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 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 &#176;C.</li>
</ol>
2. Transfection
NOTE: Gal3-GFP is applied as an indicator for live-cell imaging of lysosomal rupture.
<ol>
	<li>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.</li>
	<li>Dilute 300 ng of Gal3-GFP plasmid in 25 &#181;L of reduced serum medium, and then add 0.6 &#181;L of P3000 reagent. Gently mix. Dilute 0.45 &#181;L of Lipofectamine 3000 reagent in 25 &#181;L of reduced serum medium. Gently mix.</li>
	<li>Incubate for 5 min at room temperature. Mix diluted DNA and diluted Lipofectamine 3000, and then incubate for 10 min at room temperature.</li>
	<li>Mix the DNA-Lipofectamine mixture, 3 x 105 suspended HeLa cells, and 200 &#181;L of DMEM+10% FBS, and then seed the cells on the central glass region of a 35 mm glass button dish.</li>
</ol>
3. AlPcS2a staining
<ol>
	<li>To allow cell attachment, incubate the cells at 37 &#176;C in the incubator supplied with 5% CO2&#160;for at least 4 h.</li>
	<li>Dilute AlPcS2a in DMEM supplemented with 10% FBS to make a 1 &#181;M AlPcS2a solution. Pre-warm the AlPcS2a-containing medium in a 37 &#176;C water bath. Replace the culture medium with the AlPcS2a-containing medium.</li>
	<li>Incubate the cells at 37 &#176;C in the incubator supplied with 5% CO2 overnight (16-18 h).</li>
	<li>The following day, wash the cells twice with PBS to remove extracellular AlPcS2a. Replace the medium with fresh medium, and then incubate at 37 &#176;C for 4 h to allow residual dye along the endocytic pathway to accumulate into lysosomes. 
	&#8203;NOTE: To stain endosomes, incubate cells in 1 &#181;M AlPcS2a in DMEM containing 10% FBS for 15 min at 37 &#176;C. Next, wash cells twice with PBS and incubate them in pre-warmed medium before imaging .</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63962/63962fig01.jpg" /> 
Figure 1. A schematic figure representing the selective IV damage with AlPcS2a. The figure shows the schematics of selective IV damage. <a href="https://www.jove.com/files/ftp_upload/63962/63962fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63962/63962fig02.jpg" /> 
Figure 2. Lysosomal staining with AlPcS2a. Lysosomes labeled overnight with 1 &#181;M AlPcS2a in HeLa cells are positively stained with 50 nM green fluorescent dye. Scale bar: 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63962/63962fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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.
<ol>
	<li>Manipulate single cells by damaging lysosomes within a single cell, as described below.
	<ol>
		<li>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.</li>
		<li>Circle a 5 x 5 &#181;m2 region of interest (ROI) on the AlPcS2a-labeled vesicles which are selected to be damaged.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Damage all labeled cells in a culture dish as described below.
	<ol>
		<li>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).</li>
		<li>Place the culture dish on the microscope and monitor the indicators of membrane permeabilization as mentioned in step 4.1.3.</li>
	</ol>
	</li>
	<li>Assess injury of IVs using the following indicators.
	<ol>
		<li>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.</li>
		<li>The size of the wound can be determined by the release of membrane-impermeable dye as described in21.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63962/63962fig03.jpg" /> 
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 &#181;M 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 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63962/63962fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors declare no conflict of interest.

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...

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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 ...

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Research

JoVE Journal

Immunology and Infection

1.0K Views.  Academia Sinica. AlPcS2a-mediated chromophore-assisted laser inactivation (CALI) is a powerful tool for studying spatiotemporal damage of intracellular vesicles (IVs) in live cells.

Video: A Photodynamic Approach to Study Function of Intracellular Vesicle Rupture

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes

The structural protein of HIV-1, Pr55Gag (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an essential step for virus particle formation, since a defect in Gag membrane binding results in severe impairment of viral particle production. To gain mechanistic details of Gag-lipid membrane interactions, in vitro methods based on NMR, protein footprinting, surface plasmon resonance, liposome flotation centrifugation, or fluorescence lipid bead binding have been developed thus far. However, each of these in vitro methods has its limitations. To overcome some of these limitations and provide a complementary approach to the previously established methods, we developed an in vitro assay in which interactions between HIV-1 Gag and lipid membranes take place in a &#34;cell-like&#34; environment. In this assay, Gag binding to lipid membranes is visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique. Here we describe the background and the protocols to obtain myristoylated full-length Gag proteins and GUV membranes necessary for the assay and to detect Gag-GUV binding by microscopy.

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle GUV Membranes

We illustrate here an in vitro membrane binding assay in which interactions between HIV-1 Gag and lipid membranes are visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique.

The structural protein of HIV-1, Pr55Gag (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

Evaluation of Extracellular Vesicle Function During Malaria Infection

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible for most malaria-related deaths globally. Several factors including parasite sequestration in tissues, vascular dysfunction, and inflammatory responses influence the evolution of the disease in malaria-infected people. P. falciparum-infected red blood cells (iRBCs) release small extracellular vesicles (EVs) containing different kinds of cargo molecules that mediate pathogenesis and cellular communication between parasites and host. EVs are efficiently taken up by cells in which they modulate their function. Here we discuss strategies to address the role of EVs in parasite-host interactions. First, we describe a straightforward method for labeling and tracking EV internalization by endothelial cells, using a green cell linker dye. Second, we report a simple way to measure permeability across an endothelial cell monolayer by using a fluorescently labeled dextran. Finally, we show how to investigate the role of small non-coding RNA molecules in endothelial cell function.

In this work, we describe protocols to investigate the role of extracellular vesicles (EVs) released by Plasmodium falciparum infected erythrocytes. In particular, we focus on the interactions of EVs with endothelial cells.

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible ...