The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.
In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.
Protein misfolding and aggregation are recognized as a hallmark of several neurodegenerative diseases such as AD, PD, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and many others. For instance, α-synuclein assemblies into amyloid fibrils that accumulate as Lewy bodies particularly in the substantia nigra of PD patients, while in ALS patients TDP-43 or FUS misfold to form cytoplasmic aggregates in degenerating motor neurons. In each of these neurodegenerative disorders, mechanisms maintaining protein homeostasis or proteostasis fail to prevent the accumulation of misfolded proteins, consequently leading to disease.
Proteostasis is critical to ensure cellular functions and under normal conditions these regulatory mechanisms tightly control the rate of protein synthesis, folding, and degradation. Several studies demonstrate that with ageing, the ability of many cells and organs to preserve protein homeostasis is gradually compromised and the physiological deterioration of the proteostasis networks with age is an important aggravating factor for neurodegenerative diseases (reviewed in references1,2,3). The fact that the protein quality control and the cellular response to unfolded protein stress are compromised with age suggests that protein misfolding and aggregation could be a general consequence of aging. Indeed, we and others have demonstrated that protein aggregation is not restricted to disease and instead part of the proteome becomes highly detergent-insoluble in aged animals4,5,6,7,8,9,10. Computational and in vivo analysis revealed that these physiological age-related aggregates resemble disease aggregates in several aspects5. The discovery of endogenous, age-dependent protein aggregation gives us the opportunity to dissect the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. At present, only limited information exists about the regulation of widespread protein insolubility and about the effects of this dysregulation on the health of the organism.
The nematode C. elegans is one of the most extensively studied model organisms in aging research as these animals have a relatively short lifespan and show many characteristic aging features observed in higher organisms. The effects of aging on protein insolubility have been studied in C. elegans by sequential biochemical fractionation based on differential solubility, which is widely used to extract disease aggregates in the field of neurodegeneration research11. By quantitative mass spectrometry, several hundred proteins were shown to become aggregation-prone in C. elegans in the absence of disease5. Here we describe in detail the protocol to grow large numbers of worms in liquid culture and the sequential extraction to isolate aggregated proteins for quantification by mass spectrometry and analysis by Western blot. Because misfolded and aggregation-prone proteins accumulate in aged C. elegans gonads and masks changes in other somatic tissues5,12,13, we use a gonad-less mutant to focus the analysis on protein insolubility in non-reproductive tissues. The method presented enables the analysis of highly-insoluble, large aggregates that are insoluble in 0.5% SDS and pelleted by relatively low centrifugal speed. Alternatively, a less stringent extraction protocol to collect also smaller and more soluble aggregates has been published elsewhere10. In addition, we describe the method used to assess aggregation in vivo in C. elegans.
Overall, these methods in combination with RNA interference (RNAi) can evaluate the role of a gene of interest in modulating age-dependent protein aggregation. For this we describe the analysis of extracts from young and aged worms with and without knockdown of a specific protein of interest using RNAi. These methods should be a powerful tool to determine which components of the proteostasis network regulate protein insolubility. Several interventions such as reduced insulin/insulin-like growth factor (IGF) 1 signaling (IIS) have been shown to dramatically delay C. elegans aging14. Longevity pathways often induce protein-quality control mechanisms and thus these pathways could be actively influencing the rate of protein aggregation. As an example, we demonstrate reduced inherent protein aggregation in long-lived animals upon inhibition of the IIS pathway7.
NOTE: For a better understanding of the procedure, a schematic of the workflow (Figure 1) is attached.
1. Growth of Large Numbers of Young and Aged C. elegans Subjected to RNAi Targeting a Gene of Interest
NOTE: Use C. elegans temperature-induced sterile gon-2(q388) mutants (CF2253) to obtain large aged-synchronized populations. During all steps, it is important to work under semi-sterile conditions with an open flame and to check that no contaminations (for example, with fungi or bacteria) are present. Perform the steps (also centrifugations) at room temperature if the temperature is not described.
