Name: applying live cell imaging and cryo-electron tomography to resolve spatiotemporal features of the legionella pneumophila dot/icm secretion system
Uploaded: 2020-03-10T13:30:00
Description: 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

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:...

<ol>
	<li>Franco, I. S., Shuman, H. A., Charpentier, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+perplexing+functions+and+surprising+origins+of+Legionella+pneumophila+type+IV+secretion+effectors.">The perplexing functions and surprising origins of Legionella pneumophila type IV secretion effectors.</a> Cellular Microbiology. 11, 1435-1443 (2009).</li><li>Burstein, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-scale+identification+of+Legionella+pneumophila+effectors+using+a+machine+learning+approach.">Genome-scale identification of Legionella pneumophila effectors using a machine learning approach.</a> PLOS Pathogens. 5, 1000508 (2009).</li><li>Ninio, S., Roy, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effector+proteins+translocated+by+Legionella+pneumophila:+strength+in+numbers.">Effector proteins translocated by Legionella pneumophila: strength in numbers.</a> Trends in Microbiology. 15, 372-380 (2007).</li><li>Vogel, J. P., Andrews, H. L., Wong, S. K., Isberg, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugative+transfer+by+the+virulence+system+of+Legionella+pneumophila.">Conjugative transfer by the virulence system of Legionella pneumophila.</a> Science. 279, 873-876 (1998).</li><li>Isberg, R. R., O'Connor, T. J., Heidtman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Legionella+pneumophila+replication+vacuole:+making+a+cosy+niche+inside+host+cells.">The Legionella pneumophila replication vacuole: making a cosy niche inside host cells.</a> Nature Reviews Microbiology. 7, 13-24 (2009).</li><li>Roy, C. R., Berger, K. H., Isberg, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Legionella+pneumophila+DotA+protein+is+required+for+early+phagosome+trafficking+decisions+that+occur+within+min+of+bacterial+uptake.">Legionella pneumophila DotA protein is required for early phagosome trafficking decisions that occur within min of bacterial uptake.</a> Molecular Microbiology. 28, 663-674 (1998).</li><li>Archer, K. A., Roy, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MyD88-Dependent+Responses+Involving+Toll-Like+Receptor+2+Are+Important+for+Protection+and+Clearance+of+Legionella+pneumophila+in+a+Mouse+Model+of+Legionnaires'+Disease.">MyD88-Dependent Responses Involving Toll-Like Receptor 2 Are Important for Protection and Clearance of Legionella pneumophila in a Mouse Model of Legionnaires' Disease.</a> Infection and Immunity. 74, 3325-3333 (2006).</li><li>Nagai, H., Kubori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+IVB+Secretion+Systems+of+Legionella+and+Other+Gram-Negative+Bacteria.">Type IVB Secretion Systems of Legionella and Other Gram-Negative Bacteria.</a> Frontiers in Microbiology. 2, 136 (2011).</li><li>Kubori, T., Nagai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Type+IVB+secretion+system:+an+enigmatic+chimera.">The Type IVB secretion system: an enigmatic chimera.</a> Current Opinion in Microbiology. 29, 22-29 (2016).</li><li>Chetrit, D., Hu, B., Christie, P. J., Roy, C. R., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+cytoplasmic+ATPase+complex+defines+the+Legionella+pneumophila+type+IV+secretion+channel.">A unique cytoplasmic ATPase complex defines the Legionella pneumophila type IV secretion channel.</a> Nature Microbiology. 3, 678-686 (2018).</li><li>Merriam, J. J., Mathur, R., Maxfield-Boumil, R., Isberg, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+Legionella+pneumophila+fliI+gene:+intracellular+growth+of+a+defined+mutant+defective+for+flagellum+biosynthesis.">Analysis of the Legionella pneumophila fliI gene: intracellular growth of a defined mutant defective for flagellum biosynthesis.</a> Infection and Immunity. 65, 2497-2501 (1997).</li><li>Feeley, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charcoal-yeast+extract+agar:+primary+isolation+medium+for+Legionella+pneumophila.">Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila.</a> Journal of Clinical Microbiology. 10, 437-441 (1979).