Name: Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions (Video) | JoVE Journal
Uploaded: 2014-09-09T18:00:00.000+00:00
Duration: 12 min 52 s
Description: Learn protocols to study autophagy-cytoskeleton interactions during Shigella flexneri infection using HeLa cells and zebrafish larvae. These methods enable real-time visualization of septin cages and autophagy dynamics.

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Work in the SM laboratory is supported by a Wellcome Trust Research Career Development Fellowship [WT097411MA].

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</ol>

The authors declare that they have no competing financial interests.

When monitoring autophagy and the cytoskeleton in vitro using tissue culture cells, the protocols described in sections 1 and 2 can be applied to a wide variety of tissue culture cell types. Moreover, to follow autophagy (e.g., ATG8/LC3+ve autophagosomes) and cytoskeleton (e.g., actin tails, septin cages) dynamics in real-time during Shigella infection using live imaging, tissue culture cells can be transiently transfected using GFP-, RFP- or CFP-tagged constructs as previously described7,8. To increase the percentage of cells infected by Shigella (i.e., generally desirable for real-time analysis considering that Shigella can invade 5-30% of HeLa cells at 100:1 MOI), directly add 400 &#181;l of Shigella (OD600 = 0.3-0.6) to cells in 2 ml MEM (serum-starved) and wait at least 1.5 hr post infection for sufficient bacterial entry, escape from the phagosome, replication, autophagy recognition and septin caging. Alternatively, one may use the Shigella M90T AfaI strain that expresses the adhesin AfaE and have much higher invasion abilities in epithelial cells compared to the M90T strain28. Of note, the M90T AfaI strain has not yet been tested in vivo using zebrafish. Plates of Shigella colonies can be kept at 4 &#176;C for 2-3 days and used for several experiments. However, over time, colonies of Shigella that have lost the virulence plasmid can also absorb the Congo Red and appear to have retained their virulence plasmid. For this reason we recommend to use fresh bacterial stocks when possible.
When monitoring the cell biology of infection in vivo, protocols described in sections 3 and 4 use wildtype AB line zebrafish. To monitor Shigella-leukocyte interactions, transgenic zebrafish lines can be used, e.g., mpx:GFP or lyz:DsRed to visualize neutrophils19,29,30 or mpeg1:mcherry to visualize macrophages19,31. To visualize autophagy in vivo, the GFP-Lc3 zebrafish transgenic line19,24 can be used as described in section 3.8.
To perturb autophagy in vivo, the effective morpholino oligonucleotide dose has to be assessed experimentally based on its efficiency to inhibit transcript splicing or protein translation. It is advisable to perform a titration experiment and to confirm the depletion by RT-PCR (for splice morpholino oligonucleotide) or by SDS-PAGE (for translational morpholino oligonucleotide)32. RNA isolation from zebrafish embryos or larvae can be performed using guanidinium thiocyanate-phenol-chloroform extraction. To extract protein from zebrafish larvae (8 to 15 larvae/tube), mechanically homogenize using a pestle in 200 &#181;l lysis buffer (1 M Tris, 5 M NaCl, 0.5 M EDTA, 0.01% octylphenol ethylene oxide condensate, and protease inhibitor). Centrifuge tubes at 19,000 x g at 4 &#176; C for 15 min and transfer the supernatant to a new tube. Add Laemmli buffer and heat the sample at 95 &#176; C for 15 min. Lysates can be stored at -80 &#176; C until needed, and can be evaluated by Western blotting as described in section 2.3.
The zebrafish is an excellent model for in vivo drug application. Analysis using morpholino oligonucleotides can be complemented with established drugs to manipulate autophagy (e.g., rapamycin and bafilomycin). Uninfected and/or infected larvae can be treated with rapamycin (1.5 &#181;M) or bafilomycin (80 nM) diluted in E2 and autophagic flux can be evaluated by Western blotting as described in 25,33. The consequence of autophagy manipulation on the outcome of the infection and survival of the infected larvae can be evaluated as described in section 3.5.
In addition to studying host cell determinants, in vitro and in vivo protocols can be applied to assess bacterial determinants required for autophagy recognition, using bacterial mutant strains that are differentially recognized by autophagy, e.g., Shigella&#916;icsA (the Shigella protein IcsA recruits N-WASP and then Arp2/3 for actin tail and septin cage formation; in its absence there can be no actin tails, no septin cages) and Shigella&#916;icsB (Shigella avoids the autophagic response via the bacterial effector protein IcsB, which prevents the recruitment of autophagy machinery to IcsA; in its absence there can be more septin cages, more autophagy)7,8.
Shigella is not a natural pathogen of zebrafish and grows optimally at 37 &#176;C. However, work has shown that virulence factors required for Shigella invasion, escape from the phagocytic vacuole and replication in the cytosol can be expressed and are functional in zebrafish larvae at 28 &#176;C19. 28 &#176;C is the most commonly used temperature for zebrafish rearing and standard temperature to ensure normal zebrafish development23. Strikingly, the major pathogenic events that lead to shigellosis in humans (i.e., macrophage cell death, invasion and multiplication within epithelial cells, cell-to-cell spread, inflammatory destruction of the host epithelium) are faithfully reproduced in the zebrafish model of Shigella infection19.
Autophagy and cytoskeleton genes are ubiquitously expressed and have a wide range of biological functions. Mouse studies have shown that knockout of essential autophagy26 or septin genes5 are embryonic lethal, and it is likely that some of these genes will also be essential for zebrafish development (although this problem may be reduced by the fact that zebrafish have multiple paralogous genes33). If so, there are several alternatives to overcome this issue, including (i) the use of pharmacological reagents to regulate autophagy and the cytoskeleton, (ii) morpholinos can be titrated down, (iii) knockout of genes can be designed for only specific cell types, and/or (iv) genes involved in autophagic recognition that are not essential for animal development (e.g., p62) may be targeted.
While the zebrafish is an ideal model system to investigate autophagy and the cytoskeleton during Shigella infection, molecular tools are currently lacking. The field needs to generate new tools and drive cell specific expression of the proteins of interest. To knock down expression of autophagy/cytoskeleton genes, new morpholino sequences are required, and novel methods for genome engineering (e.g., TALEN, CRISPR/Cas9) can also be used. In the meantime, several tools previously generated for human or mouse studies may equally work for zebrafish.
The intracellular bacteria S. flexneri has emerged as an exceptional model organism to address key issues in biology, including the ability of bacteria to be recognized by the immune system1,2. The host cell employs septins to restrict the motility of S. flexneri and target them to autophagy, a critical component of cell autonomous immunity7,8. These observations suggest a new molecular framework to study autophagy and its ability to degrade cytosolic bacteria. A major issue is now to fully decipher the underlying molecular and cellular events, and to validate these events analyzed in vitro during bacterial infection in vivo using relevant animal models. To this end, the zebrafish has been established as a valuable new host for the analysis of S. flexneri infection19. Interactions between bacteria and host cells can be imaged at high resolution, and the zebrafish model should prove useful for understanding the cell biology of Shigella infection in vivo. Zebrafish larvae can be used to investigate the role of bacterial autophagy in host defense, and work has shown that that the perturbation of autophagy can adversely affect host survival in response to Shigella infection19.
The observations generated from study of Shigella, septin caging and autophagy in vitro using tissue culture cells and in vivo using zebrafish larvae might provide fundamental advances in understanding host defense. They could also suggest the development of new strategies aimed at combating infectious diseases.
A critical aim of this report is to make sense of the molecular and cellular events analyzed in vitro (i.e., autophagy, actin tails, septin caging) during bacterial infection in vivo in the context of an entire organism, using zebrafish larvae. If not familiar with zebrafish biology and handling, one may refer to in depth protocols for proper zebrafish husbandry23 and in vivo analysis of zebrafish infection19,35.

