Butler, Victoria et al. (2009) International Worm Meeting "Worm Tracker 2.0: Generating a phenome database."

Sternberg, Paul, Schafer, William, Yemini, Eviatar, Cronin, Chris, Butler, Victoria, Jucikas, Tadas

[International Worm Meeting, 2009]

The nematode Caenorhabditis elegans is widely used for the genetic analysis of nervous system function. This analysis relies on the precise description of behavioural phenotypes but standard methods for classifying the behavioural patterns of mutants are qualitative and imprecise. Together with the Sternberg lab, we have developed the Worm Tracker 2.0 system; a low cost, feature-rich single worm tracker that allows the rapid and consistent quantification of the effects of mutations on behaviour. This system allows us to obtain measurements of a wide range of morphological and behavioural features, including velocity, flex, bending frequency, track amplitude and track wavelength, and we will present data collected from crawling and swimming assays. A standardized phenotyping system makes it possible to compare behavioural data collected by different researchers in different labs and we are using this system to start the generation of a comprehensive C. elegans phenotypic database that could be used to explore the clustering and relative similarities of mutant phenotypes.

Butler, Victoria et al. (2015) International Worm Meeting "Progranulin and its cleavage products, the granulins, are reciprocal regulators of lysosome function and organismal stress response."

Butler, Victoria, Judy, Meredith, Salazar, Dominique, Hsu, Tsung-Yuan, Kao, Aimee

[International Worm Meeting, 2015]

Progranulin is a highly conserved, cysteine-rich, secreted protein. Elevated progranulin production is associated with cancer growth and metastases while progranulin insufficiency is implicated in neurodegenerative and lysosomal storage diseases. Therefore, proper regulation of progranulin levels appears to be critical to healthy homeostasis. Previously, we showed that C. elegans mutants lacking the progranulin (pgrn-1) gene exhibit resistance to environmental stressors (Judy PLoS Genet 2013) and accelerated engulfment of apoptotic cells (Kao PNAS 2011). Full-length progranulin can be cleaved into granulin fragments, yet little is known about granulin production and function. We are utilizing C. elegans to investigate the potentially reciprocal relationship between the progranulin pro-protein and its cleavage products.To understand regulation of progranulin production and cleavage, we performed stress assays at different ages. We showed that full-length progranulin increases with age and heat stress. Interestingly, progranulin cleavage into granulins also increased with age and stress. We next assessed the effects of individual granulins on stress response. Transgenic expression of individual granulins impaired stress response. In contrast, over-expression of full-length human progranulin promoted stress resistance. How do granulin fragments achieve their toxic effect? To investigate the molecular function of granulins we performed co-immunoprecipitation experiments with C. elegans granulin 3 and pulled down two intriguing lysosome-associated proteins: an aspartate protease, ASP-3, and a cytoplasmic subunit of the V-ATPase proton pump, VHA-12. Confocal imaging demonstrated that granulin expression disrupted lysosome morphology and altered expression of lysosomal-associated membrane protein 1 (LAMP-1). Thus, progranulin and granulins likely regulate stress response via signals emanating from the endo-lysosomal system.Our data suggest a model in which progranulin haploinsufficiency leads to neuronal injury and neurodegeneration via a relative imbalance in the protective effects of intact progranulin and the toxic effects of cleaved granulins. Likewise, tumors may utilize uncleaved progranulin to promote their own growth and survival. If so, then progranulin proteases may represent new therapeutic targets in both diseases. In future studies, we will identify these proteases and further delineate the timing and mechanism of progranulin and granulin function.

Salazar, Dominique et al. (2015) International Worm Meeting "The progranulin cleavage products, granulins, exacerbate toxicity in a C. elegans model of TDP-43 neurodegeneration."

William, Seeley, Argouarch, Andrea, Ayumi, Nakamura, Hsu, Tsung-Yuan, Kao, Aimee, Miller, Bruce, Salazar, Dominique, Butler, Victoria

[International Worm Meeting, 2015]

Mutations in the human progranulin gene resulting in protein haploinsufficiency cause frontotemporal lobar degeneration with TDP-43 inclusions (FLTD-TDP). While progress has been made in understanding the normal functions of progranulin and TDP-43, the molecular interactions between these proteins remain unclear. Progranulin is proteolytically processed into granulins, but the role of granulins in the pathogenesis of neurodegenerative disease is unknown. We utilized a Caenorhabditis elegans model of neuronal TDP-43 proteinopathy to specifically interrogate the contribution of granulins to the neurodegenerative process. Complete loss of the progranulin gene did not worsen TDP-43 toxicity while progranulin heterozygosity did. Interestingly, expression of individual granulins alone had little effect on the behaviors measured. In contrast, when granulins were individually co-expressed with TDP-43, they exacerbated its toxicity in a variety of behaviors including motor coordination, development, lifespan, progeny production, and egg laying. These same granulins increased TDP-43 levels via a post-translational mechanism. We further found that in human neurodegenerative disease subjects, cleaved progranulin fragments accumulated specifically in diseased regions of the brain. To our knowledge, this is the first demonstration of a toxic role for granulin fragments in a neurodegenerative disease model. Furthermore, this toxicity appeared to be circuit-specific, preferentially affecting circuits involving cholinergic neurons. These studies suggest that presence of cleaved granulins, rather than or in addition to loss of full-length progranulin, may contribute to disease in TDP-43 proteinopathies.

Wang, A. et al. (2019) International Worm Meeting "Progranulin Polymorphisms in Aging and Stress Resistance."

Kao, A, Cortopassi, W, Butler, V, Jacobson, M, Wang, A.

[International Worm Meeting, 2019]

The cysteine-rich protein progranulin (PGRN) has been implicated in the pathobiology of several diseases including neurodegeneration and lysosomal storage related diseases (Baker, Nature 2006; Cruts, Nature 2006; Neumann, Science 2006; Smith, Am J Hum Genet 2012). Progranulin can be processed into bioactive granulin peptides, the number of which vary from species to species. C. elegans progranulin contains 3 granulins. Despite the variance in number of domains, the granulins share a stable, compact structure of beta-pleated sheets connected by disulfide bonds(Palfree, Plos One 2015). Studies have shown that loss of progranulin leads to features of accelerated aging, such as lipofuscin deposits and decreased lysosome protease activity (Ahmed, The American Journal of Pathology 2010; Ward, Science Translational Medicine 2017). In killifish, progranulin polymorphisms segregate with a short-lived aging phenotype (H151Q, C449W, R158H) (Valenzano, Cell 2015). The cysteine to tryptophan polymorphism is particularly interesting due to the cysteine's structural contribution to disulfide bonds. In addition, the histidine to glutamine polymorphism could potentially alter pH sensitivity. We have shown that pgrn-1(-) animals demonstrate a robust, stress-resistant phenotype that interestingly, can be decoupled from lifespan extension (Butler, J Neurosci 2019; Meredith, Plos Genetics 2013). Furthermore, progranulin acts in the lysosome and directly increases the activity of cathepsin-D (CTSD), a crucial lysosomal protease (Butler, Human Molecular Genetics 2018). We hypothesize that while complete absence of progranulin and granulin does not affect lifespan, that the polymorphisms in the granulin domains will impact aging and lifespan as well as stress response via regulation of lysosomal protease activity. To address this, we have created single copy insertions using MosSCI of these progranulin SNPs into C. elegans. We plan to test lifespan of these worms. We will also measure markers of aging and test stress response. To better understand potential molecular mechanisms, we will use molecular modeling to elucidate changes to progranulin and CTSD/ASP-3 interactions and test these in vivo. We anticipate that certain progranulin polymorphisms will increase lifespan and healthspan. We also predict that stress resistance will improve. We anticipate these will be a result of progranulin polymorphisms enhancing proteostasis via improved lysosomal protease activity.

David Alan Wharton (2010) Evolutionary Biology of Caenorhabditis and Other Nematodes "Panagrolaimus davidi, an Antarctic nematode model for the survival of extreme environmental stress"

David Alan Wharton

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2010]

Nematodes are found in almost all environments, including those where they are often exposed to extreme environmental stress. Panagrolaimus davidi is an Antarctic nematode living associated with moss and algae in terrestrial habitats on the Victoria Land coast that are free of snow and ice for part of the year. It has to survive very variable thermal and hydric environments where liquid water and temperatures suitable for growth are only periodically available. P. davidi can survive complete water loss (anhydrobiosis) and is the only organism that has been shown to survive intracellular ice formation throughout its tissues. It has several cold tolerance strategies, including; freeze avoidance, cryoprotective dehydration, freezing tolerance and anhydrobiosis. The mechanisms involved may include the production of trehalose, ice active proteins and the control of ice nucleation. Do the different survival strategies of P. davidi represent the expression of different gene sets or does the production of stress-related compounds provide protection against a variety of environmental challenges? Other nematodes, including Caenorhabditis elegans, are not so resistant to desiccation and freezing. Comparing the genomes of P. davidi and C. elegans may thus highlight the adaptations that are necessary for the survival of extreme environmental stress.

Wen, Quan et al. (2011) International Worm Meeting "Bending waves during C. elegans locomotion are driven by proprioceptive coupling."

Whitesides, George, Butler, Victoria, Gershow, Marc, Liu, Xinyu, Schafer, William, Leifer, Andrew, Fang-Yen, Christopher, Chen, Sway, Wen, Quan, Hulme, Elizabeth, Chklovskii, Dmitri, Samuel, Aravinthan, Wyart, Matthieu

[International Worm Meeting, 2011]

Locomotion requires mechanisms for coordinating motor activity throughout an animal's body. Here, using microfluidic devices to bend specific body regions while monitoring the consequences on the rest of the body, we show that proprioceptive coupling drives and organizes forward locomotion in C. elegans. Proprioceptive feedback compels each body region to bend after and in the same direction as the bending in the anterior neighboring region. To understand how proprioception is integrated into the neuromuscular network, we performed calcium imaging of muscle cells to directly visualize the motor activity along the body of a worm trapped in these microfluidic devices. We used optogenetic stimulation of the motor circuit to interrogate the cellular mechanism of proprioceptive feedback. We found that the cholinergic motor neurons both generate and propagate the proprioceptive signal from anterior to posterior body regions. Moreover, body wall muscles in C. elegans can sustain contraction without synaptic input, thereby requiring motor neurons only to trigger changes in bending states but not to maintain bending. Quantifying our experimental observations enabled us to build a simple mathematical model of locomotion, in which we show that proprioceptive coupling not only organizes the bending wave during locomotion, but also explains gait adaptation when the external load on a moving worm is gradually increased.

Butler, Jeffrey J. et al. (2011) International Worm Meeting "'Gerontometabolites' Define A Unique Metabolic Signature In Long-Lived Mit Mutants."

Rea, Shane L., Butler, Jeffrey J.

[International Worm Meeting, 2011]

