A Photodynamic Approach to Study Function of Intracellular Vesicle Rupture

Yu-Hsien Hung; Hsiu-Jung Wang; Jen-Shuang Wang; Chia-Hao Hsu; Huan-Yuan Chen

doi:10.3791/63962

JoVE Journal > Immunology and Infection

Imunologia e Infecção

A Photodynamic Approach to Study Function of Intracellular Vesicle Rupture

Published: March 17, 2023

doi:

10.3791/63962

Yu-Hsien Hung¹, Hsiu-Jung Wang¹, Jen-Shuang Wang¹, Chia-Hao Hsu¹, Huan-Yuan Chen¹

¹Inflammation Core Facility, Institute of Biomedical Sciences,Academia Sinica

Summary

AlPcS_2a-mediated chromophore-assisted laser inactivation (CALI) is a powerful tool for studying spatiotemporal damage of intracellular vesicles (IVs) in live cells.

Abstract

Intracellular vesicles (IVs) are formed through endocytosis of vesicles into cytoplasm. IV formation is involved in activating various signal pathways through permeabilization of IV membranes and the formation of endosomes and lysosomes. A method named chromophore-assisted laser inactivation (CALI) is applied to study the formation of IVs and the materials in controlling IV regulation. CALI is an imaging-based photodynamic methodology to study the signaling pathway induced by membrane permeabilization. The method allows spatiotemporal manipulation of the selected organelle to be permeabilized in a cell. The CALI method has been applied to observe and monitor specific molecules through the permeabilization of endosomes and lysosomes. The membrane rupture of IVs is known to selectively recruit glycan-binding proteins, such as galectin-3. Here, the protocol describes the induction of IV rupture by AlPcS_2aand the use of galectin-3 as a marker to label impaired lysosomes, which is useful in studying the downstream effects of IV membrane rupture and their downstream effects under various situations.

Introduction

Endosomes, a type of intracellular vesicle (IV), are formed by endocytosis and then mature into lysosomes. Various intracellular signal pathways are involved in the formation of IVs; additionally, different intrinsic and extrinsic stimuli can damage IVs (e.g., pathogens can escape from the bounded membrane during infection and enter into cytoplasm¹). This is usually accompanied by the rupture of endocytotic vesicles². Therefore, the techniques for targeting and damaging IVs can be used in related studies³.

Photodynamic therapy (PDT) is a light-dependent therapy to combat diseases by killing tumors or pathogens⁴. In PDT, targeted cells are labeled with non-toxic chromophores, called photosensitizers, that can be locally activated by light illumination⁵^,⁶. Photosensitizers absorb energy from light and transform into an excited singlet state, leading to the long-lived excited triplet state. Photosensitizers of the triplet state can undergo electron or energy transfer and form reactive oxygen species (ROS) in the presence of oxygen, and can spatially destroy labeled cells within the illumination region⁷. The consequence varies depending on the power of light⁸. By controlling the concentration of photosensitizers and the intensity of light illumination, targeted biomolecules can be selectively inactivated without cell lysis, termed as chromophore-assisted light inactivation (CALI)⁹. With the significant development of photosensitizers that can selectively label various subcellular targets, CALI has become a valuable tool to control light-mediated inactivation of biomolecules for small biomolecules such as nucleotides and proteins, as well as organelles such as mitochondria and endo-lysosomes³^,¹⁰^,¹¹^,¹²^,¹³.

