Apfeld, Javier et al. (2009) International Worm Meeting "Relating cellular and organismal aging phenotypes in C. elegans."

Fontana, Walter, Apfeld, Javier

[International Worm Meeting, 2009]

Aging appears to be a universal phenomenon in living systems leading to functional changes affecting the onset of disease and the timing of death. Living systems comprise multiple structural and organizational levels, from sub-cellular to whole organism. Aging may occur at any of these levels. In the past two decades aging research has begun to map how organismal lifespan depends on genetic and environmental factors. Yet, the relationship between cellular and organismal aging phenotypes is rarely investigated and poorly understood. We have developed and deployed indicators of cellular state to probe causality between processes leading to failure in individual neurons and processes that determine organismal lifespan in C. elegans. We perform longitudinal measurements in individual neurons utilizing fluorescent probes in both wild-type animals and transgenic animals commonly used as models of neurodegenerative disease. We find that the states of certain neurons become more uncertain as animals get older. In addition, time-dependent correlations between some pairs of neurons develop from initially uncorrelated states. The existence of correlations between specific states of different cells suggests the existence of processes of inter-cellular communication mediating those dependencies. Notably, these neuronal aging processes are independent of processes that determine organismal lifespan. We find that mutations in components of the insulin signaling pathway that increase lifespan promote neuronal aging processes in some neurons yet inhibit them in others. The action of insulin signaling in these neuronal processes is independent of its influence on organismal lifespan. Together, these results show that C. elegans undergoes independent aging processes at different organismal levels.

Romero, Catalina et al. (2011) International Worm Meeting "Investigating the spatial and temporal dynamics of protein oxidation in C. elegans."

Romero, Catalina, Fontana, Walter, Apfeld, Javier

[International Worm Meeting, 2011]

Increased protein oxidation is a phenomenon common to aging in animals in diverse phyla. Protein oxidation is often associated with loss of protein function, which may, in turn, impair cellular protein homeostasis and contribute to the organism's functional decline with age. We are interested in investigating the temporal and spatial dynamics of protein oxidation in different tissues during aging, a question that is difficult to tackle through biochemical approaches. To achieve this goal, we have created transgenic animals expressing a genetically encoded redox sensor, which reports the level of oxidation of specific cysteine residues via changes in fluorescence. We have validated our approach by showing that: i) the sensor responds quickly and reversibly to exposure of the worm to cell-permeable oxidants and reductants; ii) the sensor correctly reports differences in the oxidative environments of specific cellular compartments (nucleus, cytosol and endoplasmic reticulum). We are currently investigating whether mutations that affect lifespan also affect the level of protein oxidation, under normal and oxidative-stress conditions.

Romero, Catalina et al. (2009) International Worm Meeting "In vivo determination of mitochondrial state during the C. elegans aging process."

Romero, Catalina, Apfeld, Javier, Fontana, Walter

[International Worm Meeting, 2009]

Several mutations and RNAi knockdowns of mitochondrial genes affect the lifespan of C. elegans. Furthermore, environmental and genetic perturbations that increase lifespan, such as caloric restriction and reduction in insulin signaling, increase mitochondrial activity (Bishop & Guarente, Nature 447, 545-9, 2007; Houthoofd et al., Aging Cell 4, 87-95, 2005). We are interested in studying how alterations in mitochondrial activity lead to metabolic and physiological changes that result in an extended lifespan. We are quantifying mitochondrial matrix pH in vivo by targeting a pH-sensitive GFP variant to the mitochondrial matrix of muscle cells. This allows us to follow changes in mitochondrial function and morphology in single cells over time. We are currently investigating how (i) differences between individuals and (ii) differences between cells within each individual correlate with the large variation on lifespan that is observed in isogenic populations of worms. We hope that these studies will allow us to quantify the dependencies between changes in mitochondrial activity at the cellular level and the aging phenotype at the organismic level, both in wild type animals and in mutants with extended lifespans.

Stroustrup, Nicholas et al. (2009) International Worm Meeting "The Lifespan Machine: Automated C. elegans lifespan acquisition on agar plates."

Fontana, Walter, Stroustrup, Nicholas, Apfeld, Javier

[International Worm Meeting, 2009]

The current method for identifying individual death times within C. elegans populations is both labor-intensive and repetitive, limiting the quality and scope of demographic aging data available to the C. elegans research community. In response, we have developed a scalable and low-cost imaging platform to allow fully-automated acquisition of nematode mortality data. Our system captures month-long, time-lapse videos of entire C. elegans populations, with images taken once every two hours at 8 mm resolution. Animals are maintained on temperature-controlled NGM agar plates seeded with E. coli, allowing straightforward integration of machine-acquired results with mortality and behavioral data collected via traditional manual techniques. Through utilization of consumer-grade optical equipment, we have produced an inexpensive system suitable for both small, single-researcher installations and large, high-throughput clusters capable of monitoring tens of thousands of worms simultaneously. To complement the new hardware, we have developed a software package that analyzes time-lapse videos to extract survival curves in a fully-automated fashion. We present survival curves acquired with our technique of large populations (>500 animals each) at high temporal resolution (observations made every two hours). We use this high-resolution mortality data as a basis for the quantitative comparison of aging demographics between various long-lived mutant strains. We also demonstrate the suitability of our method for performing RNAi screens. We hope to share this technology as a platform for the collection and analysis of more and higher quality demographic aging data in the C. elegans community.

Stroustrup, Nicholas et al. (2011) International Worm Meeting "The Lifespan Machine comes of age: Time-dependent changes in the action of insulin signaling are revealed by high-resolution mortality data."

Apfeld, Javier, Stroustrup, Nicholas, Fontana, Walter

[International Worm Meeting, 2011]

Aging C. elegans populations show characteristic changes in mortality rate and tolerance to stresses including exposure to high temperature and oxidizing agents. We are interested in how individual genes determine the variability and temporal dynamics of these age-dependent phenotypes. We have developed an automated, high-throughput microscopy platform (the "lifespan machine") capable of assaying several thousand worms for movement on agar plates every ten minutes over several weeks. In addition to acquiring high-resolution lifespan distributions at 20 deg C and 25 deg C, our method also produces consistent survival curves under stress regimes including 35 deg C thermotolerance assays and tert-butyl hydroperoxide resistance assays. By subjecting mutant populations to these stressors starting at different times of adulthood, we probe how individual genes affect the age-dependency of stress resistance. In isogenic populations, thermotolerance and oxidant resistance can be quite variable between individuals, reminiscent of the wide variation observed in lifespan under standard conditions. We find that genetic determinants of the mean and variation in lifespan also affect the temporal dynamics of stress resistance as animals age. Strikingly, we find that mutations in several pathway components of insulin signaling known to affect lifespan only affect thermotolerance and resistance to oxidative stress at certain times during adulthood. These findings indicate that worms experience age-dependent changes in the action of insulin signaling.

Stroustrup, Nicholas et al. (2013) International Worm Meeting "Life in the hot seat: Comparing aging and stress resistance."

Nash, Zachary, Apfeld, Javier, Stroustrup, Nicholas, Fontana, Walter

[International Worm Meeting, 2013]

To address the current limitations of manual survival assays with regard to throughput, statistical power, and reproducibility, we developed an automated system for the acquisition and analysis of C. elegans lifespan data. Our technology -- dubbed the "Lifespan Machine" - provides a standardized method for accurate measurement of lifespan, while being low-cost and scalable to any desired statistical resolution. Our lab's installation uses an array of fifty flatbed scanners to monitor up to 30,000 individuals across 2.5 square meters of agar lawn distributed across 800 plates every fifteen minutes at 8 mm optical resolution. We have developed software that analyzes this large volume of image data to identify the death times of individual worms based on their spontaneous movement, with a focus on subtle, late-life postural changes. Our toolset permits the rapid visual validation of automatic measurements, which constitutes a crucial step for the routine production of rigorous mortality statistics. By using standard agar Petri plates, the lifespan machine can automate a variety of assays including feeding RNAi experiments, stress- and pathogen-resistance assays, as well as compound testing. The statistical and temporal resolution afforded by automation allowed us to take a first look at the time-dependent hazard rate of C. elegans populations dying across the full temperature range from 20 deg C to 36.5 deg C at fraction of degree intervals. We identified striking similarities between the hazard functions of animals dying over 14 hours at 33 deg C and those dying over two weeks at 25 deg C. This calls into question the separation between stress resistance (time to death at high temperatures) and aging (time to death at room temperature), widely considered as qualitatively distinct phenomena. Our finding that the shape of the hazard function is universal across temperature is especially surprising when considering that the observed scaling relationship (mean lifespan vs temperature) suggest that distinct physiological transitions occur as temperature increases.

Stroustrup, Nicholas et al. (2015) International Worm Meeting "Slowing Down: Diverse interventions extend C. elegans lifespan through temporal scaling."

Fontana, Walter, Stroustrup, Nicholas, Apfeld, Javier, Anthony, Winston

[International Worm Meeting, 2015]

Many interventions alter C. elegans lifespan: changes in diet, temperature, exposure to xenobiotics, and the disruption of many genes. Yet, individuals vary enormously in their lifespan even within isogenic populations in controlled environments. To probe this stochastic component of aging, we used the Lifespan Machine, our automated system for capturing lifespan data, to collect multiple replicates of high-resolution survival curves across different classes of interventions. We find that most but not all interventions alter lifespan distributions through what appears to be a perfect temporal scaling: the simple stretching of survival curves along the time axis.This temporal scaling provides a strong hint as to the physiological consequences of the interventions studied. Quantitatively, a "stretching" of survival curves seems to require that a "stretching" occurs in each individual's physiology, that every physiologic process's contribution to lifespan is altered in unison. Indeed, we observe that exposure to moderate increases in temperature during just the first three days of adulthood shortens lifespan to the extent expected if animals were uniformly "sped-up" during exposure. The magnitude of this youthful, transient, apparent acceleration in aging corresponds closely to that predicted by the temporal scaling of survival curves across temperatures.Previous work has demonstrated that the rates of decline in feeding behaviors, stress resistance, fertility, and other "healthspan" phenotypes are decoupled by lifespan-extending perturbations. Yet, our results suggest that the specific subset of aging processes causal to death must in fact be altered to exactly the same extent, in young and old animals alike, across a broad class of interventions including changes in diet, temperature, oxidative stress, and disruption of daf-2, daf-16, hsf-1, and hif-1.

Burglin TR et al. (2016) Chromosoma "Homeodomain proteins: an update."

Affolter M, Burglin TR

[Chromosoma, 2016]

Here, we provide an update of our review on homeobox genes that we wrote together with Walter Gehring in 1994. Since then, comprehensive surveys of homeobox genes have become possible due to genome sequencing projects. Using the 103 Drosophila homeobox genes as example, we present an updated classification. In animals, there are 16 major classes, ANTP, PRD, PRD-LIKE, POU, HNF, CUT (with four subclasses: ONECUT, CUX, SATB, and CMP), LIM, ZF, CERS, PROS, SIX/SO, plus the TALE superclass with the classes IRO, MKX, TGIF, PBC, and MEIS. In plants, there are 11 major classes, i.e., HD-ZIP (with four subclasses: I to IV), WOX, NDX, PHD, PLINC, LD, DDT, SAWADEE, PINTOX, and the two TALE classes KNOX and BEL. Most of these classes encode additional domains apart from the homeodomain. Numerous insights have been obtained in the last two decades into how homeodomain proteins bind to DNA and increase their specificity by interacting with other proteins to regulate cell- and tissue-specific gene expression. Not only protein-DNA base pair contacts are important for proper target selection; recent experiments also reveal that the shape of the DNA plays a role in specificity. Using selected examples, we highlight different mechanisms of homeodomain protein-DNA interaction. The PRD class of homeobox genes was of special interest to Walter Gehring in the last two decades. The PRD class comprises six families in Bilateria, and tinkers with four different motifs, i.e., the PAIRED domain, the Groucho-interacting motif EH1 (aka Octapeptide or TN), the homeodomain, and the OAR motif. Homologs of the co-repressor protein Groucho are also present in plants (TOPLESS), where they have been shown to interact with small amphipathic motives (EAR), and in yeast (TUP1), where we find an EH1-like motif in MAT2.

Roxani Gatsi et al. (2005) International Worm Meeting "Suppressor screens for the dissection of the IP3 signalling pathway in the pharynx"

Roxani Gatsi, Howard Baylis

[International Worm Meeting, 2005]

IP3 mediated calcium signalling in C. elegans determines specificity of cellular responses to extracellular stimuli. Specialized IP3 receptors (IP3Rs), which are located on the endoplasmic reticulum membrane, regulate cytoplasmic calcium concentrations that determine various cellular functions. One of these functions is the up-regulation of pharyngeal pumping in response to food (Walker et al, 2002a). Relatively little is known about the IP3-mediated regulation of the rhythmic pumping of the pharynx. Walter et al (2002b) has shown that interaction between IP3Rs and myosin are required for regulation of pharynx pumping but not for other physiological rythmic functions in C. elegans, indicating the importance of protein interactions for determining specificity. In order to identify additional components of the IP3 signalling pathway in the pharynx we performed genetic suppressor screens using an IP3R mutant (sa73), which shows reduced pharyngeal pumping. We have isolated various mutants that suppress various defects of the sa73 phenotype. The results from the characterization of these mutants, as well as the approach to map the mutations will be presented. Walker DS., et al. (2002a) Mol Biol Cell 13, 1329-1337. Walker DS., et al. (2002b) Curr Biol 12, 951-956

Gattegno T et al. (2007) Mol Biol Cell "Genetic control of fusion pore expansion in the epidermis of Caenorhabditis elegans."

Valansi C, Podbilewicz B, Hall DH, Gattegno T, Chernomordik LV, Mittal A, Nguyen KC

[Mol Biol Cell, 2007]

Monitoring Editor: Peter Walter Developmental cell fusion is found in germlines, muscles, bones, placentae and stem cells. In C. elegans 300 somatic cells fuse during development. While there is extensive information on the early intermediates of viral-induced and intracellular membrane fusion, little is known about late stages in membrane fusion. To dissect the pathway of cell fusion in C. elegans embryos, we use genetic and kinetic analyses using live-confocal and electron microscopy. We simultaneously monitor the rates of multiple cell fusions in developing embryos and find kinetically distinct stages of initiation and completion of membrane fusion in the epidermis. The stages of cell fusion are differentially blocked or retarded in eff-1 and idf-1 mutants. We generate kinetic cell fusion maps for embryos grown at different temperatures. Different sides of the same cell differ in their fusogenicity: while the left and right membrane domains are fusion-incompetent, the anterior and posterior membrane domains fuse with autonomous kinetics in embryos. All but one cell pair can initiate the formation of the largest syncytium. The first cell fusion does not trigger a wave of orderly fusions in either direction. Ultrastructural studies show that epidermal syncytiogenesis require eff-1 activities to initiate and expand membrane merger.