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Method Article
A step-by-step guide to probe loss of lysosomal acidity in the intestine of C. elegans using the pH-sensitive vital dye 5(6)-carboxy-2',7'-dichlorofluorescein diacetate (cDCFDA)
The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are facilitated by the transparency of the animal, the ability to do forward and reverse genetic assays, the relative ease of generating fluorescently labeled proteins, and the use of fluorescent dyes that can either be microinjected into the early embryo or incorporated into its food (E. coli strain OP50) to label cellular organelles (e.g. 9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1'H-2,2'-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron). Here, we present the use of a fluorescent pH-sensitive dye that stains intestinal lysosomes, providing a visual readout of dynamic, physiological changes in lysosomal acidity in live worms. This protocol does not measure lysosomal pH, but rather aims to establish a reliable method of assessing physiological relevant variations in lysosomal acidity. cDCFDA is a cell-permeant compound that is converted to the fluorescent fluorophore 5-(and-6)-carboxy-2',7'-dichlorofluorescein (cDCF) upon hydrolysis by intracellular esterases. Protonation inside lysosomes traps cDCF in these organelles, where it accumulates. Due to its low pKa of 4.8, this dye has been used as a pH sensor in yeast. Here we describe the use of cDCFDA as a food supplement to assess the acidity of intestinal lysosomes in C. elegans. This technique allows for the detection of alkalinizing lysosomes in live animals, and has a broad range of experimental applications including studies on aging, autophagy, and lysosomal biogenesis.
The appearance of protein aggregates is widely accepted to be a hallmark of aging in eukaryotic cells1,2,3, and the formation of which is thought to be among the principle drivers of cellular senescence4,5,6,7. There is growing evidence that as cells age, protein catabolism is impaired, leading to an increase in protein aggregation. The collapse of proteolysis in aging cells involves an impairment of autophagy8 as well as proteasome-mediated protein degradation9. Finally, irreversible protein oxidation is increased in old cells, further impairing protein catabolism10.
Autophagy was initially thought to be a non-selective process for bulk degradation of damaged proteins, but recent studies have indicated that autophagy is highly selective to the catabolism of protein aggregates and dysfunctional organelles that are not amenable to degradation via other protein clearance mechanisms11. During the process of autophagy, damaged and aggregated proteins are sequestered into a double-membrane vesicle called the autophagosome. This autophagosome then fuses with the acidic organelles called lysosomes, which leads to degradation of the autophagosome cargo12. Lysosomes represent the end-point of the autophagic pathway and participate in different cellular processes such as membrane repair, transcriptional control and nutrient sensing; highlighting their centralized role in cellular homeostasis (reviewed in ref. 13). Several studies have shown an association between an age-dependent decrease in lysosomal function and various neurodegenerative disorders13. Consistently, restoring lysosomal function in older cells can delay the onset of aging-related phenotypes14,15. Studies of the composition of the intralumen milieu suggest that the collapse of lysosomal function in older cells is not due to a reduction in the production of lysosomal proteases16. Alternatively, it has been proposed that loss of intralysosomal acidity, a critical requirement of its enzymatic activity, may underlie the drop in lysosome-mediated proteolysis17. To be able to explore this hypothesis, it is essential to develop reagents and protocols to probe dynamic changes in lysosomal pH in live cells in a replicable and consistent manner.
The intestine of C. elegans is the major metabolic tissue in worms and it is a critical regulator of systemic homeostasis and lifespan. We have developed assays to evaluate changes in the acidity of the lumen of intestinal lysosomes of worms to determine how lysosome-mediated proteolysis contributes to aging. Though pH-sensitive fluorophores have been used previously in C. elegans to mark intestinal lysosomes, there hasn't yet been an effort to establish a successful protocol that can detect small increases in lysosomal pH in vivo18. Here, we provide a protocol that can be used to detect loss of lysosomal acidity in the intestinal cells of C. elegans using a simple and convenient feeding protocol that incorporates a pH-sensitive fluorophore (cDCFDA) into OP50 food.
1. Stain and Image intestinal lysosomes
cDCFDA stains lysosomes in a pH-dependent manner, and its low pKa and ready uptake into lysosomes makes it an ideal pH sensor21. cDCFDA staining intensity is inversely proportional to lysosomal pH (i.e. staining intensity increases as pH decreases)18,22. cDCFDA signals are consistently weak in lysosomes of animals treated with 20 mM of chloroquine, an inhibitor of lysosomal acidification, and in wo...
A variety of cellular and molecular events contribute to aging, influenced by life history traits and genetic factors. Our recent study22 suggests that the reproductive cycle plays an important role in controlling the fitness of the soma through the regulation of lysosomal pH dynamics. We showed that lysosomal-mediated proteolysis is promoted while animals actively reproduce by upregulation of v-ATPase transcription, which in turn ensures acidic lysosomes. Upon the end of reproduction, v-ATPase ex...
The authors declare no conflict of interest.
We would like to thank the Caenorhabditis Genetics Center for strains, the Natural Sciences and Engineering Research Council (NSERC), and the Canada Foundation for Innovation (CFI) for funding. We would like to thank Dr. Lizhen Chen (Department of Cell Systems and Anatomy, UT Health San Antonio) for allowing unrestricted use of her lab facilities for all C. elegans experiments as well as Dr. Exing Wang (Associate Director, Optical Imaging Facility, UT Health San Antonio) for assistance with confocal microscopy. We would also like to thank Dr. Myron Ignatius for providing support and encouragement to facilitate the video shoot.
Name | Company | Catalog Number | Comments |
OP50 (E. coli) | Caenorhabditis Genetics Center | Order online at https://cgc.umn.edu/strain/OP50 | |
5(6)-carboxy-2’,7’-dichlorofluorescein diacetate | ThermoFisher | C369 | Commonly known as cDCFDA |
9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1'H-2,2'-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron | ThermoFisher | L7528 | Commonly known as Lysotracker Red |
Confocal microscope (e.g. Zeiss LSM 510) | |||
ImageJ | Download for free from https://imagej.nih.gov/ij/download.html | ||
LB Broth powder | ThermoFisher | 22700041 | |
Bacto Agar | Sigma | A5306-1KG | |
NaCl | Sigma | S9888 | |
Bacto Peptone | Fisher Scientific | S71604 | |
Cholesterol powder | Sigma | C3045 | |
CaCl2 | Sigma | 449709 | |
MgSO4 | Sigma | M7506 | |
K3PO4 | Sigma | P5629 | |
Sodium Azide | Sigma | S2002 | |
DMSO | Sigma | D8418 | |
Microscope Slides | VWR | 48311-703 | |
Cover Slips | ThermoFisher | 3406 | |
Agarose | Sigma | A6013 | |
Incubator | |||
Mirror or other smooth flat surface |
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