Merrill, Sean et al. (2013) International Worm Meeting "Locating synaptic calcium channels."

Watanabe, S., Jorgensen, E.M., Richards, J.R., Merrill, Sean, Frokjaer-Jensen, C.

[International Worm Meeting, 2013]

Calcium is the essential trigger for synaptic vesicle fusion. When open, calcium channels create steep calcium gradients that fuse nearby vesicles while sparing distal vesicles. The extent and duration of the gradients depends on the type and location of activated calcium channels. Similarly, vesicles must be primed and positioned to respond to the calcium influx. UNC-13 is required for priming vesicles in presynaptic active zones, while RIM (UNC-10) delivers primed vesicles specifically to dense projections where they can most rapidly respond to calcium. Mammalian calcium channels can be divided into four major classes, encoded by at least 10 genes and thousands of unique isoforms. C. elegans has just three calcium channels at the presynaptic neuromuscular junction: N-type (UNC-2), L-type (EGL-19), and the ryanodine receptor (UNC-68). Determining calcium channel organization has been difficult because of the small size of synapses. Nano-fEM is a new tool developed in our lab that correlates super-resolution fluorescence microscopy with scanning electron microscopy to localize proteins at nanometer resolution. We have transgenically tagged each channel with tdEos, a photoconvertible fluorophore used for fEM. We are determining the precise location of these calcium channels within neuromuscular synapses to build a model of their respective synaptic functions. Finally, we will investigate the effect of mutations in unc-13 and unc-10 on the location of both synaptic vesicles and their adjoining calcium channels.

Merrill, Sean et al. (2015) International Worm Meeting "Locating synaptic calcium channels."

Cherry, Alex, Merrill, Sean, Jorgensen, Erik, Watanabe, Shigeki

[International Worm Meeting, 2015]

Neurotransmission occurs when calcium triggers exocytosis of synaptic vesicles primed at release sites. The number, position and activity of nearby calcium channels determine the perdurance of free calcium at a release site. To understand the synapse we must identify the location of calcium channels in relation to synaptic vesicles, fusion proteins, and subcellular structures such as the dense projection. In the simplest model, calcium enters through a single source at the center of the synapse. However, calcium entry through multiple sources within a synapse has been studied only indirectly. In C. elegans unc-68 (RyR), egl-19 (L-type), and unc-2 (N-type) channels are each encoded by a single gene and contribute the calcium for synaptic vesicle exocytosis at neuromuscular junctions. We are determining the colocalization of calcium channels with other synaptic proteins at nanometer resolution. CRISPr and miniMos create single-copy transgenes that attach to each channel an enzymatic tag that covalently binds organic fluorophores suitable for imaging by biplane 3D super-resolution fluorescence microscopy. Transgenic animals are crossed with mutations that test the contributions of the vesicle priming proteins unc-13, unc-10 (RIM), and rimb-1 (RIM-binding protein) in localizing each synaptic calcium channel and its adjoining vesicles.

Chang JT et al. (2020) Methods Mol Biol "Correction to: Assessing Tissue-Specific Autophagy Flux in Adult Caenorhabditis elegans."

Chang JT, Kumsta C, Hansen M

[Methods Mol Biol, 2020]

Correction to: Chapter 17 in: Sean P. Curran (ed.), Aging: Methods and Protocols, Methods in Molecular Biology, vol. 2144.

Eddy SR (1994) Worm Breeder's Gazette "Three Families of Reverse Transcriptase Elements in C. elegans"

Eddy SR

[Worm Breeder's Gazette, 1994]

Three families of reverse transcriptase elements in C. elegans Sean Eddy, MRC-LMB, Hills Road, Cambridge CB2 2QH, UK

Hujber, Edward J. et al. (2015) International Worm Meeting "Three-dimensional super-CLEM."

Hobson, Robert J., Merrill, Sean A., Jorgensen, Erik M., Hujber, Edward J.

