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Cold-tolerance is a fast and easy method to identify neuronal dysfunction in C. elegans

Christopher D. Link1, Colson P. Tomberlin1, Nicole F. Liachko2,3
1Institute for Behavioral Genetics, University of Colorado, Boulder, CO 80309, USA.
2Geriatrics Research Education and Clinical Center, Veterans Affairs Puget Sound Health Care System, Seattle, WA 98108, USA.
3Division of Gerontology and Geriatric Medicine, Department of Medicine, University of Washington, Seattle, WA 98104, USA.
Correspondence to: Nicole F. Liachko (nliachko@uw.edu)
Wild-type C. elegans raised at cooler temperatures (16°C) can survive a sudden shift to a cold temperature (4°C). However, animals raised at warmer temperatures (20-25°C) die following a similar sudden cold stress. A single pair of neurons, ASJL and ASJR, are required for sensing warm temperature. Active ASJ neurons initiate a signaling cascade that results in lipid changes in the gut and decreased cold tolerance. In the absence of signaling from these neurons, worms maintain a cold-resistant lipid profile. Mutants that affect neuronal signaling broadly or ASJ signaling specifically are resistant to cold temperatures (Ohta et al., 2014). Testing C. elegans ability to survive a sudden drop in temperature is a rapid and robust method to detect changes in signaling from the ASJ nerve pair, and may provide a surrogate screen for broader neuronal dysfunction.
Many C. elegans models of neurodegenerative disease have been developed with a range of phenotypes, from mild to severe neuronal dysfunction and degeneration. To determine whether these transgenic models of neurodegenerative disease display defects in sensory signaling, we assayed their level of cold-tolerance. We found that two different models of amyotrophic lateral sclerosis (ALS) are resistant to cold. TDP-43 M337V (bkIs423[Psnb-1::TDP-43(M337V); Pmyo-2::dsRED]) has a severe phenotype including progressive age-related neurodegeneration. TDP-43 WT (bkIs410[Psnb-1::TDP-43(WT); Pmyo-2::dsRED]) has a mild phenotype and no neurodegeneration (Liachko et al., 2010). Interestingly, both strains exhibited robust cold-tolerance (Figure 1). We extended this observation to a third TDP-43 model (dvIs62[Psnb-1::TDP-43 wt]) (Ash et al., 2010), as well as transgenic strains with neuronal expression of α-synuclein, Aβ, and tau (Figure 2) (Dosanjh et al., 2010). While neuronal expression of these toxic transgenic proteins also resulted in cold tolerance, muscle-specific expression of Abeta or TDP-43 did not (Figure 3). Cold sensitivity is able to detect neuronal dysfunction in strains with mild phenotypes, and is specific to changes in neuronal function.
Cold sensitivity may be broadly applicable for rapidly characterizing neuronal function in specific genetic or transgenic backgrounds. In addition, this method may also be useful for larger scale forward and reverse genetic screening. For example, mutagenesis followed by multiple rounds of selection for cold tolerance might be expected to yield a high percentage of mutants with neurological defects. The setup and equipment needed to perform these assays are minimal, and any laboratory already utilizing C. elegans has the needed resources. To test your own strains for cold-tolerance, raise a stage-synchronized population of worms at 20-25°C to day 1 adult on standard NGM plates seeded with OP-50 bacteria. Spread C. elegans plates in a single layer on a tray to minimize temperature differentials. Do not expose worms to cooler temperatures (16-18°C) prior to the shift to cold. Transfer the worm plates to a 4°C refrigerator for 24-28 hours. We used an upright refrigerator that maintained an internal temperature range of 1-11°C. After their cold exposure, return the plates to 20-25°C and allow the worms to recover for 2-4 hours before scoring survival.

Figures



Figure 1: Worms expressing wild-type (WT) or mutant (M337V) human TDP-43 in neurons are cold resistant. N=941(N2), 936(bkIs423), 432(bkIs410).