2. Insoluble Protein Extraction with Young and Aged Animals Subjected to RNAi Targeting a Gene of Interest
3. Comprehensive Identification and Quantification of Changes in Protein Insolubility with Age Induced by RNAi Targeting a Gene of Interest
4. In Vivo Evaluation of the Influence of a Gene of Interest on the Aggregation Pattern During Aging
We used the methods presented here to evaluate how long-lived animals with reduced IIS modulate age-dependent protein aggregation. By Western blot (see step 2.2, Quick insoluble protein extraction for Western blot analysis), we analyzed the total and the insoluble protein content of young (day 3 of adulthood) and aged (day 18 of adulthood) worms on control RNAi and on RNAi targeting the insulin/IGF-1-like receptor daf-2. We observed no large changes in total protein levels with age or between control and daf-2 RNAi conditions (Figure 2A). As expected, we detected a strong age-related increase in levels of highly-insoluble proteins in control animals (Figure 2B). In comparison in the long-lived animals on daf-2 RNAi, aggregation propensity was greatly reduced. Whole protein staining of the insoluble extracts revealed a less complex protein band pattern in extracts from 18-day-old worms on daf-2 RNAi compared to age-matched controls. Quantitative mass spectrometry analysis demonstrated that long-lived animals with reduced IIS have overall less age-associated protein aggregation and importantly, daf-2 inhibition completely abrogated the age-dependent insolubility of a specific subset of proteins7. To follow-up on these findings, we focused on one of these aggregation-prone proteins, PAB-1. PAB-1 is an RNA-binding protein with a predicted "prion-like" domain. Antibody staining confirmed that long-lived animals maintained PAB-1 soluble with age (Figure 2C).
For in vivo analysis of PAB-1 aggregation, we produced transgenic animals expressing tagRFP::PAB-1 in the pharyngeal muscles. In young animals, tagRFP::PAB-1 was mainly diffusely located throughout the pharynx (Figure 3A, "low" aggregation level) but with age, we observed a progressive accumulation in bright puncta starting in the posterior pharyngeal bulb (Figure 3A, "intermediate" aggregation level). During aging, we also observed aggregation in the anterior pharyngeal bulb (Figure 3A, "high" aggregation level) in an increasing number of animals. By grouping animals into these different categories, we scored the rate of PAB-1 aggregation in a population. At day 2 of adulthood most of the worms (88%) had none or very few puncta and no animals with high aggregation levels were detected. Already at day 5, 19% of the population showed intermediate and 16% high aggregation levels, while at day 7 more than 70% of the worms presented intermediate and high aggregation levels. To analyze the effect of longevity on the age-dependent aggregation of tagRFP:PAB-1, we generated transgenic tagRFP::PAB-1 worms with a daf-2 mutation. These animals showed significantly reduced tagRFP:PAB-1 puncta formation with aging, with less than 15% of the population exhibiting intermediate and high aggregation levels even at day 7 (Figure 3B).
By using the different approaches described here, we revealed that reduced IIS prevents age-dependent protein aggregation to different extents and long-lived animals are particularly efficient in restoring the dynamics of PAB-17.
Figure 1: Schematic of the workflow to study changes in inherent protein aggregation with age in C. elegans. Main steps are bold. Please click here to view a larger version of this figure.
Figure 2: Protein insolubility increases with age in control animals, but not in animals with reduced daf-2 signaling. (A) Total protein staining of total fraction from day 3 and day 18 gon-2(-) mutants subjected to control and daf-2 RNAi (grown at 25 °C). (B) Total protein staining of SDS-insoluble fraction from day 3 and day 18 gon-2(-) mutants subjected to control and daf-2 RNAi (grown at 25 °C). Panel republished from reference7. (C) Immunoblot detecting PAB-1 in SDS-insoluble fraction, relevant protein bands indicated by red arrow. Panel republished from reference7. Please click here to view a larger version of this figure.
