Name: avidity-based extracellular interaction screening (avexis) for the scalable detection of low-affinity extracellular receptor-ligand interactions
Uploaded: 2012-03-05T13:00:00
Duration: 12 min 30 s
Description: Watch this Scientific Journal Video about Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions at JoVE.com

This work was supported by the Wellcome Trust grant number [077108] awarded to GJW.

1. Compiling a Library of Bait and Prey Plasmid-expression Vectors
<ol>
	<li>Compile a list of cell surface receptor and secreted proteins of interest to be screened for interactions and retrieve their sequences from a suitable protein sequence database. Most proteins containing an N-terminal secretory signal peptide are suitable (these include type I, GPI-linked and secreted proteins). Typically, a protein family (e.g. immunoglobulin superfamily or all receptors restricted to a specific cell type such as the erythrocyte) would be selected.</li>
	<li>Determine the extent of the extracellular regions by identifying the location of the signal peptide and transmembrane region using protein feature prediction software. We typically use SignalP v3.05 and TMHMM v2.06.</li>
	<li>Design primer sets that amplify the entire ectodomain fragment for each receptor. We have a suite of bait and prey vectors that are suitable for this purpose which are available from Addgene (<a href="http://www.addgene.org/">http://www.addgene.org/</a>). Design the sense primer around the start methionine to include a NotI restriction enzyme site and an optimal Kozak sequence; this is followed by 25 base pairs of exact match gene-specific sequence (Fig. 2A).</li>
	<li>Design the antisense primer to truncate type I receptor proteins just prior to the predicted transmembrane region so as to express the entire ectodomain as a soluble recombinant protein. Include an AscI restriction site in this primer which should then translate in frame to a C-terminal rat Cd4d3+4 protein tag7 (Fig. 2A).</li>
	<li>Amplify the ectodomain fragments from all genes using these primers and clone them into the bait or prey vector. We sometimes find it helpful to first clone them into shuttle vectors before subcloning into an appropriate mammalian expression vector, we use a derivative of the pTT3 vector3,4. Both the bait and prey vectors contain C-terminal protein tags as follows (Fig. 2B).
	<ol>
		<li>Baits: A tag containing domains 3 and 4 of the rat Cd4 (Cd4d3+4) protein followed by a peptide sequence that is a substrate for the E.coli BirA enzyme and can therefore be monobiotinylated on a specific lysine residue (Fig. 2B). The Cd4d3+4 tag is recognised by the OX68 monoclonal antibody and is used to quantify the expression of the bait proteins by ELISA. The bait proteins are therefore monomeric and monobiotinylated. Additional bait protein vectors where the biotinylatable peptide is replaced by a 6-his tag for purification are available.</li>
		<li>Preys: The preys also contain a rat Cd4d3+4 tag followed by a peptide sequence from the rat cartilage oligomeric matrix protein (COMP) and a C-terminal &beta;-lactamase enzyme. The COMP peptide ensures the prey forms pentamers once expressed (Fig. 2B).</li>
	</ol>
	</li>
</ol>
2. Prey- and Bait-protein Expression
We use a convenient high-level expression system using suspension culture of the HEK293E cell line4 to produce our protein libraries, but any mammalian expression system should be suitable. A commercially available alternative is the Freestyle system. Cells are routinely cultured on a shaking platform (125 r.p.m.) at 37 &deg;C, 5% CO2 at 70% relative humidity. Both positive and negative control proteins must also be expressed. The rat Cd4d3+4 tag-only fragment is available as both a bait and prey and are suitable negative controls. Similarly, bait and prey vectors that encode a positive control are available (Addgene); we typically use the rat Cd200- Cd200R interaction8.
<ol>
	<li>Seed the HEK293E cells the day prior to transfection at a density of 2.5 x 105 cells mL-1. We routinely culture the cells in volumes of 50 mL in Freestyle293 media following the manufacturer's recommendations. To ensure efficient biotinylation, supplement the cell culture medium used to produce bait proteins with D-biotin to a final concentration of 100 &mu;M. Medium used to express the prey proteins does not need to be supplemented with additional D-biotin.</li>
	<li>The following day, transfect cells using a suitable transfections reagent (e.g. 293fectin). Use 25 &mu;g of the bait or prey plasmid constructs to transfect a 50 mL culture. To produce the biotinylated baits, co-transfect the plasmid encoding the bait construct with a plasmid that encodes a secreted form of the E.coli protein-biotin ligase (BirA) which is available from Addgene. Co-transfect bait plasmids at a 10:1 ratio (2.5 &mu;g) of the plasmid encoding the secreted BirA protein.</li>
	<li>Harvest cultures five days post-transfection by first pelleting the cells by centrifugation at 3000 xg for 20 minutes followed by filtration of the supernatant through a 0.22 &mu;M filter.</li>
</ol>
[Tip: Bait and prey proteins should be stored undiluted at 4&deg;C and as a general guideline should be used within a year. Add bacteriostatics such as 50 &mu;g/mL polymyxin B sulphate or 10 mM NaN3 to prevent bacterial contamination of the supernatants.]
3. Normalisation of the Bait and Prey Samples
One of the key features of the AVEXIS assay is that because the recombinant proteins are secreted they can be diluted or concentrated (normalised) to within established threshold activities prior to being used in the assay. We have found that the levels at which the recombinant proteins are expressed span a wide range - up to 4 orders of magnitude - and so the normalisation step significantly reduces the number of false positives during the screens.
3.1 Bait normalisation
Note: Unconjugated D-biotin must be removed from the media (typically by dialysis) since it will compete with the biotinylated bait protein for binding to streptavidin binding sites.
<ol>
	<li>Transfer bait supernatants into dialysis tubing, label, clip securely and dialyse extensively against a suitable buffer such as PBS to remove free biotin.</li>
</ol>
[Tip: We have found that sufficient dialysis is normally achieved for 6 x 50 mL bait supernatants against a volume of 4.5 L of PBS with 4 changes over a 24 hour period. Insufficient dialysis can be observed by an inhibition of protein binding at low dilutions during the bait normalisation step (Fig. 3A.)
<ol start="2">
	<li>Perform an ELISA to determine the relative levels at which the bait proteins are expressed. Prepare a dilution series of the biotinylated bait proteins and capture them on a streptavidin-coated microtitre plate. Use the rat Cd4d3+4 tag to detect and quantify the captured bait proteins using a monoclonal antibody (OX68). Concentrate or dilute bait proteins to the minimum amount required to saturate all the biotin binding sites in the well of a streptavidin-coated plate.</li>
	<li>Block the wells of a streptavidin-coated plate with phosphate buffered saline/0.1% Tween-20 (PBST)/2% bovine serum albumin (BSA) for 15 minutes.</li>
	<li>In a separate plate, make a dilution series (four 1:3 dilutions are usually sufficient) of the bait proteins in PBST/2% BSA and transfer to wells of a streptavidin-coated plate. Incubate for 1 hour at room temperature.</li>
	<li>Wash 3x in PBST. Add 100 &mu;L of 2 &mu;g/mL mouse anti-rat Cd4d3+4 IgG (OX68) incubate for 1 hour at room temperature.</li>
	<li>Wash 3x in PBST. Add 100 &mu;L of anti-mouse alkaline phosphatase at 1:5,000 dilution in PBST/2% BSA and incubate for 1 hour at room temperature.</li>
	<li>Wash 3x in PBST and perform a final wash in PBS to remove any residual detergent. Add 100 &mu;L of 1 mg/mL Substrate 104 in diethanolamine buffer (10% diethanolamine (v/v), 0.5mM MgCl2, 10 mM NaN3, pH9.8, stored protected from light at 4 &deg;C).</li>
	<li>After one hour, measure absorbance at 405 nm. Representative bait normalisation results are shown in Figure 3A.</li>
	<li>Bait proteins that are expressed at levels that are insufficient to saturate all the biotin-binding sites of a streptavidin-coated plate, when used undiluted, should be concentrated using Vivaspin concentrators (Vivascience, 10kDa MWCO). As a guide, we estimate that ~5 &mu;g of a typical bait protein (~80 kDa) is sufficient to saturate a 96 well plate.
	<ol>
		<li>Bait proteins expressed at high levels can be diluted in PBST/2% BSA to a concentration sufficient to saturate the streptavidin-coated plate. The activity of the baits should be rechecked after dilution or concentration to ensure that they saturate all the biotin binding sites in the streptavidin-coated plate.</li>
	</ol>
	</li>
</ol>
3.2 Prey normalisation
<ol>
	<li>Quantify the relative levels at which the prey proteins are expressed using the &beta;-lactamase enzyme activity.</li>
	<li>First, make a dilution series of the supernatant containing the prey proteins in PBST/2%BSA buffer. Then add 20 &mu;L of the dilutions to 60 &mu;L of a 242 &mu;M (0.125 mg/mL) nitrocefin solution (Calbiochem).</li>
	<li>Immediately transfer the plate to a microtitre plate reader that takes an absorbance measurement at 485 nm every minute for 20 minutes at room temperature. Representative prey normalisation results are shown in Figure 3B.</li>
	<li>Concentrate samples using centrifugal concentrators or dilute them in PBST/2%BSA to reach a threshold activity of ~2 nmol min-1 turnover of nitrocefin. Practically, this is achieved if all the nitrocefin is hydrolysed within ~7 minutes.</li>
</ol>
4. AVEXIS Screening Procedure
<ol>
	<li>Wash a streptavidin-coated plate in PBST.</li>
	<li>Add 100 &mu;L of PBST/2%BSA to each well and incubate for 30 minutes to block any spurious protein binding sites within each well.</li>
</ol>
[Tip: to remove any residual wash buffer after wash steps, the plate is tapped - vigorously - on paper towels]
<ol start="3">
	<li>Remove the blocking solution with a smart flick of the plate over a sink and add 100 &mu;L normalised biotinylated monomeric bait to the appropriate wells and incubate for 1 hour at room temperature.</li>
	<li>Remove the bait samples from the plate by upturning the plate and smartly flicking over a sink, and wash 3 times in PBST.</li>
	<li>Add 100 &mu;L of the normalised pentamerized prey construct to each well and incubate at room temperature for 1 hour, wash as in step 4.4 and perform one final wash with PBS only.</li>
	<li>Add 60 &mu;L of 242 &mu;M nitrocefin solution (dissolve 5 mg of nitrocefin in 500 &mu;L of DMSO, mix thoroughly and then further dilute the nitrocefin in 40 mL PBS and pass through a 0.22 &mu;M filter before adding to the plates) and incubate at room temperature. Positive interactions can be observed by eye since the yellow nitrocefin is hydrolysed to a red colour. Quantify by reading absorbance at 485 nm using a plate reader. A typical plate and quantitation is shown in Fig. 4.</li>
</ol>
[Tip: Positive interactions are usually observed within 2 hours at room temperature although plates should be placed at 4&deg;C for 16 hours to ensure all potential interactions are detected. We have also found that precipitated nitrocefin and imperfections on the plastic of the microtitre plate such as scratches/fingerprints can occasionally lead to artefactual false positives at 485 nm despite no overt nitrocefin hydrolysis. It is therefore advisable to take photographs of the plates to visually record nitrocefin turnover in the wells.]
5. Representative Results
An example of a representative AVEXIS screening plate is shown in Figure 4. One should observe hydrolysis (yellow to red) of the nitrocefin substrate in the positive control wells (both the antibody-mediated prey capture control and also positive control interaction) within about ten minutes. Some interactions within the screen are also observed within this time scale, but others may take longer (a few hours) to appear. Typically, a hit-rate of around 0.4-0.6% (a positive interaction for every 150-250 interactions being screened) is typical, depending on the protein libraries being screened.
<img alt="Figure 1" src="/files/ftp_upload/3881/3881fig1.jpg" /> 
Figure 1. Identification of receptor-ligand interactions mediating cellular recognition processes using AVEXIS . The schematic shows two interacting cells (A and B) exchanging information through interactions made between membrane-embedded receptor proteins. AVEXIS is a scalable protein interaction assay specifically designed to identify novel receptor-ligand pairs which are characterised by very weak interaction strengths. Recombinant soluble protein libraries representing the whole ectodomain of the cell surface receptor repertoire of both cells are compiled either as pentameric &beta;-lactamase-tagged preys (cell type A) or monomeric biotin-tagged baits (for cell type B). The pentamerisation of the preys increases the local concentration of the ectodomains to effect an overall avidity gain so that even very transient interactions can be detected. The bait library is arrayed on streptavidin-coated plates and systematically probed for interactions with the prey proteins. After a brief wash, any captured preys are detected using the &beta;-lactamase activity associated with the prey.
<img alt="Figure 2" src="/files/ftp_upload/3881/3881fig2.jpg" /> 
Figure 2. Bait and prey construct design. (A) The rat Cd200 receptor protein is provided as an example of how the entire ectodomains of cell surface receptor proteins are determined and primers are designed to make both bait and prey expression constructs. The cDNA and translated protein sequence are shown from the N-terminal signal peptide to the membrane-spanning transmembrane region of rat Cd200. A sense primer is designed to include a 5' NotI restriction enzyme site and optimal Kozak sequence followed by 25 bases of exact match sequence to the Cd200 cDNA sequence. The antisense primer contains an AscI site and 25 bases of exact match sequence located immediately upstream of the selected ectodomain truncation residue. To keep the ectodomain in frame with the rat Cd4d3+4 tag, the AscI site should translate as Gly-Ala-Pro. (B) Schematic representations of the bait and prey expression constructs. Each contains receptor ectodomains flanked by NotI and AscI sites and a Cd4d3+4 tag. The baits contain a C-terminal peptide sequence that can be specifically monobiotinylated by cotransfection with a plasmid expressing a secreted BirA enzyme. The preys are followed by the pentamerisation sequence from the cartilage oligomeric matrix protein (COMP) and the &beta;-lactamase enzyme.
<img alt="Figure 3" src="/files/ftp_upload/3881/3881fig3.jpg" /> 
Figure 3. Normalisation of the bait and prey proteins. (A) Representative examples of bait normalisation. A dilution series of four different dialysed bait supernatants were detected by ELISA using an anti-Cd4 tag monoclonal antibody. The four baits are expressed at different levels 1&gt;2&gt;3&gt;4. Baits should be normalised to a threshold level that saturates the biotin binding sites in each well. Highly-expressed baits can be diluted to conserve protein (e.g. bait 1 could be diluted ~1:100), and poorly-expressed baits concentrated. Decreased readings at low dilutions are sometimes observed in some baits (e.g. bait 2) which is attributable to the presence of unconjugated D-biotin due to incomplete dialysis. (B) The levels at which prey proteins are expressed is quantified using the hydrolysis rate of a &beta;-lactamase substrate, nitrocefin. In the example shown, the three preys are expressed at different levels: prey 1&gt;2&gt;3. Preys should be normalised to an activity of ~2 nmol min-1, which corresponds to complete hydrolysis of 14.5 nmol nitrocefin in ~7 minutes (e.g. prey 1). The other preys in this example should be concentrated before use in screening.
<img alt="Figure 4" src="/files/ftp_upload/3881/3881fig4.jpg" /> 
Figure 4. Representative AVEXIS screening results. (A) A photograph of a representative AVEXIS screening plate. A prey protein is probed against 41 different baits and a positive interaction is detected in well E2. Controls used in the screening plates include: a biotinylated anti-Cd4 antibody to capture all the prey protein added to the well (G6), a control interaction: rat Cd200 (bait)-Cd200R (prey) with the Cd200R prey used at threshold dilution (H3), 1:10 (H4) and 1:100 (H5). Negative controls were: a Cd4d3+4 bait probed with the prey protein being screened (H1) and the Cd200R prey (H2). (B) Quantification of the absorbance values of the same screen performed in triplicate. Data points are mean &plusmn; SD.

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The authors have nothing to disclose.

When living cells are observed in vivo, their plasma membranes exhibit highly dynamic behaviours: extending and retracting membrane processes as they contact and communicate with their neighbours2. The physical interactions between the membrane-embedded receptor proteins which mediate many of these contacts have evolved to be extremely weak so as to permit this highly motile behaviour. It has been estimated that monomeric interaction strengths as weak as 50 &mu;M could be sufficient to drive spontaneous interactions at physiological receptor densities9 which makes detecting novel interactions extremely challenging2,10. The AVEXIS method described here provides a sensitive method for detecting transient extracellular protein:protein interactions that can be implemented in a scalable manner with a low false positive rate. While other scalable methods have been developed to detect extracellular interactions11-15, one advantage of AVEXIS is that the experimental parameters such as the bait and prey activities have been quantified to detect even the weakest of interactions (t1/2 &le; 0.1 s) while retaining a low false positive rate3. In addition, the streamlined and robust sample preparation procedures have enabled this assay to be implemented on a much larger scale than other assays and we have recently described a systematic interaction screen totalling over ~16,500 potential interactions16.
The assay can be performed on any protein for which it is possible to express a soluble recombinant protein fragment. This therefore includes proteins that contain an N-terminal signal peptide such as type I, GPI-linked and secreted proteins. We have recently designed a suite of vectors that now enable us to express type II proteins as baits17. The assay is not generally suitable for multipass membrane proteins such as ion transporters or pumps since they are unlikely to be correctly folded outside the context of a plasma membrane. We have applied this to a variety of different systems and have successfully expressed extracellular proteins from zebrafish3,16,18, human, mouse and P. falciparum for interaction screens.
In terms of the resources required to set up an AVEXIS screen, we have found that a significant bottleneck is the selection, construction, and full sequencing of the expression plasmids. This aspect of the method can take up to a year to make ~100 bait and prey constructs; however, with the decreasing cost of gene synthesis, we now favour commercially outsourcing this aspect of the project. The mammalian expression system together with a large shaking tissue culture incubator (we use an INFORS HT Multitron Cell) enables a single researcher to express and normalise up to ~100 bait and prey proteins a month. Once the proteins are produced and normalised, screening for interactions is rapid with up to twelve 96-well plates being conveniently processed per day. The only bespoke equipment that we have developed to facilitate the production of such a large number of proteins is a compressed air-driven piston which is used to pass up to five supernatants through a 0.22&mu;m filter in parallel.
The main class of interactions that might be missed when compared to interactions expected from the literature (false negatives) are homophilic interactions, although some of these interactions can be detected3. In the cases where expected interactions are not detected, we believe that this is due to the formation of homophilic interactions by the prey proteins (a prey "masking" effect) which then prevents further interactions with immobilised bait proteins. The frequency at which interactions are detected depends upon the content of the protein library being screened. As a guide to the reader, a screen of &gt;30,000 interactions within the zebrafish immunoglobulin superfamily resulted in 188 positives - a frequency of 0.6%3,16,18. A very rapid check that can be performed to verify any positive hits is to determine whether the interaction can be detected in both bait-prey orientations; that is, can the same interaction be detected irrespective of whether the binding proteins are present as either a bait or a prey. Whenever we have observed this reciprocation behaviour, the binding has subsequently been shown to be specific by demonstrating direct saturable binding of purified proteins using surface plasmon resonance. It should be stressed that because we increase binding avidity by artificially pentamerising the prey proteins, we do not consider the assay suitable for quantitatively comparing interaction strengths. To obtain biophysical binding parameters of interactions we use monomeric proteins and surface plasmon resonance. Finally, once an interaction has been identified, the high throughput nature of the assay makes it relatively easy to adapt the assay to screen for reagents such as antibodies or small molecules that block the interaction.

<table border="1">
<tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Cat. Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Freestyle media</td>
 <td>Invitrogen</td>
 <td>12338018</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nitrocefin</td>
 <td>Calbiochem</td>
 <td>484400</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>SA-coated microtitre plates</td>
 <td>Nunc</td>
 <td>734-1284</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>OX68 antibody</td>
 <td>AbDSerotec</td>
 <td>MCA1022R</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dialysis tubing</td>
 <td>Pierce</td>
 <td>PN68100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Polymyxin B</td>
 <td>Sigma</td>
 <td>P4932</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>D-biotin</td>
 <td>Sigma</td>
 <td>B4639-5G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Substrate-104</td>
 <td>Sigma</td>
 <td>1040-506</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Vivaspin-20 centrifugal concentrators</td>
 <td>Sartorius</td>
 <td>VS2002</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.22 &mu;m filters</td>
 <td>Nalgene</td>
 <td>190-2520</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

avidity-based extracellular interaction screening (avexis) for the scalable detection of low-affinity extracellular receptor-ligand interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Watch this Scientific Journal Video about Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions at JoVE.com

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

avidity-based-extracellular-interaction-screening-avexis-for-scalable

Research

JoVE Journal

Biology

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick&#8217;s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

A BW Reporter System for Studying Receptor-Ligand Interactions

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native receptor and the ligand are challenging since the ligand of a specific receptor may be unknown and experimental procedures with the native ligand can be technically complicated. To address these obstacles, we describe a reporter system to detect the binding and activation of a specific receptor by a ligand of interest. In this reporter system, the extracellular domain of a specific receptor is conjugated to mouse CD3&#950; and this chimeric protein is then expressed in mouse BW cells. These transfected BW cells can then be incubated with different targets (e.g., cells or antibodies). Activation of a transfected receptor leads to the secretion of mouse interleukin-2 (mIL-2) which can be detected by enzyme-linked immunosorbent assay (ELISA). This reporter system has the advantages of being sensitive and specific to a single receptor. In addition, the activation level of a specific receptor can easily be quantified and can be used even in cases where the ligand of the receptor is unknown. This system has been implemented successfully in many of our studies to characterize receptor-ligand interactions. We have recently employed this system to study the activation of human Fc&#947; receptors (Fc&#947;Rs) by different monoclonal anti-CD20 antibodies in clinical use.

This protocol describes how to establish a reporter system that can be used to identify and quantify receptor-ligand interactions.

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native ...

Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving area of research. There are many different methods for EV isolation, each with distinct advantages and disadvantages that affect the downstream yield and purity of EVs. Thus, characterizing the EV prep isolated from a given source by a chosen method is important for interpretation of downstream results and comparison of results across laboratories. Various methods exist for determining the size and quantity of EVs, which can be altered by disease states or in response to external conditions. Nanoparticle tracking analysis (NTA) is one of the prominent technologies used for high-throughput analysis of individual EVs. Here, we present a detailed protocol for quantification and size determination of EVs isolated from mouse perigonadal adipose tissue and human plasma using a breakthrough technology for NTA representing major advances in the field. The results demonstrate that this method can deliver reproducible and valid total particle concentration and size distribution data for EVs isolated from different sources using different methods, as confirmed by transmission electron microscopy. The adaptation of this instrument for NTA will address the need for standardization in NTA methods to increase rigor and reproducibility in EV research.

We demonstrate how to use a novel nanoparticle tracking analysis instrument to estimate the size distribution and total particle concentration of extracellular vesicles isolated from mouse perigonadal adipose tissue and human plasma.

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving ...

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular communication in various research fields; cancer biology, neuroscience, and drug delivery. Many EV uptake assays have been reported in the literature; however, there is a lack of practical, detailed experimental methodology. EV uptake can be assessed by fluorescently labeling EVs to detect their location within cells. Distinguishing between internalized EVs in cells and the superficial EVs on cells is difficult, yet critical, to accurately determine the EV uptake. Therefore, an assay that efficiently quantifies EV uptake through three-dimensional (3D) fluorescence confocal microscopy is proposed in this work. Fluorescently labeled EVs were prepared using a nano-filtration-based microfluidic device, visualized by 3D confocal microscopy, and then analyzed through advanced image-processing software. The protocol provides a robust methodology for analyzing EVs on a cellular level and a practical approach for efficient analysis.

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

Extracellular vesicles (EVs) contribute to cellular biology and intercellular communications. There is a need for practical assays to visualize and quantify EVs uptake by the cells. The current protocol proposes the EV uptake assay by utilizing three-dimensional fluorescence imaging via confocal microscopy, following EV isolation by a nano-filtration-based microfluidic device.

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular ...