M.A. was supported by a Schr&#246;dinger fellowship of the Austrian Science Fund (FWF, J3086-B11) and thanks the Max-Planck-Society for financial and administrative support. G.S. and J.H. were supported by the Vienna Science and Technology Fund (WWTF, LS13-030).

<ol><li>Garcia, K. C., Adams, J. J., Feng, D., Ely, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+molecular+basis+of+TCR+germline+bias+for+MHC+is+surprisingly+simple%5BTitle%5D)+AND+%22Nat+Immunol.%22%5BJournal%5D)">The molecular basis of TCR germline bias for MHC is surprisingly simple.</a> Nat Immunol.. 10, 143-147 (2009).</li>
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<li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell-substrate+contacts+illuminated+by+total+internal+reflection+fluorescence%5BTitle%5D)+AND+%22The+Journal+of+cell+biology%22%5BJournal%5D)">Cell-substrate contacts illuminated by total internal reflection fluorescence.</a> The Journal of cell biology. 89, 141-145 (1981).</li>
<li>Grakoui, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+immunological+synapse%3A+a+molecular+machine+controlling+T+cell+activation%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The immunological synapse: a molecular machine controlling T cell activation.</a> Science. 285, 221-227 (1999).</li>
<li>Kaizuka, Y., Douglass, A. D., Varma, R., Dustin, M. L., Vale, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+for+segregating+T+cell+receptor+and+adhesion+molecules+during+immunological+synapse+formation+in+Jurkat+T+cells%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">Mechanisms for segregating T cell receptor and adhesion molecules during immunological synapse formation in Jurkat T cells.</a> Proc Natl Acad Sci USA. 104, 20296-20301 (2007).</li>
<li>Varma, R., Campi, G., Yokosuka, T., Saito, T., Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((T+cell+receptor-proximal+signals+are+sustained+in+peripheral+microclusters+and+terminated+in+the+central+supramolecular+activation+cluster%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">T cell receptor-proximal signals are sustained in peripheral microclusters and terminated in the central supramolecular activation cluster.</a> Immunity. 25, 117-127 (2006).</li>
<li>Purbhoo, M. A., Irvine, D. J., Huppa, J. B., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((T+cell+killing+does+not+require+the+formation+of+a+stable+mature+immunological+synapse%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">T cell killing does not require the formation of a stable mature immunological synapse.</a> Nat Immunol. 5, 524-530 (2004).</li>
<li>Binz, H. K., Stumpp, M. T., Forrer, P., Amstutz, P., Pluckthun, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Designing+repeat+proteins%3A+well-expressed%2C+soluble+and+stable+proteins+from+combinatorial+libraries+of+consensus+ankyrin+repeat+proteins%5BTitle%5D)+AND+%22J+Mol+Biol%22%5BJournal%5D)">Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins.</a> J Mol Biol. 332, 489-503 (2003).</li>
</ol>

The authors declare that they have no competing financial interest.

Measuring protein-protein interactions in situ is highly desirable especially when dealing with low affinity interactions such as TCR-pMHC binding 11. This is because the on-rate as well as the stability of such interactions are significantly influenced by the particular circumstances under which binding takes place. Minimally-invasive FRET-based imaging approaches are thus in principle perfectly suited for such tasks, yet involve a number of hurdles that must first be overcome. Noise generated by cellular autofluorescence limits the sensitivity of the measurements and should therefore be kept at a minimum. TIRF microscopy serves this need very well 12 but requires the functionalization of glass-slides, ideally in the form of a planar glass-supported lipid bilayer decorated with proteins of choice 13-15. Another advantage of a partly reconstitutive approach is that recombinant bilayer-resident FRET partners can be much more easily labeled in a quantitative, site-specific and rational manner with smaller and brighter fluorophores than would be possible with cell surface-expressed proteins. TCRs are tagged with recombinant scFVs, which do not influence T-cell recognition, as was tested previously 2. Moreover, the protein composition of the SLB, for example the density of pMHCs and the choice of accessory factors can be adjusted to one&#8217;s specific needs. We have previously performed experiments with varying densities of stimulatory pMHCs, but have not detected significant differences in 2D-koff and 2D-KD2.
So far here the recognition of MHC class II molecules has been dealt with only, mainly because of the nature of their peptide binding cleft, which is open at both ends and thus accommodates larger peptides including a linker for fluorophore attachment. In some cases such approach might also work for labeling MHC class I molecules 16 but great caution should be taken to verify their use in experiments. The sensitivity of T-cells towards antigens, which can be measured via T-cell proliferation assays, as well as the pMHC-TCR binding kinetics as measured in vitro by surface plasmon resonance should not be affected by the addition of the linker and fluorophore to the peptide. Alternatively, MHC class I molecules themselves can be labeled in a site-specific manner with the introduction of an unpaired cysteine within the sequence of the heavy chain (unpublished observations).
With the use of appropriate molecular probes any synaptic protein-protein interaction can in principle be studied in a fashion described herein. Such probes, e.g., scFVs or Designed Ankyrin Repeat Proteins (DARPins)17, should be monovalent and should bind their target stably without affecting the interaction of interest. Of course, structural information is highly desirable for rational probe design but not absolutely required. When establishing a new pair of FRET partners, it is recommended to record and analyze FRET in bulk first. Sites of label attachment may be varied considerably to maximize the FRET signal and also to verify that measured FRET yields differ based on the inter-dye distance. Once the system is optimized, single molecule FRET signals may be recorded by limiting the labeling of the high abundance FRET partner to 10-30% and bleaching the low abundance FRET partner until single molecules are resolvable in the field of illumination.
Last but not least it should be noted that SLBs approximate some but not all aspects of a physiological plasma membrane. Qualities such as membrane curvature and flexibility, domain compartmentalization, cytoskeletal rearrangements and cell motility as well as a high variety of surface-expressed membrane proteins are not represented by SLBs but might influence the process under investigation. Much effort will need to be invested to establish imaging modalities that allow monitoring protein-protein interactions with single molecule resolution in physiological synapses, which are inaccessible to TIRF imaging.