The Caenorhabditis elegans mitochondrial (Mit) mutants have disrupted mitochondrial electron transport chain (ETC) functionality yet are long-lived. ByBy mutants also have disrupted mitochondrial ETC functionality but are short-lived. In an effort to identify mechanisms responsible for Mit mutant life extension, we recently established a novel approach that monitored the C. elegans exometabolism as a surrogate marker for internal metabolic events. Using HPLC-UV-based metabolomics and multivariate statistical analyses, we showed that long-lived clk-1(qm30) and isp-1(qm150) Mit mutants have a common metabolic profile that is distinct from that of either aerobically- or anaerobically-cultured wild-type animals. Moreover, we showed that two ByBy mutants, mev-1(kn1) and ucr-2.3(pk732), also shared a common metabolic signature that is unique (Butler, et. al, 2010). In this study we have used gas-chromatography-mass spectrometry (GC-MS) to fully dissect the excretome of these four mutants. We have now obtained single metabolite resolution and identified over 80 compounds that are excreted by worms. From these profiles we have identified a unique metabolic pathway operative in long-lived Mit mutants that involves the production of several carboxylated end-products. We have used genetic methods to recapitulate the Mit profile and find that, accordingly, lifespan is increased. Finally, our findings reveal an unexpected role for miRNAs in the control of the Mit mutant metabolic profile.

Zhu X et al. (1995) International C. elegans Meeting "INTEGRIN EXPRESSION PATTERN IN C. elegans"

Zhu X, Hedgecock EM

[International C. elegans Meeting, 1995]

Integrins are a family of extracellular matrix receptors involved in cell adhesion, signal transduction and cytoskeletal organization. Three genes for integrin subunits have been identified in C. elegans to date. Two a-subunit genes were discovered by the genome sequencing project. One b-subunit gene was cloned by degenerate PCR based on the known insect and vertebrate sequences, later identified as the product of pat-3 (Gettner et al., 1992). Genetic and immunohistochemical studies indicate that pat-3 is essential for muscle development, canal outgrowth and some cell migrations. These and other studies in higher organisms have led us to speculate that pat-3 might play diverse roles in development, both embryonically and post- embryonically. To further study bPAT-3 function, we sought to establish the spatial and temporal expression pattern in more detail using A. victoria GFP as a tag. We have inserted GFP into the bPAT-3 reading frame immediately upstream of the normal terminator. When this construct was injected into pat-3 null mutants, the animals were fully rescued by a heritable extrachromosomal array. Rescued animals had bright green fluorescence in specific patterns including most sites previously detected by immunofluorescence with anti-integrin antibodies. Hence, the fusion protein bPAT-3::GFP is functional and behaves much like the wildtype protein. bPAT-3::GFP is expressed continuously in muscle cells in larvae and adults. In body wall muscles, it is localized at Z-disc (dense body), M-line and dense plaque attachments. Vulval, uterine , anal depressor and sphincter muscles, but not pharynx, also express strongly. Muscle arms are also seen projecting to the nerve cords and ring. In addition, sheet-like structures lining the inner surface of the nerve ring are visible. Tentatively, these are the processes of mesoglial cells GLR. bPAT-3::GFP is also expressed transiently in many tissues. Larval neuroblasts such as ALM and Q descendants express during migration. Weaker signals are seen in the gonad, including distal tip cells and sheath cells. Finally, various cells express transiently in the late embryo. In conclusion, our data shows that both transient and continuous expression of pat-3 play roles in many different cell types.

Papp, Andy et al. (2009) International Worm Meeting "Investigation of Low-cost GFP Microscopy."

Papp, Andy, Aldrich, Chris, Bangalore, Srikanth, Perry, David

[International Worm Meeting, 2009]

