Name: immunofluorescence analysis of stress granule formation after bacterial challenge of mammalian cells
Uploaded: 2017-07-03T13:00:00.000+00:00
Duration: 11 min 37 s
Description: Watch this Scientific Journal Video about Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells at JoVE.com

PS is a recipient of the Bill and Melinda Gates Grand Challenge Grant OPP1141322. PV was supported by a Swiss National Science Foundation Early Postdoc Mobility fellowship and a Roux-Cantarini postdoctoral fellowship. PJS is supported by an HHMI grant and ERC-2013-ADG 339579-Decrypt.

<ol>
	<li>Reineke, L. C., Lloyd, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversion+of+stress+granules+and+P-bodies+during+viral+infection.">Diversion of stress granules and P-bodies during viral infection.</a> Virology. 436 (2), 255-267 (2013).</li><li>Kedersha, N., Ivanov, P., Anderson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+granules+and+cell+signaling:+more+than+just+a+passing+phase.">Stress granules and cell signaling: more than just a passing phase.</a> Trends Biochem Sci. 38 (10), 494-506 (2013).</li><li>Vonaesch, P., Campbell-Valois, F. -. X., Dufour, A., Sansonetti, P. 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C., Musch, M. W., Chang, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+granule+formation+mediates+the+inhibition+of+colonic+Hsp70+translation+by+interferon-+and+tumor+necrosis+factor.">Stress granule formation mediates the inhibition of colonic Hsp70 translation by interferon- and tumor necrosis factor.</a> Am J Physiol Gastointest Liver Physiol. 298 (4), 481-492 (2010).</li><li>Barry, E. M., Pasetti, M. F., Sztein, M. B., Fasano, A., Kotloff, K. L., Levine, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+pitfalls+in+Shigella+vaccine+research.">Progress and pitfalls in Shigella vaccine research.</a> Nat Rev Gastroenterol Hepatol. 10 (4), 245-255 (2013).</li><li>Lanata, C. F., Fischer-Walker, C. L., 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=Global+Causes+of+Diarrheal+Disease+Mortality+in+Children.">Global Causes of Diarrheal Disease Mortality in Children.</a> PloS One. 8 (9), 72788 (2013).</li><li>Ashida, H., Ogawa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shigella+deploy+multiple+countermeasures+against+host+innate+immune+responses.">Shigella deploy multiple countermeasures against host innate immune responses.</a> Curr Opin Microbiol. 14 (1), 16-23 (2011).</li><li>Ashida, H., Ogawa, M., Mimuro, H., Kobayashi, T., Sanada, T., Sasakawa, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shigella+are+versatile+mucosal+pathogens+that+circumvent+the+host+innate+immune+system.">Shigella are versatile mucosal pathogens that circumvent the host innate immune system.</a> Curr Opin Immunol. 23 (4), 448-455 (2011).</li><li>Kedersha, N., Anderson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+stress+granules+and+processing+bodies.">Mammalian stress granules and processing bodies.</a> Methods Enzymol. 431, 61-81 (2007).</li><li>Ray, K., Marteyn, B., Sansonetti, P. J., Tang, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+on+the+inside:+the+intracellular+lifestyle+of+cytosolic+bacteria.">Life on the inside: the intracellular lifestyle of cytosolic bacteria.</a> Nat Re. Micro. 7 (5), 333-340 (2009).</li></ol>

The authors have nothing to disclose.

The protocol outlined here describes the induction, localization, and analysis of SGs in non-infected cells and cells infected with the cytosolic pathogen S. flexneri in the presence or absence of exogenous stress. Using free imaging software, the protocols allows for the precise qualitative and quantitative analysis of SG formation to identify and statistically address differences in given phenotypes.
There are several critical steps within the protocol for the infection, SG-inductio...