Compared to CALI, chemical or physical methods are also used to impair membranes, such as bacterial toxin¹⁴^,¹⁵ and Leu-Leu-OMe¹⁶ treatment for lysosomal damage. However, these methods display bulk impairment of IVs within cells. Robust photosensitizers (i.e., Al(III) phthalocyanine chloride disulfonic acid (AlPcS_2a)) are used in CALI; AlPcS_2a, targeting the lysosomes through endocytosis, is used to rupture endosomes or lysosomes in a controlled region¹⁷. AlPcS_2a is a cell membrane-impermeable phthalocyanine-based chromophore that binds to lipid on plasma membrane and is internalized through endocytosis and eventually accumulates within the lysosome through the endocytic pathway¹⁸. It absorbs light within a near-infrared spectral region and generates singlet oxygen, a major ROS generated by excited AlPcS_2a¹⁸. Singlet oxygen decaying rapidly limits its diffusion and reaction distance within a tiny region in cells (approximately 10-20 nm)¹⁹. By adjusting the duration of AlPcS_2a incubation and light illumination, spatiotemporal control of the damage of IVs within a subcellular area is allowed. CALI therefore becomes a powerful tool for examining the consequences of IV damage, and the formation and regulation of IVs.

In this study, a specific protocol of CALI using AlPcS_2aas a photosensitizer is addressed. This protocol can be applied to various types of IVs, including endosomes and lysosomes, and used to examine the follow-up responses after membrane rupture. HeLa cells expressing fluorophore-conjugated galectin-3¹⁶^,²⁰ revealed after lysosome rupture are used to demonstrate this protocol.

Protocol

1. AlPcS_2a stock preparation

Dissolve 10 mg of AlPcS_2a in 400 µL of 0.1 M NaOH. To improve solubility, heat the solution at 50 °C and vortex.
Mix the solution with 4 mL of phosphate-buffered saline (PBS). Then, filter the solution with a 0.22 µm filter to remove the insoluble precipitates.
Measure the concentration of the solution through a UV-Vis spectrophotometer. The extinction coefficient of AlPcS_2a at 672 nm is 4 x 10⁴ cm^-1 M^-1. Dilute the AlPcS_2a solution with PBS to make a 1 mM solution. Make 1 mL aliquots and store at -20 °C.

2. Transfection

NOTE: Gal3-GFP is applied as an indicator for live-cell imaging of lysosomal rupture.

Maintain HeLa cell culture in DMEM supplemented with 10% Fetal Bovine Serum (FBS). After washing the cells with PBS, trypsinize the cells, then resuspend the cells in DMEM supplemented with 10% FBS.
NOTE: The method is applicable for most of the attached cell lines, including HeLa and A549 cell lines. In principle, despite not testing suspension cells, CALI assays could be performed with them.
Dilute 300 ng of Gal3-GFP plasmid in 25 µL of reduced serum medium, and then add 0.6 µL of P3000 reagent. Gently mix. Dilute 0.45 µL of Lipofectamine 3000 reagent in 25 µL of reduced serum medium. Gently mix.
Incubate for 5 min at room temperature. Mix diluted DNA and diluted Lipofectamine 3000, and then incubate for 10 min at room temperature.
Mix the DNA-Lipofectamine mixture, 3 x 10⁵ suspended HeLa cells, and 200 µL of DMEM+10% FBS, and then seed the cells on the central glass region of a 35 mm glass button dish.

3. AlPcS2a staining

To allow cell attachment, incubate the cells at 37 °C in the incubator supplied with 5% CO₂ for at least 4 h.
Dilute AlPcS_2a in DMEM supplemented with 10% FBS to make a 1 µM AlPcS_2a solution. Pre-warm the AlPcS_2a-containing medium in a 37 °C water bath. Replace the culture medium with the AlPcS_2a-containing medium.
Incubate the cells at 37 °C in the incubator supplied with 5% CO₂ overnight (16-18 h).
The following day, wash the cells twice with PBS to remove extracellular AlPcS2a. Replace the medium with fresh medium, and then incubate at 37 °C for 4 h to allow residual dye along the endocytic pathway to accumulate into lysosomes.
NOTE: To stain endosomes, incubate cells in 1 µM AlPcS_2a in DMEM containing 10% FBS for 15 min at 37 °C. Next, wash cells twice with PBS and incubate them in pre-warmed medium before imaging .

Figure 1. A schematic figure representing the selective IV damage with AlPcS_2a. The figure shows the schematics of selective IV damage. Please click here to view a larger version of this figure.

Figure 2. Lysosomal staining with AlPcS2a. Lysosomes labeled overnight with 1 µM AlPcS_2a in HeLa cells are positively stained with 50 nM green fluorescent dye. Scale bar: 10 µm. Please click here to view a larger version of this figure.

4. Sample imaging and light illumination

NOTE: IV damage within a subcellular region of a single cell or all the AlPcS_2a-labeled cells in a culture dish can be performed. Bulk illumination of the whole culture dish allows quantitative study of this damage, including biochemical studies.

Manipulate single cells by damaging lysosomes within a single cell, as described below.
1. Place the culture dish on the stage of a confocal microscope. Use the 488 nm and 561 nm lasers for exciting GFP and AlPcS2a, respectively.
2. Circle a 5 x 5 µm² region of interest (ROI) on the AlPcS_2a-labeled vesicles which are selected to be damaged.
3. Pulse illuminate the ROI with a 633 nm laser of 0.21 mW for 70 repeats. Monitor the formation of Gal3 puncta as an indicator of lysosomal membrane permeabilization.
Damage all labeled cells in a culture dish as described below.
1. Place the culture dish on a platform. Illuminate the dish with 660 nm of collimated LED light (near-infrared light is ideal base on the excitation spectrum of AlPcS2a).
2. Place the culture dish on the microscope and monitor the indicators of membrane permeabilization as mentioned in step 4.1.3.
Assess injury of IVs using the following indicators.
1. The contents of endosome or lysosome are most highly glycosylated, which are exposed to cytosol upon IV rupture. Rapidly label these using cytosolic glycan-binding galectins, including galectin-1, galectin-3, galectin-8, and galectin-9¹⁶^,²⁰.
2. The size of the wound can be determined by the release of membrane-impermeable dye as described in²¹.
3. The difference between endosomes and lysosomes are their luminal pH value. Lysosomal rupture perturbs the luminal pH, which can be measured by using pH-sensitive dye, such as FITC-dextran³.

Figure 3. AlPcS_2a-mediated CALI induces local recruitment of TagRFP-galectin-3 (Gal3) to the lysosomes within the illumination region. Lysosomes in TagRFP-galectin-3-expressed HeLa cells were stained overnight with 1 µM AlPcS_2a, followed by focusing illumination with near-infrared light (633 nm) within the yellow square. AlPcS_2a signal within the yellow square is photobleached, accompanied by the formation of TagRFP-galectin-3 puncta. Scale bar: 10 µm. Please click here to view a larger version of this figure.

Representative Results

A schematic figure representing the AlPcS_2a-induced damage of IV, including endosome and lysosome, has been shown (Figure 1).

Commercially available markers can be used to determine the AlPcS_2a staining conditions. For example, AlPcS_2a puncta and green fluorescent dye²² colocalization (Figure 2).

Fluorophore-labeled galectin-3 can be applied as an indicator for monitoring IV damage (Figure 3). Moreover, the location of Gal3 puncta could also be tracked for assaying the downstream signaling pathway, including lysosome repair and lysophagy³^,²³. The rapid accumulation of Gal3 from cytosol to damaged IV would significantly increase the intensity of Gal3, which would make the intensity of the Gal3 puncta saturated if the contrast of the images is adjusted to show cytosolic Gal3. In fact, the images also showed a slight decrease in cytosolic Gal3.

In summary, these results indicate that AlPcS_2a-based CALI is able to control local lysosomal rupture within the ROI, leaving the rest of the lysosomes intact.

Discussion

AlPcS_2a binds to the plasma membrane, then is internalized by endocytosis and eventually accumulates in lysosomes. AlPcS_2a can thus be localized in the subcellular compartments by adjusting the incubation duration. A limitation of this methodology is that only a sub-population of IVs could be labeled by AlPcS_2a through endocytosis because there are many other membrane sources of IVs, such as ER and Golgi apparatus. In addition, the selective labeling of AlPcS_2a into early or late endosomes would be challenging, however, fluorescent markers can be used to differentiate early-formed endosomes from late-formed ones during subcellular imaging. CALI-induced IV damage can be induced with a variety of dyes³^,²⁴.

The intensity of damage can be controlled by the power and illumination duration of light. Higher intensity of damage may result in cell shrinking or necrosis. Here, several indicators of IV damage have been shown, which could be used to determine the experimental conditions. Additionally, these indicators may also serve as markers for damaged-IVs and provide information about their subcellular distribution over time.

Clinically, AlPcS_2a is designed to improve the delivery of therapeutic agents in a site and time-specific manner through photochemical internalization (PCI)²⁵. However, unavoidable side effects, such as cell apoptosis or differentiation, may also accompany the PCI treatment²⁶. Since such effects may be the consequence of induction of lysosomal damage, the provided assays present a potential tool for preclinical use.

In this study, the AlPcS_2a-mediated CALI protocols describe the selective control of the damage size (local and part of endosomes or lysosomes). Scientists can monitor their subcellular behavior, while the rest of the IVs remain intact and could be treated as a control. This methodology has been applied in several studies. For example, damaged lysosomes are labeled sequentially with ubiquitin and microtubule-associated protein light chain 3 (LC3), and undergo autophagic clearance³. Furthermore, galectin-3 and galectin-8 labeling onto damaged endosomes are controlled by cell surface glycan²⁷. Since AlPcS_2a has been used in vivo for PCI, AlPcS2a-mediated CALI could also be used in vivo. This protocol provides a robust tool to study cell biology using AlPcS_2a-mediated CALI. These studies support that the AlPcS_2a-mediated CALI is a robust tool for cell biology studies.

Declarações

The authors have nothing to disclose.

Acknowledgements

The authors wish to thank the Academia Sinica Inflammation Core Facility, IBMS for research support. The core facility is funded by the Academia Sinica Core Facility and Innovative Instrument Project (AS-CFII-111-213). The authors thank the Common Equipment Core Facility of the Institute of Biomedical Sciences (IBMS), Academia Sinica (AS) for assisting the image acquisition.

Materials

Reagent
Al(III) Phthalocyanine Chloride Disulfonic acid (AlPcS_2a)	Frontier Scientific	P40632
Culture dish	ibidi	812128-200
Culture Medium			DMEM supplemented with 10% FBS and 100 U/mL penicillin G and 100 mg/mL Streptomycin
DMEM	Gibco	11965092
FBS	Thermo Fisher Scientific	A4736301
Gal3-GFP plasmid	addgene
Lipofectamine 3000 kit	Thermo Fisher Scientific	L3000008
LysoTracker Green DND-26	Thermo Fisher Scientific	L7526	green fluorescent dye
Multiwall plate	perkinelmer	PK-6005550
NaOH	Thermo Fisher Scientific	Q15895
OptiMEM	Thermo Fisher Scientific	31985070
Penicillin-streptomycin	Gibco	15140163
Phosphate-Buffered Saline (PBS)	Gibco	21600-069	137 mM NaCl, 2.7 mM KCl, 10mM Na2HPO4, 1.8 mM KH2PO4
Cell line
HeLa Cell Line	ATCC	CCL-2	The methods are applicable for most of the attached cell lines. Conditions must be determined individually.
Equipments
0.22 µm Filter	Merck	SLGV013SL
Collimated LED Light (660nm)	Thorlabs	M660L3-C1 and DC2100	Near-infared light is ideal base on the excitation spectrum of AlPcS2a.
Confocal microscopy	Carl Zeiss	LSM 780	An incubation system is required for long-term imaging.
NanoDrop 2000/2000c Spectrophotometers	Thermo Fisher Scientific
Red LED light	Tholabs	M660L4-C1

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Hung, Y., Wang, H., Wang, J., Hsu, C., Chen, H. A Photodynamic Approach to Study Function of Intracellular Vesicle Rupture. J. Vis. Exp. (193), e63962, doi:10.3791/63962 (2023).