Churgin, M. et al. (2017) International Worm Meeting "Longitudinal imaging of C. elegans with the WorMotel reveals variation in behavioral decline during aging."

Yu, C.C., Jung, S.K., Fang-Yen, C., Churgin, M., Chen, X., Raizen, D.

[International Worm Meeting, 2017]

The roundworm C. elegans is a mainstay of aging research due to its short lifespan and easily manipulable genetics. Standard methods for lifespan measurements in C. elegans are tedious and dependent on manual observation. In addition, they are largely performed as population assays and do not have access to potentially rich information contained in individual aging trajectories. To address these limitations, we developed the WorMotel, a microfabricated device for long-term cultivation and automated longitudinal imaging of large numbers of C. elegans confined to individual wells. Each WorMotel, which is fabricated by PDMS molded from a 3D printed master, consists of an array of 240 wells, each 3 mm in diameter, with a shape optimized for worm cultivation and imaging. Wells contain agar, bacteria, and a single worm. Residents of the WorMotel exhibit behaviors and lifespan very similar to those on standard plates. We use custom machine vision analysis software to automatically quantify aging behavior and lifespan for each animal simultaneously. An aversive blue light stimulus enables measurements of stimulated behaviors in addition to spontaneous behaviors. We apply the WorMotel to characterize differences in behavioral decline across individuals and strains with differences in lifespan. Previous work has shown that population lifespan curves scale uniformly under different genetic and environmental conditions, suggesting that the shape of behavioral decline might also scale. We find that short-lived and long-lived mutant strains exhibit patterns of behavioral decline that do not temporally scale between individuals or populations, but instead resemble the shortest and longest lived individuals in a wild type population, respectively. We show that the shape of behavioral decline differs significantly between individuals with different lifespans, and that the shape of decline follows a universal dependence on lifespan across multiple genetic backgrounds. We also apply our method to quantify aging in worms experiencing external oxidative stress. We find that behavioral trajectories of worms subject to oxidative stress, which reduces mean survival by about twenty-fold, resemble trajectories observed during aging. Therefore, our results suggest a complex relationship between genes, environment, and the shape of the aging process. Current work aims to identify molecular mechanisms of inter-individual variability in aging.

Churgin, M. et al. (2017) International Worm Meeting "Antagonistic serotonergic and octopaminergic neural circuits mediate food-dependent locomotory behavior in Caenorhabditis elegans."

McCloskey, R., Peters, E., Fang-Yen, C., Churgin, M.

[International Worm Meeting, 2017]

Animals must tune their physiology and behavior appropriately to the environment. How such adaptations are achieved remains largely unknown. Biogenic amines are conserved signaling molecules that link food cues to behavior and metabolism in a wide variety of organisms. The biogenic amines serotonin (5-HT) and octopamine regulate a number of food-related behaviors in C. elegans, including locomotion and feeding. We use long-term quantitative behavioral imaging in a multi-well device (WorMotel) to investigate the role of 5-HT and octopamine in food-dependent locomotor behavior states. We find that well-fed and fasting animals exhibit distinct patterns of locomotion characterized by differences in locomotion behaviors known as roaming, dwelling, and quiescence. Worms lacking endogenous 5-HT, such as tph-1 mutants, exhibit fasting-like locomotion even under fed conditions, whereas worms lacking octopamine, such as tbh-1 mutants, exhibit feeding-like locomotion under fasting conditions. Therefore, we conclude that 5-HT and octopamine promote feeding-like and fasting-like locomotion patterns, respectively. We find that exogenous treatment of fasting worms with 5-HT produces a reduction of quiescent behavior and increase in dwelling. 5-HT produced by the NSM neurons acts on the MOD-1 receptor to promote low-amplitude locomotor behavior characteristic of well-fed animals. Interestingly, 5-HT produced by the ADF neurons acts through the SER-5 receptor to suppress quiescent behavior and promote roaming in fasting worms. Therefore, 5-HT can act both to promote high-amplitude and low-amplitude locomotion through the SER-5 and MOD-1 receptors, respectively. Octopamine, produced by the RIC neurons, acts through SER-3 and SER-6 receptors to promote roaming behaviors characteristic of fasting animals. Both the SER-3 and SER-6 receptors are required for normal behavioral responses to octopamine. Overall, we find that 5-HT signaling is required for worms to assume food-related locomotion behavior, whereas octopamine signaling is required for worms to assume fasting-related behaviors. The requirement for both neurotransmitters in both the feeding and fasting states enables increased behavioral adaptability. Our results define the molecular and neural pathways through which parallel biogenic amine signaling tunes behavior appropriately to nutrient conditions. Current experiments aim to further identify the specific tissues and neurons in which these receptors act to exert their effects.