Visualizing host-pathogen interactions on a cellular level is a powerful method for gaining insights into pathogenic strategies and for identifying key cellular pathways. Indeed, pathogens can be used as tools to pinpoint important cellular targets or structures, as pathogens have evolved to subvert central cellular processes as a strategy for their own survival or propagation. Visualization of cellular components can be achieved by recombinantly expressing fluorescently-tagged host proteins. While this allows for real-time analysis, the generation of cell lines with specifically-tagged host proteins is highly laborious and may result in undesirable side effects. More convenient is the detection of cellular factors using specific antibodies, because multiple host factors can be analyzed simultaneously and one is not limited to a particular cell type. A drawback is that only a static view can be captured as immunofluorescence analysis necessitates host cell fixation. However, an important advantage of immunofluorescence imaging is that it readily lends itself to both qualitative and quantitative analysis. This in turn can be used to obtain statistically significant differences to provide new insights into host-pathogen interactions.
Fluorescent image analysis programs are powerful analytical tools for performing 3D and 4D analysis. However, the high cost of software and its maintenance make methods based on free open source software more widely attractive. Careful image analysis using bio-analysis software is valuable as it substantiates visual analysis and, when assigning statistical significances, increases confidence in the correctness of a given phenotype. Previously, SGs have been analyzed using the free ImageJ software, which necessitates the manual identification of individual SGs4. Here we provide a protocol for the induction and analysis of cellular SG formation in the context of bacterial infections using the free open source bio-image analysis software ICY (http://icy.bioimageanalysis.org). The bio-image analysis software has a built-in spot detector program that is highly suitable for SG analysis. It allows the fine-tuning of the automated detection process in specified Regions Of Interest (ROIs). This overcomes the need for manual analysis of individual SGs and removes sampling bias.
Many environmental stresses induce the formation of SGs, which are phase dense cytosolic, non-membranous structures of 0.2 - 5 &#181;m in diameter5,6. This cellular response is evolutionarily conserved in yeast, plants and mammals and occurs when global protein translation is inhibited. It involves aggregation of stalled translation initiation complexes into SGs, which are considered holding places for translationally-inactive mRNAs, allowing selective translation of a subset of cellular mRNAs. Upon removal of the stress, SGs dissolve and global rates of protein synthesis resume. SGs are composed of translation elongation initiation factors, proteins involved in RNA metabolism, RNA-binding proteins, as well as scaffolding proteins and factors involved in host cell signaling2, although the exact composition can vary depending on the stress applied. Environmental factors that induce SG formation include amino acid starvation, UV irradiation, heat shock, osmotic shock, endoplasmic reticulum stress, hypoxia and viral infection2,7,8. Much progress has been made in understanding how viruses induce and also subvert SG formation, while little is still known about how other pathogens, such as bacterial, fungal or protozoan pathogens, affect this cellular stress response1,7.
Shigella flexneri is a gram-negative facultative cytosolic pathogen of humans and the causative agent of severe diarrhea or shigellosis. Shigellosis is a major public health burden and leads to 28,000 deaths annually in children under 5 years of age9,10. S. flexneri infects the colonic epithelium and spreads cell-to-cell by hijacking the host&#39;s cytoskeletal components11,12. Infection of the epithelium supports the replication of S. flexneri within the cytosol but infected macrophages die through an inflammatory cell death process called pyroptosis. Infection leads to a massive recruitment of neutrophils and severe inflammation that is accompanied by heat, oxidative stress and tissue destruction. Thus, while infected cells are subject to internal stresses induced by infection, such as Golgi disruption, genotoxic stress and cytoskeletal rearrangements, infected cells are also subjected to environmental stresses due to the inflammatory process.
Characterization of the effect of S. flexneri infection on the ability of cells to respond to environmental stresses using a number of SG markers has demonstrated that infection leads to qualitative and quantitative differences in SG composition3. However, little is known about other bacterial pathogens. Here we describe a methodology for the infection of host cells with the cytosolic pathogen S. flexneri, the stressing of cells with different environmental stresses, the labeling of SG components, and the qualitative and quantitative analysis of SG formation and composition in the context of infected and non-infected cells. This method is widely applicable to other bacterial pathogens. In addition, the image analysis of the SG formation may be used for infections by viruses or other pathogens. It can be used to analyze SG formation upon infection or the effect of infection on SG formation in response to exogenous stresses.

