Current methods to analyze lysosome morphology, positioning, motility and function

Abstract

Since the discovery of lysosomes more than 70 years ago, much has been learned about the functions of these organelles. Lysosomes were regarded as exclusively degradative organelles, but more recent research has shown that they play essential roles in several other cellular functions, such as nutrient sensing, intracellular signalling and metabolism. Methodological advances played a key part in generating our current knowledge about the biology of this multifaceted organelle. In this review, we cover current methods used to analyze lysosome morphology, positioning, motility and function. We highlight the principles behind these methods, the methodological strategies and their advantages and limitations. To extract accurate information and avoid misinterpretations, we discuss the best strategies to identify lysosomes and assess their characteristics and functions. With this review, we aim to stimulate an increase in the quantity and quality of research on lysosomes and further ground-breaking discoveries on an organelle that continues to surprise and excite cell biologists.

Keywords: TFEB; endolysosomes; lysosomal storage diseases; lysosome biogenesis; lysosome exocytosis; lysosome-related organelles; lysosomes; mTOR; membrane contact sites.

FIGURE 1

Transmission electron micrographs of electron‐dense lysosomes containing endocytosed gold particles. Primary porcine retinal pigment epithelial cells were incubated with bovine serum albumin (BSA)‐gold for 2 hours, followed by overnight chase to load lysosomes. Gold particles aggregate in the acidic environment of the lysosome after degradation of the BSA. Lysosomes are electron dense and sometimes contain membrane whorls. Scale bar, 200 nm

FIGURE 2

Lysosome heterogeneity and the lysosome cycle. Lysosomes accumulate near the nucleus, where they fuse with endosomes, phagosomes or autophagosomes to generate highly acidic degradative organelles (endolysosomes, phagolysosomes or autolysosomes). A process of lysosome reformation then occurs and can involve tubulation and budding to form less acidic protolysosomes, which may require replenishing with lysosomal enzymes. Lysosomes are also found peripherally, where they tend to be smaller and less acidic, and can be stimulated to fuse with the plasma membrane during lysosome exocytosis

FIGURE 3

Dextran assay. Representative high content images obtained with Opera (40x water objective) of (A) uptake kinetics (from 5 to 20 minutes) in HAP‐1 cells (cell line derived from chronic myelogenous leukaemia) loaded with Alexa Fluor 568‐dextran (Thermo Fisher, D22912) (in red) and (B) uptake kinetics (from 30 minutes to 3 hours) in mouse embryonic fibroblasts (MEF). Nuclei stained with Hoechst are shown in blue. Scale bar, 20 μm

FIGURE 4

DQ‐BSA assay. Representative high content images of DQ‐BSA puncta in ARPE‐19 human retinal pigment epithelial cells treated with DMSO or Torin‐1 (1 μM for 3 hours) and incubated for 16 hours with 10 μg/mL of DQ‐BSA. Lower row images show the same cells subjected to analysis for the selection of DQ‐BSA puncta (selected spots traced in green; discarded spots with very low fluorescent intensity traced in red). More details in Data S1. Scale bar, 20 μm

FIGURE 5

Transmission electron micrographs showing lysosome membrane contact sites (MCS). (A) Lysosome:ER MCS in HeLa cells. (B) Lysosome: mitochondria MCS in HeLa cells incubated with horseradish peroxidase for 6 hours prior to incubation with DAB and preparation for EM. Orange arrows, MCS. Scale bars, 200 nm

FIGURE 6

Globotriaosylceramide (Gb3) accumulation in HeLa cells knockout (KO) for α‐galactosidase, treated with 1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol (PDMP). Gb3 lipid accumulation in GLA KO Hela cells treated with PDMP (glucosylceramide synthase inhibitor) or DMSO, as a control. This read‐out has been used to develop a Gb3 lipid accumulation assay. PDMP was used as a positive control for Shiga toxin (STX). ***P ≤ .0001, as determined by Student's t‐test. Scale bar, 20 μm

FIGURE 7

Lysosomal morphometric assay. Results were obtained by the quantitative high content image analysis of normal ARPE‐19 human retinal pigment epithelial cells (WT) and ARPE‐19 Niemann‐Pick C (NPC) 1 KO cells generated by genome editing, as a model of NPC1, a lysosomal storage disease in which lysosomes accumulate cholesterol. The increase in the number of lysosomes and the percentage of cells showing lysosomal aggregation was quantified in NPC1 KO cells and compared to WT cells. Scale bar, 20 μm

FIGURE 8

Intracellular galectin 3 (GAL3) accumulation upon treatment with LLOMe. Human ARPE‐19 retinal pigment epithelial cells were stained for GAL3 using a specific antibody (green). Untreated cells show no GAL3 puncta, whereas after 30 minutes of treatment with LLOMe (a potent inducer of lysosome membrane permeabilization), the number of GAL3 spots increase, indicating accumulation. Washout reverts the GAL3 accumulation. The graphs show the number of GAL3 spots per cell. Scale bar, 20 μm

FIGURE 9

Lysosomal permeabilization assay. In control conditions (CTRL), Acridine Orange stains lysosomes (red puncta), while the treatment with concanamycin A (Conc. A, a specific inhibitor of v‐ATPase activity; 100 nM, 1 hour) dramatically reduces lysosomal staining. With the change in lysosomal pH, there is also an increase in green intensity. The plots show the number of red spots per cell, and the intensity ratio between red and green emission. The assay is developed with live cells and in order to perform a high content analysis, a stable HeLa cell clone expressing the histone protein H2B fused with cyan fluorescent protein (CFP; blue) to detect the nuclei (n = 500 cells per condition) is used. Scale bar, 20 μm