Smart, Joelle et al. (2017) International Worm Meeting "Touch neurons can toss out mitochondria."

Arnold, Meghan, Toth, Marton, Melentijevic, Ilija, Driscoll, Monica, Harinath, Girish, Smart, Joelle, Guasp, Ryan

[International Worm Meeting, 2017]

Mitochondria provide energy, execute key steps of metabolism, control calcium, and modulate cellular decisions for life/death. Given these critical functions in cell, tissue, and organism health, it is not surprising that mitochondrial functionality plays an essential role in neuronal maintenance in everyday biology, aging, and late-onset neurodegenerative disease. Mitochondrial quality control is thought to be primarily executed through cell-internal elimination via mitophagy and lysosome degradation. However, the Driscoll lab has discovered, and recently published (Melentijevic, 2017 Nature 542:367) that mitochondria can be thrown out of neurons in large membrane bound vesicles we call exophers. Mitochondria in exophers budding from C. elegans touch neurons tend to have elevated oxidation of mitoROGFP reporters localized to the matrix. Genetic and pharmacological treatments that compromise mitochondria can increase numbers of exophers produced by touch neurons, suggesting that throwing away defective mitochondria may be a mechanism for neuronal quality control. Indeed, some mammalian neurons can throw out their mitochondria for degradation by neighboring astrocytes (Davis, PNAS 11:9633), suggesting relevance across phyla. In C. elegans, beautiful work on degradation of sperm mitochondria upon fertilization have been published (Sato, Science 334:1141). We will present data on our initial efforts to characterize mito-exopher production and the factors that prompt neurons to extrude mitochondria. Our hope is that our findings will be relevant to understanding neuronal maintenance and neuronal degeneration, especially as associated with perturbed mitochondrial quality as may occur in Parkinson's disease and many other human disorders.

Smart, Joelle et al. (2021) International Worm Meeting "Neuronal mitochondria utilize a novel fission mechanism during extrusion into exophers"

Driscoll, Monica, Nguyen, Ken, Park, Eunchan, Hall, Dave, Smart, Joelle, Rongo, Chris

[International Worm Meeting, 2021]

The Driscoll lab discovered a previously unknown capacity of C. elegans adult neurons to extrude large (~5 microm) vesicles that include cytoplasmic contents (Melentijevic, 2017). These vesicles, which we call "exophers", are a microcosm of the originating soma: they can contain almost anything from the cell, including aggregated proteins, cytoskeletal components, and organelles. Importantly, I have found mitochondria are extruded into the majority of C. elegans touch neuron exophers, and recent work in mouse cardiomyocytes demonstrates elimination of defective mitochondria through exopher-genesis plays a critical role in cardiac homeostasis (Nicolás-Ávila, 2020). Mitochondrial morphology is thought to influence cell function and age-associated decline. In all animals, FZO-1/MFN1/2 and EAT-3/OPA1 respectively mediate outer and inner mitochondrial membrane fusion, while the dynamin motor DRP-1 is required for canonical mitochondrial fission. Several interesting questions arise: Are small, hyper-fragmented mitochondria (produced by inhibiting mito fusion) more amenable to trafficking into exophers? Does abolishing mitochondrial fission (knocking out DRP-1) prevent mitochondrial severance and extrusion into exophers? I have made the surprising discovery that portions of the mitochondrial network are severed and ejected into exophers even when DRP-1-mediated mitochondrial fission is abolished. Furthermore, I have shown mitochondria contained within exophers are typically intact. In fact, in a small fraction of cases, exophers and their mitochondria can remain morphologically intact for weeks following separation from the soma, suggesting mitochondrial fragmentation during exopher-genesis is not irreparably traumatic. Most extruded mitochondria, however, appear to be degraded by the surrounding hypodermis within 3 hours of exopher-genesis. I will be presenting my findings on the proteins, organelles, and physical factors governing this non-canonical mechanism of mitochondrial fission, as well as the biochemical and functional properties of both extruded and retained mitochondria. This research documents a novel method of mitochondrial sculpting and distribution that likely has important implications for mitochondrial homeostasis, trafficking, and membrane repair. Melentijevic I, et al. C. elegans neurons jettison protein aggregates and mitochondria under neurotoxic stress. Nature. 2017 Feb 16;542(7641):367-371. doi: 10.1038/nature21362. Epub 2017 Feb 8. PMID: 28178240; PMCID: PMC5336134. Nicolás-Ávila JA, et al. A Network of Macrophages Supports Mitochondrial Homeostasis in the Heart. Cell. 2020 Oct 1;183(1):94-109.e23. doi: 10.1016/j.cell.2020.08.031. Epub 2020 Sep 15. PMID: 32937105.

