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 pro....

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 th....

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

List of materials used in this article

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