<table><tbody><tr><td>&lt;strong&gt;Primary Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>eIF3b (N20), origin goat&amp;nbsp;</td><td>Santa Cruz</td><td>sc-16377</td><td>Robust and widely used SG marker. Cytosolic staining allows cell delineation. Dilution 1:300</td></tr><tr><td>eIF3b (A20), origin goat&amp;nbsp;</td><td>Santa Cruz</td><td>sc-16378</td><td>Same target as eIF3b (N20) and in our hands was identical to eIF3b (N20). Dilution 1:300</td></tr><tr><td>eIF3A (D51F4), origin rabbit (MC: monoclonal)</td><td>Cell Signaling</td><td>3411</td><td>Part of multiprotein eIF3 complex with eIF3b . Dilution 1:800</td></tr><tr><td>eIF4AI, origin goat&amp;nbsp;</td><td>Santa Cruz</td><td>sc-14211</td><td>Recommended by (Ref # 13). Dilution 1:200</td></tr><tr><td>eIF4B, origin rabbit</td><td>Abcam</td><td>ab186856</td><td>Good stress granule marker in our hands. Dilution 1:300</td></tr><tr><td>eIF4B, origin rabbit</td><td>Cell Signaling</td><td>3592</td><td>Recommended by Ref # 13. Dilution 1:100</td></tr><tr><td>eIF4G, origin rabbit</td><td>Santa Cruz</td><td>sc-11373</td><td>Widely used SG marker. (Ref # 13): may not work well in mouse cell lines. Dilution 1:300</td></tr><tr><td>G3BP1, origin rabbit (MC: monoclonal)</td><td>BD Biosciences</td><td>611126</td><td>Widely used SG marker. Dilution 1:300</td></tr><tr><td>Tia-1, origin goat&amp;nbsp;</td><td>Santa Cruz</td><td>sc-1751</td><td>Widely used SG marker. Can also be found in P bodies when SG are present (Ref # 13). Dilution 1:300</td></tr><tr><td>&lt;strong&gt;Alexa-conjugated Secondary Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>A488 anti-goat , origin donkey</td><td>Thermo Fisher</td><td>A-11055</td><td>Cross absorbed. Dilution 1:500</td></tr><tr><td>A568 anti-goat, origin donkey</td><td>Thermo Fisher</td><td>A-11057</td><td>Cross absorbed. Dilution 1:500</td></tr><tr><td>A488 anti-mouse, origin donkey</td><td>Thermo Fisher</td><td>A-21202</td><td>Dilution 1:500</td></tr><tr><td>A568 anti-mouse, origin donkey</td><td>Thermo Fisher</td><td>A10037</td><td>Dilution 1:500</td></tr><tr><td>A647 anti-mouse, origin donkey</td><td>Thermo Fisher</td><td>A31571</td><td>Dilution 1:500</td></tr><tr><td>A488 anti-rabbit, origin donkey</td><td>Thermo Fisher</td><td>A-21206</td><td>Dilution 1:500</td></tr><tr><td>A568 anti-rabbit, origin donkey</td><td>Thermo Fisher</td><td>A10042</td><td>Dilution 1:500</td></tr><tr><td>&lt;strong&gt;Other Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Shigella flexneri&lt;/em&gt;</td><td></td><td></td><td>Available from various laboratories by request</td></tr><tr><td>Tryptone Casein Soya (TCS) broth</td><td>BD Biosciences</td><td>211825</td><td>Standard growth medium for Shigella, application - bacterial growth</td></tr><tr><td>TCS agar</td><td>BD Biosciences</td><td>236950</td><td>Standard growth agar for Shigella, application - bacterial growth</td></tr><tr><td>Congo red</td><td>SERVA Electrophoresis GmbH</td><td>27215.01</td><td>Distrimination tool for Shigell that have lost the virulence plasmid, application - bacterial growth</td></tr><tr><td>Poly-L-Lysine (PLL)</td><td>Sigma-Aldrich</td><td>P1274</td><td>Useful to coating bacteria to increase infection, application - infection</td></tr><tr><td>Gentamicin</td><td>Sigma-Aldrich</td><td>G1397</td><td>Selective killing of extracellular but not cytosolic bacteria, application - infection</td></tr><tr><td>HEPES</td><td>Life