The Green Fluorescent Protein (GFP) gene from the fluorescent jellyfish Aequorea victoria, and its variants (such as EGFP), are used extensively to study the location and timing of gene expression in transgenic animals and plants (Chalfie et al, 1994). Resultant GFP fusion proteins are especially well-suited to studying in vivo gene expression patterns in the transparent nematode, C. elegans and the transparent larva of the fly D. melanogaster. Perhaps one of the most significant limitations to its use in large-scale genetic screens is the high cost of equipment needed to observe GFP microscopically - namely the Fluorescence Dissecting Stereomicroscope for screening and picking mutants and upright and inverted epi-fluorescent compound microscopes for more detailed studies. Commercially available Fluorescence Dissecting Stereomicroscopes typically sell in the midrange between US$10,000 and US$20,000. They are offered by only the "high-end" microscope companies, based upon their most expensive dissecting scopes, and incorporate their premium-priced mercury arc-lamp illuminators, power supplies and epi-fluorescence modules. This leads us to wonder whether it is possible to cut corners without sacrificing utility, and which corners can be cut. Since the inception of GFP-based expression screens, a variety of researchers have produced custom-made and home-made GFP dissecting scopes. These include, for example, Welcome Bender (Harvard Medical School, pers. comm.) and Ian Chin-Sang (Chin-Sang, 2004). There are several possibilities to consider toward lowering the cost of a Fluorescence Dissecting Stereomicroscope. It may be possible to get sufficient light from relatively inexpensive, long-lived, lower-power-consuming Light Emitting Diodes (LEDs) vs. mercury arc lamps. Depending upon the spectral specificity of the LEDs, filters or dichroic mirrors might be omitted. Getting enough light intensity of the precise wavelengths that excite GFP fluorescence focused onto the sample is important. The exciting beam can be provided directly or focused backward through the microscope using "epi-illumination". We will explore these possibilities and report (and possibly demonstrate) the results. Chin-Sang, Ian. (2004) GFP Stereoscope Using LED light source. Queen''s University, Kingston, ON, Canada. http://130.15.90.245/gfp_stereoscope.htm.

Martha Kirouac et al. (2001) International C. elegans Meeting "Identification of Cis -Regulatory Response Elements in Vulva Cell Fate Markers"

Martha Kirouac, Paul Sternberg

[International C. elegans Meeting, 2001]

After induction of primary and secondary fates by the anchor cell and lin-12 , and undergoing two rounds of cell division the final vulval cell types are specified A, B1, B2, C, D, E and F. A number of transcription factors have been identified that play a role in vulva development. However we know little about how these transcription factors interact with each other, about their interactions with the inductive pathway, or about how they are regulated. The identification of cis -regulatory regions that confer cell specificity and respond to the ras pathway would be very helpful in determining such relationships. Three such target genes are egl-17 , cdh-3 and zmp-1 . These genes offer the opportunity to find response regions for multiple vulva cell types: vulE, vulF, vulC, vulD, and vulA as well as the anchor cell. In addition, egl-17 is an early cell fate marker for the response to the inductive signal; the isolation of a cis – regulatory element that drives this early expression and identification of genes that regulate this expression would be informative. We are analyzing these regions using a PCR based strategy to generate defined deletions to test the necessity and the sufficiency of fragments to confer regulation on the naïve promoter pes-10 . egl-17::gfp comes on in P6.p, P6.p daughters and granddaughters; goes off in early L4; staying off in mid-L4; on in vulC and vulD cells in mid L4 [Burdine RD, Branda CS, and Stern MJ, Development Mar;125(6):1083,1998]. This GFP marker strain contains 3.8 kb of cis- regulatory region. Since this gene is the earliest known maker in response to the inductive signal, it makes a good candidate for a immediate response element to the ras pathway. We have found a 0.2 kb region that is necessary and sufficient for vulC and vulD cell specificity and a 1.5 kb region that confers early expression in P6.p daughters. zmp-1::gfp is expressed during late L4 in vulE and vulA cells and in the L3 animals it is also expressed in the anchor cell [J. Butler & J. Krug, personal communication]. This GFP marker also contains 3.8 kb of upstream regulatory sequence. We have defined a 0.35 kb region that is necessary and sufficient vulE, vulA and anchor cell specificity. cdh-3::gfp is expressed during L3 in vulE and vulF cells and in L4 animals it is expressed in the vulC and vulD cells. cdh-3 is also expressed in the anchor cell starting in the late L2 stage. The gfp marker strain contains 6.0 kb of upstream regulatory sequence. We have found a 0.6 kb region that confers specificity to the anchor cell element and 0.5 kb region that drives expression in vulE and vul F cells.