UNC-16 (JIP3) Acts Through Synapse-Assembly Proteins to Inhibit the Active Transport of Cell Soma Organelles to Caenorhabditis elegans Motor Neuron Axons

Abstract

The conserved protein UNC-16 (JIP3) inhibits the active transport of some cell soma organelles, such as lysosomes, early endosomes, and Golgi, to the synaptic region of axons. However, little is known about UNC-16's organelle transport regulatory function, which is distinct from its Kinesin-1 adaptor function. We used an unc-16 suppressor screen in Caenorhabditis elegans to discover that UNC-16 acts through CDK-5 (Cdk5) and two conserved synapse assembly proteins: SAD-1 (SAD-A Kinase), and SYD-2 (Liprin-a). Genetic analysis of all combinations of double and triple mutants in unc-16(+) and unc-16(2) backgrounds showed that the three proteins (CDK-5, SAD-1, and SYD-2) are all part of the same organelle transport regulatory system, which we named the CSS system based on its founder proteins. Further genetic analysis revealed roles for SYD-1 (another synapse assembly protein) and STRADa (a SAD-1-interacting protein) in the CSS system. In an unc-16(2) background, loss of the CSS system improved the sluggish locomotion of unc-16 mutants, inhibited axonal lysosome accumulation, and led to the dynein-dependent accumulation of lysosomes in dendrites. Time-lapse imaging of lysosomes in CSS system mutants in unc-16(+) and unc-16(2) backgrounds revealed active transport defects consistent with the steady-state distributions of lysosomes. UNC-16 also uses the CSS system to regulate the distribution of early endosomes in neurons and, to a lesser extent, Golgi. The data reveal a new and unprecedented role for synapse assembly proteins, acting as part of the newly defined CSS system, in mediating UNC-16's organelle transport regulatory function.KEYWORDS Caenorhabditis elegans; axonal transport; JIP3; Cdk5; Liprin; SAD-A; dynein N EURONS have a unique architecture consisting of a cell soma, one or more dendrites that receive information, and a single axon, which can be a single process or intricately branched. Part of the axon is specialized to form synapses, which integrate and transmit information to other neurons or muscle cells via synaptic vesicles and dense core vesicles. This unique architecture and cell biology places extraordinary demands on the membrane-trafficking and transport machinery. In addition to transporting synaptic vesicles and dense core vesicles long distances into axons, motor neurons must also restrict, or even prevent, the flow of some organelles, including Golgi, lysosomes, and endosomes, into the synaptic region of their axons, which, under normal conditions, are relatively devoid of these organelles compared to cell somas. However, there may be special conditions, such as the need for axon repair or growth, where neurons require cell soma organelles in their axons, so the regulatory system for organelle transport must include components that inhibit, as well as promote, axonal transport. The association of mutations in the axonal transport machinery with neurodegenerative disorders in mice and humans underscores the importance of a properly functioning transport system for the long-term viab...

Synapse-Assembly Proteins Maintain Synaptic Vesicle Cluster Stability and Regulate Synaptic Vesicle Transport in Caenorhabditis elegans

“…In the accompanying study (Edwards et al 2015), our analysis of cell soma organelle trafficking in the same motor neurons supports a general organelle transport function for SAD-1, SYD-1, SYD-2, and CDK-5, as well as STRD-1 (STRADa), a protein shown to directly interact with SAD-1 (Kim et al 2010). The demonstration that sad-1 and syd-2 null mutants have wild-type rates of coordinated locomotion (Edwards et al 2015) also suggests that the axons and dendrites in the cholinergic motor neurons that mediate this locomotion maintain their proper identity despite the transport defect. Despite the lack of evidence for an axon-identity defect in the synapse-assembly mutants in C. elegans cholinergic motor neurons, prior studies have demonstrated a clear role for SAD-1's mammalian orthologs SAD-A and SAD-B in specifying axon identity in forebrain neurons (Kishi et al 2005), where SAD-A/B act in a pathway downstream of the LKB1 kinase (Barnes et al 2007).…”

“…Previous studies have found that CDK-5 and the synapse-assembly protein SYD-2 inhibit the dynein-mediated dendritic accumulation of DCVs (Goodwin et al 2012;Goodwin and Juo 2013). CDK-5 and SYD-2 also act together in the same system to inhibit the dendritic accumulation of lysosomes in mutants lacking UNC-16 (Edwards et al 2015). To determine if CDK-5 and/or SYD-2 inhibit DCVs from accumulating in dendrites in unc-104(ce782) mutants, we quantified dendritic and cell soma DCV densities in cdk-5 and syd-2 single mutants and the corresponding doubles with unc-104(ce782).…”

“…We isolated sad-1(ce749) and syd-2(ce759) in an unrelated forward genetic screen (Edwards et al 2015). We isolated unc-104(ce515) in an unrelated forward genetic screen (K. G.…”

“…Edwards et al (2015) describes the mapping and whole-genome sequencing methods that we used to identify sad-1(ce749) and syd-2(ce759). We outcrossed the strains bearing the ce515, ce749, ce759, ce782, and ok217 mutations one, two, two, two, and three times to N2, respectively, before using them for the experiments in this article.…”

“…The mutant also contributed to our discovery that multiple synapse-assembly proteins, and not just SYD-2, have dual roles in maintaining synaptic vesicle cluster stability and in regulating SV transport. An accompanying article shows that these same synapse-assembly proteins have an even broader role in regulating the active transport of cell soma organelles in neurons (Edwards et al 2015). …”