A Photodynamic Approach to Study Function of Intracellular Vesicle Rupture

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_2a 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. AlPcS_2a stock preparation

1. 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.
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 AlPcS_2a at 672 nm is 4 x 10

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 la.......

Materials

Name	Company	Catalog Number	Comments
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