A more fundamental understanding of how T-cells recognize antigens requires looking at the right place, that is, within the immunological synapse formed between the T-cell and the APC. Here, molecular binding kinetics are not only determined by the inherent biochemical properties of the interaction partners involved but depend to a large extent on cellular parameters, which include cellular forces, membrane architecture and lateral interactions between membrane proteins as well as synapse-specific geometrical constraints 3. Biochemical approaches are limited in resolving power as they necessitate the disruption of at least one of the synaptic membranes involved. For this reason a FRET-based imaging methodology was developed to monitor binding of the TCR to antigenic pMHCs 2. Here T-cells are decorated with a recombinant and site-specifically labeled TCR&#946;-reactive single chain antibody fragment (scFV) and confronted with planar glass-supported lipid bilayers (SLBs), which harbor MHC class II molecules loaded with a fluorescently labeled antigenic peptide, costimulatory molecules and adhesion proteins. Synaptic binding between dye-labeled TCR and dye-labeled pMHC results in FRET, which can be monitored on a bulk and single molecule level by Total Internal Reflection Fluorescence (TIRF) microscopy.
In this article it is explained in detail how to utilize SLBs for assaying T-cell synapses, verify their integrity through a functional T-cell calcium-flux assay, conduct FRET measurements in bulk and with single molecule sensitivity, and analyze the acquired data. Recommendations are offered to producing properly conformed proteins required for bilayer functionalization. For more specific information regarding bilayer formation and setup of a suitable TIRF microscope please refer to an additional public access JoVE publication published back to back 4.
Nature of SLBs
Functionalizable SLBs can be readily generated from unilamellar vesicles (SUVs) containing the two lipids 1-palmitoyl-2-oleoyl-sn-gylcero-3-phosphocholine (short: POPC, 90-99%) and 1,2-dioleoyl-sn-glycero-3-{[N(5-amino-1-carboxypentyl) iminodiacetic acid] succinyl} (short: DGS NTA-Ni, 1-10%). SUVs spread on clean glass slides to form a contiguous planar bilayer 4. DGS-NTA-Ni serves to anchor polyhistidine-tagged proteins via polyhistidine-mediated complex-formation with the synthetic NTA-Ni-containing head group (Figure 1A). For stable association one typically replaces the native transmembrane domain and cytoplasmic tail of the adhesion protein ICAM-1 and the costimulatory molecule B7-1 with one tag containing twelve histidines (ICAM-1-12H, B7-1 -12H) (Figure 1B). The peptide-loaded class II molecule I-Ek contains two membrane-embedded (&#945; and &#946;) polypeptide chains. The transmembrane/cytoplasmic domains of both chains have to be replaced with a tag containing six histidines each (I-Ek&#945;6H &#946;6H or I-Ek-2x6H). As an alternative, extending the &#945;-chain with twelve histidines and leaving the extracellular domain of the &#946;-chain untagged (giving rise to I-Ek&#945;12H &#946;0H or I-Ek-12H) gives rise to satisfactory results (Figure 1B).
Site-specific labeling of pMHCs
It is important to label the pMHC stoichiometrically and site-specifically in order to be able to convert measured FRET yields into meaningful equilibrium binding constants. This can be achieved by chemical labeling of a synthetic peptide that is loaded into the peptide-binding cleft of recombinant histidine-tagged MHC class II molecules 2,5. The peptide includes all residues of the T-cell epitope as well as a short C-terminal linker (GGS) followed by cysteine (e.g. in the moth cytochrome c (MCC) peptide ANERADLIAYLKQATK-GGSC, the linker is marked in bold). This cysteine is used to label the peptide stoichiometrically with the use of maleimide-dye derivatives. At this point extra care should be devoted to verifying quantitative dye-coupling to the cysteine-containing peptide. HPLC-purification of the peptide-dye adduct is recommended and has to be followed by electrospray ionization mass spectroscopy. Any recorded masses corresponding to the peptide educt (without dye) reflect incomplete labeling. If this is true, the HPLC-purified peptide should be subjected to consecutive rounds of dye-labeling until labeling is deemed quantitative. Note that MALDI-TOF mass spectroscopy should be avoided as this method involves laser radiation for sample ionization. This treatment disintegrates the attached sensitive fluorophores before peptide mass is read out and thus underrepresents degrees of dye-conjugation.
Indirect yet site-specific labeling of cell-bound TCRs with the use of monovalent single chain FV fragments
It is still challenging to attach dyes to cell surface-associated proteins of living cells in a site-specific manner. To overcome this hurdle for surface-exposed TCRs, a monovalent single chain version (scFV) from the genes of the TCR&#946; -reactive monoclonal antibody H57-197 2has been constructed. The crystal structure of this antibody in complex with the TCR allows to rationally design a version, in which a serine residue in close proximity to the C-terminus of the MHC associated peptide (where the corresponding FRET partner dye is attached) is substituted for a cysteine residue. This mutant cysteine then serves as an acceptor for dye conjugation (Figure 2).
Methodologies to record FRET
Bulk FRET values are best suited to verify the relationship between chosen inter-dye distances and FRET efficiencies measured in this TCR-pMHC binding system 2. In addition, bulk FRET measurements reveal qualitative and quantitative differences in synaptic TCR-pMHC affinities (see below and protocol section 3.2). Various approaches for quantifying FRET efficiencies have been introduced in the literature 6. In this article FRET is recorded via
(a) donor recovery after acceptor bleaching, and via
(b) sensitized FRET acceptor emission.
The first method (a) requires the use of a FRET acceptor that can be easily photobleached, and a donor, which is rather photostable. In addition it is important to ensure that the photobleached acceptor is no longer capable of quenching the donor fluorescence. As the same detection channel (donor) is used for quantification, no correction factors and no chromatic aberrations have to be considered, which renders this methodology simple and reliable. However, quantitative measurements cannot be repeated on the same specimen spot and changes in FRET cannot be recorded over time. To avoid effects caused by molecular diffusion or cellular motility a fast bleaching step should be aimed for, which minimizes the time passing between the first FRET donor (before acceptor bleaching) and the second FRET donor image acquisition (after FRET acceptor bleaching). It is recommend to employ a powerful laser light source of the FRET acceptor excitation wavelength in order to minimize illumination and bleaching times.
In contrast, in the approach of sensitized FRET emission measurement (b) the FRET donor is excited and the emission of the FRET acceptor is observed in the FRET acceptor channel. Changes in FRET acceptor signal can be recorded over time but emission of the FRET donor into the red-shifted acceptor channel (termed bleedthrough) and FRET acceptor cross-excitation via donor excitation have to be accurately determined and subtracted from the recorded FRET acceptor channel. For this the corresponding FRET donor and FRET acceptor images have to be spatially aligned.
Detection of single molecule (sm) FRET events
With the use of lasers as excitation source, a sensitive camera and noise-attenuated TIRF microscopy the fluorescence of single fluorophores can be easily traced over time. Similar is true for the detection of intermolecular smFRET events. However, complications may be caused by FRET donor bleedthrough and cross-excitation of the FRET acceptor, and thus great care has to be taken when adjusting the fluorophore densities in the smFRET experiment.
In the protocol provided below (protocol section 4) the TCR was chosen as FRET donor in high abundance and pMHC as FRET acceptor in low abundance. To attenuate FRET donor bleedthrough sufficiently, decorate 10-30% of the TCRs with fluorescent scFV and 90-70% of the TCRs with non-fluorescent scFV. Here the FRET acceptor channel was chosen as single molecule channel because it is confocal with the single molecule FRET channel. This helps to align smFRET events with single molecule FRET acceptors, which is the basis of smFRET validation.
Extracting synaptic off-rates through smFRET measurements
Photobleaching of both FRET donor and FRET acceptor have to be accounted for when extracting the half-life of interactions from single molecule FRET traces. The number of observable FRET-signals at the beginning of their appearance as single donor-acceptor pair N(0) is reduced over time by both unbinding of the receptor-ligand complex and photobleaching. The number of surviving complexes at a given time N(t) can be mathematically expressed as follows:
<img alt="Equation 1" src="/files/ftp_upload/53157/53157eq1.jpg" />
In the photobleaching term exp(-t/&#964;bleach) the time t is described by the product of the number of observations n and the illumination time till because of the non-continuous, discrete observation mode (i.e., bleaching only occurs during illumination). Within the kinetic term exp-(t/&#964;off) the time t is the product of the number of observations n and the time tlag for a single FRET observation (i.e., kinetic unbinding happens continuously). Equation 1 can be expressed as:
<img alt="Equation 2" src="/files/ftp_upload/53157/53157eq2.jpg" />
The term &#964;bleach/&#964;ill describes the number of observations until bleaching occurs and is defined as the expectation value &#60;nbleach&#62; of its exponential function. Equation 2 can be simplified as follows:
<img alt="Equation 3" src="/files/ftp_upload/53157/53157eq3.jpg" />
The expectation value &#60;n(tlag)&#62; of the number of frames N(t) with observable FRET-events after time t is directly determined from the experiment. It depends on the settable time between observations (tlag) chosen in the experiment and the unknown values for &#964;off (the inverse of the off-rate koff ) and &#60;nbleach&#62;, the expectation value of number of observations before bleaching occurs.
Thus, calculation of the expectation value &#60;n(tlag)&#62; for at least two values of tlag allows the experimental determination of &#60;nbleach&#62; and &#964;off .
Extracting synaptic 2D-KD values through FRET-based measurements
Measuring TCR occupancy a, i.e., the ratio between bound TCRs and total TCRs, is central to determining synaptic 2D-KD values. According to equation 4 this term is directly proportional to the measured FRET yield as long as TCRs serves as FRET donors and pMHCs as FRET acceptors.
<img alt="Equation 4" src="/files/ftp_upload/53157/53157eq4.jpg" />
with a = TCR occupancy, C =conversion factor
C is a constant, which depends on the FRET system and the fluorophores used. It can be determined experimentally as shown below. a can be converted into a 2D-KD according to equation 5 when the initial density of TCR ligands prior to addition of T-cells to the bilayer is known. This is because of the high mobility of SLB-attached proteins and also because SLBs provide an almost inexhaustible reservoir of ligands 2.
<img alt="Equation 5" src="/files/ftp_upload/53157/53157eq5.jpg" />
with [pMHC initial] = initial density of pMHC prior to the addition of T-cells
With equations 4 and 5 one can now easily determine the synaptic 2D-KD between TCR and pMHC. This is most reliably done with FRET measurements based on donor recovery after acceptor bleaching (see protocol section 3.1).
However, to measure C the relationship between the FRET intensity IFRET (corrected for background, FRET donor bleedthrough and FRET acceptor cross-excitation) and TCR occupancy a has to be determined. For this, one needs to know the ratio R between the average fluorescence intensity of single TCR-associated FRET donor fluorophores (e.g. Cy3 or AF555) sm IFRET donor and the average intensity of single molecule FRET events sm IFRET. R depends on the FRET system in question, emission filters and camera used for fluorescence detection.
The TCR occupancy a can then be directly determined according to equation 6.
<img alt="Equation 6" src="/files/ftp_upload/53157/53157eq6.jpg" />
with R = sm IFRET donor / sm IFRET
R was determined as 1.45 for the H57 scFv- Cy3/pMHC-Cy5 system leads to:
a= bulk IFRET/ bulk ITCR-cy3 &#8226; 1.45
The relationship between the TCR occupancy a and the FRET yield can be determined by FRET donor recovery after acceptor bleaching. For this both parameters are plotted against one another for a number TCR microclusters as shown in Figure 4A.The slope of the linear fit indicates the conversion factor C (from equation 4).
As demonstrated in Figure 4A, C amounts for (a) the H57 scFV- Cy3/pMHC-Cy5 FRET system and (b) the applied microscope system configuration to 1.995. The TCR occupancy a can be readily deduced as follows:
TCR occupancy a = FRET yield &#8226; 1.995

<table><tbody><tr><td>LB-media</td><td>Fisher Scientific</td><td>10000713</td><td>bacterial expression</td></tr><tr><td>Sf900 II</td><td>Life Technologies</td><td>10227402</td><td>insect cell media for baculo virus production</td></tr><tr><td>Insect-XPRESS with L-glutamine (Lonza)</td><td>Fisher Scientific</td><td>10564038</td><td>insect cell media for baculo virus expression</td></tr><tr><td>Sf9 cells</td><td>Life Technologies</td><td>11496-015</td><td>cells for virus production and expansion</td></tr><tr><td>High Five Cells</td><td>Life Technologies</td><td>B855-02</td><td>cells for potein expression</td></tr><tr><td>LB-media</td><td>Fisher Scientific</td><td>10000713</td><td>bacterial expression</td></tr><tr><td>Centramate System</td><td>Pall</td><td></td><td>protein concentartion from large volumes</td></tr><tr><td>Centramate cassette 10kDa cutoff</td><td>Pall</td><td>OS010T12</td><td>protein concentartion from large volumes</td></tr><tr><td>Amicon Ultra-15 Centrifugal Filter Units</td><td>EMD Millipore</td><td>UFC900308</td><td>protein concentartion</td></tr><tr><td>Amicon Ultra-4 Centrifugal Filter Units</td><td>EMD Millipore</td><td>UFC800308</td><td>protein concentartion</td></tr><tr><td>Amicon Stirred Ultrafiltration Cell Model 200 mL</td><td>EMD Millipore</td><td>5123</td><td>protein concentartion</td></tr><tr><td>&amp;Auml;kta pure 25L</td><td>GE Healthcare</td><td>29-0182-24</td><td>protein purification</td></tr><tr><td>Superdex 200 10/300 GL</td><td>GE Healthcare</td><td>17-5175-01</td><td>protein purification</td></tr><tr><td>Superdex 75 10/300 GL</td><td>GE Healthcare</td><td>17-5174-01</td><td>protein purification</td></tr><tr><td>Mono Q 5/50GL</td><td>GE Healthcare</td><td>17-5166-01</td><td>protein purification</td></tr><tr><td>Ni Sepharose 6 Fast Flow</td><td>GE Healthcare</td><td>17-5318-01</td><td>protein purification</td></tr><tr><td>Tricorn 10/20 column</td><td>GE Healthcare</td><td>28-4064-13</td><td>protein purification</td></tr><tr><td>Gilson HPLC system</td><td>Gilson</td><td></td><td>purificationof fluorochrome-coupled peptides</td></tr><tr><td>Pursuit XRs C18, 5 &amp;micro;m particle size, 21.2*250mm column size</td><td>Agilent</td><td>A6000250X212</td><td>purificationof fluorochrome-coupled peptides</td></tr><tr><td>Pursuit XRs C18, 5 &amp;micro;m particle size, 21.2*50 mm column size</td><td>Agilent</td><td>A6000050G212</td><td>purificationof fluorochrome-coupled peptides</td></tr><tr><td>Tricorn 10/20 column&amp;nbsp;</td><td>GE Healthcare</td><td>28-4064-13</td><td>protein purification</td></tr><tr><td>Gilson HPLC system</td><td>Gilson</td><td></td><td>purificationof fluorochrome-coupled peptides</td></tr><tr><td>Pursuit XRs C18, 5 &amp;micro;m particle size, 21.2*250mm column size</td><td>Agilent</td><td>A6000250X212</td><td>purificationof fluorochrome-coupled peptides</td></tr><tr><td>Pursuit XRs C18, 5 &amp;micro;m particle size, 21.2*50 mm column size</td><td>Agilent</td><td>A6000050G212</td><td>purificationof fluorochrome-coupled peptides</td></tr><tr><td>Cy3 maleimide</td><td>GE Healthcare</td><td>PA23031</td><td>site-specific protein labeling via mutant unpaired cysteines</td></tr><tr><td>Cy5 maleimide</td><td>GE Healthcare</td><td>PA25031</td><td>site-specific protein labeling via mutant unpaired cysteines</td></tr><tr><td>Alexa Fluor 555 C2 Maleimide</td><td>Life Technologies</td><td>A-20346</td><td>site-specific protein labeling via mutant unpaired cysteines</td></tr><tr><td>Alexa Fluor 647 C2 Maleimide</td><td>Life Technologies</td><td>A-20347</td><td>site-specific protein labeling via mutant unpaired cysteines</td></tr><tr><td>Fura-2, AM, cell permeant</td><td>Life Technologies</td><td>F-1221</td><td>calcium-sensitive dye for cell labeling</td></tr><tr><td>dimethyl sulfoxide</td><td>Sigma Aldrich</td><td>151874</td><td>for dissolving fura-2 am</td></tr><tr><td>Hank&#039;s Balanced Salt Solution plus calcium/magnesium</td><td>Fisher Scientific</td><td>10225362</td><td>imaging buffer</td></tr><tr><td>PBS</td><td>Life Technologies</td><td>14190-136</td><td></td></tr><tr><td>Bovine Serum Albumin lyophilized powder</td><td>Sigma Aldrich</td><td>A2153</td><td>supplement for imaging buffer</td></tr><tr><td>14-4-4S antibody</td><td>affimetrix eBioscience</td><td>14-5980-81</td><td>blocking antibody for H2-I-Ek (recognized by the 5c.c7, 2B4 and AND TCR)</td></tr><tr><td>5 ml polypropylene round-bottom tube</td><td>Becton Dickinson</td><td>FALCON 352063</td><td></td></tr><tr><td>0.22 &amp;mu;m Ultrafree-MC centrifugal filter unit</td><td>EMD Millipore</td><td>UFC30GV0S</td><td></td></tr><tr><td>Syringe filter 0.2&amp;micro;m</td><td>Millipore</td><td>GVWP04700</td><td></td></tr><tr><td>TetraSpeck Microspheres, 0.1 &amp;micro;m, fluorescent blue/green/orange/dark red</td><td>Life technologies</td><td>T-7279</td><td></td></tr><tr><td>Microscope for fura-2-based calcium measurements&amp;nbsp;</td><td>LEICA</td><td>DMI4000B</td><td></td></tr><tr><td>Microscope for (single molecule) FRET measurements</td><td>LEICA/ZEISS/NIKON/OLYMPUS</td><td>for details please refer to parallel JoVE contribution by Axmann et al.</td><td></td></tr><tr><td>planar supported lipid bilayers</td><td></td><td>for details please refer to parallel JoVE contribution by Axmann et al.</td><td></td></tr><tr><td>RPMI 1640, with L-Glutamine</td><td>Life Technologies</td><td>11554416</td><td>T-cell media</td></tr><tr><td>non-essential amino acid 100X</td><td>Hyclone</td><td>SH30238.01</td><td>T-cell media supplement</td></tr><tr><td>penicillin/streptomycin/L-glutamine 100x</td><td>Life Technologies</td><td>12000226</td><td>T-cell media supplement</td></tr><tr><td>2-mercaptoethanol</td><td>Sigma Aldrich</td><td>M6250</td><td>T-cell media supplement</td></tr><tr><td>mouse interleukin-2 recombinant protein</td><td>BPS Bioscience</td><td>90185-B</td><td>T-cell media supplement</td></tr><tr><td>Research Grade Fetal Bovine Serum</td><td>Hyclone</td><td>SV30160.03</td><td>T-cell media supplement</td></tr><tr><td>Origin (analysis program)</td><td>OrigenLab</td><td>http://www.originlab.com/</td><td>non-linear fitting of two parameters (tauoff, [ntlag])</td></tr></tbody></table>

measuring tcr-pmhc binding in situ using a fret-based microscopy assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

The recording of intracellular calcium via the fura-2 calcium dye as well as the subsequent cellular analysis to verify the stimulatory potency and hence functionality of SLBs are shown in Figure 4. As becomes evident, calcium levels rise in T-cells (expressed as the normalized fura-2 340nm/380nm ratio with baseline being 1) immediately as soon as they settle onto the stimulatory SLBs. Calcium levels return to baseline levels shortly after the addition of a antibody which blocks pMHCs from TCR engagement and which terminates T-cell activation.
Figure 6 depicts a typical experiment involving FRET donor recovery after acceptor bleaching, which is employed to measure FRET yields and which serves to calculate 2D-KD values (shown in Figure 4). Please note the increase in FRET donor intensity, which represents TCR labeled via AF555, after rapid and complete ablation of the FRET acceptor species (here: AF647 associated with pMHCs). Also evident is the strong reduction in the FRET channel, i.e., the FRET-acceptor channel under FRET donor excitation, after FRET acceptor bleaching. The barely visible remaining signal corresponds to FRET donor bleedthrough. FRET yields within individual TCR microclusters or entire synapses are calculated based on the indicated measured intensity values (Figure 6B).
Figure 7 depicts a trajectory and time lapse of a single molecule FRET event visible in two time frames. As outlined above in the introduction such behavior is caused by both decay of synaptic TCR-pMHC binding and photobleaching. To discriminate between these two contributions, the experimental acquisition time frames have to be varied in duration: while photobleaching remains a constant, changes in FRET event trajectory length are only caused by the binding kinetics. A quantitation of tracelengths, which forms the basis for the calculation of off-rates and bleaching shown in Figure 7, are provided in tables 1 to 3.
Determination of 2D-KDs requires recording of bulk FRET yields for FRET-donor labeled TCRs. With the use of the experimentally deduced constant C (Figure 4), a FRET yield measured for a TCR microcluster or an entire synapse can be converted into the TCR occupancy a, i.e., the ratio of pMHC-engaged and total TCRs (Figure 4C). With known pMHC densities present on the SLB prior to addition of the T-cell, a values can be applied to determine synaptic 2D-KD values (Figure 4D). On-rates can be calculated with the law of mass action (2D-kon = 2D-koff/2D-KD) from the determined synaptic off-rate and 2D-KD values.
<img alt="Figure 1" src="/files/ftp_upload/53157/53157fig1.jpg" /> 
Figure 1. Schematic outline of the planar glass-supported lipid bilayer (SLB) system. (A) SLBs are composed of POPC (90-99%) and the synthetic lipid DGS Ni-NTA (1-10%) and form spontaneously when clean glass surfaces are charged with small unilamellar vesicles (SUVs) consisting of the corresponding lipids. (B) Once formed, such SLBs can be functionalized with soluble polyhistidine-tagged extracellular portions derived from pMHCs, costimulatory B7-1 proteins and ICAM-1 adhesion proteins, to serve as APCs for T-cells. For more information on preparing SLBs refer to Axmann et al.4. <a href="https://www.jove.com/files/ftp_upload/53157/53157fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53157/53157fig2.jpg" /> 
Figure 2. F&#246;rster Resonance Energy Transfer-based assay to quantitate TCR-pMHC binding in situ. (A) A composite structure of a TCR complexed with an H57 single chain fragment engaging a pMHC illustrates the FRET-based approach described herein. Note the short distance of about 41&#197; separating the two corresponding fluorophores undergoing FRET. Acceptor sites for fluorophore-maleimides are indicated in green and red. (B) The principle of detecting TCR-pMHC interactions in situ is illustrated. Only scFV-decorated TCRs and pMHCs (here I-Ek), which form specific complexes, give rise to a measurable FRET signal. <a href="https://www.jove.com/files/ftp_upload/53157/53157fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53157/53157fig3.jpg" /> 
Figure 3. Chromatograms of the final gel filtration step giving rise to monomeric scFVs and peptide-loaded I-Ek -2x6H molecules. X-axes represent retention volume in ml, Y-axes indicate absorbance at 280 nm in arbitrary units (AU). (A) H57 scFV site-specifically labeled with Alexa Fluor 555 maleimide was subjected to S75 chromatography to separate unreacted dye from the protein (step 1.2.5). Fractions corresponding to retention interval 14 to15 ml (dashed lines) represent labeled monomeric H57 scFV. (B) I-Ek -2x6H molecules complexed with the UV-cleavable ANP-space holder peptide had been UV-irradiated, incubated with site-specifically Alex 647 maleimide-labeled peptide and finally subjected to S200 chromatography to separate the protein from free peptide (step 1.1.2.4). The interval between the dashed lines contains properly folded and monomeric pMHCs. (A, B) 0.7 ml sample was applied to the column at the start of the rum (0 ml point). Collected fractions were concentrated. The protein-to-dye ratio was determined by photospectrometry prior to dialysis against PBS/50% glycerol (for storage at -20&#176;). <a href="https://www.jove.com/files/ftp_upload/53157/53157fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53157/53157fig4.jpg" /> 
