Molecular Mechanisms for Co‐Assembly of Endocytic and Exocytic Machineries at a Synapse - Project summary At presynaptic nerve terminals, the precise nano-scale positioning of two protein machineries, the active zone and the endocytic apparatus, is critical for high fidelity synaptic transmission. The active zone, which generates release sites for synaptic vesicles, is aligned to postsynaptic receptors to ensure efficient signal transmission. The endocytic apparatus, which restores vesicles after release, is assembled adjacent to active zones. Most components of these machineries are associated with brain disorders, underlining their importance for brain function. This grant generates fundamental insight into the molecular mechanisms that position the endocytic apparatus next to the active zone at vertebrate synapses. The presynaptic scaffold Liprin-α is an ideal candidate to spatially organize these two machineries for three reasons. First, the broad synaptic interactome of Liprin-α and the generalized phenotypes caused by ablating the single invertebrate gene predict that it organizes multiple presynaptic compartments. Second, my previous work and preliminary data show that deletion of a subset of the four vertebrate Liprin-α proteins already causes mislocalization of active zone and endocytic components, which suggests co-organizing roles for vertebrate Liprin-α. Finally, Liprin-α undergoes phase separation, which is a potential synaptic organizing mechanism, and my ongoing work establishes that both active zone and endocytic proteins are recruited to Liprin-α phase condensates. Therefore, I hypothesize that Liprin-α positions the endocytic apparatus next to active zones at vertebrate synapses. I will use newly-generated Liprin-α quadruple mutants, in which all four Liprin-α genes can be deleted, to test this hypothesis. Aim 1 (training) will dissect roles for Liprin-α in active zone organization by comparing the nano- scale positioning of active zone proteins and active zone function after Liprin-α quadruple ablation in excitatory and inhibitory synapses in cultured neurons and at the Calyx of Held. Aim 2 (training) will determine roles for Liprin-α in organizing the endocytic apparatus by using the same approaches. Both aims will be complemented with experiments in cell lines to assess the ability of Liprin-α to recruit endocytic and active zone proteins. I will also do structure-function experiments to dissect the relevance of the protein sequences that mediate Liprin-α phase separation in presynaptic assembly. These aims will establish the roles of Liprin-α in the organization of active zones and endocytic assemblies, and will provide an essential training experience in the studies of the endocytic machinery and in the assessment of their in vivo organization using super-resolution microscopy. Aim 3 (independent) will test the complementary model that the endocytic apparatus mediates the spatial organization of these machineries. In particular, I will dissect structural roles for Dynamin, which is fundamental for synaptic endocytosis, and also undergoes phase separation. I will use Dynamin triple mutants and build upon the methodology implemented during (training) aims 1 & 2 to determine these roles.