Figure 3: Reduced daf-2 signaling prevents the accumulation of tagRFP::PAB-1 puncta with age. (A) Representative pictures of worms with tagRFP::PAB-1 overexpression in the pharyngeal muscles (high magnification microscope, tagRFP detection settings: excitation 558 nm, emission 583 nm). Animals with low (0-10 tagRFP::PAB-1 puncta in posterior pharyngeal bulb), intermediate (more than 10 puncta in the posterior pharyngeal bulb), or high aggregation levels (more than 10 puncta in the anterior pharyngeal bulb) are shown. Scale bar = 20 µm. (B) Quantification of different tagRFP::PAB-1 aggregation levels with age in wildtype and daf-2 mutant background reveals strongly delayed tagRFP::PAB-1 aggregation with age in long-lived animals. Day 2, day 5, and day 7 daf-2(-) versus wildtype background, low compared to intermediate and high aggregation levels: Fisher's exact test, ****p <0.0001. Panel republished from reference7. Please click here to view a larger version of this figure.
Stock solution | Amount | Final concentration | Comments/Description |
Liquid culture, total volume 300 mL | |||
S basal (For 500 mL: 5.9 g NaCl, 50 mL 1 M Potassium phosphate pH 6) | 200 mL | 1 M Potassium phosphate pH 6: For 1 L: 129 g KH2PO4 monobasic, 52 g K2HPO4 dibasic anhydrous | |
1 M Potassium citrate pH 6 (For 400 mL: 13.1 g citric acid monohydrate, 134.4 g tri-potassium citrate monohydrate) | 3 mL | 10 mM | |
Trace metals solution (For 1 L: 1.86 g Ethylenediaminetetraacetic acid (EDTA), 0.69 g FeSO4 · 7 H2O, 0.2 g MnCl2 · 4 H2O, 0.29 g ZnSO4 · 7 H2O, 0.025 g CuSO4 · 5 H2O) | 3 mL | Store stock solution in the dark at 4 °C | |
1 M MgSO4 (For 500 mL: 123.3 g) | 900 µL | 3 mM | |
1 M CaCl2 (For 500 mL: 73.5 g) | 900 µL | 3 mM | |
200 µg/mL Carbendazim (For 50 mL: 10 mg in Methanol) | 150 µL | 100 ng/mL | |
5 mg/mL Cholesterol (For 100 mL: 0.5 g in Ethanol) | 300 µL | 5 µg/mL | |
Reassembly (RAB) high-salt extraction buffer, 30 mL | |||
1 M 4-Morpholineethanesulfonic acid (MES) | 3 mL | 100 mM | RAB stock buffer component |
0.5 M Ethyleneglycoltetraacetic acid (EGTA) | 60 µL | 1 mM | RAB stock buffer component |
0.5 M Ethylenediaminetetraacetic acid (EDTA) | 6 µL | 0.1 mM | RAB stock buffer component |
1 M MgSO4 | 15 µL | 0.5 mM | RAB stock buffer component |
5 M NaCl | 4.5 mL | 750 mM | RAB stock buffer component |
1 M Sodium fluoride (NaF) | 600 µL | 20 mM | RAB stock buffer component |
Complete to 30 mL total with ddH2O | |||
50x Protease Inhibitor Cocktail | * | 2x | *Add reagents after taking the amount of RAB stock buffer that is needed (for the extraction for mass spectrometry 6 mL). |
100 mM Phenylmethylsulfonyl fluoride (PMSF) in isopropanol, -20°C | * | 1 mM | |
10 U/µL DNaseI | * | 200 U/mL | |
4 mg/mL RNaseA | * | 100 µg/mL | |
RAB with 1 M sucrose, 30 mL | |||
2 M sucrose (For 50 mL: 34.2 g) | 15 mL | 1 M | Add the RAB buffer reagents besides DNaseI and RNaseA |
Radioimmunoprecipitation assay (RIPA) buffer, 30 mL | |||
1 M Tris(hydroxymethyl)aminomethane (Tris) pH 8 | 1.5 mL | 50 mM | RIPA stock buffer component |
5 M NaCl | 0.9 mL | 150 mM | RIPA stock buffer component |
0.5 M EDTA | 0.3 mL | 5 mM | RIPA stock buffer component |
10 % Sodium dodecyl sulfate (SDS) | 1.5 mL | 0.50% | RIPA stock buffer component |
10 % Sodium deoxycholate (SDO) | 1.5 mL | 0.50% | RIPA stock buffer component |
Nonylphenylpolyethylenglycol (NP40) | 0.3 mL | 1% | RIPA stock buffer component |
Complete to 30 mL total with ddH2O | |||
100 mM PMSF | * | 1 mM | *Add reagents after taking the amount of RIPA stock buffer that is needed (for the extraction for mass spectrometry 10 mL). |
50x Protease Inhibitor Cocktail | * | 1x | |
Urea/SDS buffer, 10 mL | |||
Urea | 4.8 g | 8 M | |
10 % SDS | 2 mL | 2% | |
Dithiothreitol (DTT) | 77 mg | 50 mM | |
1 M Tris pH 8 | 0.5 mL | 50 mM | |
Complete to 10 mL total with ddH2O | |||
Dialysis buffer, 1 L | |||
Tris | 6 g | 50 mM | |
DTT | 154 mg | 1 mM | |
100 mM PMSF | 1 mL | 0.1 mM | |
Adjust to pH 7.5 and complete to 1 L total with ddH2O |
Table 1: Buffers for liquid culture, insoluble protein extraction, and dialysis.
Here we report a methodology to isolate highly-insoluble protein aggregates from aging C. elegans subjected to RNAi for analysis by mass spectrometry and Western blotting. We show that improving proteostasis by reducing IIS greatly prevents age-dependent protein aggregation. By selecting specific aggregation-prone proteins to overexpress in C. elegans, it is possible to dissect further the mechanisms modulating inherent protein aggregation.
Inherent Age-dependent Protein Aggregation Versus Disease-associated Protein Aggregation
This current work is based on the previous findings that protein aggregation is not restricted to specific proteins aggregating in a disease context. By focusing on endogenous aggregation-prone proteins rather than ectopically expressed disease-associated proteins, the expectation is to provide novel insight into physiological regulation of the aggregation process. As age-dependent protein aggregates are found in different cellular locations5, their study should also provide insight into compartment specific quality-control mechanisms. Overall, the mechanisms that control inherent protein aggregation could also be relevant in preventing disease protein aggregation. Of note, although age-dependent protein aggregation resembles in several aspects disease protein aggregation, it still remains unclear whether these aggregates contain amyloid fibrils, a characteristic of many disease aggregates.
Choice of C. Elegans Model: Advantages and Limitations
Compared to a mammalian model, one of the greatest advantages of using C. elegans to study an aging-related characteristic is its very short lifespan. Several hundred proteins consistently become highly-aggregation-prone during aging and this large increase in insolubility is easily detectable even with a simplified biochemical extraction. However, a major limitation of using C. elegans for studying age-dependent protein aggregation is the necessity to isolate large numbers of aged animals. As C. elegans are hermaphrodites and produce approximately 220 or more progeny during their reproductive phase19, it is essential to prevent reproduction or eliminate progeny in order to age a population. Several options are available to induce sterility: One option is to use the chemical fluorodeoxyuridine (FUdR), an inhibitor of DNA synthesis. However, FUdR leads to numerous side-effects including changes in proteostasis and reduced protein aggregation20,21,22. Importantly, FUdR induces genotype-specific responses23. Another option is to use temperature-sensitive sterile mutants. For example, spe-9(hc88), fer-15(b26), fem-1(hc17), glp-1(e2141), and gon-2(q388) have been used previously for mass spectrometry analyses of the age-dependent aggregating proteome5,8.