Figure 4. Determining 2D-KDs and 2D-kons. (A) The correlation between the FRET yield as determined by donor recovery after acceptor bleaching and TCR occupancy was measured experimentally. TCR occupancy can be determined for individual TCR microclusters as explained in section 4.2. A linear fit of the data is displayed by the line, the slope of which is equal to the ratio C between TCR occupancy and the FRET yield. C is a constant specific for the FRET system and the fluorophores (here Cy3 and Cy5) employed. In this example it yielded 1.988. (B) FRET yield data were determined for individual TCR microclusters (N = 187, temperature = 24 &#176;C) through donor recovery of acceptor bleaching. Numbers below histogram bars indicate the upper limit within the interval. (C) Conversion of the data shown in (B) by multiplying measured FRET yields with the constant C determined in (A). Numbers below bars indicate the upper limit within the interval. (D) Histogram (semi-logarithmic, base = 4) depicting the distribution of 2D-KDs measured for individual TCR microclusters. The median 2D-KD is indicated in blue. Numbers below bars indicate the upper limit within the interval. (E) The histogram shown in (D) was converted into a 2D-kon -histogram (semi-logarithmic, base = 4) employing the synaptic koff for 24 &#176;C (0.41 s-1). The determined median 2D-kon value is indicated in blue. Data were originally published in Huppa et al.2 and are visualized here in a new format. <a href="https://www.jove.com/files/ftp_upload/53157/53157fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53157/53157fig5.jpg" /> 
Figure 5. Functional validation of SLBs employed for T-cell stimulation and imaging. (A) TCR-transgenic T-cell blasts loaded with fura-2 were confronted with stimulatory SLB harboring antigenic pMHCs, ICAM-1 and B-7. Cellular fura-2 emission excited at 340 nm and 380 nm as well as DIC images were recorded. As indicated, ratio values of the emission intensities excited at 340 and 380 nm are shown in the right panel. Addition of pMHC blocking antibodies 14 min into the experimental run resulted in a decrease in intracellular calcium levels comparable to that of resting T-cells. (B) A typical temporal profile of average fura-2 ratios in T-cells contacting stimulatory SLBs is characterized by a an initial rise in intracellular calcium which is 2 to 4 times higher compared to that of non-activated T-cells or T-cells deprived of antigen after antibody-mediated blockade. Green circles indicate time points illustrated in (A). <a href="https://www.jove.com/files/ftp_upload/53157/53157fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/53157/53157fig6.jpg" /> 
Figure 6. Bulk FRET yields as measured through FRET donor recovery after FRET acceptor bleaching. (A) Shown is an example of a typical synaptic FRET-measurement. As is indicated on the left and on top a series of images was acquired with the use of a emission beam splitter giving rise to a FRET donor and a FRET acceptor channel (for more detailed information on beam splitters refer to Axmann et al4). The line shown in the left DIC image indicates the boundary of the T-cell synapse. Note the loss in intensity within the FRET acceptor channel as well is the increase in intensity in the FRET donor channel after FRET acceptor bleaching (step 4). (B) FRET efficiencies can be quantified as indicated for individual synaptic regions or for entire synapses. For inspection, images before and after FRET acceptor bleaching are shown with the use of two lookup tables (LUTs, green and physics). <a href="https://www.jove.com/files/ftp_upload/53157/53157fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/53157/53157fig7.jpg" /> 
Figure 7. Single molecule FRET events appear and disappear insingle steps and are perfectly aligned with a single FRET acceptor fluorophore.The time lapse of the single molecule FRET event is shown. Images were acquired using a back-illuminated EMCCD camera. <a href="https://www.jove.com/files/ftp_upload/53157/53157fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/53157/53157fig8.jpg" /> 
Figure 8. Determining &#964;off = 1/ koff from measured smFRET trajectories. (A) The normalized cumulative sums of observable FRET signals (derived from H57 scFV-AF555 decorated 5c.c7 TCR transgenic T-cell blasts recognizing I-Ek/K3-AF647 at 24&#176;C) for four different time lags (42 msec, 490 msec, 1,007 msec, 1,989 msec) were plotted as a function of the total number of observations. Mono-exponential fit functions give rise to corresponding the negative inverse of the expectation values &#60;n(tlag)&#62;. (B) Expectation values were plotted against delays tlag and fitted using equation&#160;&#60;n(tlag)&#62; = &#964;off /{(&#964;off /&#60;nbleach&#62;) + tlag} to yield&#964;offand &#60;nbleach&#62;. <a href="https://www.jove.com/files/ftp_upload/53157/53157fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay at JoVE.com

