Name: application of fluorescent nanoparticles to study remodeling of the endo-lysosomal system by intracellular bacteria
Uploaded: 2015-01-02T14:00:00.000+00:00
Duration: 11 min 38 s
Description: Watch this Scientific Journal Video about Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria at JoVE.com

This work was supported by the Deutsche Forschungsgemeinschaft by grant Z within Sonderforschungsbereich 944 &#8216;Physiology and Dynamics of Cellular Microcompartments&#8217; and HE1964/18 within priority program 1580.

1. Synthesis of 10 nm Gold Nanoparticles (Gold NPs)15
<ol>
	<li>Prepare solution A: add 2 ml 1% aqueous gold chloride into 160 ml Milli-Q, or double distilled, water.</li>
	<li>Prepare solution B: add 8 ml 1% tri-sodium citrate x 2 H2O and 160 &#181;l 1% Tannic acid into 32 ml Milli-Q, or double distilled, water.</li>
	<li>Warm up solution A and B to 60 &#176;C and mix them while stirring. Observe a dark blue color immediately. Observe red color after about 15 min. Then heat up to 95&#176;C, keep 5 min and cool the solution to RT.</li>
	<li>Add a drop of the NP suspension onto a carbon coated grid and allow to dry in air. Check the size and morphology of NPs by transmission electron microscopy.</li>
</ol>
2. Coating of Gold NPs with BSA and Labeling with Rhodamine N-hydroxysuccinimidyl Ester (NHS)16
<ol>
	<li>Add 900 &#956;l gold NPs into a 1.5 ml Eppendorf tube, centrifuge at 15,000 x g for 30 min. Optionally, prepare multiple tubes may at once.</li>
	<li>Discard the supernatant, re-suspend the pellet in 900 &#956;l sterilized Milli-Q water and add 100 &#956;l 2 mg/ml BSA/PBS, mix on a Vortex at 800 rpm for 30 min.</li>
	<li>To remove excess BSA, centrifuge the preparation at 15,000 x g for 60 min.</li>
	<li>Discard the supernatant, and re-suspend the gold-BSA NPs in 125 &#956;l PBS, add 12.5 &#181;l of 1 M bicarbonate.</li>
	<li>Immediately before use, prepare a 10 mg/ml solution of rhodamine NHS/DMSO, add 30 &#181;l of rhodamine NHS/DMSO solution to 1ml of the gold-BSA NPs suspension. Incubate the reaction for 2 hr (or O/N) at RT during 800 rpm mixing, avoiding exposure to light.</li>
	<li>Purify the gold-BSA-rhodamine NPs through dialysis against PBS at 4 &#186;C with 5 buffer changes over the period of 36 - 48 hr.</li>
	<li>To stabilize NPs, add 10 &#956;l of 200 mg/ml BSA/PBS into each 1 ml gold-BSA-rhodamine NPs. To remove the free or released BSA-rhodamine, centrifuge at 15,000 x g for 60 min. Resuspend the pellet in 2 mg/ml BSA/PBS, measure OD520, and store at 4 &#176;C, avoiding exposure to light.</li>
	<li>Dilute the NPs 10 times with Milli-Q or double distilled water, add a drop onto a carbon-coated grid, and allow to dry in air. Check size and morphology of NPs by transmission electron microscopy (TEM). 
	NOTE: All materials used for NP preparation were sterilized and the operation was conducted in a cell culture hood or on a bench beside a flame.</li>
</ol>
3. Culture of HeLa Cells
<ol>
	<li>HeLa cells permanently expressing LAMP1-GFP are cultured in DMEM with 10% fetal calf serum (FCS) and grown at 37 &#176;C in an atmosphere containing 5% CO2.</li>
	<li>Seed the cells at a density of 50,000 per well in an 8-well chamber slide (Ibidi) and incubate O/N.</li>
</ol>
4. Culture of Bacteria
<ol>
	<li>Use Salmonella enterica serovar Typhimurium NCTC 12023 wild-type (WT) strain. For comparison, use mutant strains &#916;ssaV defective in the SPI2-T3SS and &#916;sifA lacking key effector SifA for SIF. Use strains harboring plasmid pFPV25.1 for constitutive expression of enhanced GFP. 
	NOTE: Bacterial strains are routinely cultured in Luria-Bertani broth (LB) with addition of 50 &#181;g/ml carbenicillin (WT) and LB with addition of 50 &#181;g/ml carbenicillin and/or 50 &#181;g/ml kanamycin (&#916;ssaV, &#916;sifA) of required to maintain plasmids.</li>
	<li>Inoculate a single colony of bacteria in 3 ml LB with appropriate antibiotics and grow O/N&#160;at 37 &#186;C under shaking conditions for aeration, then dilute 1:31 in fresh LB and continue growth for 3.5 hr. At this time-point, the cultures reach the late log phase and bacteria are highly invasive. A &#8216;roller drum&#8217; is convenient to incubate test tube cultures with aeration.</li>
</ol>
5. Infection of HeLa cells by Salmonella and Gold-BSA-Rhodamine NPs Pulse Chase-labeling (see Figure 1 for scheme)
<ol>
	<li>Measure OD600 of the sub-cultured bacteria and dilute to OD600 = 0.2 in 1 ml PBS (~3 &#215; 108 cfu/ml), add appropriate amounts of bacteria to HeLa cells in 8-well chamber slides to achieve a multiplicity of infection (MOI) of 100.</li>
	<li>Incubate for 30 min in the cell incubator, wash 3 times with PBS to remove non-internalized bacteria (this time point was set as 0 hr post-infection, or 0 hr p.i.). Add 300 &#181;l of fresh culture medium containing 100 &#181;g/ml gentamicin and maintain for 1 hr. Then replace the medium with fresh medium containing 10 &#181;g/ml gentamicin for the rest of the incubation time.</li>