Yet there are several limitations associated with using these mutants. First, as the sterility is temperature-dependent, it is necessary to grow the animals at 25 °C, which constitutes a mild stress and accelerates aging as well as protein aggregation. Conversely, we have observed that gon-2, glp-1 and fem-1 mutants are longer-lived compared to wild-type when maintained at 25 °C (data not shown). Second, there are specific limitations associated with each type of reproductive defect. One disadvantage of using sperm-defective mutants is the production of unfertilized oocytes (also produced in aged wild-type hermaphrodites). The quality of these oocytes greatly declines with age and they accumulate in the gonad24,25. Quantitative mass spectrometry analysis demonstrates that sperm-defective mutants have several fold higher levels of insoluble proteins accumulating with age compared to gonad-less or germline-deficient mutants. This indicates protein misfolding and aggregation located in the gonad5, in agreement with previous studies12,13 and in vivo experiments revealing aggregated proteins located in the abnormal oocyte clusters5. Therefore, excessive protein aggregation in the gonad would mask more subtle changes in protein aggregation in the somatic tissues. This should also be considered when comparing interventions that act to different extents on reproductive and somatic tissue proteostasis. Therefore, to assess only somatic protein aggregation, we favor using gonad-less gon-2 mutants. An alternative would be to use germline-deficient glp-1 mutants. However, we note that these animals have less inherent protein aggregation with age compared to gon-2 mutants5. This is likely a consequence of improved proteostasis in somatic tissues due to signaling from the germline-defective gonad26,27,28. Another alternative is to use wild-type animals and remove progeny every day by sedimentation. However, this is problematic due to the difficulty of completely removing all progeny.
Another general limitation of using C. elegans is its small size which makes specific tissue extractions challenging. As such whole animal extractions do not provide relevant information related to how different conditions modulate protein aggregation in specific tissues. Of note, this issue could potentially be circumvented by using a recently developed method based on fluorescence-activated cell sorting to isolate specific cells from C. elegans29. Yet it remains to be determined whether this method would be suitable to obtain sufficient material for insoluble protein extraction and whether it would be applicable to aged animals. Alternatively, tissue specific protein aggregation and its regulation can be investigated by additional in vivo experiments. As transgenic animals expressing a fluorescently-labeled aggregation-prone protein of choice can be rapidly generated and as the animals are transparent, it is relatively easy to examine protein aggregation in situ.
Overall, it is essential to follow up the proteomic results with in vivo experiments in a wild-type background. The combination of a biochemical and microscopy-based characterization enables a comprehensive study of protein aggregation and an assessment of the role of genes of interest. The latter is facilitated in C. elegans by RNAi mediated through feeding and the availability of whole genome RNAi libraries. In addition, a large number of characterized mutants exist to confirm results obtained by RNAi or to use instead of RNAi.
Liquid Culture and Biochemical Extraction: Advantages and Limitations
As highly-aggregated proteins make up only a small fraction of total proteins, it is necessary to start the extraction with a large number of animals. With large quantities of insoluble proteins, it is then feasible to perform extensive peptide fractionation to reduce sample complexity for mass spectrometry and thus improve identification of lower abundant proteins. If only small amounts of insoluble proteins are required, an alternative is to grow worms on plates and homogenize them with ceramic beads. One of the disadvantages of liquid culture is that in the case of a contamination, the whole culture is affected and must be discarded. In general, C. elegans aging is strongly influenced by temperature and this parameter must be tightly controlled in the liquid culture to avoid variations in age-dependent protein aggregation. Temperature control is also essential to obtain a homogenous population of gonad-less animals when using the gon-2(-) mutants.
Depending on the goal of the study, it is important to consider different extraction methods. The biochemical extraction presented here was initially adapted from protocols to isolate disease-associated aggregates11,30,31. We chose relatively high concentrations of detergents such as 0.5% SDS to isolate the more insoluble aggregates. Also by pelleting the 0.5% SDS insoluble aggregates with low centrifugal speeds, this protocol would only isolate the larger aggregates. Alternatively, a less stringent protocol was recently published10. In this study, more soluble and smaller aggregates were also extracted by omitting SDS and by using very high centrifugal speeds at 500,000 x g. A comparison of the different protocols shows a general increase in the number of proteins in the aggregating proteome using the less stringent protocol7. We note that long-lived animals with reduced daf-2 signaling efficiently prevented the accumulation of both SDS-soluble and SDS-insoluble aggregates in gonad-less animals7.
