Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

Summary

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)

Abstract

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.

Introduction

The appearance of protein aggregates is widely accepted to be a hallmark of aging in eukaryotic cells^1,2,3, and the formation of which is thought to be among the principle drivers of cellular senescence^4,5,6,7. 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 phenotypes^14,15. 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.

Protocol

1. Stain and Image intestinal lysosomes

1. Seed nematode growth media (NGM) plates with OP50
   1. Prepare NGM plates as per the recommended protocol¹⁹ and allow the closed plates to dry for 2 days at room temperature.
   2. Inoculate OP50 bacteria into sterile Luria Broth (LB) broth and grow in a shaking incubator or water bath at 37 °C for 36 h or until the OD is between 0.2 and 0.4. Avoid using a bacterial culture with OD >1 or OD <0.2.
   3. Once NGM plates are dry, place a drop (~30 µL) of OP50 inoculum onto the center of the plate, and then spread the drop into a patch roughly that is 2 cm x 2 cm in size.
      NOTE: Any remaining OP50 inoculum can be stored at 4 °C for up to a month.
   4. Invert the OP50 plates once the inoculum has dried (roughly 10 or 15 min), and then incubate the plates at 37 °C for 36 h.
   5. After 36 h, remove the plates from the incubator and store at 4 °C until later use.
      NOTE: The plates should be good for up to 1 month at 4 °C.
2. Supplementing cDCFDA to OP50
   1. To supplement cDCFDA onto OP50 plates, prepare a working stock of 10 mM cDCFDA in dimethyl sulfoxide (DMSO). Keep the cDCFDA solution protected from light and store at -20 °C for later use.
   2. Gently place 100 µL of 10 mM cDCFDA solution onto the surface of the 2 cm x 2-cm OP50 patch, such that the entire patch is uniformly covered with cDCFDA.
      NOTE: It is very important that the entire patch of OP50 is covered with cDCFDA. If required, use up to 150 µL of cDCFDA for each plate. It is also imperative that the cDCFDA solution does not extend beyond the borders of the OP50 patch, since any cDCFDA that flows out of the OP50 patch will not be incorporated into the food, and will result in reduced staining intensity.
   3. Place the OP50 plates in a dark box on the bench top until all of the cDCFDA solution is absorbed into the bacterial patch (usually about 25 to 30 min).
3. Staining worms using cDCFDA
   1. Once the cDCFDA has completely permeated the OP50 patch, place no more than 20 worms per plate and incubate the plates inverted at 20 °C for a minimum of 14 h.
      NOTE: It is recommended to place the cDCFDA plates in an opaque box to minimize exposure to light.
   2. To control for intake differences between experiments and to normalize cDCFDA signals, (recommended) co-stain with 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 (2.5 µL of a freshly made 1 mM solution), an acidotropic weak amine dye whose fluorescence is largely non-variant over the acidic pH spectrum (see Table of Materials for common names of reagents).
      NOTE: Do not stain worms for less than 14 h since this may provide inadequate cDCFDA staining intensity and give a false interpretation of lysosomal pH.
4. Preparing slides for microscopy
   1. Place two parallel strips of labeling tape about 3 inches apart on a smooth flat surface, such as a mirror (Figure 1).
   2. Coat the surface of the mirror with water repellent spray, and wipe surplus liquid off.
   3. Melt 2% agarose (dissolved in distilled H₂O) in a microwave until completely liquefied, then place a 50 µL drop of agarose between the two strips of tape and quickly cover the drop with a microscope slide, such that the slide is perpendicular to the strips of tape. After the agarose has solidified, forming a circular pad under the slide, flip the slide over and add 10 to 15 µL of 5mM sodium azide (NaN₃) onto the agarose pad.
      NOTE: NaN₃ is a cytochrome C inhibitor that when used in low concentrations, reversibly anesthetizes worms so that they will not move during imaging.
   4. Pick worms from the OP50 plate supplemented with cDCFDA, and transfer them to a clean NGM plate without any OP50. Allow the worms to move about for a few seconds to allow most of the OP50 to be removed from the surface of the worms, and then transfer the worms to the agarose pad containing 5 mM sodium azide.
   5. Immediately cover the agarose pad with a cover slip. Be sure to gently place the cover slip without applying too much pressure, since this can result in worms bursting.
      NOTE: OP50 bacteria supplemented with cDCFDA fluoresce when visualized by microscopy, hence it is important to clean the worms as best as possible before placing them on the agarose pad containing sodium azide. If prepping more than 6 strains of C. elegans (including N2, wild type control), it is advisable to prepare the samples in batches of 6, so as to ensure that the worms do not dry out on the agarose pad. In some strains, the vulva starts to rupture after extended periods of time due to pressure from the cover slip, so it is important to plan accordingly.
5. Confocal microscopy
   NOTE: Some microscopes have a built-in feature that automatically increases fluorescence intensity to compensate for variable levels of fluorescence. Such microscopes might not work well for imaging worms stained with cDCFDA, since they will inherently increase the fluorescence intensity of samples.
   1. Perform imaging on a standard confocal microscope using excitation/emission wavelengths set to 488/530 nm for cDCFDA. Take single plane images (not Z-stacks) of intestinal lysosomes and use only lysosomes that are in focus (maximal signal intensity) for intensity quantification.
      NOTE: Perhaps because of differences in dye availability, lysosomes of the intestinal cells directly posterior to the pharynx maintain cDCFDA staining intensity irrespective of genotype or age, hence care should be taken to avoid imaging lysosomes in these cells.
   2. Image the slide containing 2 day old wild type N2 worms first. While doing so, adjust the confocal imaging parameters such as laser power, pinhole, and aperture to ensure that the cDCFDA staining intensity is not oversaturated.
      NOTE: Once these settings are set, do not change them for the entire duration of the imaging for that day.
   3. Capture 1024 x 1024 pixel images of a single plane of intestine (3 to 4 images per worm) using a 60X magnification lens.
      NOTE: The cDCFDA emission spectrum in worms will yield a single prominent peak at 520 nm coinciding with its reported fluorescence spectrum²⁰, providing a specific signal readout that is distinct from intestinal autofluorescence (Figure 3).
   4. Export raw confocal image (.lsm files) as .tiff files for each channel.
   5. Next, open ImageJ and click on File| Open to open the image file to be quantified. Once the image loads, use the oval shape selection tool in ImageJ to select a region of interest (ROI).
   6. Thereafter, click on Analyze| Measure to quantify fluorescence intensity for that ROI. Select 4–5 different in-focus lysosomes per image and calculate the average relative fluorescence intensity of each region of interest (ROI).
   7. For each image, measure the background fluorescence intensity in a region of the intestine between lysosomes. Subtract the background fluorescence intensity values from the lysosomal fluorescence intensity values.
   8. In total, collect around 30 to 50 individual normalized values for cDCFDA intensity (6 to 10 animals) for each strain being tested. Use these values to plot a box and whisker plot using any appropriate statistical software.
      NOTE: Staining can vary considerably depending on factors like temperature, incubation time and the overall health/age of animals such that fluorescence intensity comparisons are only relevant within samples process in the same staining experiment. It is therefore important to process controls in every staining experiment. Calculate statistical differences (t-tests) using any appropriate statistical software.
   9. Image all strains from the same experiment (stained on the same day) in sequence, taking care not to change any of the imaging parameters.

Results

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...

Discussion

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...

Materials

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