	<li>After incubation with culture medium containing 100 &#181;g/ml gentamicin for 1 hr, the medium is replaced by imaging medium (Eagles MEM without FCS, L-glutamine, phenol red and sodium bicarbonate, with 30 mM HEPES, pH 7.4) containing 10 &#181;g/ml gentamicin. Gold-BSA-rhodamine NPs are added to HeLa cells to obtain a final concentration of OD520 = 0.1. 
	NOTE: Gold-BSA-rhodamine NPs may also be added to cells before infection, or at various time-points p.i.</li>
	<li>After 1 hr incubation, remove the medium, wash 3 times with PBS, and add 300 &#181;l of fresh imaging medium containing 10% FCS and 10 &#181;g/ml gentamicin for the rest of the incubation time. 
	NOTE: Duration of incubation may vary depending on concentration of NPs and cell lines used. For RAW264.7 macrophages, we observed that 30 min incubation with NPs at a concentration of OD520 = 0.05 allowed sufficient internalization.</li>
</ol>
6. Imaging
<ol>
	<li>Use a confocal imaging system such as a confocal laser-scanning (CLSM) or spinning disc (SD) microscope with a humidified environment chamber for high resolution imaging at different time-points.</li>
	<li>Switch on the temperature control system and wait until it is stable. Optimize the imaging settings such as magnification, scanning speed, resolution, Z-stack etc. Use appropriate excitation/emission settings for GFP and gold-BSA-rhodamine NPs. For this protocol, use a 400 Hz scanning speed, resolution of 512 x 512 pixels and Z-step size of 0.25 &#181;m. Excite GFP and gold-BSA-rhodamine using an Ar laser (488 nm) and a HeNe laser (543 nm), respectively. Include a bright-field (BF) channel for observing the shape of the cells. For other microscopy systems and infection conditions, the setting have to be adjusted accordingly. 
	NOTE: Use the same Ar laser as the light source of BF channel and signal was detected with a photo multiplier (PMT) detector.</li>
	<li>At indicated time-points p.i., mount the 8-well chamber slides containing infected cells on the microscope stage and record images.</li>
</ol>
7. Analysis of Images
<ol>
	<li>Use microscopy image analysis software (see Materials and Equipment&#8217;s Table) to analyze the remodeling of the endo-lysosomal system by intracellular Salmonella. Open the data by clicking &#8216;Open&#8217; and choosing the file. 
	NOTE: Alternatively, open source software packages such as ImageJ or FIJI may be used to quantitative image analyses.</li>
	<li>In the objects toolbar of the Surpass view click on the icon<img alt="Icon" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon1.jpg" /> to add a new surface item. Click on <img alt="Icon" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon2.jpg" /> (Next).</li>
	<li>To analyze a Region of Interest (ROI), select segment a ROI. In a viewing area, a rectangle-bordered section overlaid on image is representing the ROI. Enter the values in the corresponding x-, y-, and z- fields, or directly click on the arrows in the preview rectangle to modify size and position of the ROI. Then click <img alt="Icon" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon2.jpg" /> (Next).</li>
	<li>As a source channel, select Channel 3 (signal for Gold-BSA-Rhodamine NPs). Check the smooth option to set up the smoothness of the resulting area. Define a value manually or accept the automatically generated value. For Threshold, select the Absolute Intensity option.</li>
	<li>For the threshold adjustment, select the manual option and set a value. In the viewing area, a surface threshold preview is displayed in gray.</li>
	<li>On the tab Classify Surfaces the resulting surface can be filtered by various criteria. A default filter is &#8216;number of voxels &#62;10&#8217;, and other filters can also be included by clicking &#8216;add&#8217;. If a filtering is not necessary, then delete all filters by clicking on the delete button. Click <img alt="Icon" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon2.jpg" /> (Next).</li>
	<li>To complete Surface creation, click on <img alt="Icon" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon3.jpg" /> (Finish). In the Object list, now un-check the box for the item Volume and new created surface is displayed in viewing area.</li>
	<li>Export the statistics. Now, in the Surpass view the color of the subjects vary from purple to red according to their area. And the &#8216;Plot Numbers Area&#8217; table shows the statistics information (e.g., ID number and area of objects). Export all statistics of surface 1 to an Excel file by click <img alt="Icon" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon4.jpg" /> (Save).</li>
</ol>