Wypijewska A et al. (2012) Biochemistry "7-methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity."

Wypijewska A, Darzynkiewicz E, Davis RE, Jemielity J, Bojarska E, Lukaszewicz M, Stepinski J

[Biochemistry, 2012]

Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.

Churgin MA et al. (2014) Methods "Construction of a system for single-cell transgene induction in Caenorhabditis elegans using a pulsed infrared laser."

Murray JI, Churgin MA, He L, Fang-Yen C

[Methods, 2014]

The spatial and temporal control of transgene expression is an important tool in Caenorhabditis elegans biology. We previously described a method for evoking gene expression in arbitrary cells by using a focused pulsed infrared laser to induce a heat shock response (Churgin et al., 2013). Here we describe detailed methods for building and testing a system for performing single-cell heat shock. Steps include setting up the laser and associated components, coupling the laser beam to a microscope, and testing heat shock protocols. All steps can be carried out using readily available off-the-shelf components.

Ali Khan L et al. (1994) Worm Breeder's Gazette "cej-1 Encodes a Novel Protein With Poly-Threonine Motif"

Siddiqui SS, Fukushige T, Ali Khan L, Tsukita Sh, Tabish M, Itoh M

[Worm Breeder's Gazette, 1994]

cej-1 Encodes a Novel Protein with Poly-Threonine Motif M. L. A. Khanl, M. Tabish, T. Fukushigel1 S. Tsukita2, M. Itoh , Sh. Tsukita , and S. S. Siddiqui. (1): Lab. of Molecular Biology, Dept of Ecological Engg. Toyohashi Univ. Technology, Toyohashi 441, and (2). National Institute for Physiological Sciences, Okazaki 444, Japan.

Wood WB (1976) Worm Breeder's Gazette "maternal effects among ts zygote-defective (zyg) mutants."

Wood WB

[Worm Breeder's Gazette, 1976]

