D.C. and C.R.R. were supported by the NIH (R37AI041699 and R21AI130671). D.P., B.H., and J.L were supported by the National Institutes of Health (R01AI087946 and R01GM107629).

NOTE: All procedures involving the growth, manipulation, and imaging of L. pneumophila should be performed in a biological safety level 2 laboratory in compliance with local guidelines.
1. Insertion of sfGFP into L. pneumophila Chromosome Using Allelic Exchange and Double Selection Strategy (Figure 2, Figure 3)
<ol>
	<li>Clone into the gene replacement vector pSR47S11 the following sequence: the 1,000 bp upstream of the site of interest, then the sfGFP sequence, then the 1,000 bp downstream of the site of interest (Figure 2). The sfGFP sequence should be placed in frame to the N-terminus or C-terminus ends with a linker that contains four to eight amino acids. Transform the resulting vector into E. coli DH5&#945;&#955;pir. Later, streak L. pneumophila (the recipient) for single colonies on charcoal-yeast extract (CYE) agar12 containing 100 &#181;g/mL streptomycin and grow for 5 days at 37 &#176;C (Figure 3).</li>
	<li>Streak L. pneumophila on CYE-agar-streptomycin and grow for 2 days at 37 &#176;C (heavy patch)10. Streak E. coli DH5&#945; transformed with pRK600 helper plasmid (helper)13 on LB agar containing 25 &#181;g/mL chloramphenicol. Streak the E. coli DH5&#945;&#955;pir (donor) on LB agar containing 50 &#181;g/mL kanamycin.</li>
	<li>Perform triparental mating: incubate a colony of the helper, a colony of the donor, and the recipient by overlaying patches of the three strains on a CYE agar plate without selection and incubating for 4&#8211;8 h at 37 &#176;C. As negative controls incubate a helper+recipient strain mix and a donor+recipient strain mix for the same periods of time.</li>
	<li>Resuspend the mating reactions in 500 &#181;L of ddH2O. Plate 20 &#181;L and 50 &#181;L of the reactions on CYE agar containing 100 &#181;g/mL streptomycin and 10 &#181;g/mL kanamycin and grow for 5 days at 37 &#176;C. Streak four of the resulting clones on CYE agar containing 100 &#181;g/mL streptomycin and grow for 5 days at 37 &#176;C.</li>
	<li>Streak 16 clones on CYE agar containing 5% sucrose and 100 &#181;g/mL streptomycin and grow for 5 days at 37 &#176;C. Then, streak 32 of these clones on CYE agar containing 100 &#181;g/mL streptomycin and on CYE agar containing 100 &#181;g/mL streptomycin and 10 &#181;g/mL kanamycin and grow for 5 days at 37 &#176;C.</li>
</ol>
2. Isolation of Clones that Integrated sfGFP into the L. pneumophila Chromosome
<ol>
	<li>Streak clones that were sensitive to kanamycin on CYE-agar-streptomycin plates and confirm the insertion of sfGFP into the chromosome with colony PCR. Use primers that are complementary to the sfGFP gene and to the chromosomal region of interest to amplify the insertion junction.
	<ol>
		<li>Mix 0.5 &#181;L of each of the 10 &#181;M primers solutions and one colony to a final volume of 12.5 &#181;L and denature for 10 min at 95 &#176;C. Cool on ice for 10 min, add 12.5 &#181;L of 2x PCR master mix solution, and perform a PCR analysis.</li>
	</ol>
	</li>
	<li>Grow heavy patches of the isolated colonies on CYE-agar-streptomycin plates for 2 days at 37 &#176;C. Examine the expression levels and stability of the sfGFP fusions by immunoblotting with an anti-GFP antibody.</li>
</ol>
3. Live Cell Imaging of L. pneumophila with Fluorescently Tagged Dot/Icm Components
<ol>
	<li>Preparation of agarose pads
	<ol>
		<li>Make about 30 mL of a 1% low-melt agarose solution in water. Microwave in a glass flask for about 90 s, swirling occasionally, until the agarose is completely dissolved.</li>
		<li>Place two 22 x 22 x 0.15 mm3 glass slides on the edge of a 25 x 75 x 1.1 mm3 glass slide, one on top of the other. Stack two more 22 x 22 x 0.15 mm3 glass slides on the other edge.</li>
		<li>Pipette about 1 mL of the molten agarose into the center slide between the two upper glass slides, then place another 25 x 75 x 1.1 mm3 slide on top of the molten agarose. Try to avoid the formation of air bubbles. Cool the slides at 4 &#176;C for 15 min.</li>
		<li>Using a scalpel or razor blade, gently cut the pad into small squares, ~5 x 5 mm2. Fix a double-sided adhesive 17 x 28 x 0.25 mm3 frame on a 25 x 75 x 1.1 mm3 glass slide and place several pads on the slide.</li>
	</ol>
	</li>
	<li>Image acquisition 
	NOTE: The following steps are described for a microscope that is under the control of SlideBook 6.0 and equipped with solid state illuminators, CCD monochrome camera, and a 100x objective lens (1.4 numerical aperture). If needed, use alternative microscopy devices with appropriate hardware and software configurations that can be customized according to the protocol settings.
	<ol>
		<li>Dissolve a heavy patch of L. pneumophila in 1 mL of ddH2O, vortex and pipet 2&#8211;3 &#181;L of the dilution onto the pads. Place a 50 x 24 x 0.15 mm3 coverslip gently over the adhesive frame.</li>
		<li>In the capture window adjust the ND to 180. Adjust the binning to 2&#215;2 and use the 488 nm channel to expose the sample between 500&#8211;1,000 ms. Validate the specificity of the fluorescence signal by imaging untagged L. pneumophila with the same parameters (Figure 4).</li>
	</ol>
	</li>
</ol>
4. Quantification of Polar Localization and Dynamics of Dot/Icm Components
NOTE: The following steps are designed for images with 0.129 &#181;m per pixel that were acquired with 2 x 2 binning.
<ol>
	<li>Quantification of polarity for sfGFP fusion proteins (Figure 5)
	<ol>
		<li>Adjust the image contrast so the bacteria are clearly visible. Use the region tool to place a 0.25 x 1.3 &#181;m2 rectangle starting at the pole and extending into the cytoplasm. The rectangle must remain precisely within the bacterial borders.</li>
		<li>Mark at least 200 bacteria and use the Region to Mask button to create masks to the regions of interest. Under Mask Statistics and Mask Scope, choose Object. Then, under Features and Intensity, mark Mean Intensity and Variance.</li>
		<li>Export the data and calculate the polarity scores of each bacterium as the ratio between the variance to the mean intensity.</li>
		<li>For high-throughput applications use a phase objective and an appropriate condenser setup to acquire images with the phase and 488 nm channels. Make sure to choose fields of view where the bacteria are fully separated.</li>
		<li>Adjust the phase channel contrast of the image to a level where the bacteria are clearly visible. Open the dual channel image, launch the Create Segment Mask window and change the channel to phase.</li>
		<li>Adjust an appropriate threshold and remove small objects with the Define Objects button. Under Refine Mask choose Remove Edges Objects and later separate masks of bacteria that are adjacent to each other.</li>
		<li>Calculate the polarity scores of the signal in the 488 channel for each cell as was previously described in steps 4.1.2&#8211;4.1.3.</li>
	</ol>
	</li>
	<li>Quantification of dynamics for sfGFP fusion proteins (Figure 6) 
	NOTE: Follow the instructions in section 3 to prepare a sample for image acquisition. Dynamics are defined as changes in intensity over time and the following steps are designed for short imaging periods (i.e., several minutes). Add to the pad the appropriate supplements if longer imaging periods are desired.
	<ol>
		<li>In the Image Capture window, mark Timelapse, enter the time of intervals in the interval box, and enter 2 in the # of Time Points box. Acquire two successive images of L. pneumophila expressing the fluorescent protein of interest.</li>
		<li>Adjust the image contrast until the cells are clearly visible. Follow Figure 6A and the descriptions below to place three different masks. Use the region tool to place a 0.25 x 0.25 &#181;m square in the middle of at least 400 cells.</li>
		<li>Use the Region to Mask button to create a mask (mask 1) of the squares of interest. Create a new empty mask (mask 2) and use the pixel tool or the polygon tool to mark the entire cell area of at least 25 random cells, which will be used to calculate fluorescence bleaching. Create a new empty mask (mask 3) and use the large brush tool to mark areas between the cells, which will be used for background subtraction.</li>
		<li>Under Mask Statistics and Mask Scope, choose Object for mask 1 and mask 2. Then, under Features and Intensity, choose Mean Intensity and export the data of the two masks. For mask 3, export the mean intensity of the entire mask.</li>
		<li>Calculate the change in fluorescence intensity for each object in mask 1 using the following formulas:</li>
	</ol>
	</li>
</ol>
<img alt="Equation 1" src="/files/ftp_upload/60693/60693eq1v2.jpg" />
<img alt="Equation 2" src="/files/ftp_upload/60693/60693eq2b.jpg" />
where mask 1 is a 0.25 &#181;m &#215; 0.25 &#181;m square placed at the cell center, mask 2 covers the whole cell, mask 3 is the background between cells, t1 is the mean intensity in the first time point, and t2 is the mean intensity in the second time point.
5. Detection of sfGFP Mass Density with Cryo-ET
<ol>
	<li>Sample preparation, data collection, and reconstruction
	<ol>
		<li>Grow a heavy patch of L. pneumophila expressing a fluorescently tagged Dot/Icm protein on CYE-agar-streptomycin plates for 48 hours at 37 &#176;C. Resuspend the cells in ddH2O to OD600 ~0.7. Add 5 &#181;L of colloidal gold particles (BSA Tracer, 10 nm) to 20 &#181;L of the cell suspension.</li>
		<li>Pipette 5 &#956;L of the cell mixture onto freshly glow-discharged holey carbon grid (R 2/1 on Cu 200 mesh) and let stand for 1 min. Blot with filter paper and freeze in liquid ethane using a gravity-driven plunger apparatus as described previously14,15.</li>
		<li>Image the frozen-hydrated specimens with a 300 kV transmission electron microscope equipped with a field emission gun, an energy filter, Volta phase plate, and a direct detection device. Collect single-axis tilt series at 26,000x and 42,000x magnifications, which result in pixel sizes at the specimen level of 5.4 &#197;/pixel or 3.4 &#197;/pixel, respectively.</li>
		<li>Use the tomographic package SerialEM to collect image stacks at ~0 &#956;m defocus, with a range of tilt angles between -60&#176; and +60&#176; with 3&#176; step increment and accumulative dose of ~60 e-/&#197;2,16. Align dose-fractionated movie images in each stack using MotionCor217. Assemble drift-corrected stacks using TOMOAUTO14.</li>
		<li>Align drift-corrected stacks by the IMOD marker-dependent alignment18. Reconstruct tomograms with the SIRT method19 for segmentations and direct image analysis and the WBP method20.</li>
	</ol>
	</li>
	<li>Subtomogram analysis of sfGFP fusions samples
	<ol>
		<li>Use the tomographic package I3 (0.9.9.3) for subtomogram analysis14,21,22. 
		NOTE: The alignment proceeds iteratively, with each iteration consisting of three parts in which references and classification masks are generated, subtomograms are aligned and classified, and class averages are aligned to each other.</li>
		<li>Use 4 x 4 x 4 binned subtomograms for an initial alignment. Merge particles that belong to class averages and show electron density corresponding to sfGFP. After sorting particles with sfGFP fusions, use 2 x 2 x 2 binned subtomograms for a focused alignment of a region of interest (like the Dot/Icm cytoplasmic ATPase complex) to obtain a high-resolution structure.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Elucidating the functions of bacterial secretion systems is key to a complete understanding of host-pathogen interactions. Secretion systems are complex machines that can inject effectors proteins into host cells, and in some cases promote establishment of a subcellular niche that supports bacterial replication. The above method provides important new tools for studying the Dot/Icm secretion system of the respiratory bacterial pathogen Legionella pneumophila, yielding clues to the mechanisms of effector transloc...

Legionella pneumophila (L. pneumophila), the etiological agent of Legionnaires&#39; disease, inhabits freshwater reservoirs, where the bacteria propagate by infecting and replicating within aquatic free-swimming protozoa. L. pneumophila causes disease outbreaks in humans when inhalation of aerosolized bacteria from potable water sources occurs. In infected cells, subversion of host pathways allows L. pneumophila to delay endocytic maturation of the vacuole in which it resides and to promote biogenesis of a cellular compartment that supports bacterial replication. This process is driven by a specialized bacterial type IVB secretion system (T4BSS) known as Dot/Icm and its repertoire of over 300 &#34;effector&#34; proteins that are translocated into the host cytosol during infection to facilitate manipulation of cellular functions1,2,3,4,5. Mutants lacking a functional Dot/Icm apparatus fail to deliver effectors into the host cytosol, are defective for intracellular replication, and are avirulent in animal models of disease6,7.
Many bacterial species have developed extremely complex and dynamic multicomponent machines that are required for infection processes. Other T4BSS like the Dot/Icm system are also essential for intracellular replication of bacterial pathogens such as Coxiella burnetii and Rickettsiella grylli. Although T4BSS are evolutionarily related to prototypical type IVA systems, which mediate DNA transfer and can deliver a limited repertoire of effector proteins, the Dot/Icm system has nearly twice as many machine components and delivers a wide variety of effectors. Presumably, this expansion in the number of components has enabled the Dot/Icm apparatus to accommodate and integrate new effectors easily8,9.
We recently used cryo-electron tomography (cryo-ET) to solve the structure of the Dot/Icm apparatus in situ and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Further analysis revealed that the cytosolic ATPase DotB associates with the Dot/Icm system at the L. pneumophila cell pole through interactions with the cytosolic ATPase DotO. We have discovered that DotB displays a cytosolic movement in most bacterial cells, indicating that this ATPase is present in a dynamic cytosolic population but also associates with the polar Dot/Icm complexes. In addition, DotO forms a hexameric assembly of DotO dimers associated with the inner membrane complex, and a DotB hexamer joins to the base of this cytoplasmic complex. The assembly of the DotB-DotO energy complex creates a cytoplasmic channel that directs the translocation of substrates through the T4SS (Figure 1)10.
Despite these recent advances, little is known about how the Dot/Icm system functions and how each protein assembles to form an active apparatus8. Uncovering the regulatory circuitry of the Dot/Icm T4SS is fundamental to understanding the molecular mechanisms of host-pathogen interactions. Therefore, we discuss how to use live cell microscopy and cryo-ET to detect and characterize essential L. pneumophila Dot/Icm system components that are tagged with super-folder GFP (sfGFP). Using quantitative fluorescence microscopy, the polar localization of DotB will be defined in a wild type background or when the type IV system is deleted. Time-lapse microscopy will be used to quantify differences in localization and dynamics between the Dot/Icm cytosolic ATPases.
The combined application of two complementary approaches such as live imaging and cryo-ET provides an advantage compared to other in vitro systems. Both methods are performed in intact cells and preserve the natural environment of the T4BSS, thus minimizing disruption of the native structure during sample preparation. Because overexpression of proteins may impair the stoichiometry of the secretion apparatus, sfGFP fusions are returned via allelic exchange to the Legionella chromosome so that each fusion is encoded in single copy and the expression is driven by the endogenous promoter. Visualization of chromosomally-encoded fusions enables quantification of the exact level of protein being expressed at a defined time point. Cryo-ET also has many advantages for determining the structure of secretion systems. The most notable advantage is that cryo-ET samples are comprised of frozen intact cells that preserve native complexes in the context of bacterial cell architecture. Consequently, cryo-ET may be preferable to biochemical purification approaches, which extract membrane complexes and may strip peripheral proteins from the core apparatus or modify the overall structure. In addition, tagging a protein of interest with a bulky protein such as sfGFP adds a mass that is detectable by cryo-ET and can assist with mapping the different subcomplexes of the Dot/Icm apparatus onto the structure obtained by cryo-ET.
This approach is a powerful tool for uncovering structural information about multimolecular complexes that assemble in the bacterial cell membrane. The interpretation of structures elucidated using these techniques will help the field understand how T4BSS components function, why so many components are required for function, how the components interact within the greater complex, and what functions these subassemblies perform.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 nm colloidal gold particles</td>
			<td>Aurion</td>
			<td>25486</td>
			<td></td>
		</tr>
		<tr>
			<td>100x Plan Apo objective (1.4 NA)</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ACES</td>
			<td>Sigma-Aldrich</td>
			<td>A9758</td>
			<td></td>
		</tr>
		<tr>
			<td>Activated charcoal</td>
			<td>Sigma-Aldrich</td>
			<td>C5510</td>
			<td></td>
		</tr>
		<tr>
			<td>Agaroze GPG/LMP, low melt</td>
			<td>American bioanalytical</td>
			<td>AB00981</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto dehydrated agar</td>
			<td>BD</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>CoolSNAP EZ 20 MHz digital monochrome camera</td>
			<td>Photometrics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gene Frame, 1.7x2.8 cm, 125 &#181;L</td>
			<td>Fisher Scientific</td>
			<td>AB-0578</td>
			<td></td>
		</tr>
		<tr>
			<td>Holey Carbon grid R 2/1 Cu 200 mesh</td>
			<td>Quantifoil</td>
			<td>Q225-CR1</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron(III) nitrate nonahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>216828</td>
			<td></td>
		</tr>
		<tr>
			<td>K2 Summit camera for cryo-EM</td>
			<td>GATAN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>C7352</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover slides 22x22 mm</td>
			<td>Fisher Scientific</td>
			<td>12-542B</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover slides 24x50 mm</td>
			<td>Fisher Scientific</td>
			<td>12-545K</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides 25x75x1 mm</td>
			<td>Globe Scientific</td>
			<td>1380</td>
			<td></td>
		</tr>
		<tr>
			<td>SlideBook 6.0</td>
			<td>Intelligent Imaging Innovations</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectra X light engine</td>
			<td>Lumencor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Taq 2X Master Mix</td>
			<td>New England BioLabs</td>
			<td>M0270</td>
			<td></td>
		</tr>
		<tr>
			<td>Titan Krios</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>BD</td>
			<td>212750</td>
			<td></td>
		</tr>
	</tbody>
</table>

applying live cell imaging and cryo-electron tomography to resolve spatiotemporal features of the legionella pneumophila dot/icm secretion system

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

Homologous recombination with double selection in two steps was used to construct the defined insertion of sfGFP. In the first step, triparental mating was performed, where the pRK600 conjugative plasmid (an IncP plasmid) from the E. coli helper strain MT616 was mobilized to the donor E. coli strain with the suicide vector pSR47S containing the sfGFP gene flanked by the two homologous regions, the origin of transfer oriT and the Bacillus subtilis counterselection gene sacB. Next, the c...

Watch this Scientific Journal Video about Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System at JoVE.com

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

applying-live-cell-imaging-cryo-electron-tomography-to-resolve

Research

JoVE Journal

Immunology and Infection

6.8K Views.  Yale University School of Medicine. Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

Video: Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread.

Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.

Advances in live cell fluorescence microscopy  techniques, as well as the construction of recombinant viral strains that  express fluorescent fusion ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

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 ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has a broad spectrum of applications. Detection of antigens allows for characterization and identification of multiple cell types. Located above the kidneys and encapsulated by a layer of mesenchymal cells, the adrenal gland is an endocrine organ composed by two different tissues with different embryological origins, the mesonephric intermediate mesoderm-derived outer cortex and the neural crest-derived inner medulla. The adrenal cortex secretes steroids (i.e., mineralocorticoids, glucocorticoids, sex hormones), whereas the adrenal medulla produces catecholamines (i.e., adrenaline, noradrenaline). While conducting adrenal research, it is important to be able to distinguish unique cells with different functions. Here we provide a protocol developed in our laboratory that describes a series of sequential steps required for obtaining immunofluorescence staining to characterize the cell types of the adrenal gland. We focus first on the dissection of the mouse adrenal glands, the microscopic removal of periadrenal fat followed by the fixation, processing and paraffin embedding of the tissue. We then describe sectioning of the tissue blocks with a rotary microtome. Lastly, we detail a protocol for immunofluorescent staining of adrenal glands that we have developed to minimize both non-specific antibody binding and autofluorescence in order to achieve an optimal signal.

Here we present a method to isolate adrenal glands from mice, fix the tissues, section them, and perform immunofluorescence staining.

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has ...