[International Worm Meeting, 2015]

Correlative light and electron microscopy (CLEM) can reveal a protein's localization relative to cellular features on the nano-scale. We have made several improvements to our protocol to improve imaging resolution in all three dimensions. Among the most important changes has been using organic dyes as fluorophores, instead of fluorescent proteins. This allows us to localize tagged proteins to high resolution in three dimensions using a biplane imaging approach. Optimizing embedding resins, fixatives, and staining protocols has enabled use of transmission electron tomography instead of scanning electron microscopy, providing us with sub-nanometer cellular context in three dimensions. Using a fiducial-based alignment, we have correlated proteins with cellular structures at ~30 nm lateral and ~50 nm axial resolution.

Rich, Matthew S. et al. (2021) International Worm Meeting "unc-44 (Ankyrin) is required for axon stability in C. elegans"

Hsu, Sharlei, Labella, Matthew L., Rich, Matthew S., Bastiani, Michael J., Merrill, Sean A., Jorgense, Erik M.

[International Worm Meeting, 2021]

After an axon reaches and forms synapses with its target, it must become stable and persist potentially for the life of the organism. It is becoming clear that regulation of the axonal cytoskeleton is necessary for this process; mutations in known cytoskeletal components like unc-44 (ankyrin) cause highly disordered nervous systems in C. elegans. Axons in unc-44(-) animals branch, sprout ectopic growth cones, and exhibit severe degeneration. Historically, unc-44 has been categorized as being required for axon outgrowth. Using longitudinal and time-lapse imaging during larval development, we show that UNC-44 is not required for proper outgrowth in GABA neurons. In unc-44(-) animals these axons extend normally toward their targets, but continue to sprout growth cones. These defects increase in frequency as the animal ages, implying a defect in maturation. We recently showed that the expression of a long, 6,994aa isoform of UNC-44 is required for axon stability, but it is still unclear how this protein stabilizes the axon. Using CRISPR, we have shown that only the C-terminal ~850aa are required for UNC-44's neuron maturation function; deleting this region phenocopies unc-44 null mutations. This region contains three domains conserved in nematodes. Based on further truncations it appears that these act combinatorially; deleting individual conserved domains still yield nulls. We are currently mutagenizing this region further and using protein biochemistry to precisely map the sequences in UNC-44's C-terminus and the binding partners that are required for neuron maturation.

Zhang H et al. (2008) Mol Biol Cell "RAB-11 permissively regulates spindle alignment by modulating metaphase microtubule dynamics in Caenorhabditis elegans early embryos."

Zhang H, White JG, Squirrell JM

[Mol Biol Cell, 2008]

Monitoring Editor: Sean Munro Alignment of the mitotic spindle along a pre-formed axis of polarity is crucial for generating cell diversity in many organisms (Segal and Bloom, 2001; Betschinger and Knoblich, 2004), yet little is known about the role of the endomembrane system in this process. RAB-11 is a small GTPase enriched in recycling endosomes. When we depleted RAB-11 by RNAi in C. elegans, the spindle of the one-cell embryo failed to align along the axis of polarity in metaphase and underwent violent movements in anaphase. The distance between astral microtubules ends and the anterior cortex was significantly increased in rab-11(RNAi) embryos specifically during metaphase, possibly accounting for the observed spindle alignment defects. Additionally, we found that normal ER morphology requires functional RAB-11, particularly during metaphase. We hypothesize that RAB-11, in conjunction with the ER, acts to regulate cell cycle specific changes in astral microtubule length to ensure proper spindle alignment in C. elegans early embryos.

Prasad BC et al. (1997) International C. elegans Meeting "THE C. ELEGANS unc-3 GENE ENCODES A HOMOLOGUE OF THE O/E FAMILY OF MAMMALIAN TRANSCRIPTION FACTORS"

Prasad BC, Reed RR, Seydoux GC

[International C. elegans Meeting, 1997]

The expression of specialized signal transduction components in mammalian olfactory neurons is thought to be regulated by the O/E (Olf-1/EBF) family of transcription factors. The O/E proteins are expressed in cells of the olfactory neuronal lineage throughout development, and are also expressed transiently in neurons in the developing nervous system during embryogenesis. We have identified a C. elegans homologue of the mammalian O/E proteins, which displays greater than 80% similarity over 350 amino acids. The CeO/E cDNA maps to cosmid F42D1, previously shown to encode unc-3(1). We demonstrate that CeO/E is the product of the unc-3 gene, mutations in which cause defects in the axonal outgrowth of motor neurons as well as defects in dauer formation, a process requiring chemosensory input. Like its mammalian homologues, CeO/E is expressed in certain chemosensory neurons (ASI amphid neurons) throughout development, and is also expressed transiently in developing motor neurons when these cells undergo axonal outgrowth. Currently, we are defining the DNA sequence binding specificity of the CeO/E protein. These observations suggest that the O/E family of transcription factors play a central and evolutionarily conserved role in the expression of proteins essential for axonal pathfinding and neuronal differentiation. (1) Sean Eddy, WBG 12(5):86 (February 1, 1993)

McEwen JM et al. (2008) Mol Biol Cell "UNC-18 promotes both the anterograde trafficking and synaptic function of syntaxin."

Kaplan JM, McEwen JM

[Mol Biol Cell, 2008]

Monitoring Editor: Sean Munro The SM protein UNC-18 has been proposed to regulate several aspects of secretion, including synaptic vesicle (SV) docking, priming, and fusion. Here we show that UNC-18 has a chaperone function in neurons, promoting anterograde transport of the plasma membrane SNARE protein Syntaxin-1. In unc-18 mutants, UNC-64 (C. elegans Syntaxin-1) accumulates in neuronal cell bodies. Colocalization studies and analysis of carbohydrate modifications both suggest that this accumulation occurs in the endoplasmic reticulum. This trafficking defect is specific for UNC-64 Syntaxin-1, as 14 other SNARE proteins and 2 active zone markers were unaffected. UNC-18 binds to Syntaxin through at least two mechanisms: binding to closed Syntaxin, or to the N-terminus of Syntaxin. It is unclear which of these binding modes mediates UNC-18 function in neurons. The chaperone function of UNC-18 was eliminated in double mutants predicted to disrupt both modes of Syntaxin binding, but was unaffected in single mutants. By contrast, mutations predicted to disrupt UNC-18 binding to the N-terminus of Syntaxin caused significant defects in locomotion behavior and responsiveness to cholinesterase inhibitors. Collectively, these results demonstrate the UNC-18 acts as a molecular chaperone for Syntaxin transport in neurons, and that the two modes of UNC-18 binding to Syntaxin are involved in different aspects of UNC-18 function.

Zhu GD et al. (2009) Mol Biol Cell "SPE-39 family proteins interact with the HOPS complex and function in lysosomal delivery."

Doucette MM, Salazar G, Faundez V, Fiza B, L'Hernault SW, Heilman CJ, Zhu GD, Zlatic SA, Levey AI

[Mol Biol Cell, 2009]

Monitoring Editor: Sean Munro Yeast and animal homotypic fusion and vacuole protein sorting (HOPS) complexes contain conserved subunits, but HOPS-mediated traffic in animals might require additional proteins. Here we demonstrate that SPE-39 homologues, which are found only in animals, are present in RAB5-, 7- and 11-positive endosomes where they play a conserved role in lysosomal delivery and probably function via their interaction with the core HOPS complex. While C. elegans spe-39 mutants were initially identified as having abnormal vesicular biogenesis during spermatogenesis, we show that these mutants also have disrupted processing of endocytosed proteins in oocytes and coelomocytes. C. elegans SPE-39 interacts in vitro with both VPS33A and VPS33B while RNAi of VPS33B causes spe-39-like spermatogenesis defects. The human SPE-39 ortholog, C14orf133, also interacts with VPS33 homologues and both coimmunoprecipitates and cosediments with other HOPS subunits. SPE-39 knockdown in cultured human cells altered the morphology of syntaxin 7-, 8-, and 13-positive endosomes. These effects occurred concomitantly with delayed mannose 6-phosphate receptor-mediated cathepsin D delivery and degradation of internalized epidermal growth factor receptors. Our findings establish that SPE-39 proteins are a previously unrecognized regulator of lysosomal delivery and that C. elegans spermatogenesis is an experimental system useful for identifying conserved regulators of metazoan lysosomal biogenesis.