Smart, Joelle et al. (2019) International Worm Meeting "Touch and dopaminergic neurons eject mitochondria under native and genetic stress conditions."

Rongo, Chris, Wang, Guoqiang, Driscoll, Monica, Smart, Joelle, Brennan, Rachel, Melentijevic, Ilija, Grant, Barth, Park, Eunchan, Vergalasov, Edward

[International Worm Meeting, 2019]

Mitochondrial quality control is thought to be primarily executed through cell-internal elimination via mitophagy and lysosome degradation. However, we discovered, and recently published (Melentijevic, Nature 542:367) that mitochondria can be thrown out of neurons in large membrane-bound vesicles we call exophers. Genetic and pharmacological treatments that compromise mitochondria can increase numbers of exophers produced by touch neurons, suggesting that throwing away defective mitochondria may be a mechanism for neuronal quality control. Some mammalian neurons can throw out their mitochondria for degradation by neighboring astrocytes (Davis, PNAS 11:9633), suggesting relevance across phyla. Mitochondrial dysfunction is considered a central factor in the progressive loss of substantia nigra pars compacta dopaminergic neurons in Parkinson's disease. Mutations in genes encoding the mitophagy proteins PINK1 and Parkin are directly linked to autosomal recessive PD. The major cause of late-onset autosomal dominant PD is a gain-of-kinase function mutation in LRRK2, which is involved in mitochondrial dynamics, turnover, and DA neuron oxidative stress response (Saha, J Neurosci 29:9210). Furthermore, chemical disruption of mitochondrial electron transport chain complexes can selectively kill dopaminergic neurons in rats and C. elegans, with mutations in PINK1 and Parkin enhancing this vulnerability. We have reported that pink RNAi disruption and Parkin deletion in C. elegans can increase exopher production. However, our initial studies involved strains in which aggregating proteins were expressed in neurons studied via high copy number transgenes. We have found that mitochondria can still be extruded in exophers in the absence of foreign aggregating proteins, and that genetic manipulation of mitochondrial fission/fusion and PD-related genes can significantly impact mito-exopher rates. We will present data on our initial efforts to characterize mito-exopher production from touch and DA neurons and the factors that prompt neurons to extrude mitochondria. Our hope is that our findings will be relevant to understanding neuronal maintenance and neuronal degeneration, especially as associated within relation to the perturbed mitochondrial quality as may occurobserved in Parkinson's disease and many other human disorders.

Martin, Olivier et al. (2021) International Worm Meeting "A faster and more efficient RNASeq protocol supports new approaches for studying gene regulation and tissue composition in C. elegans"

Stroustrup, Nicholas, Martin, Olivier, Eder, Matthias

[International Worm Meeting, 2021]