</li><li>Andrews, H. L., Vogel, J. P., Isberg, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+linked+Legionella+pneumophila+genes+essential+for+intracellular+growth+and+evasion+of+the+endocytic+pathway.">Identification of linked Legionella pneumophila genes essential for intracellular growth and evasion of the endocytic pathway.</a> Infection and Immunity. 66, 950-958 (1998).</li><li>Morado, D. R., Hu, B., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Tomoauto:+A+Protocol+for+High-throughput+Automated+Cryo-electron+Tomography.">Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography.</a> Journal of Visualized Experiments. (107), e53608 (2016).</li><li>Hu, B., Lara-Tejero, M., Kong, Q., Galan, J. E., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Situ+Molecular+Architecture+of+the+Salmonella+Type+III+Secretion+Machine.">In Situ Molecular Architecture of the Salmonella Type III Secretion Machine.</a> Cell. 168, 1065-1074 (2017).</li><li>Mastronarde, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+electron+microscope+tomography+using+robust+prediction+of+specimen+movements.">Automated electron microscope tomography using robust prediction of specimen movements.</a> Journal of Structural Biology. 152, 36-51 (2005).</li><li>Zheng, S. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MotionCor2:+anisotropic+correction+of+beam-induced+motion+for+improved+cryo-electron+microscopy.">MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy.</a> Nature Methods. 14, 331-332 (2017).</li><li>Kremer, J. R., Mastronarde, D. N., McIntosh, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+visualization+of+three-dimensional+image+data+using+IMOD.">Computer visualization of three-dimensional image data using IMOD.</a> Journal of Structural Biology. 116, 71-76 (1996).</li><li>Gilbert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iterative+methods+for+the+three-dimensional+reconstruction+of+an+object+from+projections.">Iterative methods for the three-dimensional reconstruction of an object from projections.</a> Journal of Theoretical Biology. 36, 105-117 (1972).</li><li>Radermacher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Weighted+Back-projection+Methods.">Weighted Back-projection Methods.</a> Electron Tomography. , 245-273 (2007).</li><li>Winkler, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomographic+subvolume+alignment+and+subvolume+classification+applied+to+myosin+V+and+SIV+envelope+spikes.">Tomographic subvolume alignment and subvolume classification applied to myosin V and SIV envelope spikes.</a> Journal of Structural Biology. 165, 64-77 (2009).</li><li>Winkler, H., Taylor, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+marker-free+alignment+with+simultaneous+geometry+determination+and+reconstruction+of+tilt+series+in+electron+tomography.">Accurate marker-free alignment with simultaneous geometry determination and reconstruction of tilt series in electron tomography.</a> Ultramicroscopy. 106, 240-254 (2006).</li><li>Prevost, M. S., Waksman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+crystal+structures+of+the+type+IVb+secretion+system+DotB+ATPases.">X-ray crystal structures of the type IVb secretion system DotB ATPases.</a> Protein Science. 27, 1464-1475 (2018).</li><li>Miklos, G. L., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+genome+project+in+determining+gene+function:+insights+from+model+organisms.">The role of the genome project in determining gene function: insights from model organisms.</a> Cell. 86, 521-529 (1996).</li><li>Reyrat, J. M., Pelicic, V., Gicquel, B., Rappuoli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counterselectable+markers:+untapped+tools+for+bacterial+genetics+and+pathogenesis.">Counterselectable markers: untapped tools for bacterial genetics and pathogenesis.</a> Infection and Immunity. 66, 4011-4017 (1998).</li><li>Yu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Molecule+Studies+in+Live+Cells.">Single-Molecule Studies in Live Cells.</a> Annual Review of Physical Chemistry. 67, 565-585 (2016).</li><li>Stewart, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-electron+microscopy+and+cryo-electron+tomography+of+nanoparticles.">Cryo-electron microscopy and cryo-electron tomography of nanoparticles.</a> Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology. 9, (2017).</li></ol>

