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Özet
Abstract
Introduction
Protocol
Sonuçlar
Discussion
Açıklamalar
Acknowledgements
Materials
Referanslar
İmmünoloji ve Enfeksiyon

A Photodynamic Approach to Study Function of Intracellular Vesicle Rupture

Published: March 17, 2023
doi:
10.3791/63962
, , , ,
1Inflammation Core Facility, Institute of Biomedical Sciences,Academia Sinica

Özet

AlPcS2a-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 AlPcS2a and 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 (AlPcS2a)) are used in CALI; AlPcS2a, targeting the lysosomes through endocytosis, is used to rupture endosomes or lysosomes in a controlled region17. AlPcS2a 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 AlPcS2a18. 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 AlPcS2a 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 AlPcS2a as 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. AlPcS2a stock preparation

  1. Dissolve 10 mg of AlPcS2a in 400 µL of 0.1 M NaOH. To improve solubility, heat the solution at 50 °C and vortex.
  2. 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.
  3. Measure the concentration of the solution through a UV-Vis spectrophotometer. The extinction coefficient of AlPcS2a at 672 nm is 4 x 104 cm-1 M-1. Dilute the AlPcS2a 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.

  1. 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.
  2. 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.
  3. Incubate for 5 min at room temperature. Mix diluted DNA and diluted Lipofectamine 3000, and then incubate for 10 min at room temperature.
  4. 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

  1. To allow cell attachment, incubate the cells at 37 °C in the incubator supplied with 5% CO2 for at least 4 h.
  2. Dilute AlPcS2a in DMEM supplemented with 10% FBS to make a 1 µM AlPcS2a solution. Pre-warm the AlPcS2a-containing medium in a 37 °C water bath. Replace the culture medium with the AlPcS2a-containing medium.
  3. Incubate the cells at 37 °C in the incubator supplied with 5% CO2 overnight (16-18 h).
  4. 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 AlPcS2a 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
Figure 1. A schematic figure representing the selective IV damage with AlPcS2a. The figure shows the schematics of selective IV damage. Please click here to view a larger version of this figure.

Figure 2
Figure 2. Lysosomal staining with AlPcS2a. Lysosomes labeled overnight with 1 µM AlPcS2a 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 AlPcS2a-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.

  1. 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 AlPcS2a-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.
  2. 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.
  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
Figure 3. AlPcS2a-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 AlPcS2a, followed by focusing illumination with near-infrared light (633 nm) within the yellow square. AlPcS2a 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 AlPcS2a-induced damage of IV, including endosome and lysosome, has been shown (Figure 1).

Commercially available markers can be used to determine the AlPcS2a staining conditions. For example, AlPcS2a 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 AlPcS2a-based CALI is able to control local lysosomal rupture within the ROI, leaving the rest of the lysosomes intact.

Discussion

AlPcS2a binds to the plasma membrane, then is internalized by endocytosis and eventually accumulates in lysosomes. AlPcS2a 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 AlPcS2a through endocytosis because there are many other membrane sources of IVs, such as ER and Golgi apparatus. In addition, the selective labeling of AlPcS2a 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, AlPcS2a 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 AlPcS2a-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 AlPcS2a 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 AlPcS2a-mediated CALI. These studies support that the AlPcS2a-mediated CALI is a robust tool for cell biology studies.

Açıklamalar

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 (AlPcS2a) 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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Referanslar

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  4. Sharma, S. K., et al. Photodynamic therapy for cancer and for infections: what is the difference. Israel Journal of Chemistry. 52 (8-9), 691-705 (2012).
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  8. Lavie, G., et al. A photodynamic pathway to apoptosis and necrosis induced by dimethyl tetrahydroxyhelianthrone and hypericin in leukaemic cells: possible relevance to photodynamic therapy. British Journal of Cancer. 79 (3-4), 423-432 (1999).
  9. Jay, D. G. Selective destruction of protein function by chromophore-assisted laser inactivation. Proceedings of the National Academy of Sciences. 85 (15), 5454-5458 (1988).
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  12. Hsieh, C. W., Yang, W. Y. Triggering mitophagy with photosensitizers. Methods in Molecular Biology. 1880, 611-619 (2019).
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  18. Berg, K., et al. Photochemical internalization: a novel technology for delivery of macromolecules into cytosol. Kanser Araştırmaları. 59 (6), 1180-1183 (1999).
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  20. Thurston, T. L., Wandel, M. P., von Muhlinen, N., Foeglein, A., Randow, F. Galectin 8 targets damaged vesicles for autophagy to defend cells against bacterial invasion. Nature. 482 (7385), 414-418 (2012).
  21. Repnik, U., et al. L-leucyl-L-leucine methyl ester does not release cysteine cathepsins to the cytosol but inactivates them in transiently permeabilized lysosomes. Journal of Cell Science. 130 (18), 3124-3140 (2017).
  22. Griffiths, G., Hoflack, B., Simons, K., Mellman, I., Kornfeld, S. The mannose 6-phosphate receptor and the biogenesis of lysosomes. Cell. 52 (3), 329-341 (1988).
  23. Jia, J., et al. Galectin-3 Coordinates a cellular system for lysosomal repair and removal. Developmental Cell. 52 (1), 69-87 (2020).
  24. Chu, Y. P., Hung, Y. H., Chang, H. Y., Yang, W. Y. Assays to monitor lysophagy. Methods in Enzymology. 588, 231-244 (2017).
  25. Nguyen, L., Madsen, S. J., Berg, K., Hirschberg, H. An improved in vitro photochemical internalization protocol for 3D spheroid cultures. Lasers in Medical Science. 36 (8), 1567-1571 (2021).
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Etiketler

Photodynamic ApproachIntracellular Vesicle RuptureEndocytosisSignal PathwaysChromophore-assisted Laser InactivationSubcellular DamageHeLa CellsGal3-GFPAlPcS2aLysosomeConfocal Microscopy

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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).
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