Technologies</td><td>15630-056</td><td>PH buffer useful when cells are incubated at RT, application - cell culture</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Life Technologies</td><td>31885</td><td>Standard culture medium for HeLa cells, application - cell culture</td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>Biowest</td><td>S1810-100</td><td>5% supplementation used for HeLa cell culture medium, application - cell culture</td></tr><tr><td>Non-Essential Amino Acids (NEAA)</td><td>Life Technologies</td><td>11140</td><td>1:100 dilution used for HeLa cell culture medium, application - cell culture</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D2650</td><td>Reagent diluent, application - cell culture</td></tr><tr><td>Sodium arsenite</td><td>Sigma-Aldrich</td><td>S7400</td><td>Potent stress granule inducer (Note: highly toxic, special handling and disposal required), application - stress inducer</td></tr><tr><td>Clotrimazole</td><td>Sigma-Aldrich</td><td>C6019</td><td>Potent stress granule inducer (Note:health hazard, special handling and disposal required), application - stress inducer</td></tr><tr><td>Paraformaldehyde (PFA</td><td>Electron Microscopy Scences</td><td>15714</td><td>4% PFA is used for standard fixation of cells, application - fixation</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>Used at 0.03% for permeabilizationof host cells before immunofluorescent staining, application - permeabilization</td></tr><tr><td>A647-phalloidin</td><td>Thermo Fisher</td><td>A22287</td><td>Dilution is at 1:40, best added during secondary antibody staining, application - staining</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td>Nucleid acid stain used to visualize both the host nucleus and bacteria, application - staining</td></tr><tr><td>Parafilm</td><td>Sigma-Aldrich</td><td>BR701501</td><td>Paraffin film useful for immunofluorescent staining of coverslips, application - staining</td></tr><tr><td>Prolong Gold</td><td>Thermo Fisher</td><td>36930</td><td>Robust mounting medium that works well for most fluorophores , application - mounting</td></tr><tr><td>Mowiol</td><td>Sigma-Aldrich</td><td>81381</td><td>Cheap and robust mounting medium that works well for most fluorophores, application - mounting</td></tr><tr><td>24-well cell culture plate</td><td>Sigma-Aldrich</td><td>CLS3527</td><td>Standard tissue culture plates, application - cell culture</td></tr><tr><td>12 mm&amp;nbsp; glass coverslips</td><td>NeuVitro</td><td>1001/12</td><td>Cell culture support for immunofluorescent applications, application - cell support</td></tr><tr><td>forceps</td><td>Sigma-Aldrich</td><td>81381</td><td>Cheap and robust mounting medium that works well for most fluorophores, application - mounting</td></tr><tr><td>&lt;strong&gt;Programs and Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Prism</td><td>GraphPad Software</td><td></td><td>Data analysisand graphing program with robust statistical test options, application - data analysis</td></tr><tr><td>Leica SP5</td><td>Leica Microsystems</td><td></td><td>Confocal microsope, application - image acquisition</td></tr><tr><td>Imaris</td><td>Bitplane</td><td></td><td>Professional image analysis program, application - data analysis</td></tr><tr><td>Excel</td><td>Microsoft</td><td></td><td>Data analysis and graphing program, application - data analysis</td></tr></tbody></table>

immunofluorescence analysis of stress granule formation after bacterial challenge of mammalian cells

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

To explain and demonstrate the protocol described in this manuscript, we characterized the image of clotrimazole-induced SGs in HeLa cells infected or not with the cytosolic pathogen S. flexneri. An outline of the procedure is presented in Figure 1, and includes virulent and avirulent S. flexneri streaked on Congo Red plates, bacteria preparation, infection, addition of environmental stress, sample fixation and staining, sample imaging and q...

Watch this Scientific Journal Video about Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells at JoVE.com

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...

immunofluorescence-analysis-stress-granule-formation-after-bacterial

Research

JoVE Journal

Immunology and Infection

10.1K Views.  Institut Pasteur. We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

Video: Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

Bioengineering

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion (STED) Microscopy

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure that optimizes B-cell receptor (BCR) signaling and BCR-mediated antigen acquisition. Both the remodeling of the actin cytoskeleton and the reorientation of the microtubule network towards the antigen contact site are essential for immune synapse formation. Remodeling of the actin cytoskeleton into a dense peripheral ring of F-actin is accompanied by polarization of the microtubule-organizing center towards the immune synapse. Microtubule plus-end binding proteins, as well as cortical plus-end capture proteins mediate physical interactions between the actin and microtubule cytoskeletons, which allow them to be reorganized in a coordinated manner. Elucidating the mechanisms that control this cytoskeletal reorganization, as well as understanding how these cytoskeletal structures shape immune synapse formation and BCR signaling, can provide new insights into B cell activation. This has been aided by the development of super-resolution microscopy approaches that reveal new details of cytoskeletal network organization. We describe here a method for using stimulated emission depletion (STED) microscopy to simultaneously image actin structures, microtubules, and transfected GFP-tagged microtubule plus-end binding proteins in B cells. To model the early events in immune synapse formation, we allow B cells to spread on coverslips coated with anti-immunoglobulin (anti-Ig) antibodies, which initiate BCR signaling and cytoskeleton remodeling. We provide step-by-step protocols for expressing GFP fusion proteins in A20 B-lymphoma cells, for anti-Ig-induced cell spreading, and for subsequent cell fixation, Immunostaining, image acquisition, and image deconvolution steps. The high-resolution images obtained using these procedures allow one to simultaneously visualize actin structures, microtubules, and the microtubule plus-end binding proteins that may link these two cytoskeletal networks.

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy

We present a protocol for using STED microscopy to simultaneously image actin structures, microtubules, and microtubule plus-end binding proteins in B cells that have spread on coverslips coated with antibodies to the B-cell receptor, a model for the initial phase of immune synapse formation.

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure ...

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types&#39; responses to matrix stiffness have been characterized, how adherent cells&#39; susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.

We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, ...

Substructure Analyzer: A User-Friendly Workflow for Rapid Exploration and Accurate Analysis of Cellular Bodies in Fluorescence Microscopy Images

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in live-cell imaging and high-throughput microscopy techniques. This led to a constant increase in the amount and complexity of the microscopy data for a single experiment. Because manual analysis of microscopy data is very time consuming, subjective, and prohibits quantitative analyses, automation of bioimage analysis is becoming almost unavoidable. We built an informatics workflow called Substructure Analyzer to fully automate signal analysis in bioimages from fluorescent microscopy. This workflow is developed on the user-friendly open-source platform Icy and is completed by functionalities from ImageJ. It includes the pre-processing of images to improve the signal to noise ratio, the individual segmentation of cells (detection of cell boundaries) and the detection/quantification of cell bodies enriched in specific cell compartments. The main advantage of this workflow is to propose complex bio-imaging functionalities to users without image analysis expertise through a user-friendly interface. Moreover, it is highly modular and adapted to several issues from the characterization of nuclear/cytoplasmic translocation to the comparative analysis of different cell bodies in different cellular sub-structures. The functionality of this workflow is illustrated through the study of the Cajal (coiled) Bodies under oxidative stress (OS) conditions. Data from fluorescence microscopy show that their integrity in human cells is impacted a few hours after the induction of OS. This effect is characterized by a decrease of coilin nucleation into characteristic Cajal Bodies, associated with a nucleoplasmic redistribution of coilin into an increased number of smaller foci. The central role of coilin in the exchange between CB components and the surrounding nucleoplasm suggests that OS induced redistribution of coilin could affect the composition and the functionality of Cajal Bodies.

We present a freely available workflow built for rapid exploration and accurate analysis of cellular bodies in specific cell compartments in fluorescence microscopy images. This user-friendly workflow is designed on the open-source software Icy and also uses ImageJ functionalities. The pipeline is affordable without knowledge in image analysis.

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in ...

Multi-scale Analysis of Bacterial Growth Under Stress Treatments

Analysis of the bacterial ability to grow and survive under stress conditions is essential for a wide range of microbiology studies. It is relevant to characterize the response of bacterial cells to stress-inducing treatments such as exposure to antibiotics or other antimicrobial compounds, irradiation, non-physiological pH, temperature, or salt concentration. Different stress treatments might disturb different cellular processes, including cell division, DNA replication, protein synthesis, membrane integrity, or cell cycle regulation. These effects are usually associated with specific phenotypes at the cellular scale. Therefore, understanding the extent and causality of stress-induced growth or viability deficiencies requires a careful analysis of several parameters, both at the single-cell and at the population levels. The experimental strategy presented here combines traditional optical density monitoring and plating assays with single-cell analysis techniques such as flow cytometry and real time microscopy imaging in live cells. This multiscale framework allows a time-resolved description of the impact of stress conditions on the fate of a bacterial population.

This protocol allows a time-resolved description of bacterial growth under stress conditions at the single-cell and the cell population levels.

Analysis of the bacterial ability to grow and survive under stress conditions is essential for a wide range of microbiology studies. It is relevant to ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

Biology

Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly and fate is important to understand the cellular response to stress. The deficiency in key factors of SGs like G3BP (RasGAP SH3 domain Binding Protein) leads to developmental defects in mice&#160;and alterations of the Central Nervous System. To study the dynamics of SGs in cells from an organism, one can culture primary cells and follow the localization of a transfected tagged component of SGs. We describe time-lapse experiment to observe G3BP1-containing SGs in Mouse Embryonic Fibroblasts (MEFs). This technique can also be used to study G3BP-containing SGs in live neurons, which is crucial as it was recently shown that these SGs are formed at the onset of neurodegenerative diseases like Alzheimer&#39;s disease. This approach can be adapted to any other cellular body and granule protein component, and performed with transgenic animals, allowing the live study of granules dynamics for example in the absence of a specific factor of these granules.

Stress granules (SGs) are cytoplasmic RNA granules containing stalled ribonucleoprotein particles (RNPs), and important in cellular response to various stresses. Dynamics of SGs can be followed in live cells by visualizing the localization of a tagged component of SGs in transfected primary cells after stress.

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly ...

Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to stress conditions, are implicated in various aspects of cell metabolism and survival. SGs modulate cellular signaling pathways, post-transcriptional gene expression, and stress response programs. The formation of these mRNA-containing granules is directly connected to cellular translation. SG assembly is triggered by inhibited translation initiation, and SG disassembly is promoted by translation activation or by inhibited translation elongation. This relationship is further highlighted by SG composition. Core SG components are stalled translation pre-initiation complexes, mRNA, and selected RNA-binding Proteins (RBPs). The purpose of SG assembly is to conserve cellular energy by sequestering translationally stalled housekeeping mRNAs, allowing for the enhanced translation of stress-responsive proteins. In addition to the core constituents, such as stalled translation preinitiation complexes, SGs contain a plethora of other proteins and signaling molecules. Defects in SG formation can impair cellular adaptation to stress and can thus promote cell death. SGs and similar RNA-containing granules have been linked to a number of human diseases, including neurodegenerative disorders and cancer, leading to the recent interest in classifying and defining RNA granule subtypes. This protocol describes assays to characterize and quantify mammalian SGs.

Stress Granules (SGs) are nonmembranous cytoplasmic structures that form in cells exposed to a variety of stresses. SGs contain mRNAs, RNA-binding proteins, small ribosomal subunits, translation-related factors, and various cell signaling proteins. This protocol describes a workflow that uses several experimental approaches to detect, characterize, and quantify bona fide SGs.

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to ...

An Immunofluorescence Assay to Characterize and Quantify Mammalian Stress Granules

Source: Aulas, A. et al., <a href="https://app.jove.com/v/55656">Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates an assay for characterizing and quantifying mammalian stress granules, which consist of mRNAs, RNA-binding proteins (RBPs), and various other proteins.

1. Cell Preparation

	Add autoclaved coverslips to 12 wells of a 24-well plate, as indicated in&#160;Figure 1A; this can be done using a sterile Pasteur ...