Salazar, Christopher et al. (2021) International Worm Meeting "A Rab-like GTPase Restricts Dendritic Branching"

Buelow, Hannes, Salazar, Christopher, Grant, Barth, Diaz-Balzac, Carlos

[International Worm Meeting, 2021]

Dendrite development depends on numerous extracellular and intracellular cues to ensure proper structure and function. However, the regulatory mechanisms of dendrite development remain incompletely understood. To better understand dendrite development, we are utilizing the PVD somatosensory neuron with its highly stereotyped 'menorah'-like dendrites. The Menorin pathway consists of several factors that function from different tissues to promote PVD dendrite development and have been heavily studied. However, much less is known about factors that restrict branching. During a genetic screen, we have identified a locus that encodes for a putative, uncharacterized Rab-like GTPase, which we name rabl-3. Time course analyses determined that the number of branching points was significantly increased in adult animals, suggesting rabl-3 suppresses dendrite growth. A transcriptional reporter shows expression in the epidermis from early embryonic through adult stages and transgenic expression of a rabl-3 cDNA in the epidermis is sufficient to rescue the mutant phenotype, possibly in manner that is independent of the GTPase activity of RABL-3. We found that a rescuing N-terminal translational RABL-3 fusion displays intracellular, perinuclear localization, indicative of typical Rab GTPases. Genetic analyses show that the Menorin pathway is largely epistatic to rabl-3, indicating it may function in the same genetic pathway. In addition, RNAi knockdown of rab-5 phenocopies the mutant rabl-3 excessive branching phenotype, suggesting sorting into the early endosome could play a role in suppression of dendrite branching. Current experiments aim to (1) determine whether mutations in rabl-3 affect localization of components of the Menorin pathway, (2) determine the subcellular localization of rabl-3, and (3) identify potential molecular interactions to build a mechanism by which RABL-3 restricts dendritic branching.

Christopher J Cronin et al. (2005) International Worm Meeting "The Combined UC San Diego/Caltech Quantitative Behavior Analysis System"

William R Schafer, Zhaoyang Feng, Kuang-man Huang, Paul W Sternberg, Christopher J Cronin

[International Worm Meeting, 2005]

California Institute of Technology and University of California, San Diego have each developed practical, automated movement analysis systems to quantify aspects of worm behavior and morphology (see An imaging system for standardized quantitative analysis of C. elegans behavior, Zhaoyang Feng, et al, BMC Bioinformatics 2004, 5:115, An automated system for measuring parameters of nematode sinusoidal movement, Christopher J Cronin, et al, BMC Genetics 2005, 6:5, and previous meeting abstracts). We have consolidated our development efforts and merged our independent system designs to create a single, unified behavior analysis system. The combined system provides researchers with all of the quantification tools and database functionality of the individual UCSD and Caltech systems, but also establishes a common, open software and hardware foundation for continued development by Caltech, UCSD and the community. Current efforts include expanding system capabilities for the automated analysis of mating behavior with the challenge of tracking multiple moving individuals, both in contact with and separated from each other.

Pena-Gonzalez, I. et al. (2013) International Worm Meeting "Suppressors of TDP-1 toxicity in Caenorhadbitis elegans."

Link, CD., Pena-Gonzalez, I.

[International Worm Meeting, 2013]

Suppressors of TDP-1 toxicity in Caenorhadbitis elegans Ilana Pena-Gonzalez1 and Christopher D Link1, 2 1 Integrative Physiology, University of Colorado, Boulder, CO 80309, USA 2 Institute for Behavioral Genetics, University of Colorado, Boulder, CO 80309, USA RNA binding protein TDP-43 forms damaging aggregates in multiple neurodegenerative diseases. We have found that the deletion of tdp-1, the C. elegans ortholog of TDP-43, leads to increased accumulation of double stranded RNA. TDP-1 is not believed to bind to dsRNA itself; however the marked increase in dsRNA accumulation in worms deleted for tdp-1 implies TDP-1 normally participates in limiting the stability and structure of dsRNA. Although worms deleted for TDP-1 do not have a severe phenotype, overexpressed nuclear TDP-1 is toxic in worms. One possible explanation for this is that too much TDP-1 leads to excessive disruption of structured RNAs needed for normal RNA metabolism. Making use of this overexpression model we have established a heat shock induction system to help identify components associated with TDP-1's prevention of dsRNA accumulation. Identifying mutations that suppress the neurotoxic effects of overexpressed TDP-1 could help identify others factors contributing to the complex mechanism of disassembling dsRNA. A mutagenesis screen has identified four mutant strains that partially suppress the toxicity of overexpressed TDP-1. We have also tested suppressors of other toxic proteins. Further identifying the pieces of the mechanism regulating dsRNA breakdown could help better explain the pathogenic pathway of TDP-1 and consequently TDP-43 in disease.

