Churgin, Matthew A et al. (2013) International Worm Meeting "Efficient single-cell transgene induction in Caenorhabditis elegans using a pulsed infrared laser."

Murray, John I, Fang-Yen, Christopher, He, Liping, Churgin, Matthew A

[International Worm Meeting, 2013]

The coupling of transgenes to heat shock promoters is a common method for regulating C. elegans gene expression. Gene induction can be performed in a temporally defined manner, through timing of heat shock, and in a tissue-specific manner, via targeted rescue in hsf-1 mutants. However, cell-specific targeting methods are limited by the availability of cell-specific promoters. Here we present a method for evoking gene expression in single cells by local activation of heat shock. Our technique builds on the IR-LEGO method by Kamei et al (Nat. Meth. 2009) in which a continuous-wave, focused infrared laser beam is used to locally induce a heat shock response. However, continuous irradiation generates significant heating away from the laser focus due to the diffusion of heat, which increases the total heated volume. We have developed an improved method using a pulsed infrared laser that minimizes off-target heating by allowing time for thermal energy to dissipate between laser pulses. The resultant total heated volume is ~32 times smaller than that of IR-LEGO. Using a Phsp-16.2::GFP transgene as a reporter of heat shock we have optimized parameters to achieve a single cell gene induction rate of over 75% in seam cells (n=30), almost doubling the previously reported efficiency. To test the spatial resolution of our method, we successfully used it to induce GFP expression in the nerve ring neuron ADL in L2 larvae, and we observed no off-target induction despite the presence of neighboring neurons within 5 microns. To explore applications in lineage tracing, we used our method to individually heat shock each cell of the four-cell embryo. Resulting GFP expression patterns at bean stage were consistent with the expected positions of the descendants of the heat-shocked cell. In addition to direct transgene expression through heat shock, we also used an Phsp-16.2::FLP construct in combination with an FRT-flanked expression cassette to induce permanent GFP expression in single cells. Our laser-induced heat shock method is modest in cost and similar in technical complexity to widely used laser ablation systems. We therefore expect that our technique will be a broadly useful tool for C. elegans research.

Churgin, Matthew et al. (2015) International Worm Meeting "Analysis of quiescence during aging in insulin/insulin-like signaling mutants using a microfabricated WorMotel multi-well device."

Churgin, Matthew, Fang-Yen, Christopher

[International Worm Meeting, 2015]

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 the potentially rich information contained in individual behavioral trajectories. To address these limitations, we developed the WorMotel, a PDMS device cast from a 3D-printed mold, which consists of an array of 240 wells, each optimized for culturing and imaging a single worm on agar media. We use this system to continuously monitor many worms throughout their lifespans. We use machine vision algorithms to quantify movement in each well, and thereby reconstruct longitudinal behavior and lifespan curves in an automated manner. Machine vision identification of time of death agrees within about 1 day with manual observations of the same worm. The WorMotel is based on standard microplate dimensions, making it compatible with automation tools including the COPAS Biosort worm sorter and plate handling robots. We used the WorMotel system to study behavior during aging in wild-type worms and mutants in the insulin/insulin-like signaling (IIS) pathway, an important regulator of lifespan in nematodes, flies, and mammals. Some groups have noticed a quiescent behavior in adult daf-2 mutants, which harbor a reduction in the IIS pathway, but this behavior has not been well-studied. Using the WorMotel, we were able to more fully characterize the adult quiescence in IIS mutants. In Class II daf-2(e1370) mutants, quiescence is accompanied by posture changes and elevated sensory arousal threshold, two characteristic properties of sleep. The DAF-16 transcription factor is completely required for this behavior, as daf-16(mu86) and daf-16;daf-2 worms exhibit no quiescence prior to end stages of life. N2 worms exhibit a moderate degree of behavioral quiescence during intermediate stages of life, suggesting that quiescence may be actively regulated by daf-16. We find that for both N2 and daf-2 animals, the fraction of lifespan spent in a quiescent state is correlated with lifespan, suggesting that behavioral quiescence may be protective against aging. We hypothesize that behavioral quiescence may be mediated by DAF-16 translocation to the nucleus, and current experiments aim to correlate behavior state in wild type and daf-2 worms with DAF-16 nuclear localization using a daf-16::gfp translational fusion strain. In addition, we are testing candidate pathways mediating other forms of quiescence as observed between molts or after stress, such as the cGMP, EGF, PKA, and neuropeptide signaling pathways, to identify the mechanisms underlying aging quiescence.

Van Buskirk, Cheryl (2017) International Worm Meeting "A forward genetic screen for mutants defective in recovery sleep."

Van Buskirk, Cheryl

[International Worm Meeting, 2017]

In response to damaging conditions such as noxious heat or UV exposure, C. elegans enters a period of behavioral quiescence known as stress-induced sleep or recovery sleep (RS), during which sensory responses are dampened and feeding and movement cease1. Recovery sleep is mediated by activation of EGF receptors on the peptidergic ALA interneuron and subsequent release of a collection of neuropeptides1-3. At this time it is not known how cellular damage leads to the initiation of EGF signaling, and gaps remain in our understanding of signal transduction events within ALA as well as in the target tissues affected by ALA peptides. In order to uncover genes required for recovery sleep we have initiated an EMS screen for sleepless F2 animals, using the pore-forming toxin Cry5B as our damage-inducing agent. Progeny of sleepless candidates are tested for responses to other known sleep-inducing stressors, such as heat and UV light. Mutants that are found to be generally defective in recovery sleep are kept for SNP mapping, complementation as needed, and eventual whole-genome sequencing. We will present our detailed methods, some obstacles that have been overcome in our mapping of sleep mutants, and information on candidate identity if available. We also describe how this project has been implemented within the context of an undergraduate laboratory course called BIOL447 FIRE: Full Immersion Research Experience. 1. Hill AJ, Mansfield R, Lopez JMNG, Raizen DM, Van Buskirk C. 2014. Cellular Stress Induces a Protective Sleep-like State in C. elegans. Curr. Biol. 24, 2399-2405. 2. Nelson MD, Lee KH, Churgin MA, Hill AJ, Van Buskirk C, Fang-Yen C, Raizen DM. 2014. FMRFamide-like FLP-13 neuropeptides promote quiescence following heat stress in Caenorhabditis elegans. Curr. Biol. 24:2406-2410. 3. Nath RD, Chow ES, Wang H, Schwarz EM, Sternberg PW. 2016. C. elegans stress- induced sleep emerges from the collective action of multiple neuropeptides. Curr. Bio.l 26:2446-2455.

Munoz-Lobato, Fernando et al. (2021) International Worm Meeting "Sleep is required for odor exposure to consolidate memory and remodel olfactory synapses."

Miller, Julia, Churgin, Matthew, Bremer, Martina, Chang, Eric, Varshney, Aruna, Nordquist, Sarah, Tran, Alan, Bokka, Anirudh, Tsujimoto, Bryan, Jimenez, Vanessa, VanHoven, Miri, L'Etoile, Noelle, Chandra, Rashmi, Brueggemann, Chantal, Baradwaj, Anjana, Andersen, Kristine, Kato, Saul, Saifuddin, Fatema, Fang-Yen, Chris, Benedetti, Kelli, Li, Joy, Dunn, Raymond, Farah, Fatima, Munoz-Lobato, Fernando, Duong, Alex

[International Worm Meeting, 2021]

Sleep is not only an essential function of humans but remains conserved across metazoans. Here we demonstrate that wild-type C. elegans sleep after repeated odor trainings This provides us with a platform to dissect how sleep affects memory at a synaptic resolution. We identified that sleep immediately after training is required for the animal to retain a long-term memory of the odor. We found that if animals do not sleep in the first two hours after training, memory is not consolidated. After identifying the neurons that are required for the memory, we show that the sensory-interneuron connections within the circuit are downscaled after sleep. Therefore, we found a time-specific requirement of sleep that modulates synaptic downscaling to preserve memory. Conversely, lack of sleep post-training erases the long-term memory and destabilizes the synaptic downscaling, indicating that modulating the amount of sleep is sufficient to modulate memory. These results make C. elegans an excellent tool to ask what molecular mechanisms, cell biological processes and circuit level reorganizations are engaged during sleep to promote memory. This understanding will provide insights into the functions of sleep that contributes to our health and well-being.