron synapse DPs appear to be polymers of the smaller NMJ DP unit with the same geometric arrangement of branch points and bays, indicating that DPs at functionally distinct synapses in C. elegans employ a common principle of structural assembly. To gain insights into the mechanisms that control synapse assembly, we analyzed the DP morphology in mutants lacking AZ proteins by HPF EM. Previous studies have proposed the C. elegans Liprin-α homolog synapse defective 2 (SYD-2) to be a key regulator in synapse formation by recruiting other synaptic proteins to nascent release sites. I demonstrate that SYD-2 together with a second AZ protein, ELKS-1, determines DP size in C. elegans NMJs. Whereas loss of SYD-2 results in significantly reduced DP size and inefficient vesicle recruitment, a gain-of-function (GF) mutation leads to the formation of elongated DPs. However, the ability of SYD-2 GF to promote DP elongation strictly depends on the presence of ELKS-1. A negative regulator of synapse formation, RSY-1, has recently been discovered to impair SYD-2 functionality. In this study I provide evidence indicating that RSY-1 acts as a counteracting negative regulator of DP size. I therefore implicate SYD-2/Liprin-α in the dynamic polymerization of DPs in cooperation with other regulators, rather than in the generation of DPs per se. To gain deeper insight into how axonal transport contributes to synapse assembly, I analyzed mutants with defective motor proteins known to be involved in synaptic protein transport. Kinesin-3 UNC-104 has been identified as a neuron-specific anterograde motor protein required for the transport of SVs and DCVs to synaptic sites. Loss of UNC-104 results in the accumulation of vesicles in the cell bodies. To understand if and how this transport defect affects AZ assembly, I analyzed C. elegans unc-104 mutants by confocal and electron microscopy. My initial analysis provides further evidence that the UNC-104 cargo binding domain is responsible for the attachment of SV precursors and DCVs to UNC-104. The formation of AZ DPs is, however, only moderately affected in unc-104 mutants, implying that organelles associated with AZ components may utilize alternative transport mechanisms. For the first time, I report the formation of ectopic, but functional, synapses in unc-104 mutant cell bodies. Furthermore, the vesicles accumulating in the cell bodies were characterizes as DCV-like vesicles distinct from synaptic DCVs in wild type. We suggest that these DCV-like vesicles are SV precursors that transport SV proteins to AZs, where mature SVs can then be assembled by local endocytosis.