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No conflicts of interest declared.

The endo-lysosomal system of mammalian cells controls important physiologic processes, including nutrient absorption, hormone-mediated signal transduction, immune surveillance, and antigen presentation17. Up to now, a variety of markers have been used for labeling of the endocytic pathway and tracking studies. For example, LysoTracker probes are fluorescent acidotropic probes developed by Molecular Probes (Life Technologies, USA) for lysosome labeling, which can selectively accumulate in cellular compartments with low internal pH and effectively label living cells at nanomolar concentrations18. However, long time incubation of LysoTracker probes with cells may induce an increase in lysosomal pH and lead to potential physiological changes of lysosomes19. Some large biomolecules, such as fluorophore-labeled dextrans, are also frequently used for endosome/lysosomes labeling. However, low-photostability, short-circulating life, and poor retention during fixation hinder their applications in long-term live cell imaging and correlative microscope studies16,19. Here, gold-BSA-rhodamine NPs were used as fluid tracers to label the host cellular endo-lysosomal system. High uptake efficiency and strong intracellular fluorescence signals were observed. The almost complete colocalization of NPs with lysosomal glycoprotein LAMP1, which is highly enriched on the membrane of late endosomes/lysosomes, verified proper labeling of the endo-lysosomal system by the NPs. Combining the use of fluorescent NPs with the other markers such as Lysotracker may provide complementary information about host cell vesicular trafficking and maturation.
It is worth to mention that cellular internalization of NPs is highly dependent on their physical dimensions and chemical components. We have tested gold-BSA-rhodamine NPs with a size of 5 nm, 10 nm, 15 nm and 30 nm, and similar intracellular distribution inside HeLa cells were observed (results not shown). We used HeLa cells as convenient cell line for Salmonella infection studies. However, gold-BSA-rhodamine NPs can also be readily internalized by various other cell lines and primary cells are amenable. For example, we observed Salmonella-induced tubular structures and labeling by NP in CHO, COS-7, CaCo2, or 3T3 cells, as well as in Interferon-&#947;-stimulated RAW264.7 macrophage-like cell line cells or primary murine macrophages and dendritic cells. However, each cell line will require adjustment of the infection and pulse/chase protocols.
It has been reported that dye-entrapped silica NPs can be used as a biocompatible, long-living, and highly photostable lysosome marker19, and we also compared the behavior of rhodamine-doped silica NPs with varying size (30 - 1,000 nm). Proper labeling of late endosomes/lysosomes by silica NPs in non-infected cells and accumulation of NPs in SCV and SIF in Salmonella-infected cells was observed. However, due to the formation of aggregates of NPs or the large size of single silica NPs, distribution of NPs along with Salmonella-induced tubular structures was not uniform (data not shown).
A characteristic feature of SCV as well as SIF is the presence of highly abundant lysosomal glycoproteins such as LAMP112. Here, a HeLa cell line stably expressing LAMP1-GFP was used for the convenience of live cell imaging of the endo-lysosomal system and the SCV and SIF structures. Salmonella strains constitutively expressing enhanced GFP were used in the infection experiments to indicate location of bacteria. Due to the uniform size and shape of the intracellular bacteria, the bacterial GFP fluorescence in general is easily distinguishable from the GFP label on endo-lysosomal membranes. However, for future experiments in which fluorescent signal from bacteria and membrane proteins need to be precisely differentiate, other fluorescent fusion proteins, e.g., mTurquoise, are recommended to be integrated. This, however, results in additional rounds of illumination and image acquisition, bearing the risk of higher photo-damage in live cell experiments.
To find out essential genes and mechanisms for bacterial pathogens to manipulate host-cell pathways, numerous mutants need to be created and their performances need to be carefully compared. For example, Salmonella mutant strains &#916;ssaV and &#916;sifA are highly attenuated in systemic virulence and intracellular replication20,21. Both &#916;ssaV and &#916;sifA strains fail to induce SIF, and additionally &#916;sifA Salmonella lose the SCV membrane during the course of infection12. Comparing phenotypes of WT and mutant strains is instrumental in understanding the ability of Salmonella to remodel host cell vesicular traffic. Based on microscopic examination, it is obvious that WT Salmonella subvert host cellular endocytic pathways to a much higher extent compared to &#916;ssaV and &#916;sifA mutant strains. This function might enable intracellular Salmonella to obtain more nutrients for their intracellular survival and replication.
Confocal microscopy images clearly demonstrate the remodeling of host cellular endo-lysosomal system by intracellular bacteria. However, quantitative analysis of the images is required for precise comparison. In the method described here, Imaris software is used for extracting 3D objects which have fluorescence intensity in Channel 3 &#62; 15 and number of voxels &#62; 10. These objects are supposed to be intracellular membrane structures containing Gold-BSA-Rhodamine NPs. The fluorescence intensity threshold and filters can be defined according to the quality of images, but should be kept constant for comparing different conditions or strains. Shown here are examples of a non-infected cell and a WT Salmonella-infected cell at 8 hr p.i., with 431 and 67 objects extracted, respectively. Despite the apparent difference, it is not rational to compare only the number of the objects, since the size of the cells are not the same. One solution is to normalize the number of the objects to the summed area (in this example, 2,252 &#181;m2 and 1,975 &#181;m2, respectively) or the sum of fluorescent intensities of the objects inside cells. Alternatively, it is also possible to track one cell before infection and at different time-points p.i. in order to compare the distribution of NPs at different stages of infection.
In conclusion, in this method gold-BSA-rhodamine NPs were applied to label the endo-lysosomal system of eukaryotic cells. The manipulation of the host cellular endo-lysosomal system by an intracellular pathogen was observed by live cell CLSM and quantitative analysis results indicated extreme rearrangements of late endosomes/lysosomes by WT Salmonella. Salmonella was used as a model pathogen in this study, but intracellular behavior of other bacterial pathogens, such as Shigella flexneri and Listeria monocytogenes could also be investigated using this approach. In principle, gold-BSA-rhodamine NPs could be internalized by a large variety of cell lines, therefore are promising being broadly applied in studies investigating interaction of the endocytic pathway with divergent endogenous or exogenous substances.