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 ...

This protocol is useful for characterizing the assembling functions of bacterial secretion complexes and can lead to a fundamental understanding of the molecular mechanisms that are required for host pathogen interactions. This technique integrates complementary approaches that preserve the native structure of the sample and allows and see to visualization of proteins complexes in intact bacterial cells. It increases our understanding of the structural function relationships of bacterial secretion systems.This method can be adapted to study other types of secretion systems or other cellular complexes in a wide variety of bacterial species. Start by making agarose pads for live cell imaging. Prepare about 30 mL of 1%low melt agarose solution in water and microwave it in a glass flask for about 90 seconds swirling it occasionally until the agarose is completely dissolved.Place two 22 by 22 by 0.15mm glass slides on the edge of a 25 by 75 by 1.1mm glass slide one on top of the other. Then, stack two more of the smaller slides on the other edge. Pipette about 1 mL of the molten agarose into the center slide between the two upper glass slides and place another large slide on top of the molten agarose making sure to avoid formation of air bubbles.Cool the slides at four degrees celsius for 15 minutes then use a scalpel or razor blade to gently cut the pad into small squares. Fix a double-sided adhesive frame on a 25 by 75 by 1.1mm glass slide and place several pads on the slide. Dissolve a heavy patch of Legionella Pneumophila in 1 mL of double-distilled water.Then vortex and pipette two to three microliters of the dilution onto the pads. Gently place a 58 by 24 by 0.15 mm cover slip over the adhesive frame. Select cells for imaging using bright field lighting.Then, in the capture window of the microscope's software, Adjust the ND to 180 and the binning to two by two. Use the 488 nanometer channel to expose the sample for 500 to 1000 milliseconds and validate the specificity of the fluorescence by imaging untagged Legionella Pneumophila with the same parameters. To quantify polarity, adjust the image contrast so the bacteria are clearly visible.Then zoom in on the region of interest and use the region tool to place a 0.25 by 1.3 micrometer rectangle starting at the pole and extending into the cytoplasm making sure that the rectangle remains within the bacterial borders. Mark at least 200 bacteria and create masks by using the region to mask button in the analyze menu. Click on mask statistics and choose object under mask scope.Then mark mean intensity and variance under features and intensity. Export the data as a text file and use the spreadsheet application to calculate the polarity scores of each bacterium as the ratio between the variance and the mean intensity. For high throughput applications use face contrast microscopy to acquire dual channel images.Make sure to choose fields of view where the bacteria are fully separated. Next, adjust the face channel contrast of the image to a level where the bacteria are clearly visible. Open the dual channel image and launch the create segment mask window.Adjust the appropriate threshold, then remove the small objects by clicking the define objects button and adjusting the minimum size to 100 pixels. Under refine mask, select remove edges objects and separate masks of bacteria that are adjacent to each other. To quantify the dynamics of the fusion proteins, open the image capture window and mark time lapse.Enter two in the number of time points box. Acquire two successful images of Legionella Pneumophila expressing the fluorescent protein of interest. Adjust the image contrast until the cells are clearly visible.Then use the region tool to place a 0.25 by 0.25 micrometer square in the middle of at least 400 cells. Next, use the region to mask button in the analyze menu to create a mask of the squares of interest. Create a new empty mask and use the pixel tool to mark the entire cell area of at least 25 random cells.Create another mask and use the large brush tool to mark areas between the cells. Finally, select mask statistics and make sure object is selected under mask scope for mask one. Under features and intensity, choose mean intensity.Export the data and repeat the process for mask 2. For mask 3, select the entire mask and export the mean intensity. To prepare the electron cryotomography sample, grow a heavy patch of Legionella Pneumophila on CYE Agrastreptomisum plates for 48 hours at 37 degrees Celsius.Resuspend the cells in water to an OD 600 of about 0.7. Then add five microliters of colloidal gold particles to 20 microliters of the cell suspension. Glow discharge a holey carbon grid and pipette five microliters of the cell mixture on it.Let it stand for one minute. Blot the grid with filter paper and freeze it in liquid ethane using a gravity-driven plunger apparatus. After inserting superfolder GFP into the Legionella Pneumophila chromosome, live cell fluorescent microscopy was performed to compare the fluorescent signal with that of the parental superfolder GFP negative strain.In addition, bright field microscopy was used to examine the proportion of cells with a specific fluorescent signal. Only a fraction of the cells that produced DotB superfolder GFP displayed polar localization of the fusion protein. When characterizing the distribution of DotB fluorescent signal, it was found that the intensity of DotB superfolder GFP at the poles was about two times higher than in the cytosol.To investigate if DotB superfolder GFP polar localisation was dependent on a fully assembled type four secretion system, polarity scores of DotB superfolder GFP were determined for individual cells in a wild type, and in a type four secretion system mutant. Indeed, polar positioning of DotB superfolder GFP disappeared when the Dot/ICM system was deleted. Dynamic patterns indicated that the DotB ATPase was present in a dynamic cytosolic population and was recruited to the polar localized Dot/ICM type four secretion system at a late stage assembly reaction.High throughput cryo-ET was used to visualize the intact type four secretion system apparatus in a Legionella Pneumophila strain expressing DotB superfolder GFP. Reconstruction of the cell revealed multiple Dot/ICM machines embedded in the cell envelope. The overall positioning of DotB and superfolder GFP in relation to the intact type four secretion system machine was also determined.Three-dimensional surface renderings indicated that superfolder GFP is positioned below the DotB hexamer, which assembles as a disc at the base of the cytoplasmic ATPase complex. When attempting this technique, make sure to assess the stability of the fusion proteins and the functionality of the tag secretion system before proceeding to image acquisition. Modeling and fitting can be effective ways to analyze the dynamic patterns and structural densities as well as for evaluation of conformational changes or localisation of subunits into the structure.This approach can help in understanding how different type four secret system components function, how they interact within the greater complex, and what functions different subassemblies perform.

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

Research

JoVE Journal

Immunology and Infection

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 ...

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy1 and two-dimensional (2D) electron crystallography2 have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method3, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments4, microtubules5, amyloid fibers6, tobacco mosaic viruses7, and bacteria flagella8, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.
In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid9 is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein ...

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 ...

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 ...