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

measuring-tcr-pmhc-binding-in-situ-using-a-fret-based-microscopy-assay

Research

JoVE Journal

Bioengineering

12.3K Views.  Vienna University of Technology. This manuscript describes how to conduct (single molecule) Förster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer. 

Video: Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

Immunology and Infection

Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the environment, a cell must be able to recognize and process it. This task mainly relies on the function of membrane receptors, whose role is to convert signals into the biochemical language of the cell. G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptor proteins in humans. Among GPCRs, metabotropic glutamate receptors (mGluRs) are a unique subclass that function as obligate dimers and possess a large extracellular domain that contains the ligand-binding site. Recent advances in structural studies of mGluRs have improved the understanding of their activation process. However, the propagation of large-scale conformational changes through mGluRs during activation and modulation is poorly understood. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to visualize and quantify the structural dynamics of biomolecules at the single-protein level. To visualize the dynamic process of mGluR2 activation, fluorescent conformational sensors based on unnatural amino acid (UAA) incorporation were developed that allowed site-specific protein labeling without perturbation of the native structure of receptors. The protocol described here explains how to perform these experiments, including the novel UAA labeling approach, sample preparation, and smFRET data acquisition and analysis. These strategies are generalizable and can be extended to investigate the conformational dynamics of a variety of membrane proteins.

This study presents a detailed procedure to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments on G protein-coupled receptors (GPCRs) using site-specific labeling via unnatural amino acid (UAA) incorporation. The protocol provides a step-by-step guide for smFRET sample preparation, experiments, and data analysis.

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the ...

Biochemistry

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

Biology

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

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 ...

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. It has been used in a wide range of biophysical and molecular biological experiments, including the measurement of molecular forces, characterization of conformational dynamics of biomolecules, observation of intracellular colocalization of proteins, and determination of receptor-ligand interaction times. In a widefield microscopy configuration, experiments are typically performed using surface-immobilized probes. Here, a method combining single-molecule tracking with alternating excitation (ALEX) smFRET experiments is presented, permitting the acquisition of smFRET time traces of surface-bound, yet mobile probes in plasma membranes or glass-supported lipid bilayers. For the analysis of recorded data, an automated, open-source software collection was developed supporting (i) the localization of fluorescent signals, (ii) single-particle tracking, (iii) determination of FRET-related quantities including correction factors, (iv) stringent verification of smFRET traces, and (v) intuitive presentation of the results. The generated data can conveniently be used as input for further exploration via specialized software, e.g., for the assessment of the diffusional behavior of probes or the investigation of FRET transitions.

This article presents a method for spatiotemporal analysis of mobile, single-molecule F&#246;rster resonance energy transfer (smFRET)-based probes using widefield fluorescence microscopy. The newly developed software toolkit allows the determination of smFRET time traces of moving probes, including the correct FRET efficiency and the molecular positions, as functions of time.

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. ...

Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) soluble cytokines. T cells release two kinds of effector particles in response to activation: trans-synaptic vesicles (tSVs) and supramolecular attack particles, which transfer intercellular messengers and mediate cytotoxicity, respectively. These entities are quickly internalized by APCs engaged in physical contact with T cells, making their characterization daunting. This paper presents the protocol to fabricate and use Bead-Supported Lipid Bilayers (BSLBs) as antigen-presenting cell (APC) mimetics to capture and analyze these trans-synaptic particles. Also described are the protocols for the absolute measurements of protein densities on cell surfaces, the reconstitution of BSLBs with such physiological levels, and the flow cytometry procedure for tracking synaptic particle release by T cells. This protocol can be adapted to study the effects of individual proteins, complex ligand mixtures, pathogen virulence determinants, and drugs on the effector output of T cells, including helper T cells, cytotoxic T lymphocytes, regulatory T cells, and chimeric antigen receptor-expressing T cells (CART).

Here we present the protocol for the stepwise reconstitution of synthetic antigen-presenting cells using Bead-Supported Lipid Bilayers and their use to interrogate the synaptic output from activated T cells.

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) ...

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...

High-Resolution Analysis of Mitochondrial Function in Human PBMCs

High-Resolution Fluorespirometry to Assess Dynamic Changes in Mitochondrial Membrane Potential in Human Immune Cells

Peripheral mononuclear cells (PBMCs) exhibit robust changes in mitochondrial respiratory capacity in response to health and disease. While these changes&#160;do not always reflect what occurs in other tissues, such as skeletal muscle, these cells are an accessible and valuable source of viable mitochondria from human subjects. PBMCs are exposed to systemic signals that impact their bioenergetic state. Thus, expanding our tools to interrogate mitochondrial metabolism in this population will elucidate mechanisms related to disease progression. Functional assays of mitochondria are often limited to using respiratory outputs following maximal substrate, inhibitor, and uncoupler concentrations to determine the full range of respiratory capacity, which may not be achievable in vivo. The conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) by ATP-synthase results in a decrease in mitochondrial membrane potential (mMP) and an increase in oxygen consumption. To provide a more integrated analysis of mitochondrial dynamics, this article describes the use of high-resolution fluorespirometry to measure the simultaneous response of oxygen consumption and mitochondrial membrane potential (mMP) to physiologically relevant concentrations of ADP. This technique uses tetramethylrhodamine methylester (TMRM) to measure mMP polarization in response to ADP titrations following maximal hyperpolarization with complex I and II substrates. This technique can be used to quantify how changes in health status, such as aging and metabolic disease, affect the sensitivity of mitochondrial response to energy demand in PBMCs, T-cells, and monocytes from human subjects.

Author Spotlight: New Insights into PBMC Mitochondrial Responses Using Fluorespirometry

Methods for studying mitochondrial bioenergetics under physiologically relevant substrate concentrations in immune cells are limited. We provide a detailed protocol that uses high-resolution fluorespirometry to assess changes in the response of the mitochondrial membrane potential to energy demand in human T-cells, monocytes, and peripheral mononuclear cells.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

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Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

High-Resolution Fluorespirometry to Assess Dynamic Changes in Mitochondrial Membrane Potential in Human Immune Cells