The studies were funded by the Wellcome Trust and Meningitis UK. HBMEC cell line was provided by Dr K.S. Kim. Confocal imaging and electron microscopy were performed in the Wolfson Bioimaging Facility, University of Bristol. We would also like to thank Mr Alan Leard, Dr. Mark Jepson (University of Bristol), and Mr. Alan Tilley (PerkinElmer) for their help and advice.

1. Immunofluorescence Protocol
Seeding, Infection &amp; immuno-staining
The following procedures require suitable safety level tissue culture and microbiological laboratory facilities.
DAY 1
A.Preparation of target cells for infection
<ol>
	<li>Seed HBMECs at 50% confluence (~1.5x104 cells/cm2) on glass coverslips (16 mm diameter) placed into a 12-well plate (3.8 cm2 growth area/well). Growth medium: RPMI 1640 supplemented with 15% heat-inactivated (56 &deg;C, 30min) FBS, 2 mM glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin/streptomycin, 1% (v/v) MEM non-essential amino acids solution and 1% MEM vitamins solution.</li>
	<li>Culture over night (O/N) at 37&deg;C, in a 5% CO2 incubator.</li>
</ol>
B. Bacterial culture
<ol>
	<li>Grow the strain of interest O/N (16-18 h) on the required medium e.g. Brain Heart Infusion (BHI) agar plates supplemented with 10% heated horse blood at 37&deg;C in 5% CO2 atmosphere4.</li>
</ol>
DAY 2
A. Preparation Of Bacterial (N. meningitidis) Suspension
<ol>
	<li>Using a 10 &mu;L culture loop, make a suspension of the overnight bacterial culture in 2 mL PBSB (PBS with calcium and magnesium).</li>
	<li>Allow large bacterial aggregates to settle by leaving the suspension to stand for 5 min at room temperature (RT).</li>
	<li>Without disturbing the pellet, transfer the top 1 mL of the suspension (stock inoculum) into a sterile tube.</li>
	<li>To estimate bacterial numbers in the stock inoculum, add aliquots (20-50 &mu;L) to tubes containing 1mL volumes of 1% SDS in 0.1M NaOH and mix gently to solubilise. </li>
	<li>Measure the nucleic acid content of the solution by determining the absorbance at 260nm (A260). In our hands, A260 of 1 corresponds to 5x108 colony forming units/ml (cfu/mL). This can be verified by preparing a series of dilutions of the stock inoculum in PBSB, plating out on agar plates and counting colonies after O/N incubation.</li>
	<li>Dilute an aliquot of the stock inoculum in the infection medium [(M199 supplemented with 2% decomplemented (56&deg;C, 30min) normal human serum (NHS)] to obtain the required bacterial density for the infection of target cells.
 <ol>
 	<li>In our laboratory, an infection ratio of 200-300 bacteria per target cell is routinely used.</li>
	</ol>
 </li>
</ol>
B. Cell Culture Infection
<ol>
	<li>Wash the coverslips with cultured target cells 3 times with Hank's medium to remove any trace of antibiotics. </li>
	<li>Infect cells with freshly prepared bacterial suspensions (described above) for 3 h to 8 h at 37&deg;C, in 5%CO2. </li>
	<li>At the end of infection period, wash cells three times with PBS and fix in 500 &mu;L of 2% paraformaldehyde (PFA) for 30-45 min at RT or O/N at 4&deg;C.
 <ol>
		<li>Paraformaldehyde fixation at the concentration and time shown above was found to be suitable for preservation of bacterial and cellular morphology.</li>
	</ol>
 </li>
	<li>After washing off paraformaldehyde, permeabilise the paraformaldehyde-fixed cells by incubating in 0.1%Triton X-100 diluted in PBS for 10 min. </li>
	<li>Wash the samples 3 times with PBS. </li>
	<li>Proceed to immuno-staining or, alternatively, leave samples in 500 &mu;L of 1%BSA/PBST O/N at 4&deg;C.</li>
</ol>
DAY 3
Immuno-staining
Staining of intracellular bacteria and &alpha;-actinin can be performed sequentially or simultaneously by the use of appropriate primary and secondary antibodies as follows; all procedures can be carried out in 12-well plates.
<ol>
	<li>Block the coverslips containing the permeabilised cells with 500 &mu;L of 3% BSA/PBST (PBS containing 0.05% Triton X-100) for 30-60 min at RT</li>
	<li>After washing with PBS, transfer each coverslip to a new dry well in the 12-well plate. </li>
	<li>Add the primary antibodies against bacteria and &alpha;-actinin; eighty to 100 &mu;L of antibody per coverslip is sufficient if added carefully to cover the surface of the coverslip. Incubate for 1h at room temperature.
 <ol>
 	<li>We used rabbit polyclonal antiserum against Neisseria meningitidis (laboratory-raised) and mouse monoclonal antibody (mAb) against &alpha;-actinin (Abcam) simultaneously. In some experiments, in place of anti &alpha;-actinin, mAbs against actin or vimentin were used (Sigma).</li>
 </ol>
 </li>
	<li>At the end of the incubation, add 200 &mu;L of PBS to the well, lift the coverslip and place in a new well containing 500 &mu;L PBS. </li>
	<li>After 5 min, remove PBS by pipetting and then add fresh PBS. Repeat twice. Transfer coverslips to a new dry well.</li>
	<li>Add the appropriate secondary antibodies conjugated to different fluorochromes diluted in 1% BSA/PBST; incubate at RT for 1h in the dark.
