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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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 comba....

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 10

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 ind.......

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 la.......

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

NameCompanyCatalog NumberComments
Reagent
Al(III) Phthalocyanine Chloride Disulfonic acid (AlPcS2a)Frontier ScientificP40632
Culture dishibidi812128-200
Culture MediumDMEM supplemented with 10% FBS and 100 U/mL penicillin G and 100 mg/mL Streptomycin
DMEMGibco11965092
FBSThermo Fisher ScientificA4736301
Gal3-GFP plasmidaddgene
Lipofectamine 3000 kitThermo Fisher ScientificL3000008
LysoTracker Green DND-26Thermo Fisher ScientificL7526green fluorescent dye
Multiwall plateperkinelmerPK-6005550
NaOHThermo Fisher ScientificQ15895
OptiMEMThermo Fisher Scientific31985070
Penicillin-streptomycinGibco15140163
Phosphate-Buffered Saline (PBS)Gibco21600-069137 mM NaCl, 2.7 mM KCl, 10mM Na2HPO4, 1.8 mM KH2PO4
Cell line
HeLa Cell LineATCCCCL-2The methods are applicable for most of the attached cell lines. Conditions must be determined individually.
Equipments
0.22 µm FilterMerckSLGV013SL
Collimated LED Light  (660nm)ThorlabsM660L3-C1 and DC2100Near-infared light is ideal base on the excitation spectrum of AlPcS2a.
Confocal microscopyCarl ZeissLSM 780An incubation system is required for long-term imaging.
NanoDrop 2000/2000c SpectrophotometersThermo Fisher Scientific
Red LED lightTholabsM660L4-C1

References

  1. Cossart, P., Helenius, A. Endocytosis of viruses and bacteria. Cold Spring Harbor Perspectives in Biology. 6 (8), 016972 (2014).
  2. Daussy, C. F., Wodrich, H. 34;Repair me if you can": membrane damage, response, and cont....

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

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