Shigella flexneri, a Gram-negative invasive enteropathogenic bacterium, can escape from phagosomes to the cytosol, and polymerize the host actin cytoskeleton to evade cytosolic immune responses and promote intra- and intercellular movement1,2. Despite the understanding of actin-based motility in vitro3,4, the mechanisms restricting bacterial dissemination in vivo have not been fully defined. This is critical for a more complete understanding of innate immunity and host defense.
Septins, a highly conserved family of proteins among metazoans, are guanosine triphosphate (GTP)-binding proteins that assemble into hetero-oligomeric complexes and form nonpolar filaments that associate with cellular membranes and the cytoskeleton5,6. Recent work has discovered that infected host cells can prevent Shigella actin based motility by compartmentalizing bacteria targeted to autophagy inside &#8216;septin cages&#8217;, revealing the first cellular mechanism that counteracts actin based motility7,8. A wide open field of investigation now lies in &#8216;septin biology and infection&#8217;. Septin assembly, induced by a variety of pathogens (e.g., Listeria monocytogenes7,9,10, Mycobacterium marinum7,8, Candida albicans11), may emerge as a key issue in host defense5,12.
Autophagy, a highly conserved intracellular degradation process, is viewed as a key component of cell-autonomous immunity because of its ability to deliver cytosolic bacteria to the lysosome13,14. However, the role of bacterial autophagy in vivo to restrict or promote bacterial replication remains poorly understood15,16. The zebrafish (Danio rerio) has emerged as a vertebrate model for the study of infections because it is optically accessible at the larval stages when the innate immune system is already functional17,18. Recent work has characterized the susceptibility of zebrafish larvae to S. flexneri, a paradigm for bacterial autophagy15, and has used the Shigella-zebrafish infection model to study the manipulation of autophagy for antibacterial therapy in vivo19.
This report provides new tools and assays to study S. flexneri interactions with autophagy and the cytoskeleton. In a first step, protocols to monitor autophagy-cytoskeleton interactions are described using Shigella infection of the human epithelial cell line HeLa. To assess the role of autophagy-cytoskeleton interactions on the Shigella infection process in vitro, methods to manipulate autophagy and cytoskeleton components (using siRNA or pharmacological reagents) are provided. New work has shown that by using Shigella infection of zebrafish larvae, similar assays can be applied to study the cell biology of infection in vivo. Protocols to prepare and infect zebrafish larvae are detailed, and to assess the host response to Shigella infection in vivo, protocols to determine host survival and bacterial burden of infected larvae are provided. Methods to monitor the recruitment of septin and autophagy markers to Shigella (using either fixed or living zebrafish larvae) and methods to test the role of these processes in vivo [using morpholino oligonucleotides (injected in 1-4 cell stage embryos) or pharmacological reagents (added directly to the zebrafish bath water)] are also discussed. This program&#160;of work is expected to provide insights into the mechanisms required for the control of infection by cytosolic host responses.

<table><tbody><tr><td>Bafilomycin A1</td><td>Sigma-Aldrich</td><td>B1793&amp;nbsp;</td><td></td></tr><tr><td>Blebbistatin</td><td>Sigma-Aldrich</td><td>B0560</td><td></td></tr><tr><td>Borosilicate glass microcapillars&amp;nbsp;</td><td>Harvard Apparatus</td><td>30038</td><td></td></tr><tr><td>Coarse manual manipulator&amp;nbsp;</td><td>Narishige</td><td>M-152</td><td></td></tr><tr><td>Cytochalasin D</td><td>Sigma-Aldrich</td><td>C6762</td><td></td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole (DAPI)</td><td>Molecular Probes</td><td>D1306</td><td></td></tr><tr><td>Dumont #5 fine tweezers</td><td>Fine Science Tools</td><td>11254-20</td><td></td></tr><tr><td>Forchlorfeneuron&amp;nbsp;</td><td>Sigma-Aldrich</td><td>32974</td><td></td></tr><tr><td>Goat anti-mouse IgG (H+L) antibody</td><td>Molecular Probes</td><td>N/A&amp;nbsp;</td><td></td></tr><tr><td>Goat ant-rabbit IgG (H+L) antibody</td><td>Molecular Probes</td><td>N/A</td><td></td></tr><tr><td>JetPEI transfection reagent</td><td>Polyplus transfection</td><td>101-01N</td><td></td></tr><tr><td>Latrunculin B</td><td>Sigma-Aldrich</td><td>L5288</td><td></td></tr><tr><td>LC3 antibody</td><td>Novus Biologicals</td><td>NB100-2220</td><td></td></tr><tr><td>Low melting agarose</td><td>Promega</td><td>V2111</td><td></td></tr><tr><td>MatTek glass bottom dish</td><td>MatTek corporation</td><td>P35G-1.0-14</td><td></td></tr><tr><td>MEM plus L-alanyl-L-glutamine&amp;nbsp;</td><td>GIBCO</td><td>41090028</td><td></td></tr><tr><td>MEM non-essential amino acids solution</td><td>GIBCO</td><td>11140-035</td><td></td></tr><tr><td>Microinjector&amp;nbsp;</td><td>Narishige</td><td>IM-300</td><td></td></tr><tr><td>Micropipette puller device&amp;nbsp;</td><td>Sutter Instrument Co., Novato,</td><td>P-87&amp;nbsp;</td><td></td></tr><tr><td>Mineral oil</td><td>Sigma-Aldrich</td><td>P35G-1.0-14</td><td></td></tr><tr><td>Monoclonal anti-tubulin, acetylated antibody&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T6793</td><td></td></tr><tr><td>Nocodazole</td><td>Sigma-Aldrich</td><td>M1404</td><td></td></tr><tr><td>N-phenylthiourea&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P7629</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Phalloidin&amp;nbsp;</td><td>Molecular Probes</td><td>A12379</td><td></td></tr><tr><td>Phenol red solution</td><td>Sigma-Aldrich</td><td>P0290</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Roche</td><td>4693116001</td><td></td></tr><tr><td>p62/SQSTM1 antibody</td><td>Cliniscience</td><td>PM045</td><td></td></tr><tr><td>Rapamycin</td><td>Sigma-Aldrich</td><td>R8781</td><td></td></tr><tr><td>Sodium pyruvate</td><td>GIBCO</td><td>11360039</td><td></td></tr><tr><td>Transfection reagent</td><td>Life Technologies</td><td>12252-011</td><td></td></tr><tr><td>Vectashield hard set mounting medium containing DAPI&amp;nbsp;</td><td>Vector Laboratories</td><td>H-1500&amp;nbsp;</td><td></td></tr></tbody></table>

use of shigella flexneri to study autophagy-cytoskeleton interactions

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Upon infection of tissue culture cells in vitro, S. flexneri can escape from the phagosome and invade the cytosol. In the cytosol, host cells can prevent the actin-based motility of Shigella by compartmentalizing bacteria inside septin cages (Figure 1A). Bacteria entrapped by septin cages can also be labeled by autophagy markers p62 (Figure 1B) and LC3 (Figure 1C). These observations highlight a novel mechanism of host defense that restricts dissemination of invasive pathogens, and also reveal new links between autophagy and the cytoskeleton. Strikingly, the depletion of autophagy markers significantly reduces septin caging of bacteria (Figure 2A), and work has also shown that the depletion of septin caging significantly reduces recruitment of autophagy markers8. Thus, at least in the case of Shigella, septin cage assembly and autophagosome formation can be viewed as interdependent processes. Other cellular requirements for compartmentalization of Shigella by septin cages include actin polymerization and actomyosin activity (Figure 2B).
There is no natural mouse model of shigellosis, and investigation of Shigella pathogenesis, septin biology and bacterial autophagy in vivo can benefit from a new animal model of infection, the zebrafish larvae19. It is possible to infect zebrafish larvae by injecting bacteria in various anatomical sites such as caudal intravenous injections for survival experiments, and tail muscle injections for in vivo microscopy (Figure 3A). Depending on the dose, S. flexneri injected in zebrafish larvae can either be cleared within 48 hr post-infection, or may result in a progressive and ultimately fatal infection (Figures 3B-3D). Shigella virulence factors are expressed at 28 ° C, the optimal growth temperature of zebrafish, and zebrafish infection by Shigella is strictly dependent upon its type III secretion system (T3SS)19, an essential virulence determinant in human disease. Taken together, these observations indicate that the zebrafish larva represents a valuable new host for in vivo analysis of Shigella infection.
The optical accessibility of zebrafish larvae enables visualization of septin caging in vivo (Figure 4A), an achievement that has never before been accomplished using mammalian host models. To complement evidence that septin cages entrap bacteria targeted to autophagy in vivo, one can infect transgenic zebrafish larvae expressing GFP-Lc3 and observe autophagy marker recruitment to Shigella (Figure 4B). For ultrastrucutral analysis of Shigella autophagosomes in vivo, electron microscopy can be used to clearly show the cytosolic sequestration of bacteria by double membrane vacuoles19. Autophagy is viewed as a key component of cell-autonomous immunity and a crucial defense mechanism against intracytosolic bacteria14-16. To characterize autophagy function in vivo, p62 morpholino-treated zebrafish larvae can be used. Unlike the core autophagy machinery [e.g., the 36 autophagy related proteins (ATGs)26], p62 is not essential for vertebrate development27 and thus zebrafish larvae can develop normally prior to infection. Strikingly, p62-depleted larvae inoculated with S. flexneri result in significantly increased mortality and increased bacterial burden19. In agreement with in vitro work showing that septin cage assembly is interdependent with autophagosome formation7,8, septin recruitment to Shigella is clearly reduced in p62-depleted larvae (Figure 4C). These data demonstrate that zebrafish survival depends on p62-mediated autophagy to control intracellular bacterial infection.
<img alt="Fluorescent microscopy of cell components: DAPI, F-actin, SEPT9, p62, GFP-LC3; merge analysis." fo:content-width="5in" src="/files/ftp_upload/51601/51601fig1highres.jpg" width="500" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. The septin cage in vitro. (A) HeLa cells were infected with S. flexneri for 4 hr 40 min, fixed, labeled with antibodies to SEPT9 and phalloidin, and imaged by confocal microcopy. Scale bar, 1 µm. (B) HeLa cells were infected with S. flexneri for 4 hr 40 min, fixed, labeled with antibodies to p62 and SEPT2, and imaged by fluorescent light microscopy. Scale bar, 1 µm. (C) HeLa cells were transfected with GFP-ATG8/LC3, infected with S. flexneri for 4 hr 40 min, fixed, labeled with antibodies to SEPT2, and imaged by fluorescent light microscopy. Scale bar, 1 µm. These figures have been modified from Mostowy et al7.
<img alt="Protein expression, Shigella percentage data in siRNA knockdown, bar graphs, Western blot analysis." fo:content-width="5in" src="/files/ftp_upload/51601/51601fig2highres.jpg" width="500" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
Figure 2. Cellular requirements for Shigella-septin cage formation. (A) HeLa cells were treated with control (CTRL), p62, ATG5, ATG6 or ATG7 siRNA. Whole-cell lysates of siRNA-treated cells were immunoblotted for GAPDH, p62, ATG5, ATG6, or ATG7 to show the efficiency of siRNA depletion (top). siRNA-treated cells were infected with S. flexneri for 4 hr 40 min, fixed, and labeled for quantitative microscopy. Graphs (bottom) represent the mean % ± SEM of Shigella inside SEPT2 cages from n ≥3 experiments per treatment. (B) HeLa cells were infected with S. flexneri, treated with DMSO, cytochalasin D (CytD), latrunculin B (LatB), nocodazole (Noco), or blebbistatin (Bleb) and after 4 hr 40 min were fixed and labeled for quantitative microscopy. Graphs represent the mean % ± SEM of Shigella inside SEPT2 cages from two independent experiments per treatment. These figures have been modified from Mostowy et al7.
<img alt="Zebrafish infection experiment; graphs show survival vs. time; fluorescence levels indicate infection." fo:content-width="5in" src="/files/ftp_upload/51601/51601fig3highres.jpg" width="500" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
Figure 3. The zebrafish model of Shigella infection. (A) Images to illustrate orientation of the zebrafish larva under the stereomicroscope. (Left panel) Zebrafish larvae 72 hr post fertilization were positioned laterally in the injection plate with their dorsal side facing the injection needle. (Middle panel) Bloodstream infection was performed by injecting the bacteria (red solution) in the caudal vein, posterior to the urogenital opening. (Right panel) Infection in the tail muscle was performed by injecting the bacteria (red solution) over a somite. (B) Survival curves of 72 hr post fertilization larvae injected with various doses of S. flexneri and incubated at 28 °C for 48 hr post infection. The effective inoculum was classified as low (&lt;103 CFU, open circles), medium (~4 x 103 CFU, open triangles) or high (~104 CFU, open squares). Mean % ± SEM (horizontal bars) from n ≥3 experiments per inoculum class. (C) Enumeration of live bacteria in homogenates from individual larvae at various times post infection measured by plating onto LB. Note, only larvae having survived the infection are included in enumeration analysis. Mean ± SEM (horizontal bars) also shown. (D) Distribution of GFP-Shigella determined by live imaging using a fluorescent stereomicroscope at various times post infection using a low, medium, or high dose inoculum (caudal intravenous injections). Overlay of transmission image (grey) and GFP fluorescence (green). (B)-(D)These figures have been modified from Mostowy et al19.
<img alt="Fluorescence microscopy, GFP-tagged Shigella, SEPT7, protein colocalization, overlay analysis." fo:content-width="5in" src="/files/ftp_upload/51601/51601fig4highres.jpg" width="500" data-altupdatedat="1787630408000" data-alt="Figure 4"> 
Figure 4. The cell biology of Shigella infection in vivo. (A) Zebrafish larvae were infected in the tail muscle with GFP-Shigella (low dose) for 24 hr, fixed, labeled with antibodies against SEPT7 (red) and to GFP (green), and imaged by confocal microscopy. Scale bar, 5 µm. (B) GFP-Lc3 zebrafish larvae were infected with mCherry-Shigella (medium dose) for 4 hr, fixed, labeled with antibodies against mCherry (red) and to GFP (green), and imaged by confocal microscopy. Scale bar, 1.5 µm. (C) Zebrafish larvae treated with either control (CTRL; left image) or p62 (right image) morpholinos were infected with GFP-Shigella for 4 hr (medium dose), fixed, labeled with antibodies against SEPT7 (red) and to GFP (green), and imaged by confocal microscopy. Arrows highlight some examples of Shigella entrapped in septin cages (CTRL) or not (p62 depleted) a 4 hr post infection. Scale bar, 5 µm. These figures have been modified from Mostowy et al19.

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

The overall goal of this procedure is to investigate intracellular mechanisms that control bacterial dissemination during infection in vitro and in vivo by using tissue culture cells in zebra fish larvae. The in vitro method begins with preparation of cha flexner, a gram-negative and enteroinvasive Bacterial pathogen Heller are then infected and irna or pharmacological reagents are used to manipulate the host autophagy machinery and cytoskeleton during infection. To study autophagy cytoskeleton interactions caused by cha in vivo zebrafish embryos are infected three days post fertilization for either method.The final step is to label tissues with autophagy and cytoskeletal markers. Ultimately, results will provide insights into the mechanisms required for the control of infection by cytosolic host responses. The advantage of using gel to study bacterial autophagy over other bacteria such as listeria, is that gel targeted autophagy can also be entrapped incepting cages.This method can help answer key questions in the autophagy field, such as what is the role of the cytoskeleton in autophagy and what is the role of autophagy in the restriction or promotion of intracellular bacteria? The implications of this technique can extend towards other processes of autophagy, such as the autophagy of viruses or mitochondria, and can also have therapeutic implications for human diseases with a septin or autophagy component. These are demonstration of zebra fish.Infection by shige is critical as the steps involving injection of zebra fish with bacteria are difficult to learn because needle preparation and proper injection takes time and high resolution. Microscopy of zebra fish infection takes experience For this protocol. Use the wild type strain of cha Flexner M nine DT from a frozen glycerol stock streak bacteria onto Congo Red triptych soy plates, and incubate them at 37 degrees Celsius overnight.Also, seed hella cells in six well plates in MEM media with 10%fetal calf serum the following day. Pick an individual colony and grow it up in TCS media overnight with shaking at 37 degrees Celsius the next day. Set up subcultures by making one to 80 dilution in fresh TCS.When the optical density of the subcultures at 600 nanometers is between 0.3 and 0.6. Collect the bacteria by spinning them down at 1000 G for five minutes. Then reconstitute the bacterial pellet in MEM to the same optical density.Hello, cells should be infected with a multiplicity of infection of 100 to one directly. Add the chag diluted in MEM to the plated hella cells and bring the total MEM volume to two milliliters per well. Then to maximize bacterial adherence to the host cells, centrifuge the plates at 700 G for 10 minutes and at room temperature, incubate the plates for 30 minutes so the bacteria can infect the cells after the infection period.Gently wash the cells twice. Using fresh MEM to further eliminate extracellular bacteria, incubate the cells in Gentamycin containing complete media for one to four hours based on empirical observations. Next, fix and label the infected cells.Image them by epi fluorescent or confocal microscopy using a 63 x or 100 x objective to resolve the DAPI labeled cha septin cages can be visualized as ring-like structures about 0.6 microns and diameter surrounding cytosolic bacteria polymerizing acton. The bacteria inside the septin cages can also be recognized by autophagy markers like P 62 and lc three a TG eight. Conceptually, the most difficult aspect of this procedure is the identification of bacterial autophagosome and sing cages in vivo and in vitro, which requires careful microscopy and analysis.Moreover, one must ensure that the proper number of events over several biological replicates are being analyzed and ensure statistical significance After infecting the hella cells with chila. First, allow the bacteria at least two and a half hours to find their way to the cytosol. Next, the drug treatment can be made to treat the cytoskeleton.Add between one to five microliters of diluted drug stock to each two milliliters of media with cells as a negative control. Treat one set of cells with DMSO in MEM, then incubate the cells for 30 minutes to manipulate the autophagic flux of the cells. Use 20 nano molar rapamycin or 160 nanomolar bafi mycin.Then incubate the cells for four to 12 hours after the treatment. Fix and label the cells as described in the text protocol. This technique models gel infection and autophagy cytoskeleton interactions in vivo To begin subculture and collect gel as previously described, wash the bacteria with one XPBS and centrifuge them at 1000 G for 10 minutes.Then resuspend the bacteria in 80 microliters of 0.1%Phenol red in PBS Keep this preparation on ice. It should have about 2000 bacteria per nanoliter 