 <ol>
		<li>For bacterial detection, we used anti-rabbit Ig conjugated to TRITC and for &alpha;-actinin and other cytoskeletal protein detection, we used anti-mouse Ig conjugated to either FITC- (Sigma) or Alexa Fluor 488- (Invitrogen).</li>
 </ol>
 </li>
	<li>At the end of the incubation, wash as in steps 4 and 5.</li>
	<li>Counter stain with 0.6 &mu;g/mL DAPI in PBS (DNA stain) for 5 min at RT in the dark.</li>
	<li>Rinse with PBS.</li>
	<li>Mount coverslips (cells facing down) on slides with a drop of mounting medium (e,g. Mowiol or Vectashield)</li>
	<li> Store in the dark at 4&deg;C.</li>
	<li>Samples are ready for observation under the microscope.</li>
</ol>
2. Confocal Laser Scanning Microscopy (CLSM)
To observe and capture images of intracellular bacteria and cytoskeletal elements, we used immunolabelled samples and captured images using a Leica SP5-AOBS confocal laser scanning microscope attached to a Leica DM I6000 inverted epifluorescence microscope. All images were collected using a 63x NA 1.4 oil immersion objective and process with Leica software.
CLSM procedure:
<ol>
	<li>To begin the CLSM procedure, add a drop of immersion oil to the objective and place the specimen slide, coverslip-side down, on the microscope stage.</li>
	<li>Set the microscope to a visual mode and find the area of interest using the eye pieces of the microscope. </li>
	<li>Using Leica Software, select the xyz acquisition mode. </li>
	<li>Select the 512 x 512 format (frame size). For colocalisation studies, the higher resolution, the more accurate the image; keeping in mind the resolution limit of the microscope. Then select the bidirectional X mode, which will increase the scanning speed and help reduce photo-bleaching.</li>
	<li>Set up sequential scanning settings. Click in the "seq" function and choose one of the scanning modes. We use "between lines" mode.</li>
	<li>Select laser beams according to the fluorochromes conjugated to the secondary antibodies: 405 nm for DAPI (blue), 488 nm for Alexa Fluor 488 (green) and 561 nm for TRITC (red). Activate Photomultiplier Tube (PMT) 1, 2, and 3, respectively. Adjust PMT settings to detect the correct emission wavelength. </li>
	<li>Set up top ("begin") and bottom ("end") of z-stacks or series. Next, set the required "z-step size". 
	<ol>
		<li>Neisseria meningitidis is a diplococcus (Figure 1G) and since each coccus has an approximate diameter of 0.5 &mu;m, the z-step size of 0.20 &mu;m was chosen to enhance the probability of scanning each coccus at least twice. It is also within the step size for optimum resolution of 0.1 - 0.2 &mu;m.</li>
 </ol>
 </li>
	<li>Set the final scan parameters by selecting line averaging of 3 to improve the signal to noise ratio.</li>
	<li>By clicking "start" double- or triple-stained images are obtained by sequential scanning at different wavelengths to eliminate cross-talk between different chromophores.</li>
	<li>For an indication of colocalisation of two fluorochromes, select the "overlay" function to merge selected channels into a single image; for example, when both Alexa 488 (green) and TRITC (red) fluorochromes colocalise, yellow color will appear in the overlaid image. </li>
	<li>Compile z-stacks or series using the "maximum projection" function for the construction of a 2D image required for visualising possible colocalisation. More detailed analysis of colocalisation can be obtained by analysis of each optical section.</li>
	<li>After acquiring the z-stacks or series, process your data to obtain an orthogonal image for visualisation of intracellular localisation of various elements (Figure 1E).</li>
</ol>
3. Quantification of Colocalisation
Statistical analyses of the confocal scanning microscope images are performed with Volocity software (Improvision, PerkinElmer). This software provides a tool designed specifically for colocalisation analysis as described by Manders et al.(1993)5. Colocalisation in the context of digital fluorescence imaging can be described as the detection of a signal at the same voxel (pixel volume) location in each channel. The two channels are made up of images of two different fluorochromes taken from the same sample area (Volocity user guide). Statistical analyses are performed with Volocity software (Improvision, PerkinElmer) using Quantitative Colocalisation Analysis described below.
Quantitative Colocalisation Analysis
<ol>
	<li>Create a library with CLSM images using Volocity software. </li>
	<li>Select "extended focus" from image in the top bar. This tool will compile z-stacks in a 2D image to be analyzed.</li>
	<li>Select "Colocalization" tool. The two channels to be analyzed should have the same color depth.</li>
	<li>Select the area that it is going to be quantified. Set the threshold to remove any background.</li>
	<li>Create the colocalisation output by selecting "generate colocalization". Colocalisation statistics are generated for regions of interest selected previously.</li>
