Financial support for this work was provided through the National Science Foundation, National Institute of Health (NIH), Materials Technology Center (MTC) and ORDA at SIUC. We thank NSF for a grant (CHE-0959568) for the purchase of an FE-SEM. We would like to thank Prof. Matthew McCarroll for helpful discussions. Julia Reyes will like to thank COLCIENCIAS, Colombian Agency and Universidad Pedagogica y Tecnologica de Colombia for her scholarship and financial support.

A. Synthesis and Characterization of PDA Liposomes4,5,6
Note 1: Protect the PDA solution from light using aluminum foil wrapping on every container throughout all the experimental steps.
Note 2: Two different sets of liposome solution (B and C) were prepared following procedure A (Synthesis and characterization of PDA liposomes).
1. Synthesis of N-hydroxysuccinimide Diacteylene (NHS-PCDA)
<ol>
 <li>To prepare liposomes, an essential ingredient PCDA-NHS is required. We have synthesized PCDA-NHS using following procedure:</li>
 <li>Add 10,12-pentacosadiynoic acid (PCDA) (0.267 g, 0.713 mmol), N-hydroxysuccinimide (0.0914 g, 0.786 mmol) and 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (0.144 g, 0.713 mmol) in dry CH2Cl2 (20 ml).</li>
 <li>Stir the solution at room temperature for 2 hr.</li>
 <li>Carefully remove the solvent by using rotary evaporator to yield a dry thin film.</li>
 <li>Use separating funnel to extract the residue with diethyl ether (25 ml) and water (25 ml) three times.</li>
 <li>Dry the organic layer with MgSO4 (1.0 g) for half an hour. Filter and remove the solvent by rotary evaporation to obtain a white solid powder (0.24 g, &gt;90%).</li>
 <li>Analyze final compound under Nuclear Magnetic Resonance (NMR).</li>
 <li>1H NMR (300 MHz, DMSO), &delta; (ppm): 0.893 (t, 3H), 1.268 (m, 26H), 1.512 (m, 4H), 1.754 (m, 2H), 2.252 (t, 4H), 2.365 (m, 1H), 2.610 (m, 1H), 2.842 (s, 2H).</li>
</ol>
2. Liposome preparation5,6,7
<ol>
 <li>Dissolve PCDA: PCDA-NHS: 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) :: 8 : 1: 1 in 20 ml of dichloromethane.</li>
 <li>Filter the solution with a filter paper to remove aggregates.</li>
 <li>Evaporate the solvent completely to yield a thin film of monomers.</li>
 <li>Dry the thin film overnight under vacuum.</li>
 <li>Hydrate the film with deionized water (50 ml) to make a liposome solution of desired concentration (0.65-1 mM).</li>
 <li>Sonicate the resultant suspension with a probe sonicator at 76 &deg;C for ~18 min.</li>
 <li>Carefully pass the solution through a paper filter to remove the lipid aggregates</li>
 <li>Cool the solution at 4 &deg;C overnight to promote self-assembly of the monomers. The final solution should be optically clear.</li>
 <li>Polymerize the self-assembled diacetylene monomers (liposome) by irradiation with 254 nm of UV radiation for ~2 min using a Pen Ray UV source (4.5 mW/cm2) in air.</li>
 <li>The liposome solution was stable at room temperature for at least two weeks. The solution was more stable when refrigerated.</li>
</ol>
B. Preparation of Rhodamine-tagged Bovine Serum Albumin (BSA-Rh) modified PCDA liposomes
1. Binding of BSA-Rh to the liposome surface
<ol>
 <li>Dissolve BSA-Rh in PBS buffer (ionic concentration was 0.01M, pH 7.2) to make the final concentration of 1.2 &mu;M of BSA-Rhodamine solution.</li>
 <li>Add 2 ml (1.2 &mu;M) of BSA-Rhodamine to 10 ml of liposome solution prepared in step A.2. (See above) at room temperature.</li>
 <li>Classical reaction for the binding of amine groups from the lysine residue of proteins to carboxylic acid activated by NHS group has been followed (Reaction scheme in Figure 2). NHS-PCDA was designed (Figure 2, step 1) for covalently binding protein molecules with liposomes using NHS-amine reactions (Figure 2, Step 2). NHS is an excellent leaving agent that drives the amine-carboxylic acid reaction in the forward direction. The yield of this reaction under appropriate condition should be quantitative.</li>
 <li>Removal of free BSA-Rh: Soak a Spectra/Por Biotech Cellulose Ester (CE) membrane (MWCO: 100,000) in deionized water for 15 min. This membrane is used for dialysis of unreacted BSA-Rhodamine (Molecular weight ~66000 Da) in deionized water.</li>
 <li>Carefully transfer the solution in to the dialysis membrane.</li>
 <li>Change the water at 2 hr, 8 hr, 14 hr, 24 hr, and 36 hr during dialysis.</li>
 <li>Collect the final solution in a vial covered with aluminum foil.</li>
</ol>
C. Preparation of SR-diamine and Biotin-tagged Liposomes
1. Instead of using DMPC in step 2.1, we will use biotin-tagged- (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (biotin-DOPE).
<ol>
 <li>Follow all the steps through 2.2 to 2.9.</li>
 <li>Instead of BSA-Rh in step B, use Diamine tagged Sulphorhodamine (SR-diamine).</li>
 <li>Follow all further steps in B.</li>
 <li>The liposomes in this preparation contained biotin and SR-diamine on their surfaces. Subsequent steps were similar to A and B (see above) with one exception: streptavidin was added to the solution to investigate biotin-streptavidin interactions through changes in the FRET efficiency.</li>
</ol>
D. Representative Results
<img alt="Title Figure" src="/files/ftp_upload/3805/3805figtitle.jpg" /> 
Title Figure. A cartoon explaining the reaction and FRET process occurring at the liposome surface (prepared by following step B).
A. Monitoring of protein attachment to liposomes using FRET1
Monitoring of FRET between Rhodamine and PDA liposomes prepared in step B.
Excitation and emission spectra of BSA-Rh and absorption spectrum of PDA are taken (Figure 3A). We can clearly see that the emission spectrum of BSA-Rh overlaps with the absorption spectrum of PDA. This satisfies the resonance requirement for the FRET mechanism. BSA-Rhodamine tagged liposomes before and after polymerization were analyzed with UV-Vis and fluorescence spectroscopy. For the isolated donor and acceptor, the FRET efficiency is highly dependent on donor-acceptor distance (r) and J1 (j-value). The quenching in the emission is observed (Figure 3B) because of FRET between rhodamine and PDA due to the appearance of electronic absorption spectrum of blue PDA after photopolymerization. In our case, FRET efficiency is zero for unpolymerized liposomes and Rhodamine because J=0 for unpolymerized liposomes in the visible region.
We performed similar experiments with only PDA liposomes that did not contain NHS on their surface. In these cases, BSA-Rh was not tagged to the surface of the liposomes. In that case, the average distance between rhodamine and PDA (raverage) was much larger than Forster radius (R0 = 2.8 nm). Thus, we didn't observe a large decrease in the fluorescence intensity. This observation also suggests that the fluorescence quenching is dominant when r &lt; 2.8 nm.
J and R0 values were calculated using following formulas:[1]
J(&lambda;)=&int;FD(&lambda;)&epsilon;A(&lambda;)&lambda;4d&lambda;
R0=0.211[k2n-4QDJ(&lambda;)]1/6
The extinction coefficient (&epsilon;) of blue-PDA is in the range of ~ 2000-10000 M-1 cm-1 prior to addition of streptavidin. The extinction coefficient of blue-PDA is dependent on the photo-polymerization condition. [2] For example, &epsilon; values will be larger for a solution that was polymerized a longer time. Further, the self-assembly of the diacetylene monomers may also affect the &epsilon; values. The J calculations take into account of these changes, and they reflect changes in the PDA extinction coefficients due to biotin-streptavidin molecular interactions. J values are calculated using experimental PDA absorption and SR-101 emission data. The changes in the J values are due to shift in the electronic absorption spectrum of PDA following the addition of streptavidin to the solution (Figure 4C). The unit of J values is M-1cm-1nm4.
B. Monitoring of FRET with addition of streptavidin to the biotin-tagged liposome solution
Note 1: Monitoring of FRET between Rhodamine and PDA liposomes prepared in step C above.
Excitation and emission spectra of Sulphorhodamine-tagged diamine (SR-diamine) and absorption spectrum of PDA are taken (Figure 4A). Unpolymerized and polymerized biotin tagged liposomes were analyzed using UV-Vis and fluorescence spectroscopy. The emission of rhodamine (SR-101) is decreased by about 45% after polymerization (Figure 4B) suggesting emission quenching due to FRET. Add 40 &mu;l aliquots (1 &mu;M) of streptavidin solution to 2 ml of liposome solution. With the addition of streptavidin to the solution, changes in J value is observed (Figure 4C). As biotin binds to streptavidin, the blue peak intensity of PDA liposome (centered at ~645 nm in Figure 1) was decreased whereas an increase in the absorption peak at 540 nm is observed (Figure 1S). Figure 4D shows changes in the FRET efficiency. The FRET efficiency decreased with the increase in streptavidin concentration is also consistent with our prediction.
Record the SR emission after every 40 &mu;l aliquot addition of streptavidin. We observed a steady increase in the rhodamine emission after the addition of streptavidin (Figure 5). This increase in rhodamine emission is due to a decrease in the J value for the sulphorhodamine emission spectrum and PDA absorbance spectrum following biotin-streptavidin interactions. At the molecular level, the biotin-streptavidin interactions lead to subtle changes in the effective conjugation length of the PDA which results in a decrease in the blue-PDA form to a more thermodynamically stable red-PDA form2. This is the basis of changes in J values. Interestingly, the subtle differences in the molecular interactions for covalently or non-covalently bonded biotin to PDA liposome can be probed using our sensing assay4.
We have also performed control experiments and have monitored the emission of the control samples under the same experimental conditions as those for streptavidin-biotin system. The control experiments consist of: (1) Liposome solutions that contained biotin on their surface were added buffer solution of same volume and concentration; and (2) Liposome solution without biotin receptors on their surface were added streptavidin of same volume and concentration. The intensity of the biotin-tagged liposome solution after the addition of streptavidin showed enhanced intensity but the intensity of the liposome solution of the control experiments (for example, please see Figure 2S), on the other hand, exhibited a decreased in emission intensity. This is attributed to dilution of the solution. These experiments clearly indicated that the enhanced emission of the solution was due to specific molecular interactions.
Forster radius (R0) for rhodamine and PDA pair is calculated (eq.2) to be ~2.80 nm. This means that for isolated PDA-rhodamine pairs, 50% of the excited state rhodamine molecules will have their energy transferred to PDA when r is 2.80 nm.
We observed that when biotin is covalently attached to the PDA backbone, the emission increase was 2-3 times larger than that of non-covalently bonded biotin to liposomes.4 These results strongly suggest that our proposed system is sensitive to distinguish subtle differences in the interactions at the transducer (linker between biotin and liposome bilayer) due to covalently and non-covalently bonded receptors attached to liposomes. Depending upon the scanning and data acquisition capabilities of the spectrophotometer, real-time monitoring (in millisecond to second time scale) of protein interactions (in the UV-Vis spectroscopy) are possible with this sensing system.
<img alt="Figure 1" src="/files/ftp_upload/3805/3805fig1.jpg" /> 
Figure 1. Absorption spectra of blue and red PDA solutions. (Inset) optical micrographs taken with a digital camera.
<img alt="Figure 2" src="/files/ftp_upload/3805/3805fig2.jpg" /> 
Figure 2. Reaction scheme for the PCDA-NHS synthesis (step 1). Reaction of PCDA-NHS to the amine substituent of proteins (step 2). Step 2 is the basis of binding of lysine residue of proteins to the carboxylic acid of PCDA monomer. <a href="https://www.jove.com/files/ftp_upload/3805/3805fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 3A" src="/files/ftp_upload/3805/3805fig3A.jpg" /> 
Figure 3A. Change in observed FRET efficiency is due to the changes in the absorption spectrum of the PDA. Before polymerization there is no overlap between BSA-Rh emission and PDA absorption but after polymerization PDA absorption overlaps with BSA-Rh emission which is the requirement for FRET. Rhodamine is a donor (red) and polymerized PDA liposomes act as an acceptor (blue).
<img alt="Figure 3B" src="/files/ftp_upload/3805/3805fig3B.jpg" /> 
Figure 3B. Fluorescence spectra of BSA-Rh tagged liposomes before (blue) and after PCDA polymerization (red). A large decrease in the rhodamine emission was observed due to FRET between rhodamine and PDA.
<img alt="Figure 4A" src="/files/ftp_upload/3805/3805fig4A.jpg" /> 
Figure 4A. Spectral overlap (J) change for PDA (blue or red) absorption spectrum and Sulphorhodamine emission spectrum (orange). Figure 4A is the representative spectrum for extreme condition; that is, when an excess of streptavidin was added to the solution. Figure 4A shows an almost complete blue-to-red PDA transformation following the addition of an excess of streptavidin. It is clearly seen that J (spectral overlap) increases with blue-shift of the PDA absorption spectrum
<img alt="Figure 4B" src="/files/ftp_upload/3805/3805fig4B.jpg" /> 
Figure 4B. Fluorescence spectra before (blue) and after (red) liposome polymerization.
<img alt="Figure 4C" src="/files/ftp_upload/3805/3805fig4C.jpg" /> 
Figure 4C. Condition for FRET: J change between donor (sulphorhodamine) and acceptor (PDA) with the streptavidin addition to the liposome solution.
<img alt="Figure 4D" src="/files/ftp_upload/3805/3805fig4D.jpg" /> 
Figure 4D. FRET efficiency change between the donor (sulphorhodamine) and acceptor (PDA) with the streptavidin addition to the liposome solution.
<img alt="Figure 5" src="/files/ftp_upload/3805/3805fig5.jpg" /> 
Figure 5. Rhodamine emission spectrum after the addition of streptavidin aliquots to the PDA liposome solution. Inset shows a larger view of emission changes in the SR-101 spectra.

<ol>
	<li>Lakowicz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Fluorescence Spectroscopy. , (1999).</li><li>Charych, D. H., Nagy, J. O., Spevak, W., Bednarski, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Colorimetric+Detection+of+a+Receptor-Ligand+Interaction+by+a+Polymerized+Bilayer+Assembly.">Direct Colorimetric Detection of a Receptor-Ligand Interaction by a Polymerized Bilayer Assembly.</a> Science. 261, 585-588 (1993).</li><li>Yoon, B., Lee, S., Kim, J. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Conceptual+and+Technological+Advances+in+Polydiacetylene-based+Supramolecular+Chemosensors.">Recent Conceptual and Technological Advances in Polydiacetylene-based Supramolecular Chemosensors.</a> Chem. Soc. Rev. 38, 1958-1968 (2009).</li><li>Xuelian, L. i., Kohli, P. u. n. i. t. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+Molecular+Interactions+in+Biosensors+Based+on+Fluorescence+Resonance+Energy+Transfer.">Investigating Molecular Interactions in Biosensors Based on Fluorescence Resonance Energy Transfer.</a> J. Phys. Chem. C. 114, 6255-6264 (2010).</li><li>Li, X., Matthews, S., Kohli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+Resonance+Energy+Transfer+in+Polydiacetylene+Liposomes.">Fluorescence Resonance Energy Transfer in Polydiacetylene Liposomes.</a> J. Phys. Chem. B. 112, 13263-13272 (2008).</li><li>Li, X., McCarroll, M., Kohli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulating+Fluorescence+Resonance+Energy+Transfer+in+Conjugated+Liposomes.">Modulating Fluorescence Resonance Energy Transfer in Conjugated Liposomes.</a> Langmuir. 22, (2006).</li><li>New, R. R. C., New, R. R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Liposomes: A Practical Approach. , 33-104 (1990).</li><li>Chen, X., Lee, J., Jou, M. J., Kim, J. -. M., Yoon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+and+Fluorometric+Detection+of+Cationic+Surfactants+Based+on+Conjugated+Polydiacetylene+Supramolecules.">Colorimetric and Fluorometric Detection of Cationic Surfactants Based on Conjugated Polydiacetylene Supramolecules.</a> Chem. Commun. , 3434-3436 (2009).</li><li>Yarimaga, O., Im, M., Choi, Y. -. K., Kim, T. W., Jung, Y. K., Park, H. G., Lee, S., Kim, J. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Color+Display+System+Based+on+Thermochromic+Conjugated+Polydiacetylene.">A Color Display System Based on Thermochromic Conjugated Polydiacetylene.</a> Macromolecular Research. 18, 404-407 (2010).</li></ol>

No conflicts of interest declared.

We have performed selective binding of lysine residue of protein on liposome surface using NHS-amine reaction. This FRET based method is capable of doing Real-time monitoring of biotin-streptavidin binding and protein (BSA) binding to the liposome surface. Similar procedure can be applied to study the binding dynamics of various protein interactions with their selective receptors. There is flexibility in choosing fluorophores that will provide changes in the J values depending upon the spectral characteristics o...

<table border="1">
	 <tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>10,12-pentacosadiynoic acid (PCDA)</td>
 <td>GFS chemicals</td>
 <td>3261</td>
 <td>Light sensitive</td>
 </tr>
 <tr>
 <td>N-hydroxysuccinimide (NHS)</td>
 <td>Acros organics</td>
 <td>157270250</td>
 <td>Moisture sensitive</td>
 </tr>
 <tr>
 <td>1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC)</td>
 <td>Chem-impex International</td>
 <td>00050</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)</td>
 <td>Avanti Polar lipids</td>
 <td>850345P</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Rhodamine-tagged Bovine Serum Albumin (BSA-Rh)</td>
 <td>Sigma Aldrich</td>
 <td>A4537</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl)(biotin-DOPE)</td>
 <td>Avanti Polar lipids</td>
 <td>870282</td>
 <td>&nbsp;</td>
 </tr>
	 </tbody>
 </table>

real-time monitoring of ligand-receptor interactions with fluorescence resonance energy transfer

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.

Watch this Scientific Journal Video about Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer at JoVE.com

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

real-time-monitoring-ligand-receptor-interactions-with-fluorescence

Research

JoVE Journal

Biology

14.4K Views.  Southern Illinois University. We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

Video: Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

Real-Time Monitoring of Aurora kinase A Activation using Conformational FRET Biosensors in Live Cells

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon its autophosphorylation on Thr288. AURKA abundance peaks in mitosis, where it controls the stability and the fidelity of the mitotic spindle, and the overall efficiency of mitosis. Although well characterized at the structural level, a consistent monitoring of the activation of AURKA throughout the cell cycle is lacking. A possible solution consists in using genetically-encoded F&#246;rster&#8217;s Resonance Energy Transfer (FRET) biosensors to gain insight into the autophosphorylation of AURKA with sufficient spatiotemporal resolution. Here, we describe a protocol to engineer FRET biosensors detecting Thr288 autophosphorylation, and how to follow this modification during mitosis. First, we provide an overview of possible donor/acceptor FRET pairs, and we show possible cloning and insertion methods of AURKA FRET biosensors in mammalian cells. Then, we provide a step-by-step analysis for rapid FRET measurements by fluorescence lifetime imaging microscopy (FLIM) on a custom-built setup. However, this protocol is also applicable to alternative commercial solutions available. We conclude by considering the most appropriate FRET controls for an AURKA-based biosensor, and by highlighting potential future improvements to further increase the sensitivity of this tool.

The activation of the multifunctional Ser/Thr kinase AURKA is hallmarked by its autophosphorylation on Thr288. Fluorescent probes relying on FRET can discriminate between its inactive and activated states. Here, we illustrate some strategies for probe engineering, together with a rapid FRET protocol to follow the kinase activation throughout mitosis.

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon ...

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. Upon wounding of a leaf, an increase in cytosolic calcium ion concentration (Ca2+ signal) occurs at the wound site. This signal is rapidly transmitted to undamaged leaves, where defense responses are activated. Our recent research revealed that glutamate leaking from the wounded cells of the leaf into the apoplast around them serves as a wound signal. This glutamate activates glutamate receptor-like Ca2+ permeable channels, which then leads to long-distance Ca2+ signal propagation throughout the plant. The spatial and temporal characteristics of these events can be captured with real-time imaging of living plants expressing genetically encoded fluorescent biosensors. Here we introduce a plant-wide, real-time imaging method to monitor the dynamics of both the Ca2+ signals and changes in apoplastic glutamate that occur in response to wounding. This approach uses a wide-field fluorescence microscope and transgenic Arabidopsis plants expressing Green Fluorescent Protein (GFP)-based Ca2+ and glutamate biosensors. In addition, we present methodology to easily elicit wound-induced, glutamate-triggered rapid and long-distance Ca2+ signal propagation. This protocol can also be applied to studies on other plant stresses to help investigate how plant systemic signaling might be involved in their signaling and response networks.

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Extracellular glutamate-triggered systemic calcium signaling is critical for the induction of plant defense responses to mechanical wounding and herbivore attack in plants. This article describes a method to visualize the spatial and temporal dynamics of both these factors using Arabidopsis thaliana plants expressing calcium- and glutamate-sensitive fluorescent biosensors.

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. ...