Figure 2: Worms expressing human WT TDP-43, A30P mutant α-synuclein, Aβ, or 4-repeat (4R) tau in neurons are cold resistant. N=346(N2), 428(dvIs62), 268(dvIs162), 65(dvIs50), 146(dvIs39).


Figure 3: Worms expressing human Aβ in muscle are not cold resistant. N=346(N2), 34(dvIs104), 61(dvIs2).

References

Ash, P.E., Zhang, Y.J., Roberts, C.M., Saldi, T., Hutter, H., Buratti, E., Petrucelli, L. & Link, C.D. (2010). Neurotoxic effects of TDP-43 overexpression in C. elegans. Hum Mol Genet, 19(16):3206-3218.  PubMed

Dosanjh, L.E., Brown, M.K., Rao, G., Link, C.D. & Luo, Y. (2010). Behavioral phenotyping of a transgenic Caenorhabditis elegans expressing neuronal amyloid-beta. J Alzheimers Dis, 19(2):681-690.  PubMed

Liachko, N.F., Guthrie, C.R. & Kraemer, B.C. (2010). Phosphorylation Promotes Neurotoxicity in a Caenorhabditis elegans Model of TDP-43 Proteinopathy. J Neurosci, 30(48):16208-16219.  PubMed

Ohta, A., Ujisawa, T., Sonoda, S. & Kuhara, A. (2014). Light and pheromone-sensing neurons regulates cold habituation through insulin signalling in Caenorhabditis elegans. Nat Commun, 5:4412.  PubMed

TEM view on midbody organization in late cytokinesis and during abscission

Irina Kolotuev
UNIL – Université de Lausanne, Faculté de biologie et de médecine, Electron Microscope Facility, Biophore – Quartier Sorge, Ch-1015 Lausanne, Switzerland
Correspondence to: Irina Kolotuev (irina.kolotueva@unil.ch)

Electron microscopy is a tool that is extensively used in C. elegans studies. However, some questions are difficult to address by commonly available methods, since they require a tedious search for an elusive feature. Such difficulty is evident when embryonic stages are considered, as tiny embryos are difficult to manipulate. In addition, identity of the cells and their appearance will highly depend on sectioning orientation that is hard to master under the ultramicrotome. An EM procedure that involves flat embedding and tight trimming can facilitate the search for the desired feature (Kolotuev, 2014). In this procedure samples are embedded in resin that after polymerization remains easy to observe under the microscope. This way samples can be selected and oriented according to the project needs. Tight trimming of the samples permits to concentrate on precise region of interest, significantly reducing working effort.One example that requires such approach is the study of cell division. C. elegans early embryo provided important information on mechanisms of cell division that are valid also in additional systems. On light microscopy level abscission process was demonstrated to be a multistep internalization process involving ESCRT complex components and Septins (Green et al., 2013). Nevertheless, the appearance of abscission site and intermediates of abscission on TEM level are not yet known. The difficulty might be explained by the fact that the process is transitory and happens inside the large embryo making it difficult to find.

Using flat embedding-tight trimming procedures numerous sections were mounted on one grid, which enabled a convenient screen for the midbody in ball stage embryos (Figures 1A and 1B). The midbody at the end of cell division lacks microtubuli or Fleming body and does not resemble the cytokinetic bridge of cells in culture (Figures 1C and 1D). These reference images for the first time give a description of cytokinetic features on TEM level and might facilitate further studies and characterization of abscission processes in C. elegans. The same strategy can be applied in the search for other rare cellular events or separate cells localization related to precise time point (such as anchor cell fusion, ventral enclosure), improving the success rate of their localization.

Figures



Figure 1: TEM micrographs of abscission intermediates in C. elegans ball-stage embryos. (High resolution images are available on wormatlas).
A. A holy TEM grid with over forty two serially collected sections. Scale bar 100 µm.
B. Low magnification image of dividing cells. Scale bar 500 nm.
C. Higher magnification on the inset from panel B. Scale bar 500 nm.
D. Three serial sections of embryonic midbody cytokinetic bridge that still connects two cells. Scale bar 200 nm.