	<li>Select Manders' coefficients R (overlap coefficient) and My (colocalisation coefficient). 
	<ol>
		<li>Manders' coefficients are not sensitive to the intensity of staining as they are normalized against total pixel intensity; thus they can be employed when the staining of one antigen is stronger than the other.</li>
		<li>Overlap coefficient R according to Manders5,6 represents the true degree of colocalisation, i.e. the number of pixels that colocalise compared with the total number of pixels.</li>
		<li>On the other hand, the colocalisation coefficient, My, describes the fluorescence contribution of the more abundant moiety (in this case &alpha;-actinin, green) to the less abundant moiety (in this case bacteria, red), i.e. the number of red pixels that overlap with green pixels compared with the total number of red pixels.</li>
		<li>Manders' coefficients range between 1 and 0, with 1 being high colocalisation, 0 being none; but they can be expressed as percentage for easier interpretation.</li>
	</ol>
 </li>
	<li>Export statistics values to an Excel document for data presentation.</li>
</ol>
4. Representative Results
Intracellular localisation of Opc-expressing Neisseria meningitidis and &alpha;-actinin
Confocal imaging of human brain microvascular endothelial cells infected with Nm for 3 and 8 hours as described above indicated colocalisation of &alpha;-actinin and Nm which appeared to be less frequent in 3 h infection experiments (not shown) compared with cultures infected for 8 h (Figure 1 A-F). A demonstrable colocalisation of &alpha;-actinin with Opc-expressing meningococci was observed each time in &gt;5 replicate experiments. Statistical analysis of colocalisation using several confocal images was carried out as described above. Overall, in HBMEC infected with Opc-expressing meningococci, >25% overlap of the green (&alpha;-actinin) and red (Nm) pixels was obtained (Figure 2B, Overlap coefficient R). In contrast to &alpha;-actinin, experiments in which labeling of internalised bacteria and either actin or vimentin was performed, occasional colocalisation was observed with actin but that with vimentin was rare (Figure. 2B).
The data were also analyzed using the coefficient My, which takes into account the relative abundance of each moiety. My is a measure of the frequency of occurrence of the more abundant signal (in this case green, &alpha;-actinin) each time the less abundant signal (in this case red, bacteria) occurs. This measure shows a striking level of occurrence of &alpha;-actinin in the vicinity of internalised meningococci (Figure 2A and C).
<img src="/files/ftp_upload/2045/2045fig1.jpg" alt="Figure 1" /> Figure 1. Confocal laser scanning microscopy to assess intracellular interactions of N. meningitidis with &alpha;-actinin. A-H. Confluent endothelial monolayers grown on coverslips were infected with the Opc-expressing (A-F) N. meningitidis. After 8 h, non-adherent bacteria were washed off, cells fixed with paraformaldehyde and permeabilised with 0.1% Triton X-100. Subsequently, bacteria and &alpha;-actinin were stained as described above (&alpha;-actinin, green; bacteria, red).
A-C. One field showing x-y images of the location of Nm (A) or &alpha;-actinin (B). The overlay image in (C) indicates several regions in which yellow-orange color appears suggesting colocalisation. Arrows in (A) and (B) show regions where a high degree of &alpha;-actinin accumulation appears to have occurred around bacteria.
D. Optical dissection of an infected HBMEC monolayer indicating colocalisation around intracellular bacteria located at the base of a cell.
Again, this colocalisation is not due to accidental proximity of &alpha;-actinin, as the general stain of &alpha;-actinin in this region is low.
E and F. Three-dimensional images of infected HBMEC monolayers processed as above. An oblique view of the apical surface (E) shows adherent bacteria stained red (red arrow) whereas several bacteria located towards the basal surfaces of endothelial cells (yellow arrow) are distinctly orange/yellow in color. Basal location can be more clearly seen in (F) which is an end-on X-Z cross-section.
G. A negatively stained electron microscope image of N. meningitidis showing its predominant diplococcal from. Each coccus is approximately 0.5 &mu;m in diameter.
<img src="/files/ftp_upload/2045/2045fig2.jpg" alt="Figure 2" /> Figure 2. Localisation and distribution of &alpha;-actinin, actin and vimentin in HBMEC cells.
A. Infected monolayers of HBMEC were treated as described in the legend above but in addition to &alpha;-actinin, some coverslips were used for the detection of actin or vimentin by procedure similar to that for &alpha;-actinin. As above, &alpha;-actinin concentrated around several internalised bacteria (white arrows). Vimentin and actin did not colocalise with bacteria to appreciable levels. Bar represents 20 &mu;m.
B. &amp; C. The values for the coefficients R and My were obtained from more than three experiments using Volocity software as described above.