Fluorescent nanoparticles (NPs), including metal NPs, quantum dots, polymer NPs, silica NPs, carbon dots, etc., have attracted considerable attention during the past decades1,2. Compared to traditional organic dyes, fluorescent NPs show desirable chemical, optical and mechanical properties, such as strong signal strength, resistance to photobleaching and high biocompatibility3,4. These advantages make them the method of choice for intracellular sensing and live cell imaging. Furthermore, a variety of electron-dense NPs are visible by electron microscopy (EM), facilitating their use for correlated microscopic analysis, which allows combination of live cell tracking with light microscopy (LM) and higher resolution at ultrastructural level with EM5. For example, gold NPs have been long time efficiently used as biosensors in living cells for sensitive diagnosis as well as in the field of immuno-labeling6. Recent studies indicate that gold NPs with different size and shape can be readily uptake by a large variety of cell lines and routinely transport through the endosomal pathway, therefore have great potential being applied for intracellular vesicle transportation tracking and the endo-lysosomal system labeling7,8.
Microbial pathogens, such as Salmonella enterica, Shigella flexneri and Listeria monocytogenes, have developed different mechanisms to invade non-phagocytic host cells9. After being internalized, the pathogens, either localized in the cytosol or sequestered in membrane-bound compartments, interact extensively with their host environments and modulate these to favor their own survival10. For instance, Salmonella enterica resides and replicates within an intracellular phagosomal compartment termed the Salmonella-containing vacuole (SCV) upon infection11. The maturing SCV traffics towards the Golgi apparatus, undergoing continuous interactions with the endocytic pathway, and induces formation of extensive tubular structures, such as Salmonella-induced filaments (SIF), sorting nexin tubules, Salmonella-induced secretory carrier membrane protein 3 (SCAMP3) tubules, etc.12-14. Studying how these bacterial pathogens manipulate host-cell pathways is essential to understanding infectious disease.
Here, gold-BSA-rhodamine NPs were used as fluid tracers to label the host cellular endo-lysosomal system, and the human gastrointestinal pathogen Salmonella enterica serovar Typhimurium (Salmonella) was used as a model bacterium to study the interactions of the pathogen with the host endocytic pathway. Intracellular gold-BSA-rhodamine NPs in non-infected cells and cells infected with WT Salmonella or mutant strains were imaged by a confocal laser-scanning microscope (CLSM). Then Imaris software was used to quantify the distribution of NPs, indicating that Salmonella infection induced extreme rearrangement of the endosomes/lysosomes. Following the description of this method, analogous experiments can be designed to track long-term fate of the internalized NPs and to investigate the influence of various exogenous substances or endogenous factors on the endocytic pathway of eukaryotic cells.

<table><tbody><tr><td>Gold chloride</td><td>Sigma-Aldrich</td><td>520918</td><td></td></tr><tr><td>Tannic acid</td><td>Sigma-Aldrich</td><td>403040</td><td></td></tr><tr><td>Tri-sodium citrate</td><td>Sigma</td><td>C8532</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A2153</td><td></td></tr><tr><td>NHS-Rhodamine</td><td>Pierce</td><td>46406</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>Gentamicin</td><td>Applichem</td><td>A1492</td><td></td></tr><tr><td>Kanamcyin</td><td>Roth</td><td>T832</td><td></td></tr><tr><td>Carbenicillin</td><td>Roth</td><td>6344</td><td></td></tr><tr><td>8-well chamber slides</td><td>Ibidi</td><td>80826</td><td>tissue culture treated, sterile</td></tr><tr><td>Imaris Software</td><td>Bitplane</td><td>version 7.6</td><td>various configurations available</td></tr></tbody></table>

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.

