Name: a photodynamic approach to study function of intracellular vesicle rupture
Uploaded: 2023-03-17T14:45:00
Description: Watch this Scientific Journal Video about A Photodynamic Approach to Study Function of Intracellular Vesicle Rupture at JoVE.com

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.

1. AlPcS2a stock preparation
<ol>
	<li>Dissolve 10 mg of AlPcS2a in 400 &#181;L of 0.1 M NaOH. To improve solubility, heat the solution at 50 &#176;C and vortex.</li>
	<li>Mix the solution with 4 mL of phosphate-buffered saline (PBS). Then, filter the solution with a 0.22 &#181;m filter to remove the insoluble precipitates.</li>
	<li>Measure the concentration of the solution through a UV-Vis spectrophotometer. The extinction coefficient of AlPcS2a at 672 nm is 4 x 10<su...

<ol>
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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporally+controlled+initiation+of+Parkin-mediated+mitophagy+within+single+cells.">Spatiotemporally controlled initiation of Parkin-mediated mitophagy within single cells.</a> Autophagy. 7 (10), 1230-1238 (2011).</li><li>Molinari, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vacuoles+induced+by+Helicobacter+pylori+toxin+contain+both+late+endosomal+and+lysosomal+markers.">Vacuoles induced by Helicobacter pylori toxin contain both late endosomal and lysosomal markers.</a> Journal of Biological Chemistry. 272 (40), 25339-25344 (1997).</li><li>Prince, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subversion+of+a+lysosomal+pathway+regulating+neutrophil+apoptosis+by+a+major+bacterial+toxin,+pyocyanin.">Subversion of a lysosomal pathway regulating neutrophil apoptosis by a major bacterial toxin, pyocyanin.</a> Journal of Immunology. 180 (5), 3502-3511 (2008).</li><li>Aits, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+detection+of+lysosomal+membrane+permeabilization+by+lysosomal+galectin+puncta+assay.">Sensitive detection of lysosomal membrane permeabilization by lysosomal galectin puncta assay.</a> Autophagy. 11 (8), 1408-1424 (2015).</li><li>Prasmickaite, L., Hogset, A., Berg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+different+photosensitizers+for+use+in+photochemical+gene+transfection.">Evaluation of different photosensitizers for use in photochemical gene transfection.</a> Photochemistry and Photobiology. 73 (4), 388-395 (2001).</li><li>Berg, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemical+internalization:+a+novel+technology+for+delivery+of+macromolecules+into+cytosol.">Photochemical internalization: a novel technology for delivery of macromolecules into cytosol.</a> Cancer Research. 59 (6), 1180-1183 (1999).</li><li>Moan, J., Berg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+photodegradation+of+porphyrins+in+cells+can+be+used+to+estimate+the+lifetime+of+singlet+oxygen.">The photodegradation of porphyrins in cells can be used to estimate the lifetime of singlet oxygen.</a> Photochemistry and Photobiology. 53 (4), 549-553 (1991).</li><li>Thurston, T. L., Wandel, M. P., von Muhlinen, N., Foeglein, A., Randow, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galectin+8+targets+damaged+vesicles+for+autophagy+to+defend+cells+against+bacterial+invasion.">Galectin 8 targets damaged vesicles for autophagy to defend cells against bacterial invasion.</a> Nature. 482 (7385), 414-418 (2012).</li><li>Repnik, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-leucyl-L-leucine+methyl+ester+does+not+release+cysteine+cathepsins+to+the+cytosol+but+inactivates+them+in+transiently+permeabilized+lysosomes.">L-leucyl-L-leucine methyl ester does not release cysteine cathepsins to the cytosol but inactivates them in transiently permeabilized lysosomes.</a> Journal of Cell Science. 130 (18), 3124-3140 (2017).</li><li>Griffiths, G., Hoflack, B., Simons, K., Mellman, I., Kornfeld, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mannose+6-phosphate+receptor+and+the+biogenesis+of+lysosomes.">The mannose 6-phosphate receptor and the biogenesis of lysosomes.</a> Cell. 52 (3), 329-341 (1988).</li><li>Jia, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galectin-3+Coordinates+a+cellular+system+for+lysosomal+repair+and+removal.">Galectin-3 Coordinates a cellular system for lysosomal repair and removal.</a> Developmental Cell. 52 (1), 69-87 (2020).</li><li>Chu, Y. 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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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Al(III) Phthalocyanine Chloride Disulfonic acid (AlPcS2a)</td>
			<td>Frontier Scientific</td>
			<td>P40632</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture dish</td>
			<td>ibidi</td>
			<td>812128-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Medium</td>
			<td></td>
			<td></td>
			<td>DMEM supplemented with 10% FBS and 100 U/mL penicillin G and 100 mg/mL Streptomycin</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>A4736301</td>
			<td></td>
		</tr>
		<tr>
			<td>Gal3-GFP plasmid</td>
			<td>addgene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 3000 kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>L3000008</td>
			<td></td>
		</tr>
		<tr>
			<td>LysoTracker Green DND-26</td>
			<td>Thermo Fisher Scientific</td>
			<td>L7526</td>
			<td>green fluorescent dye</td>
		</tr>
		<tr>
			<td>Multiwall plate</td>
			<td>perkinelmer</td>
			<td>PK-6005550</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Thermo Fisher Scientific</td>
			<td>Q15895</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140163</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>21600-069</td>
			<td>137 mM NaCl, 2.7 mM KCl, 10mM Na2HPO4, 1.8 mM KH2PO4</td>
		</tr>
		<tr>
			<td>Cell line</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa Cell Line</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td>The methods are applicable for most of the attached cell lines. Conditions must be determined individually.</td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m Filter</td>
			<td>Merck</td>
			<td>SLGV013SL</td>
			<td></td>
		</tr>
		<tr>
			<td>Collimated LED Light&#160; (660nm)</td>
			<td>Thorlabs</td>
			<td>M660L3-C1 and DC2100</td>
			<td>Near-infared light is ideal base on the excitation spectrum of AlPcS2a.</td>
		</tr>
		<tr>
			<td>Confocal microscopy</td>
			<td>Carl Zeiss</td>
			<td>LSM 780</td>
			<td>An incubation system is required for long-term imaging.</td>
		</tr>
		<tr>
			<td>NanoDrop 2000/2000c Spectrophotometers</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Red LED light</td>
			<td>Tholabs</td>
			<td>M660L4-C1</td>
			<td></td>
		</tr>
	</tbody>
</table>

a photodynamic approach to study function of intracellular vesicle rupture

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.

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...

Watch this Scientific Journal Video about A Photodynamic Approach to Study Function of Intracellular Vesicle Rupture at JoVE.com

A Photodynamic Approach to Study Function of Intracellular Vesicle Rupture

AlPcS2a-mediated chromophore-assisted laser inactivation (CALI) is a powerful tool for studying spatiotemporal damage of intracellular vesicles (IVs) in live cells.

Intracellular vesicles (IVs) are formed through endocytosis of vesicles into cytoplasm. IV formation is involved in activating various signal pathways ...

Endocytosis of vesicle into cytoplasia results in the formation of intracellular vesicles. Permeabilization of vesicle activates various signal pathways. Chromophore-assisted laser inactivation is used to study the consequence of intracellular vesicle rupture.A spatial and temporal control of subcellular damage to intracellular vesicles can be achieved by adjusting the standing condition and light illumination. To begin, maintain HeLa cell culture in DMEM supplemented with 10%FBS. After washing the cells with PBS, Trypsinize the cells.Then resuspend the cells in DMEM supplemented with 10%FBS. Dilute 300 nanograms of Gal3-GFP plasmid in 25 microliters of reduced serum medium. Then add 0.6 microliters P3000 reagent.And gently mix, followed by vortexing the solution. Dilute 0.45 microliters of Lipofectamine 3000 reagent in 25 microliters of reduced serum medium and gently mix. Incubate for five minutes at room temperature.Then mix diluted DNA and diluted Lipofectamine 3000 and incubate for 10 minutes at room temperature. After mixing the DNA lipofectamine mixture, 300, 000 suspended HeLa cells and 200 microliters of DMEM with 10%FBS, seed the cells on the central glass region of a 35-millimeter glass button dish. To allow cell attachment, incubate the cells at 37 degrees Celsius in the incubator supplied with 5%carbon dioxide for at least four hours.Dilute AlPcS2a in DMEM supplemented with 10%FBS to make a one micromolar AlPcS2a solution. Prewarm the AlPcS2a containing-medium in a 37 degrees Celsius water bath and replace the culture medium with the AlPcS2a-containing medium. Incubate the cells at 37 degrees Celsius in the incubator supplied with 5%carbon dioxide overnight.The following day, wash the cells twice with PBS to remove extracellular AlPcS2a after replacing the medium with fresh medium. Incubate at 37 degrees Celsius for four hours to allow residual dye along the endocytic pathway to accumulate into lysosomes. To manipulate single cells by damaging lysosomes, place the culture dish on the stage of a confocal microscope.Use the 488 nanometer and 561 nanometer lasers for exciting GFP and AlPcS2a, respectively. Circle a five-by-five micrometer square region of interest on the AlPcS2a labeled vesicles which are selected to be damaged. Then pulse illuminate the region of interest with a 633 nanometer laser of 0.21 milliwatt, or 70 repeats.Monitor the formation of Gal3 puncta as an indicator of lysosomal membrane permeabilization. Lysosomal staining with AlPcS2a in HeLa cells was performed using commercially-available markers, and were positively stained with green fluorescent dye. Lysosomes in TagRFP-Galectin3 expressed HeLa cells were stained with AlPcS2a.Followed by focusing illumination with near infrared light, the results indicate that AlPcS2a-based chromophore-assisted laser inactivation was able to control local lysosomal rupture within the region of interest, leaving the rest of the lysosomes intact. This step is important because the incubation time will determine standing endosomes or lysosomes. For example, people can determine the size of by the release of membrane-impermeable dye.Also can use pH sensitive dyes such as FITC dextran to difference endosomes and lysosomes. This technique is useful starting the downstream effects of intracellular vesicle membrane rupture. And there are downstream effects under various situations.

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