<ol>
	<li>Virji, M., Makepeace, K., Moxon, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+mechanisms+of+interactions+of+Opc-expressing+meningococci+at+apical+and+basolateral+surfaces+of+human+endothelial+cells;+the+role+of+integrins+in+apical+interactions.">Distinct mechanisms of interactions of Opc-expressing meningococci at apical and basolateral surfaces of human endothelial cells; the role of integrins in apical interactions.</a> Molecular Microbiology. 14, 173-184 (1994).</li><li>Virji, M., Makepeace, K., Peak, I. R., Ferguson, D. J., Jennings, M. P., Moxon, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opc-+and+pilus-+dependent+interactions+of+meningococci+with+human+endothelial+cells:+molecular+mechanisms+and+modulation+by+surface+polysaccharides.">Opc- and pilus- dependent interactions of meningococci with human endothelial cells: molecular mechanisms and modulation by surface polysaccharides.</a> Molecular Microbiology. 18, 741-754 (1995).</li><li>Sa E Cunha, C., Griffiths, N. J., Murillo, I., Virji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neisseria+meningitidis+Opc+invasin+binds+to+the+cytoskeletal+protein+alpha-actinin.">Neisseria meningitidis Opc invasin binds to the cytoskeletal protein alpha-actinin.</a> Cellular Microbiology. 11, 384-405 (2009).</li><li>Virji, M., Kayhty, H., Ferguson, D. J., Alexandrescu, C., Alexandrescu, J. E., Heckels, E. R. <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+pili+in+the+interactions+of+pathogenic+Neisseria+with+cultured+human+endothelial+cells.">The role of pili in the interactions of pathogenic Neisseria with cultured human endothelial cells.</a> Molecular Microbiology. 5, 1831-1841 (1991).</li><li>Manders, E. M. M., Verbeek, F. J., Aten, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+colocalization+of+objects+in+dual+color+confocal+images.">Measurement of colocalization of objects in dual color confocal images.</a> Journal of Microscopy. 6, 357-382 (1993).</li><li>Zinchuk, V., Zinchuk, O., Okada, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+colocalization+analysis+of+multicolor+confocal+immunofluorescence+microscopy+images:+pushing+pixels+to+explore+biological+phenomena.">Quantitative colocalization analysis of multicolor confocal immunofluorescence microscopy images: pushing pixels to explore biological phenomena.</a> Acta Histochemica et Cytochemica. 40, 101-111 (2007).</li></ol>

No conflicts of interest declared.

The possibility of binding of internalised Opc-expressing Neisseria meningitidis to &alpha;-actinin was explored using HBMEC by the examination of the colocalisation of bacteria and the cytoskeletal protein in infected cells after 3 and 8 h incubation period. By confocal microscopy, colocalisation of Neisseria meningitidis with &alpha;-actinin could be demonstrated. Notably, although bacteria were internalised at 3 h, there was little colocalisation with &alpha;-actinin at this time point. Bacterial association with the cytoskeletal protein appeared to require a longer period of intracellular residence as after 8 h infection period, significant numbers of bacteria had &alpha;-actinin apparently in close association. Alpha-actinin is a multifunctional protein, and bacterial interactions with the cytoskeletal element could have significant influence on the target cell function which is a subject of current studies.
Quantification of colocalisation as described above requires meticulous specimen preparation. Particular attention should be given to specimen fixation, blocking period and antibody dilutions. For the best signal to noise ratio, each primary and secondary antibody should be titrated in preliminary experiments to determine the optimum concentrations. In our experience, the mounting medium Mowiol produced better images.

<table><tbody><tr><td>12 well plate</td><td>Corning</td><td>BC311</td><td></td></tr><tr><td>Glass Coverslips</td><td>VWR international</td><td>631-0152</td><td></td></tr><tr><td>BHI AGAR</td><td>LAB M</td><td>LAB048</td><td></td></tr><tr><td>M199</td><td>Sigma-Aldrich</td><td>M2154</td><td></td></tr><tr><td>RPMI 1640</td><td>Lonza Inc.</td><td>BE12-167E</td><td></td></tr><tr><td>MEM Non Essential Aminoacids</td><td>Sigma-Aldrich</td><td>M7145</td><td></td></tr><tr><td>HANK&amp;rsquo;S balanced salt solution</td><td>Sigma-Aldrich</td><td>H9269</td><td></td></tr><tr><td>FBS</td><td>BioWhittaker</td><td>DE14-801F</td><td></td></tr><tr><td>Glutamine</td><td>Sigma-Aldrich</td><td>G7513</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>S8636</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P0781</td><td></td></tr><tr><td>MEM Vitamin solution</td><td>Sigma-Aldrich</td><td>M6895</td><td></td></tr><tr><td>PBSB</td><td>Lonza Inc.</td><td>BE17-513F</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A9418</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Fisher Scientific</td><td>P/0840/53</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X-100</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D-9564</td><td></td></tr><tr><td>Vectashield</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>Mowiol 4-88</td><td>Calbiochem</td><td>475904</td><td></td></tr><tr><td>Anti&amp;#8209;Nm antiserum</td><td>Laboratory raised</td><td></td><td>Rabbit serum</td></tr><tr><td>TRITC anti-rabbit Ig</td><td>Sigma-Aldrich</td><td>T6778</td><td></td></tr><tr><td>Anti &amp;alpha;&amp;#8209;actinin mAb [7H6]</td><td>Abcam</td><td>Ab32816</td><td>IgG</td></tr><tr><td>Anti actin mAb</td><td>Sigma-Aldrich</td><td>A4700</td><td>IgG2a</td></tr><tr><td>Anti vimentin mAb</td><td>Sigma-Aldrich</td><td>V6630</td><td>IgG1</td></tr><tr><td>ALEXA FLUOR 488 anti&amp;#8209;mouse IgG</td><td>Invitrogen</td><td>A11001</td><td></td></tr><tr><td>FITC anti-mouse IgG</td><td>Sigma-Aldrich</td><td>F-2012</td><td></td></tr><tr><td colspan="4">&lt;p class=&quot;jove_title&quot;&gt;1. Confocal Laser Scanning Microscopy (CLSM):&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;Leica SP5 confocal imaging system: This system, by using a combination of AOTF (acousto-optical tuneable filter) and an AOBS (acousto-optical beam splitter), simplifies excitation with specific wave lengths.&lt;/p&gt;&lt;p class=&quot;jove_title&quot;&gt; 2-Software:&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt; Leica confocal software LCS, Leica Microsystems, Germany.&lt;br/&gt;Volocity 5, Improvision, PerkinElmer, USA.&lt;/p&gt;</td></tr></tbody></table>

visualisation and quantification of intracellular interactions of neisseria meningitidis and human &alpha;-actinin by confocal imaging

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an effective invasin for human epithelial and endothelial cells. We have identified endothelial surface-located integrins as major receptors for Opc, a process which requires Opc to first bind to integrin ligands such as vitronectin and via these to the cell-expressed receptors1. This process leads to bacterial invasion of endothelial cells2. More recently, we observed an interaction of Opc with a 100kDa protein found in whole cell lysates of human cells3. We initially observed this interaction when host cell proteins separated by electrophoresis and blotted on to nitrocellulose were overlaid with Opc-expressing Nm. The interaction was direct and did not involve intermediate molecules. By mass spectrometry, we established the identity of the protein as &alpha;-actinin. As no surface expressed &alpha;-actinin was found on any of the eight cell lines examined, and as Opc interactions with endothelial cells in the presence of serum lead to bacterial entry into the target cells, we examined the possibility of the two proteins interacting intracellularly. For this, cultured human brain microvascular endothelial cells (HBMECs) were infected with Opc-expressing Nm for extended periods and the locations of internalised bacteria and &alpha;-actinin were examined by confocal microscopy. We observed time-dependent increase in colocalisation of Nm with the cytoskeletal protein, which was considerable after an eight hour period of bacterial internalisation. In addition, the use of quantitative imaging software enabled us to obtain a relative measure of the colocalisation of Nm with &alpha;-actinin and other cytoskeletal proteins. Here we present a protocol for visualisation and quantification of the colocalisation of the bacterium with intracellular proteins after bacterial entry into human endothelial cells, although the procedure is also applicable to human epithelial cells.

Watch this Scientific Journal Video about Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human Î±-actinin by Confocal Imaging at JoVE.com

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &#945;-actinin by Confocal Imaging

Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human &alpha;-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular &alpha;-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs).

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &alpha;-actinin by Confocal Imaging

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an ...

visualisation-quantification-intracellular-interactions-neisseria

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

18.1K Views.  University of Bristol, UK. Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human α-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular α-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs). 

Video: Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human α-actinin by Confocal Imaging

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 ...

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.

Neisseria meningitidis is a human specific pathogen that infects blood vessels. In this protocol human microvessels are introduced into a mouse by grafting human skin onto immunocompromised mice. Bacteria adhere extensively to the human vessels, leading to vascular damage and development of the purpuric rash typically observed in human cases.

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood ...

Microtensiometer for Confocal Microscopy Visualization of Dynamic Interfaces

Adsorption of surface-active molecules to fluid-fluid interfaces is ubiquitous in nature. Characterizing these interfaces requires measuring surfactant adsorption rates, evaluating equilibrium surface tensions as a function of bulk surfactant concentration, and relating how surface tension changes with changes in the interfacial area following equilibration. Simultaneous visualization of the interface using fluorescence imaging with a high-speed confocal microscope allows the direct evaluation of structure-function relationships. In the capillary pressure microtensiometer (CPM), a hemispherical air bubble is pinned at the end of the capillary in a 1 mL volume liquid reservoir. The capillary pressure across the bubble interface is controlled via a commercial microfluidic flow controller that allows for model-based pressure, bubble curvature, or bubble area control based on the Laplace equation. Compared to previous techniques such as the Langmuir trough and pendant drop, the measurement and control precision and response time are greatly enhanced; capillary pressure variations can be applied and controlled in milliseconds. The dynamic response of the bubble interface is visualized via a second optical lens as the bubble expands and contracts. The bubble contour is fit to a circular profile to determine the bubble curvature radius, R, as well as any deviations from circularity that would invalidate the results. The Laplace equation is used to determine the dynamic surface tension of the interface. Following equilibration, small pressure oscillations can be imposed by the computer-controlled microfluidic pump to oscillate the bubble radius (frequencies of 0.001-100 cycles/min) to determine the dilatational modulus The overall dimensions of the system are sufficiently small that the microtensiometer fits under the lens of a high-speed confocal microscope allowing fluorescently tagged chemical species to be quantitatively tracked with submicron lateral resolution.

This manuscript describes the design and operation of a microtensiometer/confocal microscope to do simultaneous measurements of interfacial tension and surface dilatational rheology while visualizing the interfacial morphology. This provides the real-time construction of structure-property relationships of interfaces important in technology and physiology.

Adsorption of surface-active molecules to fluid-fluid interfaces is ubiquitous in nature. Characterizing these interfaces requires measuring surfactant ...

Engineering

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and regulated by different suites of binding proteins unique for each polymer. Many studies now demonstrate that the dynamics of both cytoskeletal polymers are intertwined and that this crosstalk is required for most cellular behaviors. A number of proteins involved in actin-microtubule interactions have already been identified (i.e., Tau, MACF, GAS, formins, and more) and are well characterized with regard to either actin or microtubules alone. However, relatively few studies showed assays of actin-microtubule coordination with dynamic versions of both polymers. This may occlude emergent linking mechanisms between actin and microtubules. Here, a total internal reflection fluorescence (TIRF) microscopy-based in vitro reconstitution technique permits the visualization of both actin and microtubule dynamics from the one biochemical reaction. This technique preserves the polymerization dynamics of either actin filament or microtubules individually or in the presence of the other polymer. Commercially available Tau protein is used to demonstrate how actin-microtubule behaviors change in the presence of a classic cytoskeletal crosslinking protein. This method can provide reliable functional and mechanistic insights into how individual regulatory proteins coordinate actin-microtubule dynamics at a resolution of single filaments or higher-order complexes.

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence TIRF Microscopy

This protocol is a guide for visualizing dynamic actin and microtubules using an in vitro total internal fluorescence (TIRF) microscopy assay.

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and ...

Biochemistry

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, ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes depend on the advances of growth cones that located at the tips of neurites. Axonal growth cones generate driving forces by sensing their local microenvironment and modulating cytoskeletal dynamics and actin-adhesion coupling (clutch coupling). Decades of research have led to the identification of guidance molecules, their receptors, and downstream signaling cascades for regulating neuronal migration and axonal guidance; however, the molecular machineries required for generating forces to drive growth cone advance and navigation are just beginning to be elucidated. At the leading edge of neuronal growth cones, actin filaments undergo retrograde flow, which is powered by actin polymerization and actomyosin contraction. A clutch coupling between F-actin retrograde flow and adhesive substrate generates traction forces for growth cone advance. The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging. Importantly, when combined with an F-actin marker Lifeact, this technique can quantify 1) the F-actin polymerization rate and 2) the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate. Both are critical variables for generating forces for growth cone advance and navigation. In addition, the present study describes a detailed protocol of traction force microscopy, which can quantify 3) traction force generated by growth cones. Thus, by coupling the analyses of&#160;single speckle imaging and traction force microscopy, investigators can monitor the molecular mechanics underlying growth cone advance and navigation.

To advance, growth cones must exert traction forces against the external environment. The generation of traction forces is dependent on actin dynamics and clutch coupling. The present study describes methods for analyzing actin dynamics, clutch coupling and traction forces for growth cone advance.

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes ...

Neuroscience

Visualizing Proteins in Primary Hippocampal Neurons by Live&#45;Cell Imaging

Using Live&#45;Cell Imaging to Measure the Effects of Pathological Proteins on Axonal Transport in Primary Hippocampal Neurons

Bidirectional transport of cargos along the axon is critical for maintaining functional synapses, neural connectivity, and healthy neurons. Axonal transport is disrupted in multiple neurodegenerative diseases, and projection neurons are particularly vulnerable because of the need to transport cellular materials over long distances and sustain substantial axonal mass. Pathological modifications of several disease-related proteins negatively affect transport, including tau, amyloid-&#946;, &#945;-synuclein, superoxide dismutase, and huntingtin, providing a potential common mechanism by which pathological proteins exert toxicity in disease. Methods to study these toxic mechanisms are necessary to understand neurodegenerative disorders and identify potential therapeutic interventions.
Here, cultured primary rodent hippocampal neurons are co-transfected with multiple plasmids to study the effects of pathological proteins on fast axonal transport using live-cell confocal imaging of fluorescently-tagged cargo proteins. We begin with the harvest, dissociation, and culturing of primary hippocampal neurons from rodents. Then, we co-transfect the neurons with plasmid DNA constructs to express fluorescent-tagged cargo protein and wild-type or mutant tau (used as an exemplar of pathological proteins). Axons are identified in live cells using an antibody that binds an extracellular&#160;domain of neurofascin, an axon initial segment protein, and an axonal region of interest is imaged to measure fluorescent cargo transport.
Using KymoAnalyzer, a freely available ImageJ macro, we extensively characterize the velocity, pause frequency, and directional cargo density of axonal transport, all of which may be affected by the presence of pathological proteins. Through this method, we identify a phenotype of increased cargo pause frequency associated with the expression of pathological tau protein. Additionally, gene-silencing shRNA constructs can be added to the transfection mix to test the role of other proteins in mediating transport disruption. This protocol is easily adaptable for use with other neurodegenerative disease-related proteins and is a reproducible method to study the mechanisms of how those proteins disrupt axonal transport.