Gold NPs were generated through a well-established method via reduction of chloroauric acid by citrate and tannic acid. As shown in Figure 2A, the synthesized gold NPs were quasi-spherical in shape with a size of approximately 10 nm. BSA-coating and rhodamine-labeling did not influence their morphology or size (Figure 2B).
It has been reported that gold NPs can be readily taken up by various mammalian cells and ended up in the endocytic systems7. In accordance with previous works, bright red fluorescence signals were observed in HeLa cells after 1 hr incubation with gold-BSA-rhodamine NPs (Figure 3, WT 5 hr p.i.), indicating effective internalization of the NPs by cells. Most of the red signals were found colocalized with green signals, showing that the NPs were confined in late endosomes or lysosomes. However, unlike uniform distribution of NPs in late endosomes/lysosomes in non-infected cells (Figure 3, mock), their locations were largely rearranged in WT Salmonella-infected cells. In the early phase of infection (Figure 3, WT 5 hr p.i.), Salmonella replicates within the SCV and SIF undergo fast extension or contraction movement. At this time point, a fraction of the NPs was found in SCV and SIF, while another fraction was still located in free late endosomes/lysosomes. At 8 hr p.i. (Figure 3, WT 8 hr p.i.), a stabilized network of SIF was formed, and most NPs were found accumulated within the tubular structures, while only a very small portion remained located in free late endosomes/ lysosomes. The mutant strains &#916;ssaV and &#916;sifA exhibited distinct behaviors. As shown in Figure 3 (&#916;ssaV), at 8 hr p.i., &#916;ssaV was confined inside SCV while no SIF structures were formed. Some NPs were observed surrounding Salmonella inside the SCV, but the majority of NPs were still located in free late endosomes/lysosomes. For the &#916;sifA strain (Figure 3, &#916;sifA), escape of Salmonella into cytoplasm occurred, and no association between NPs and bacteria was observed.
In our previous study it was found that SIF display highly dynamic properties in the early phase of infection (3 - 5 hr p.i.), but become stabilized in the later period (&#62;8 hr p.i.)12. Therefore, we are wondering whether Salmonella in early and late phases of infection have comparable ability to rearrange distribution of intracellular NPs. As shown in Figure 4, gold NPs were incubated with HeLa cells O/N&#160;before infection, or at different time-points after infection (1 - 7 hr p.i.) for 1 hr, in all cases most of the internalized NPs were observed accumulated in SCV or SIF.
Microscopic observations revealed that infection by Salmonella results in massive rearrangement of the endo-lysosomal system of host cells. As a next step, we used the Imaris software package to analyze Salmonella-induced host cell phenotypes in a quantitative manner. Using the image of one infected HeLa cell at 8 hr p.i. as an example, a step-by-step instruction for quantitative analysis is depicted in Figure 5. Through an intensity threshold of 15 and a &#8216;number of voxels &#62;10&#8217; filter, 3D objects were extracted from the original image file. The objects were supposed to be intracellular structures in which the gold-BSA-rhodamine NPs located with. In this example, 67 objects were extracted in total, with a summed area as 1,974.64 &#181;m2. The largest object, which colocalized with the SIF network, has an area of 1,836.23 &#181;m2, while the others are smaller than 50 &#181;m2. For comparison, one example showing the quantitative analysis result of a non-infected cell was given in. Here, 431 objects were extracted in total, within which the average value of area is 5 &#181;m2 and the largest object has an area of 34 &#181;m2. The summed area of the objects is 2,252 &#181;m2, which is comparable to the infected cell in Figure 4 (1,975 &#181;m2). However, the number of objects is much larger than the other one (431 and 67 objects, respectively), which indicates large extent fusion of vesicles with the Salmonella-induced tubular structures.
<img alt="Figure 1" src="/files/ftp_upload/52058/52058fig1highres.jpg" width="700" /> 
Figure 1. Scheme for preparation of HeLa cells for live cell imaging. HeLa cells were seeded in chamber slides, mock-infected, or infected with various Salmonella strains for 30 min, washed 3 times with PBS, then incubated with medium containing 100 &#181;g/ml gentamicin for 1 hr. Subsequently, the medium was replaced by imaging medium containing 10 nm gold-BSA-rhodamine NPs (OD520 = 0.1) and 10 &#181;g/ml gentamicin, incubated for 1 hr, and then chased by NP-free medium for the rest of the experiment. Live cell imaging was performed at different time points post infection (p.i.). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52058/52058fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52058/52058fig2highres.jpg" width="500" /> 
Figure 2. TEM images of colloidal Gold NPs before (a) and after labeling (b). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52058/52058fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52058/52058fig3highres.jpg" width="500" /> 
Figure 3. Remodeling of the host cellular endo-lysosomal system by intracellular Salmonella. HeLa cells were mock-infected, or infected with Salmonella WT, &#916;ssaV or &#916;sifA mutant strains and pulse/chase with 10 nm gold-BSA-rhodamine NPs (OD520 = 0.1) was performed as described in Figure 1. Maximal intensity projections of CLSM Z-stacks are shown. Scale bars, 10 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52058/52058fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52058/52058fig4highres.jpg" width="700" /> 
Figure 4. Accessibility of the endo-lysosomal system at various phases of Salmonella infection. HeLa cells were pulsed with 10 nm gold-BSA-rhodamine NPs (OD520 = 0.1) O/N&#160;prior infection, or for 1 hr at various time-points p.i. as indicated. Live cell imaging was performed at 8 - 9 hr p.i. Maximal intensity projections of CLSM Z-stacks are shown. Scale bars, 10 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52058/52058fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52058/52058fig5highres.jpg" width="600" /> 
Figure 5. Step by step instruction for quantitative analysis by Imaris using an image of an infected HeLa cell at 8 hr p.i. as an example. (A) Open the data by clicking &#8216;Open&#8217; and choosing the file. (B) In the objects toolbar of the Surpass view add a new surface item. (C) To analyze a ROI, then select segment a ROI. (D) Enter the values in the corresponding x-, y-, and z-fields or (E) directly click on the arrows in the preview rectangle to modify the size and the position of the ROI. In this example, a ROI was not need and the whole image was analyzed. (F) As a source channel select the Channel 3 (signal of Gold-BSA-Rhodamine NPs). Check the smooth option to set up the smoothness of the resulting area. Define a value manually or accept the automatically generated value. Here, 0.1 &#181;m was used. For &#8216;Threshold&#8217; select the &#8216;Absolute Intensity&#8217; option. (G) For the threshold adjustment select the manual option and set a value (in this example 15 was used). In the viewing area a surface threshold preview is displayed in gray. (H) On the tab &#8216;Classify Surfaces&#8217; the resulting surface can be filtered by various criteria. A default filter is &#8216;number of voxels &#62;10&#8217;. The filter can be adjusted by enter a new value and other filters can be added. In this example, we kept the filter &#8216;number of voxels &#62;10&#8217;. (I) To complete Surface creation click on &#8216;Finish&#8217;. In the Object list now un-check the box for the item Volume and new created surface is displayed in viewing area in red. (J) In the Surpass view the color of the subjects vary from purple to red according to their area. (K) The &#8216;Plot Numbers Area&#8217; table shows the statistics information. Export statistics to an Excel file. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52058/52058fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52058/52058fig6highres.jpg" width="600" /> 
Figure 6. Quantitative analysis result of a non-infected cell. (A) Confocal microscope image. (B) The new created surface. (C) The new surface in the Surpass view. (D) The &#8216;Plot Numbers Area&#8217; table shows the statistics information. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52058/52058fig6highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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 ...

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Immunology and Infection

9.9K Views.  Universität Osnabrück. 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.

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

Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells

Transport of proteins and membranes from the cell surface to the Golgi and beyond is essential for homeostasis, organelle identity and physiology. To study retrograde protein traffic, we have recently developed a versatile nanobody-based toolkit to analyze transport from the cell surface to the Golgi complex, either by fixed and live cell imaging, by electron microscopy, or biochemically. We engineered functionalized anti-green fluorescent protein (GFP) nanobodies &#8212; small, monomeric, high-affinity protein binders &#8212; that can be applied to cell lines expressing membrane proteins of interest with an extracellular GFP moiety. Derivatized nanobodies bound to the GFP reporters are specifically internalized and transported piggyback along the reporters&#39; sorting routes. Nanobodies were functionalized with fluorophores to follow retrograde transport by fluorescence microscopy and live imaging, with ascorbate peroxidase 2 (APEX2) to investigate the ultrastructural localization of reporter-nanobody complexes by electron microscopy, and with tyrosine sulfation (TS) motifs to assess kinetics of trans-Golgi network (TGN) arrival. In this methodological article, we outline the general procedure to bacterially express and purify functionalized nanobodies. We illustrate the powerful use of our tool using the mCherry- and TS-modified nanobodies to analyze endocytic uptake and TGN arrival of cargo proteins.

Retrograde transport of proteins from the cell surface to the Golgi is essential to maintain membrane homeostasis. Here, we describe a method to biochemically analyze cell surface-to-Golgi transport of recombinant proteins using functionalized nanobodies in HeLa cells.