Butler, V.J. et al. (2017) International Worm Meeting "A novel stress and age-responsive lysosomal protease inhibitor affecting proteostasis in neurodegeneration."

Ashrafi, K., Chand, S., Miller, B.L., Jacobson, M.P., Gao, F., Pierce, O.M., Coppola, G., Butler, V.J., Caballero, B., Oses-Prieto, J.A., Burlingame, A.L., Kao, A.W., Seeley, W.W., Argouarch, A.R., Vohra, M., Cuervo, A.M., Mohan, S.

[International Worm Meeting, 2017]

Progressive impairment of protein homeostasis is a universal hallmark of aging, stress and disease. One potential site for this cellular dysfunction is the lysosome, which is responsible for macromolecular breakdown and recycling. We previously showed in C. elegans that granulin peptides, the cleavage products of the neurodegenerative disease protein progranulin, selectively enhance TAR DNA-binding protein 43 (TDP-43) toxicity and impair its degradation (Salazar J. Neurosci 2015). Now, we provide genetic, biochemical, computational modeling and behavioral evidence supporting a role for C. elegans granulin 3 as a stress and age-responsive inhibitor of the lysosomal aspartyl protease, ASP-3/CTSD. C. elegans progranulin contains three granulins. We show that granulin production increases with age and stress. Transgenic expression of individual granulins impairs stress response and learning and memory. Granulins localize to the lysosome where they stimulate movement of the autophagy-promoting transcription factor HLH-30/TFEB from the cytosol to the nucleus. Co-immunoprecipitation experiments show that C. elegans granulin 3 interacts with the lysosomal aspartyl protease, ASP-3, which has previously been implicated in necrotic cell death. Protein docking models predict that granulin 3 binds to and occludes the protease active site. Supporting this, ASP-3 activity is significantly reduced in granulin expressing animals. Furthermore, the expression of granulin 3 in a deg-1(d) background delays the cell death of mechanosensory neurons and prolongs response to gentle touch. In addition, human patients carrying progranulin mutations produce more granulin fragments in degenerative brain regions than age-matched controls, and this is accompanied by a significant reduction in CTSD activity. Our results identify granulins as a novel class of endogenous peptide aspartyl protease inhibitor, which in analogy to serpins and cystatins we call aspartins. Given that age and stress can increase granulin production, we propose that the regulated production of granulin peptides can impair protein homeostasis and contribute to neurodegenerative disease pathogenesis. These results also suggest that dysregulation of lysosomal protease activity may more generally contribute to aging and age-related diseases.

Bauer CC et al. (1998) West Coast Worm Meeting "a, b AND g-FILAGENIN - NEW PROTEINS IN THE CORE'S OF THICK FILAMENTS"

Schmid MF, Ortiz I, Epstein HF, Liu FZ, Bauer CC

[West Coast Worm Meeting, 1998]

The cores of muscle thick filaments are composed of an outer layer of paramyosin surrounding an inner tubular region containing additional core proteins. In order to study the structure of the thick filament core and the mechanisms and molecules involved in the formation of this length-regulated, multi-protein structural assemblage, we have isolated and characterized three novel proteins: a, b and g-filagenin. We obtained partial peptide sequences from each of the filagenin proteins and determined their predicted protein sequences from the C. elegans Genome Project's database and correlated this information with cDNA sequence obtained from RT-PCR or phage libraries. All three filagenins are novel proteins with no significant homologies to any known proteins (although homologues in parasitic nematodes have been found in the databases). All filagenins also possess the distinction of having calculated isoelectric points of over pH 10 and having a higher than average number of aromatic amino acids. Both b and g-filagenin are predicted to have secondary structures containing only beta strands and loops, whereas a-filagenin is composed of both alpha helices and beta strands. Structural studies of the core indicate that there are seven paramyosin subfilaments crosslinked by additional internal proteins. We tested our structural model with Western blotting using an affinity-purified antibody and have showed that a, b and g-filagenins are associated with the cores. All three filagenins were localized by immunofluorescence microscopy to the A bands of body wall muscles, but there was no detectable reaction with the pharynx, with the exception of a slight reaction with anti-a-filagenin antibody. b-Filagenin has been shown by immunoelectron microscopy to be assembled with paramyosin into the tubular cores of wild-type nematodes at the same periodicity of 72-nm as observed with paramyosin. In CB1214 paramyosin null mutants, b-filagenin assembles with myosin to form abnormal tubular filaments with a periodicity identical to that of wild type. These results verify that the filagenins are core proteins that co-assemble with either myosin or paramyosin in C. elegans to form tubular filaments. Further studies on nematode muscle thick filaments are intended to elucidate the mechanisms behind the formation and regulation of these stable assemblies of myosin and associated proteins. *Supported by grants from the Muscular Dystrophy Association, the National Science Foundation and a NRSA postdoctoral fellowship for Christopher Bauer.