Author Spotlight: Understanding the Impact of Pathological Proteins on Axonal Transport in Neurodegenerative Diseases

Here, we demonstrate how to combine transfection of primary hippocampal rodent neurons with live-cell confocal imaging to analyze pathological protein-induced effects on axonal transport and identify mechanistic pathways mediating these effects.

Bidirectional transport of cargos along the axon is critical for maintaining functional synapses, neural connectivity, and healthy neurons. Axonal ...

Analyzing the &alpha;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. Despite the development of multiple differentiation protocols, the generation of iPSC cardiomyocytes resembling an adult-like phenotype remains challenging. One major aspect of cardiomyocytes maturation involves the formation of a well-organized sarcomere network to ensure high contraction capacity. Here, we present a super resolution-based approach for semi-quantitative analysis of the &#945;-actinin network in cardiomyocytes. Using photoactivated localization microscopy a comparison of sarcomere length and z-disc thickness of iPSC-derived cardiomyocytes and cardiac cells isolated from neonatal tissue was performed. At the same time, we demonstrate the importance of proper imaging conditions to obtain reliable data. Our results show that this method is suitable to quantitatively monitor the structural maturity of cardiac cells with high spatial resolution, enabling the detection of even subtle changes of sarcomere organization.

Analyzing the &#945;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. ...

Biology

Tension Gauge Tether Probes for Quantifying Growth Factor Mediated Integrin Mechanics and Adhesion

Multicellular organisms rely on interactions between membrane receptors and cognate ligands in the surrounding extracellular matrix (ECM) to orchestrate multiple functions, including adhesion, proliferation, migration, and differentiation. Mechanical forces can be transmitted from the cell via the adhesion receptor integrin to ligands in the ECM. The amount and spatial organization of these cell-generated forces can be modulated by growth factor receptors, including epidermal growth factor receptor (EGFR). The tools currently available to quantify crosstalk-mediated changes in cell mechanics and relate them to&#160;focal adhesions, cellular morphology, and signaling are limited. DNA-based molecular force sensors known as tension gauge tethers (TGTs) have been employed to quantify these changes. TGT probes are unique in their ability to both modulate the underlying force threshold and report piconewton scale receptor forces across the entire adherent cell surface at diffraction-limited spatial resolution. The TGT probes used here rely on the irreversible dissociation of a DNA duplex by receptor-ligand forces that generate a fluorescent signal. This allows quantification of the cumulative integrin tension (force history) of the cell. This article describes a protocol employing TGTs to study the impact of EGFR on integrin mechanics and adhesion formation. The assembly of&#160;the TGT mechanical sensing platform is systematically detailed and the procedure to image forces, focal adhesions, and cell spreading is outlined. Overall, the ability to modulate the underlying force threshold of the probe, the adhesion ligand, and the type and concentration of growth factor employed for stimulation make this a robust platform for studying the interplay of diverse membrane receptors in regulating integrin-mediated forces.

TGT surface is an innovative platform to study growth factor-integrin crosstalk. The flexible probe design, specificity of the adhesion ligand, and precise modulation of stimulation conditions allow robust quantitative assessments of EGFR-integrin interplay. The results highlight EGFR as a 'mechano-organizer' tuning integrin mechanics, influencing focal adhesion assembly and cell spreading.

Multicellular organisms rely on interactions between membrane receptors and cognate ligands in the surrounding extracellular matrix (ECM) to orchestrate ...

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human α-actinin by Confocal Imaging

Summary

Explore More Videos

Chapters in this video