References

Green RA, Mayers JR, Wang S, Lewellyn L, Desai A, Audhya A, Oegema K. (2013). The midbody ring scaffolds the abscission machinery in the absence of midbody microtubules. J. Cell Biol. 203, 505-520. PubMed

Kolotuev I. (2014). Positional correlative anatomy of invertebrate models organisms increases efficiency of TEM data production. Microscopy and Microanalysis, 20, 1392-1403. PubMed

C. elegans synchronization: Small- and large-scale protocols to isolate synchronized L1 larvae and beyond

Mary R. Brockett1, Patrick S. Spica1 and Jessica H. Shinn-Thomas1
1Department of Biology, Utica College, Utica, NY
Correspondence to: Jessica H. Shinn-Thomas (jhthomas@utica.edu)

It is sometimes essential to synchronize C. elegans to the same larval stage. Our lab simplified and optimized synchronization protocols using standard worm media and culture techniques to ensure that we consistently obtain a sufficient number of worms at the same larval stage for our experiments. To synchronize worm cultures, we utilize unseeded Nematode Growth Media (NGM) plates (solid media with no bacterial food source) on which isolated embryos hatch and arrest at the L1 stage in the absence of food (Johnson et al., 1984; Baugh, 2013). Embryos are isolated using an alkaline hypochlorite solution that dissolves gravid hermaphrodites without damaging their embryos. Significant interest in our approach to this technique was expressed at the 20th International C. elegans Meeting and we are therefore sharing our protocols.

Small-Scale L1 Synchronization

Standard NGM is poured into 60 mm Petri plates and dried for 48 hours at room temperature before use (Brenner, 1974; Stiernagle, 2006). The media is not seeded with OP50 E. coli. Gravid hermaphrodites (10 each) from standard, well-fed culture stocks are transferred by picking into 2-50 µl drops of freshly prepared alkaline hypochlorite solution (1 ml bleach, 250 µl of 10M NaOH, and 3.75 ml sterile deionized H2O) on opposite sides of one unseeded 60 mm NGM plate for a total of 20 gravid hermaphrodites (Stiernagle, 2006). The plate is incubated at 20°C for ~20-24 hours for L1 offspring hatching. L1 offspring (see Figure 1 for yield) are transferred to NGM with OP50 for growth to the desired synchronized developmental stage or directly to experimental conditions.

Large-Scale L1 Synchronization

Standard NGM with uracil is poured into 100 mm Petri plates and dried for 96 hours at room temperature. The media (8-100 mm plates) is seeded with 500 µl of an overnight broth culture of OP50 E. coli by spreading. The bacterial lawn is grown for 4-6 days at room temperature before transferring 15 gravid hermaphrodites from standard, well-fed culture stocks onto each of the 8-100 mm seeded plates. Worms are cultured at 20°C.

The plates are monitored daily until they are barely starved (i.e., there is a large supply of gravid hermaphrodites and minimal OP50 remaining on the plates, which can occur between 3 and 7 days). Worms are sterilely collected with M9 buffer (see Stiernagle, 2006 for recipe) by pipetting 2 ml of M9 buffer onto each plate, tilting the plate, and “washing” the worms from the top to the bottom of the plate with the 2 ml of M9 buffer using a glass Pasteur pipette. This 2 ml of buffer is repeatedly “washed” over the worms to harvest. The worm suspension is collected into a 15 ml conical bottom tube. Approximately 16 ml of worm suspension is collected from all 8 plates, which is divided equally into 2-15 ml conical bottom tubes. The volume is brought to 10 ml with M9 buffer and the worms are pelleted via centrifugation for 1 minute at 1,300 x g. The supernatant is removed and worms are resuspended in 5 ml of freshly prepared alkaline hypochlorite solution (see recipe above). The dissolution of adults and release of embryos are monitored under a stereomicroscope. The suspension is swirled every 1-2 minutes to resuspend and facilitate dissolving of the adults, which takes ~6 minutes. M9 buffer is then added to the alkaline hypochlorite solution to bring the volume to 10 ml. Embryos are pelleted via centrifugation at 1,300 x g for 1 minute. The supernatant is removed and embryos are washed with 10 ml of M9 buffer followed by centrifugation at 1,300 x g for 1 minute. All but ~100 µl of embryo suspension is removed. Approximately 3-4 drops of the embryo suspension is transferred using a glass Pasteur pipette to 3-100 mm unseeded NGM plates each for better distribution and subsequent picking of L1s. The plates are incubated at 20°C for ~20-24 hours for L1 offspring hatching. L1 offspring (see Figure 1 for yield) are transferred to NGM with OP50 for growth to the desired synchronized developmental stage or directly to experimental conditions.

Notes

We use standard concentrated bleach (8.25% sodium hypochlorite) purchased from a retail store. The alkaline hypochlorite solution is made fresh before use, but the bleach is effective in solution for ~6-8 months if stored in the dark at room temperature.

Figures



Figure 1: Figure 1. Average number of L1 offspring isolated from the small-scale (n=4) and large-scale (n=3) synchronization protocols. Error bars show standard error of the mean.


Figure 2:


Figure 3:

References

Baugh LR. (2013). To grow or not to grow: Nutritional control of development during Caenorhabditis elegans L1 arrest. Genetics 194, 539-555.   PubMed

Brenner S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71-94.  PubMed

Johnson TE, Mitchell DH, Kline S, Kemal R, Foy J. (1984). Arresting development arrests aging in the nematode Caenorhabditis elegans. Mech. Ageing Dev. 28, 23-40.   PubMed

Stiernagle T. (February 11, 2006). Maintenance of C. elegans. WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.101.1, http://www.wormbook.org.   PubMed

Worm Humor – Sh*t Worm Grad Students Say

Callista Yee
Department of Biology, McGill University
Correspondence to: Callista Yee (callista.yee@mail.mcgill.ca)
  1. “Why isn’t my CRISPR working?”
  2. “If my needle clogs one…more…time…”
  3. “I’m only three trainee lineages away from Brenner.”
  4. “I don’t get why this cross isn’t working. Maybe I need to put on some mood music like Barry White.”
  5. “I went on a date last night and I told him I spent 3 exciting hours in a dark room with lasers. He asked me if it was a cool rave.”
  6. “Ugh, no cookies at seminar? I feel tricked.”
  7. “I have a lucky pick. I only bring it out for those really hard crosses. I do a silent prayer to the Genetics Gods right before I pick F2s.”
  8. “So I posted on the WormBase forum and I didn’t get roasted…”
  9. “I emailed the author asking for help with a protocol in her paper. She replied back and sent me a 20 page Word file. The original method in her paper was only 5 sentences.”
  10. “Do you think the worms know they’re in an all-you-can-eat buffet of bacteria?”
  11. “I’m so so sad I didn’t get a selfie with Mello.”
  12. “Yeah I bought the C. elegans giant microbe. Its name is Unc-ie.”
  13. “Is it sad that I look forward to In-N-Out more than socializing at the LA worm meeting?”
  14. “Pass the EtOH. No, I meant the gin, not the 70% EtOH.”
  15. “I really hope I get to go to Madison this year, pitchers at the Memorial Union are the best.”
  16. “Why is it that during poster sessions there’s alcohol but only water during talks?”
  17. “I’m sorry, did you just say free beer?”

Modified E. coli B strain OP50 facilitates RNA interference induction in C. elegans

Isaiah Neve1, Jessica Sowa1 and Meng Wang1
1Huffington Center on Aging, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston TX
Correspondence to: Meng Wang (wmeng@bcm.edu)

In the majority of research laboratories using C. elegans as a model, we sustain our animals on the B-type E. coli strain OP50 (Brenner, 1974).  This strain has provided us with a historically key platform for defining genetic pathways. Concurrently, the technique of double stranded RNA (dsRNA) feeding to incite RNA interference (RNAi) has become a key technique in many laboratories. Two major resources, the Ahringer and Vidal libraries house thousands of E. coli clones with unique plasmids expressing dsRNA corresponding to individual C. elegans genes (Kamath and Ahringer, 2003; Rual et al., 2004). While numerous experimental insights have been facilitated using these resources, both libraries are housed in the HT115 E. coli K12 strain. Several recent studies (Brooks et al., 2009; Soukas et al., 2009; Pang and Curran, 2014) have revealed the importance of the bacterial environment in eliciting certain C. elegans phenotypes. With the realization that gene-environment interactions may play a key role in genetic regulation, we have generated an OP50 strain capable of dependably housing plasmid vectors expressing dsRNA. Furthermore, when C. elegans are fed this OP50 strain, they activate a robust RNAi response against the target gene of interest. We anticipate that this resource will allow the further characterization of gene-environment specific relationships in C. elegans. To generate an RNAi competent OP50 strain, two main steps were required. We started by replacing the wild-type (WT) OP50 allele of RNAIII RNase (rnc) with the same deletion allele found in the E. coli K12 HT115 strain. We then introduced a genomically encoded IPTG-inducible T7 RNA polymerase to drive double stranded RNA production from L4440 plasmids. To do this, we prepared P1 phage lysates from strains HT115 (rnc14::DTn10) and CH1681 (laczgA::T7pol camFRT) (Figure 1A). We initially transduced OP50 overnight (O/N) culture with the HT115 (rnc14:: DTn10) lysate and selected for tetracycline (tet) resistance. After repetitive rounds of selection, we inoculated individual colonies into LB + tet and confirmed the presence of the rnc14::DTn10 allele by PCR. Following this, we transduced OP50 (rnc14::DTn10) O/N culture with CH1681 (laczgA::T7pol camFRT) P1 lysate and used multiple rounds of Chloramphenicol (cam) positive selection to purify our background. Individual colonies were then picked, and PCR was used to confirm the presence of rnc14:: DTn10 and laczgA::T7pol camFRT.

Grown overnight, OP50 replicative timing tends to be slightly delayed in comparison to its HT115 counterpart. However, the desired ODs can be easily reached which facilitate the induction of an RNAi response (Figure 1B).

Using Pges-1::sl2::gfp as our reporter C. elegans strain, we determined that after 16h of culture, OP50 RNAi competent strains housing the L4440 plasmid targeting GFP are capable of inducing significant knock-down of the target gene. This knockdown was then repeated in both the muscles and hypodermis (Figure 2A-C).

Next we generated OP50 RNAi clones targeting two endogenous C. elegans genes: act-5 and dpy-13. In the P0 generation, OP50 derived dsRNA knockdown of act-5 is as sufficient as its contemporary HT115 strain in arresting worm development. RNAi knockdown of dpy-13 in using the OP50 background exerts an even stronger effect in the reduction of worm size than the comparable HT115 knockdown experiment (Figure 2D).

Thus we have constructed an OP50 strain which can robustly initiate RNAi reactions comparable to those generated by HT115. The knockdowns generated can be in multiple different tissues, and can target various genes. Utilizing this method we plan to further elucidate gene-environment interactions. This resource is available to the research groups requesting it.

Figures



Figure 1:


Figure 2:

References

Brenner S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71-94.   PubMed

Kamath RS, Ahringer J. (2003). Genome-wide RNAi screening in Caenorhabditis elegans. Methods 30, 313-321.  PubMed

Rual JF, Ceron J, Koreth J, Hao T, Nicot AS, et al. (2004). Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library. Genome Res. 14, 2162–2168.  PubMed

Brooks KK, Liang B, Watts JL. (2009). The influence of bacterial diet on fat storage in C. elegans. PLoS One, 4:e7545.  PubMed

Soukas AA, Kane EA, Carr CE, Melo JA, Ruvkun G. (2009). Rictor/TORC2 regulates fat metabolism, feeding, growth, and life span in Caenorhabditis elegans. Genes Dev. 23, 496–511.   PubMed

Pang S, Curran SP. (2014). Adaptive capacity to bacterial diet modulates aging in C. elegans. Cell Metab. 19, 221-31.  PubMed

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