1. AlPcS2a stock preparation
2. Transfection
NOTE: Gal3-GFP is applied as an indicator for live-cell imaging of lysosomal rupture.
3. AlPcS2a staining
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. 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.
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.
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.
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 |
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.
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.
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.