Transport of proteins and membranes from the cell surface to the Golgi and beyond is essential for homeostasis, organelle identity and physiology. To ...

Biochemistry

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Neuroscience

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.

This article provides a step-by-step guide to investigate protein subcellular localization dynamics and to monitor morphological changes using high-resolution fluorescence microscopy in Bacillus subtilis and Staphylococcus aureus.

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Biology

Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase nutrients and other material in cells. The dramatic extension of cell surface ruffles, their closure to form macropinosomes, and the maturation of internalized macropinosomes are key events in this pathway that can be difficult to capture using conventional confocal imaging based on tracking a bolus of fluorescent cargo. Fluorescent dextrans are commonly used experimentally as fluid phase markers for macropinosomes and for other endocytic pathways. A method the lab has adopted to optimize the imaging of dextran uptake involves using live imaging of cells bathed in high concentrations of fluorescent dextran in the medium, with the unlabeled cells appearing in relief (as black). The cell ruffles are highlighted to visualize ruffle closure, and internalized macropinosomes appear as fluorescent vacuoles in the cell interior. This method is optimal for visualizing macropinosome features and allows for easy segmentation and quantification. This paper describes dual-labeling of pathways with different sized dextrans and the co-expression of lipid probes and fluorescent membrane proteins to demark macropinosomes and other endosomes. The detection of internalized dextran at an ultrastructural level using correlative light and electron microscopy (CLEM) is also demonstrated. These cell processes can be imaged using multiple live imaging modalities, including in 3D. Taken together, these approaches optimize macropinosome imaging for many different settings and experimental systems.

This protocol demonstrates dextran imaging in live cells using continuous uptake and inverse images to optimize visualization of ruffling, macropinosome maturation, and analysis of dextran and other cell labelings.

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase ...

The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in the biogenesis of melanosomes, a lysosome-related organelle produced by melanocytes. REs deliver the melanocyte-specific cargo to premature melanosomes during their formation. Blockage in the generation of REs, observed in several mutants of Hermansky-Pudlak syndrome, results in hypopigmentation of skin, hair, and eye. Therefore, studying the dynamics (refer to number and length) of REs is useful to understand the function of these organelles in normal and disease conditions. In this study, we aim to measure the RE dynamics using a resident SNARE STX13.

Recycling endosomes are part of the endosomal tubular network. Here we present a method to quantify the dynamics of recycling endosomes using GFP-STX13 as an organelle marker.

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in ...

An Introduction to Endocytosis and Exocytosis

Cells can take in substances from the extracellular environment by endocytosis and actively release molecules into it by exocytosis. Such processes involve lipid membrane-bound sacs called vesicles. Knowledge of the molecular architecture and mechanisms of both is key to understanding normal cell physiology, as well as the disease states that arise when they become defective. This video will first briefly review a few pivotal discoveries in the history of endo- and exocytosis research. Next, some key questions will be examined, followed by a discussion of the prominent methods used to investigate these problems, including cell labeling, fusion assays, and fluorescence imaging. Finally, it will explore current research being conducted by scientists in the field today.

Endocytosis and Exocytosis; Clathrin Coated Pits and SNAREs

Cells can take in substances from the extracellular environment by endocytosis and actively release molecules into it by exocytosis. Such processes ...

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Visualization of Vesicle Motilities in Neurons Using Fluorescence Imaging

Source: Tsuruta, F., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/55488">Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes.</a> J. Vis. Exp. (2017).
This video demonstrates how neuronal vesicles are labeled with specific fluorescent proteins to visualize their movement. First, a plasmid coding for a fluorescent protein is incubated with a transfection agent and then introduced into a culture of neurons. Subsequently, images of the neurons are acquired and analyzed to track the vesicles&#39; movement.

1. Imaging Vesicle Motility

	Prepare the plasmids that will label each type of vesicle. For instance, use EGFP-Rab5 for early endosomes, EGFP-Rab7 for ...

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

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Chapters in this video

Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells

Study of Phagolysosome Biogenesis in Live Macrophages

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis

The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking

An Introduction to Endocytosis and Exocytosis

Visualization of Vesicle Motilities in Neurons Using Fluorescence Imaging