Individuals with identical genotypes often show high phenotypic variability. To better characterize inter-individual differences in aging, we further optimized the low-input Smart-seq2 RNASeq strategy (Picelli et al., 2014; Serra et al., 2018) to support routine collection of high-quality C. elegans transcriptomes. Compared to conventional techniques that require thousands of individuals as input material, we have validated our method to show that it can produce robust results from single individuals, and scales up to pools of thirty worms at which point it replaces existing bulk methods. This combination of single and pooled RNASeq provides a quantitative means for studying variation in gene regulation and tissue composition between individuals and across populations. Looking to increase throughput and reduce cost over existing approaches, we identified an eccentricity of the Smart-seq2 template-switching polymerase that in C. elegans leads to an aberrant amplification of specific ribosomal RNAs independent of the poly-dT primers used during reverse transcription. More generally, we find that Smart-seq2 template-switching oligomers bind non-specifically to multiple non-coding RNAs. We solve this problem by combining biotin-labeled primers with an additional bead purification step after reverse transcription. This procedure excludes all non-coding RNAs and increases our effective sequencing depth by 4.5 fold, providing pooled or single-worm transcriptomes at dramatically lower cost. We demonstrate the usefulness of this new method to identify novel downstream targets of the daf-2 insulin/IGF receptor. References Picelli, Simone, et al. "Full-length RNA-seq from single cells using Smart-seq2." Nature protocols 9.1 (2014): 171-181. Serra, Lorrayne, et al. "Adapting the Smart-seq2 protocol for robust single worm RNA-seq." Bio-protocol 8.4 (2018).

Suh, Jinkyo et al. (2013) International Worm Meeting "GExplore updated: more genome-scale data mining for worm researchers."

Suh, Jinkyo, Hutter, Harald

[International Worm Meeting, 2013]

Previously we implemented a simple web-based database interface for genome-scale data mining at the protein (rather than the DNA) level for experimental planning - hosted at: http://genome.sfu.ca/gexplore/. The interface allows individual or combinatorial searches for proteins with certain domains, expression in particular tissues or specific phenotypes. Genes in a given interval can be retrieved for vetting candidate genes in positional mapping experiments. The database, which contains also GO terms and homology information, has been updated regularly using Wormbase data. Recently new datasets including stage- and sex-specific RNAseq expression data (see Gerstein et al., Science 330:1775-87) have been processed and added. We are currently preparing a major update of GExplore to include several novel features and datasets. We included protein domain data using Pfam and SMART annotations for all core species in Wormbase (C. elegans, C. briggsae, C. brenneri, C. japonica, C. remanei and P. pacificus). We extended the manually curated list of protein domains that can be searched by simple abbreviations, e.g. 'IG' to retrieve all proteins containing Pfam or SMART domains referring to ImmunoGlobulin domains, to over 450 domains. Proteins containing a particular Pfam or SMART domain can be identified using Pfam or SMART identifiers. In addition to a graphic display of the domain organization of proteins, the protein sequences are available for display and download. A new interface allows users to observe where mutations are located within a protein of interest. The output can be limited to mutations affecting certain protein domains. Our database contains mutations accessible through Wormmart and will be updated as more recent Wormbase data will become available for general data mining. The database also includes mutations isolated by the Million Mutation Project, which have been released recently by the Moerman and Waterston labs (Thompson et al. submitted). Independently of GExplore we maintain a web-based interface to search for mutations from the Million Mutation Project (hosted at http://genome.sfu.ca/mmp/). We will present the current progress in updating the GExplore website and underlying database.

Arnold, Meghan Lee et al. (2021) International Worm Meeting "An aggresome-like collection mechanism functions in neuronal expulsion of disease-aggregates"

Guasp, Ryan, Smart, Joelle, Grant, Barth, Melentijevic, Ilija, Hall, Dave, Cooper, Jason, Nyguen, Ken, Arnold, Meghan Lee, Driscoll, Monica, Ardeshna, Sohil

[International Worm Meeting, 2021]

The accumulation of aggregated proteins is associated with aging and neurodegeneration. Accumulation and subsequent spread of misfolded proteins causes toxicity that can induce loss of neurological function. Given that aggregate accumulation and spread is a prominent feature in neurodegenerative disease pathology and functional decline, a major goal in aging biology is thus to understand the mechanisms of how cells deal with protein aggregation, accumulation, and spread. In mammalian biology, cells can handle aggregated proteins to maintain proteostasis via numerous pathways -- central aggregate collection, degradation via the ubiquitin-proteasome system or autophagy-lysosome pathway. In addition, neurons can identify, collect, and eject aggregates in large membrane-bound packages, "exophers". Mammalian and fly neurons also throw out aggregated-trash, which contributes to aggregate spreading via an unknown mechanism and is thought to promote pathology in human neurodegenerative disease. While we have documented the dynamic aggregate movement from the soma into the exopher domain, followed by a dramatic budding of neuronal contents into the exopher as key hallmarks of exopher formation, we know little about the molecular requirements for these complex tasks. I will describe the novel aggresome-like organelle in C. elegans neurons, an organelle that hosts disease aggregates. We discovered that many proteins important for aggresome-formation are also important for trash-expulsion via the exopher mechanism. Together, both aggresome-formation and exophergenesis-mechanisms represent intriguing molecular targets for improper aggregate handling and aggregate spread as seen in neurodegenerative disease pathology.

Wang, Yu et al. (2021) International Worm Meeting "Phagocytosis and processing of neuron-derived exophers by the C. elegans hypodermis."

Smart, Joelle, Driscoll, Monica, Nguyen, Ken C., Melentijevic, Ilija, Morera, Andres, Wang, Yu, Hall, David H., Grant, Barth D., Cooper, Jason, Wang, Guoqiang, Arnold, Meghan L., Guasp, Ryan J.

[International Worm Meeting, 2021]

Previous work showed that adult C. elegans neurons are capable of ejecting a large portion their cell bodies as a giant vesicle (average size 3.8 micron) loaded with aggregated protein and mitochondria, a process that becomes much more frequent upon challenge by several forms of cellular stress (Melentijevic et al., 2017). Current models posit that such ejection removes toxic materials from the neuron, enhancing neuronal stress recovery. Here we have focused on the fate of exophers ejected by the mechanosensory neuron ALMR, a neuron whose soma is completely surrounded by the syncytial skin cell hyp7. By following exophers within individual animals over time we found a characteristic pattern in which a single large ALMR exopher typically moves within hyp7 in a posterior direction, away from the neuron, but initially maintains a long thin connection to the neuronal soma. Within 3 hours of ejection most intact exophers have disappeared, with remaining material derived from the exopher broken up into many smaller vesicles within the hypodermis that we have termed "starry night". Over the next few days most starry night vesicles diminish in intensity, and in many cases fluorescent material derived from exophers appears outside of the hypodermis in coelomocyte cells. This pattern suggested that ejected exophers are phagocytosed by the much larger hypodermal cell, with the hypodermal endolysosomal machinery activated to degrade toxic exopher materials via phagosome maturation. Undigested material may then be resecreted by the hypodermis, allowing uptake and another chance at degradation by the coelomocyte scavenger cells. Work in progress supporting this model will be presented, with an emphasis on current results analyzing hypodermal uptake and phagosome processing, and the role of ARF-6, CNT-1, and RAB-35 in this process.

Groth, Amy C. (2015) International Worm Meeting "A laboratory exercise utilizing C. elegans Notch mutants for an undergraduate cancer biology course."

Groth, Amy C.

[International Worm Meeting, 2015]

A laboratory exercise was developed to demonstrate the effects of Notch inactivation and overexpression on cell proliferation. Many laboratory exercises illustrating principles of cell proliferation utilize tissue culture, which is often not feasible at primarily undergraduate institutions. An effective alternative is a new laboratory exercise utilizing C. elegans strains that harbor mutations in the germline Notch receptor, glp-1. The temperature-sensitive, gain-of-function strain, GC833 (ar202), has uncontrolled proliferation of germline stem cells at 25 degC, forming a germline tumor. The balanced loss-of-function strain, JK4862 (q46), yields ~1/3rd of offspring that are sterile, with empty germlines. During this exercise, students observed phenotypes of worms in levamisole on compound light microscopes, and imaged the worms directly through one ocular with their smart phones. In addition to learning about the Notch signaling pathway, students also learned about different types of mutation (including gain-of-function, loss-of-function, missense and nonsense), performed PCR amplification, analyzed sequence data and determined the effects of base changes on the resulting protein products. This laboratory was designed for upper level undergraduate cancer biology students, but could be utilized, with or without the sequence analysis, for a vaiety of courses, including developmental biology, genetics and cell biology. .

Theresa Grana et al. (2007) International Worm Meeting "Using C. elegans to demonstrate central concepts of gene expression."

Janet C. Batzli, Theresa Grana, Jeff Hardin, Elizabeth A. Cox, Michelle A. Harris

[International Worm Meeting, 2007]

Undergraduate students often struggle with concepts related to the central dogma of biology. With the goal of a more sophisticated, deeper understanding of those concepts, we designed a four-week inquiry and discovery based lab sequence for a sophomore level cell biology laboratory. We first developed specific learning objectives that we then aligned with the lab exercises and assessments. These assessments included pre- and post-evaluations of learning gains to examine knowledge and attitude, a series of worksheets exploring central concepts and culminated in a scientific poster where students evaluated C. elegans as a model for studying the function of their assigned human disease gene. Each laboratory group was assigned one of five genes having an ortholog involved in a human disease and went on to examine that gene in a number of ways. Students compared deletion allele and feeding RNAi phenotypes to wildtype worms and used genomic PCR to examine the size of the deletion. Bioinformatics tools including Wormbase, SMART, BLAST and JMol were used to connect sequence and structural information to the observed phenotypes. Those tools were used along with PubMed and OMIM to further explore the gene and its orthologs. Students annotated the DNA and protein sequences for their assigned gene using Geneious software, which allowed them to visualize gene structure and the effects of the deletion allele on the protein product. We will present data from student assessments with regard to learning gains.

Papp, Andy et al. (2013) International Worm Meeting "Remote Control and Observation for more meaningful Behavioral Experiments."

Papp, Andy, Biondo, John

[International Worm Meeting, 2013]

C. elegans is an excellent organism for the study of the molecular genetics of a variety of neurologically-based phenomena such as mechanosensation, chemosensation, thermosensation, photosensation, behavior, learning, and memory because it manifests all of these via its simple, well characterized 302-cell nervous system (Murakami 2007). The sensitivity and interactions of these different systems necessitate carefully controlled experiments so that a single variable can be examined and meaningful conclusions can be drawn. For example, one would not want sensitive chemotaxis-based learning experiments to be interfered with due to thermotaxis, phototaxis, or mechanical stimulation caused by an inadequately controlled environment. To create a more stable environment for these experiments, we have developed a small incubator with an integrated web-cam microscope that we call the "Telebator". It can be remotely controlled, and its contents observed, via a local or Internet computer network connection (US Pat. 7618808). This system allows behavior to be observed and recorded in an environment that is isothermal with constant lighting and no changes in mechanical stimulation. The Telebator's remote-control and monitoring features also make it ideal for various other applications, including: heat shocks, temperature shifts, knowing/controlling when a plate grown for DNA extraction is just about to starve, knowing/controlling when worms of a specific developmental stage are plentiful, knowing if/when a fluorescently marked transgene is being expressed, etc. In the event of a temperature or other alarm, the system is able to notify users by e-mail, SMS text message, and YahooIM. The incubator contents and temperature log data can be observed and the incubator temperature and other parameters can be controlled via any web browser, including a "smart phone". Murakami (2007) Caenorhabditis elegans as a model system to study aging of learning and memory. Mol Neurobiol. 35(1):85-94.