In Vivo Analysis of Age-Dependent Protein Aggregation
When constructing transgenic models for following age-dependent protein aggregation, it is relevant to consider in which tissue to overexpress the aggregation-prone protein of interest and at which expression levels. We favor expression in tissues located in the head region to avoid interference with autofluorescence, which is prominent in the aging intestine. To visualize fluorescent-labeled aggregates with low-magnification, the aggregation-prone protein should be expressed at a relatively high level. On the other hand, too high expression will cause the aggregation-prone protein to form aggregates already in young animals.
Taken together, these methods will help us to understand why part of the proteome aggregates with age and ultimately lead to the development of strategies promoting healthy aging.
The authors have nothing to disclose.
This work was supported by funding from the DZNE and a Marie Curie International Reintegration Grant (322120 to D.C.D.)
Fernbach culture flask | Corning | 4425-2XL | Pyrex, Capacity 2,800 ml, with 3 baffle indents |
Membrane Screw Cap | Schott | 1088655 | GL45 |
Nutating Mixer | VWR | 444-0148 | |
Separatory funnel | Nalgene | 4300-1000 | Capacity 1,000 ml |
1 ml syringe | BD Plastipak | 300013 | |
Gray needle, 27 G x ½ ", 0.4 mm x 13 mm | BD Microlance 3 | 300635 | |
Membrane filters 0.025 µM | Millipore | VSWP04700 | |
pH strip | Machery-Nagel | 92110 | pH-Fix 0-14 |
Protease Inhibitor Cocktail | Roche | 4693132001 | Complete Mini EDTA-free tablets |
Octoxynol-9 | Applichem | A1388 | Triton X-100 |
4-Morpholineethanesulfonic acid (MES) | Sigma-Aldrich | M1317 | |
Nonylphenylpolyethylenglycol | Applichem | A1694 | Nonidet P40 (NP40) |
DNaseI | Roche | 04716728001 | recombinant, RNase free |
RNaseA | Promega | A7973 | solution |
Total protein blot staining | Thermofisher | S11791 | Sypro Ruby protein blot stain |
Total protein gel staining | Thermofisher | S12001 | Sypro Ruby protein gel stain |
TCEP (tris (2-carboxyethyl) phosphine hydrochloride) | Serva | 36970 | |
Iodoacetamide | Serva | 26710 | |
Ammoniumbicarbonate | Sigma-Aldrich | 09830 | |
Sequencing Grade Modified Trypsin | Promega | V5111 | |
Isobaric tags for relative and absolute quantitation | Sciex | 4352135 | iTRAQ Reagents Multiplex Kit |
Centrifuge Avanti J-26XP | Beckmann Coulter | 393126 | |
Ultracentrifuge Optima Max-XP | Beckmann Coulter | 393315 | |
Centrifuge 5424R | Eppendorf | 5404000413 | |
Centrifuge 5702 | Eppendorf | 5702000329 | |
Centrifuge Megafuge 40R | Thermo Scientific | 75004518 | |
Concentrator Plus | Eppendorf | 5305000304 | Centrifugal evaporator |
Fluorescent stereo-microscope M165 FC | Leica | With Planapo 2.0x objective | |
Dissection microscope | Leica | Leica S6E | |
High magnification microscope Zeiss Axio Observer Z1 | Zeiss | With PlanAPOCHROMAT 20x objective and Zeiss Axio Cam MRm | |
Software | |||
Image analysis software | ImageJ | ||
Analysis of mass spectrometry data | Protein Prospector | http://prospector.ucsf.edu/prospector/mshome.htm | |
E.coli strain | |||
OP50 | CGC | ||
RNAi bacteria | |||
L4440 | Julie Ahringer RNAi library | ||
C. elegans mutants | |||
CF2253 | CGC, strain name: EJ1158 | Genotype: gon-2(q388) | |
C. elegans transgenics | |||
DCD214 | Della David's lab at DZNE Tübingen | Genotype: N2; uqIs24[Pmyo-2::tagrfp::pab-1] | |
DCD215 | Della David's lab at DZNE Tübingen | Genotype: daf-2(e1370) III; uqIs24[Pmyo-2::tagrfp::pab-1] | |