We have studied maternal effects in 23 zyg ts mutants to estimate the times of expression of genes whose products are required in embryogenesis. We have used the following three tests, called arbitrarily A, B, and C. A test: Heterozygous (m/+) L4's are shifted to 25 C and allowed to self-fertilize. If 100% of their eggs yield larvae (25% of which express the mutant phenotype as adults), then the mutant is scored as maternal (M). If 25% of the F1 eggs fail to hatch, then the mutant is scored as non-maternal (N). An M result indicates that expression of the + allele in the parent allows m/m zygotes to hatch and grow to adulthood. A result of N indicates the opposite: that the + allele must be expressed in the zygote for hatching to occur. Out of 23 zyg mutants tested, 3 were scored N and 20 were scored M in the A test. Therefore, for most of the genes defined by these mutants, expression in the parent is sufficient for zygote survival, even if the gene is not expressed in the zygote. B test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with N2 (+/+) males. If eggs fail to hatch at 25 C, but mated hermaphrodites shifted to 16 C produce cross progeny to give proof of mating, then the mutant is scored M. If cross progeny appear in the 25 C mating, then the mutant is scored N. An M result indicates that expression of the + allele in the zygote is not sufficient to allow m/+ progeny of an m/m hermaphrodite to survive. Conversely an N result indicates either that zygotic expression of the + allele is sufficient for survival, or that a sperm function or factor needed for early embryogenesis can be supplied paternally (see C test below). Out of the 23 zyg mutants tested, 11 were scored M and 12 were scored N. The combined results of A and B tests and their simplest interpretation are as follows. Ten mutants are M,M; the genes defined by these mutants must be expressed in the hermaphrodite parent for the zygote to survive. Ten mutants are M,N; these genes can be expressed either in the parent or in the zygote. Two mutants are N,N; these genes must be expressed in the zygote. One mutant is N,M; this gene must be expressed both in the maternal parent and in the zygote. C test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with heterozygous (m/+) males. If rescue by a +/+ male in the B test depends on the + allele, then only half the cross progeny zygotes of a C test mating (m/+ male x m/m hermaphrodite) should survive. However, if rescue depends on a function or cytoplasmic component from the male sperm, then all the cross progeny zygotes in a C test should survive. Of the 10 M,N mutants, 6 have been C tested; one exhibited paternal rescue independent of the + allele. The A and B tests also were carried out on 16 mutants that arrest before the L3 molt (acc mutants). In the A test on 2 of these mutants, all m/m progeny of m/+ parents grew to adulthood at 25 C. Therefore, parental contributions are sufficient to overcome a progeny mutational block as late as the L2 stage. All 16 acc mutants scored N in the B test.

Lemire BD et al. (2009) Mech Ageing Dev "C. elegans longevity pathways converge to decrease mitochondrial membrane potential."

Behrendt M, Decorby A, Lemire BD, Gaskova D

[Mech Ageing Dev, 2009]

Energy production via oxidative phosphorylation generates a mitochondrial membrane potential (DeltaPsi(m)) across the inner membrane. In this work, we show that a lower DeltaPsi(m) is associated with increased lifespan in Caenorhabditis elegans. The long-lived mutants daf-2(e1370), age-1(hx546), clk-1(qm30), isp-1(qm150) and eat-2(ad465) all have a lower DeltaPsi(m) than wild type animals. The lower DeltaPsi(m) of daf-2(e1370) is daf-16 dependent, indicating that the insulin-like signaling pathway not only regulates lifespan but also mitochondrial energetics. RNA interference (RNAi) against 17 genes shown to extend lifespan also decrease DeltaPsi(m). Furthermore, lifespan can be significantly extended with the uncoupler carbonylcyanide-3-chlorophenylhydrazone (CCCP), which dissipates DeltaPsi(m). We conclude that longevity pathways converge on the mitochondria and lead to a decreased DeltaPsi(m). Our results are consistent with the 'uncoupling to survive' hypothesis, which states that dissipation of the DeltaPsi(m) will extend lifespan.

DiBartolomeis SM et al. (2002) Mol Genet Genomics "Molecular analysis of the Drosophila miniature-dusky ( m-dy) gene complex: m-dy mRNAs encode transmembrane proteins with similarity to C. elegans cuticulin."

Jackson FR, Akten B, Roberts MA, DiBartolomeis SM, Genova G

[Mol Genet Genomics, 2002]

Mutations in the Drosophila miniature-dusky ( m-dy) gene complex were first reported by Morgan and Bridges about 90 years ago. m-dy mutants have abnormally small wings, a phenotype attributed to a cell-autonomous reduction in the size of the epidermal cells comprising the differentiated wing. Using a molecular genetic approach, we have characterized the m-dy chromosomal interval and identified a pair of adjacent transcription units corresponding to m and dy. A dy mutant known as dy (And) has a single base substitution within the protein-coding region that is predicted to result in an amber stop codon and premature translational termination. We show that dy mRNA is expressed at two discrete periods during the life cycle - one during embryonic development and early larval instars, the second during adult development, coincident with wing differentiation. In agreement with the phenotypic similarity of m and dy mutants, sequence comparisons reveal a similarity between the predicted MINIATURE and DUSKY proteins, and indicate that the m and dy genes are members of a larger Drosophila gene family. Both m and dy, as well as other members of this superfamily, are predicted to encode transmembrane proteins with similarity to C. elegans cuticle proteins known as cuticulins. We postulate that m, dy and other members of this protein superfamily function as structural components of the Drosophila cuticulin layer. Such a role for m and dy products in wing differentiation is sufficient to explain the morphological phenotypes associated with m-dy mutants.

Nirav Amin et al. (2008) Development & Evolution Meeting "Systematic analysis of transcription factors important in C. elegans postembryonic mesodermal development"

Zach Via, Jun Liu, Nirav Amin

[Development & Evolution Meeting, 2008]

The C. elegans postembryonic mesodermal lineage, the M lineage, is a powerful model system to study mesodermal patterning and cell fate specification at single cell resolution. The M lineage arises from a single pluripotent cell, the M mesoblast, during embryogenesis. In hermaphrodites, the M cell undergoes a series of postembryonic cell divisions to produce 18 cells: 14 body wall muscles (BWMs), 2 coelomocytes (CCs), and 2 sex myoblasts (SMs). We and others have previously identified a handful of transcription factors important for the proper development of this lineage. In order to identify additional transcription factors that play a role in the M lineage, we have generated a feeding RNAi library that targets a majority of the predicted transcription factors encoded in the C. elegans genome and conducted an RNAi screen using cell type-specific GFP reporters in the M lineage. From this screen, we identified a novel set of 32 transcription factors that, upon RNAi knockdown, give reproducible phenotypes in the M lineage. Among these 32 transcription factors, four are important for patterning and fate specification of the early M lineage, while the rest appear to play a role in fate decisions in the SM lineage. We have primarily focused on let-381, which encodes a forkhead transcription factor that is essential for C. elegans development. let-381(RNAi) causes a dorsal to ventral fate transformation in the M lineage. We have found that a let-381::gfp translational fusion is expressed in the dorsal M lineage. Previous studies from our lab have shown that SMA-9, the Sma/Mab TGF-beta and LIN-12/Notch signaling pathways are involved in dorsal/ventral patterning of the M lineage. We are currently investigating the relationship between let-381 and these pathways.

Almuhanna SA et al. (2024) Cytoskeleton (Hoboken) "Sequences in the myosin A rod interact with UNC-89/obscurin and the zinc-finger protein UNC-98 during thick filament assembly and M-line formation in C. elegans striated muscle."

Lee KM, Oishi HZ, Almuhanna SA, Hoppe PE

[Cytoskeleton (Hoboken), 2024]

The M-line of striated muscle is a complex structure that anchors myosin-containing thick filaments and also participates in signaling and proteostasis. While the physical associations among many M-line components have been defined, the mechanism of thick filament attachment is not completely understood. In Caenorhabditis elegans, myosin A is essential for viability and forms the site of M-line attachment at the center of the filament, whereas myosin B forms the filament arms. Using a mutant myosin A that forms ectopic filaments, we examined interactions between myosin A and M-line proteins in intact muscle cells. Ectopic myosin A recruits the giant kinase UNC-89/obscurin, a presumed scaffolding protein, in an interaction that requires the zinc-finger protein UNC-98, but not UNC-82/NUAK, UNC-97/PINCH, or UNC-96. In myosin A mutants, UNC-89/obscurin patterning is highly defective in embryos and adults. A chimeric myosin containing 169 residues of the myosin A C-terminal rod, coincident with the UNC-98/ZnF binding site, is sufficient for colocalization of UNC-89/obscurin and UNC-98/ZnF in M-line structures whereas a myosin chimera lacking these residues colocalizes with UNC-89/obscurin in M-lines that lack UNC-98. Thus, at least two myosin A rod regions contribute independently to M-line organization. We hypothesize that these M-line-organizing functions correspond to the essential "filament initiation function" performed by this isoform.