Stavoe, Andrea K et al. (2011) International Worm Meeting "Netrin instructs presynaptic assembly through a synaptic MIG-10 isoform and the actin cytoskeleton."

Colon-Ramos, Daniel A, Nelson, Jessica C, Stavoe, Andrea K

[International Worm Meeting, 2011]

The development of the nervous system is orchestrated with a limited number of molecular cues. For example, molecules originally identified for their roles in axon guidance are now known to play important roles during synapse formation. While examples of guidance molecules controlling synaptogenesis abound, we understand less about how these molecules instruct different neurodevelopmental outcomes in a cell-specific manner. We have previously demonstrated that Netrin, originally identified as guidance molecule, instructs presynaptic assembly in the nerve ring interneuron AIY. The Netrin receptor UNC-40 is cell-autonomously necessary for presynaptic assembly in AIY. Here we identify the mechanisms by which the axon guidance receptor UNC-40 instructs presynaptic assembly in vivo. Using genetic analysis and cell biological experiments, we find that UNC-40 is required at presynaptic sites for organizing the actin cytoskeleton through the Rac GTPase pathway, specifically CED-5/DOCK180 and CED-10/Rac1. Null or strong loss-of-function mutants in these pathway components exhibit similar presynaptic vesicle clustering phenotypes as UNC-40 in the interneuron AIY. Downstream of CED-10, we find that MIG-10/Lamellipodin is cell-autonomously required for proper presynaptic vesicle clustering in AIY. We also identify a unique MIG-10 isoform, MIG-10B, which is specifically required to regulate vesicle clustering in response to Netrin. This isoform localizes to AIY synaptic regions, as determined by MIG-10B:GFP fusion proteins. MIG-10B is required for organization of the actin cytoskeleton at these sites, as determined by protein-GFP fusions and recombineering fosmid rescue experiments. Other MIG-10 isoforms do not localize to synaptic regions, nor do they rescue the presynaptic assembly phenotype in mig-10 null mutants. Thus, our data provide a novel mechanism for presynaptic targeting in vivo. Our data also indicate that signaling modules that organize the actin cytoskeleton during guidance can be co-opted at a cell biological level to organize the actin cytoskeleton in presynaptic assembly.

Hill, Sarah et al. (2015) International Worm Meeting "Autophagy is required for Axon Outgrowth and the Specification of Presynaptic Sites during Neurodevelopment."

Colon-Ramos, Daniel, Hill, Sarah, Stavoe, Andrea

[International Worm Meeting, 2015]

Autophagy is a highly conserved multi-step pathway used by cells to degrade substrates. Autophagy is important for maintaining homeostasis in neurons, and disruption of autophagy contributes to neuronal diseases. However, the role of autophagy in neurodevelopment is not well understood. Through a forward genetic screen in C. elegans, we identified the autophagy gene, atg-9, as required for specifying presynaptic sites. Atg-9 mutant animals have disrupted synaptic vesicle clusters at presynaptic sites in the interneuron AIY and elongated axons in the sensory neuron PVD. We determined that each step of autophagosome biogenesis: initiation, nucleation, elongation, and recycling, is required for these two distinct phenotypes. Presynaptic assembly and axon outgrowth both rely on appropriate actin organization. Cell-specific disruption of F-actin phenocopied autophagy mutants. Additionally, we observed disordered F-actin localization in autophagy mutants. Our results demonstrate that autophagy is required cell-autonomously, during development, and likely acts through actin to control precise neurodevelopment.

Yang, S. et al. (2017) International Worm Meeting "Molecular mechanisms of ATG-9 localization in neurons."

Yang, S., Colon-Ramos, D., Hill, S.

[International Worm Meeting, 2017]

Autophagy is an evolutionarily conserved cellular degradative process in which cytosolic components are engulfed by double membrane structures known as autophagosomes. ATG-9 is a transmembrane protein, which is thought to be important for delivering lipids from membrane compartments for autophagosome formation. Previous work from our lab demonstrated that ATG-9 localizes to presynaptic regions and is associated with autophagosome biogenesis at the synapse (Stavoe and Hill et al., 2016). How ATG-9 localizes at presynaptic regions and traffics between membranes to regulate autophagy is not well understood. We have established an in vivo system to visualize ATG-9 in a pair of Caenorhabditis elegans interneurons called AIY. In wild type animals, ATG-9 diffusely distributes at presynaptic regions. We performed unbiased forward genetic screens and candidate screens to identify molecules required for ATG-9 localization in AIY. We identified two novel alleles which display highly penetrant, abnormal distribution of ATG-9 at presynaptic regions. We now plan to use these alleles to determine the molecular mechanism governing ATG-9 localization at the synapse to better understand the role of autophagy in neurons.

Hill, Sarah et al. (2017) International Worm Meeting "Autophagosome biogenesis at the synapse."

Hill, Sarah, Colon-Ramos, Daniel

[International Worm Meeting, 2017]

Autophagy removes bulk cytoplasm or organelles from cells by forming double-membrane autophagosomes that fuse to lysosomes for degradation. Regulation of autophagosome formation and trafficking is especially important in neurons, which have long polarized processes distant from the cell body. We and others have observed that autophagosomes form at presynaptic sites and then undergo retrograde transport towards the cell body, which contains the lysosomes (Stavoe and Hill et al , 2016). However, the function of autophagy and regulation of autophagosome transport at the synapse is not completely understood. We used neurons from the C. elegans thermotaxis circuit as a model to study autophagy at the synapse. This system allows us to visualize autophagosome biogenesis and trafficking in vivo, control temperature-dependent synaptic firing, and simultaneously monitor potential autophagic regulators and targets at single-cell resolution. Using this system, we found that loss of synaptic activity results in decreased autophagosomes at the synapse and that JIP3/UNC-16, an adaptor protein known to regulate early endosome and lysosome transport, also plays an important role in regulating autophagosome transport in C. elegans neurons.