128 Investigating Cargo Adapter Function at Nematode Presynapses

Faculty Mentor: Erik Jorgensen (School of Biological Sciences, University of Utah)

Synaptic transmission happens at a very fast rate – nematode synapses fire at about 40 Hz, but some mammalian synapses fire at 500 Hz. Each firing is caused by the fusion of synaptic vesicles to the plasma membrane of the synapse and release of neurotransmitters. To support recurrent synaptic transmission, local regeneration of synaptic vesicles must occur compensatory to vesicle fusion. Vesicle regeneration is accomplished through endocytosis of membrane and recycling of proteins into new synaptic vesicles. Ultrafast endocytosis forms an endosome within 50 ms of synaptic vesicle fusion. We hypothesize that the endosome is resolved by adapter protein (AP) complexes and clathrin into new synaptic vesicles and recycling vesicles. Adapter protein complexes bind specific cargoes based on cargo recognition sequences: AP-1 binds t-SNARES, AP- 2 binds v-SNARES, AP-3 binds lysosome-destined cargoes. However, it is not known whether these complexes function at the synaptic endosome. Genetic knockout of AP complexes burdens the animal with developmental defects. To bypass developmental effects, we aim to test cargo sorting at the synaptic endosome by adding inducible degron tags to AP complex subunits from AP-1, AP-2 and AP-3, then degrade the proteins in a developed nervous system. We will assay for cargo sorting defects in the degron animals by fluorescence microscopy, and vesicle-formation defects by electron microscopy. Using CRISPR/Cas9, each Mu subunit was tagged at the C-terminus with a short DNA sequence which is recognized as a crossover region by a recombinase. Recombinase will then insert a sequence at this site. We will use recombinase to insert auxin-inducible degron fused to GFP to tag the protein. Once tagged, we will test whether SNARE proteins are mislocalized in AP-degron strains. We will also test whether the synaptic endosome becomes stuck” in the absence of AP complexes. To validate our assay, we assayed v-SNARE trafficking. Fluorescence microscopy shows punctate synaptotagmin::GFP in the wildtype, which corresponds to the synaptic vesicle cloud at synaptic boutons. In the AP-2 null, synaptotagmin::GFP was no longer localized to synaptic varicosities, but instead was diffuse throughout the process. This result confirms previous conclusions that AP-2 recruits and sorts synaptotagmin into synaptic vesicles after endocytosis. Thus, AP-2 is required to bring v-SNAREs into the synapse to be sorted. However, whether the endosome is able to resolve remains unclear.