72 hours before the planned injection. Collect embryos from spawning zebrafish to remove any extracellular contaminants.Wash the embryos in E two media with 0.003%bleach for 10 minutes. After 10 minutes, change to E two media and wash the embryos five times in E two. Next load 10 centimeter dishes with 50 milliliters of E two and up to 50 embryos.Grow up a total of about 100 embryos at 28 degrees Celsius, 24 hours post fertilization. Treat the developing embryos with 0.003%and pheno threa in E two to prevent mein, which interferes with microscopy. Begin with anesthetizing the 72 hour larvae using a 200 microgram per milliliter trica bath in E two media.Then using a micro manipulator and fine tweezers, break the tip of the micro injection needle and check the brake under a microscope. Now load the needle first, transfer a drop of the bacterial culture to a cover slip on the microscope stage. Second, turn on the micro injector and gas cylinder.Third, lower the needle tip into the drop. And fourth, fill the needle to assess zebrafish survival during cha infection. Use a coddle intravenous injection using a fine paintbrush.Position the anesthetized larvae laterally on the injection plate with their dorsal sides facing the needle. Now the needle tip must be positioned close to the urogenital opening aimed at the coddle vein. Then pierce the skin and inject a one in five nanoliter bolus of bacteria.A successful injection into the codal vein is indicated by visualizing phenol red extending along the vein. Technically, the codal vein infection is the most difficult aspect of this procedure, and we require practice zebrafish larva. Confocal microscopy can also be challenging because the larvae require proper ing.After the injections transfer each infected larvae individually to a well of a 24 well plate containing one milliliter of E two media. Incubate the plate at 28 degrees Celsius and monitor the embryos daily. To visualize recruitment of septin and autophagy markers.Inject Ella at localized sites such as the tail muscle. Prepare to inject the larvae as shown in the previous section. Orient the zebra fish laterally and place the needle over the muscle.Somites then pierce the space between two somites and inject about one nanoliter of Ella. A successful injection into the tail muscles will show that the injected so mite colors in red. As a consequence of the phenol red buffer, have prepared low melting 0.1%agarose in E two media at 37 degrees Celsius.Add drops of agarro to a glass bottom dish individually. Transfer the injected larvae with a minimal volume of solution to the drops of agarro. Orient them there as desired and let the agarro solidify.Next, cover the whole dish with LMA followed by E two with trica. Then take Zacks every two minutes for several hours. In infected tissue culture cells, shaila escapes from the phagosome to the cytosol where the host cytoskeleton can rearrange and compartmentalize bacteria inside septin cage like structures.Bacteria and trapped in septin cages are recognized by autophagy markers such as P 62 and lc three. The zebrafish represents a valuable new host for the in vivo study of CHA infection. Depending on the infectious dose, zebrafish can either clear or low dose within 48 hours post-infection, or in the case of a high dose succumb to infection.The optical transparency of zebra fist RV enables in vivo visualization of septin cages and achievement that has not been performed using other vertebrate models to complement evidence that septin cages and trap bacteria targeted to autophagy. GFP lc three transgenic zebrafish were infected to observe the recruitment of lc three positive autophagosome to intracellular cha in vivo in real time to characterize autophagy P 62 morpho treated larvae were injected with chala control. Morpho larvae showed normal septin cage structures.However, in the P 62 morpho treated larvae, septin recruitment to chala was reduced, indicating that P 62 is an essential component to the autophagy mechanism, him Once mastered the infection, PHI larva can be done in one to two when it is performed properly Following this procedure. Other methods such as genome editing using the crisp perca system can be used to manipulate zebrafish genome. In addition, generation of novel fluorescently target zebrafish line can be engineered to follow autophagy cytoskeleton dynamics in real time during gel infection using live imaging After its discovery.The Chaga Septin cage has been used to study autophagy cytoskeleton interactions and has paved the way for researchers in infection and cell biology to illuminate new roles for the cytoskeleton in autophagy. Don't forget that working with Ella can be extremely hazardous and precautions such as working in a level two lab environment should always be taking while performing this procedure.

use-of-shigella-flexneri-to-study-autophagy-cytoskeleton-interactions

Research

JoVE Journal

Immunology and Infection

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

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

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.<br...

Study of Protein-protein Interactions in Autophagy Research

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

We present a protocol to study the distribution of mitochondria and endoplasmic reticulum in whole cells after genetic modification using correlative light and volume electron microscopy including ascorbate peroxidase 2 and horseradish peroxidase staining, serial sectioning of cells with and without the target gene in the same section, and serial imaging via electron microscopy.

Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

A protocol for imaging the dynamic microtubules in vivo using fluorescently labeled End binding protein has been presented. We described the methods to label, image, and analyze the dynamic microtubules in the Posterior lateral microtubule (PLM) neuron of C. elegans.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

Here, we present a protocol for optimized on-section correlative light-electron microscopy based on endogenous, fluorescent labeling as a tool to investigate the localization of rare proteins in relation to cellular ultrastructure. The power of this approach is demonstrated by ultrastructural localization of endogenous LC3 in starved cells without Bafilomycin treatment.

Ultrastructural Localization of Endogenous LC3 by On-Section Correlative Light-Electron Microscopy

The present protocol describes infection assays to interrogate Shigella adherence, invasion, and intracellular replication using in vitro epithelial cell lines.

Epithelial Cell Infection Analyses with Shigella

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Dual-color Correlative Light and Electron Microscopy for the Visualization of Interactions between Mitochondria and Lysosomes

Learn protocols to study autophagy-cytoskeleton interactions during Shigella flexneri infection using HeLa cells and zebrafish larvae. These methods enable real-time visualization of septin cages and autophagy dynamics.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

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