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Immunology and Infection

Real-time Live Imaging of T-cell Signaling Complex Formation

Published: June 23rd, 2013

DOI:

10.3791/50076

1The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University
* These authors contributed equally

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.

Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

1. T-cell Transfection

  1. Prepare the activated Amaxa Nucleofector solution by mixing the kit's "Supplement" solution to the Nucleofector Solution. The activated solution may be stored at 4 °C for up to three months.
  2. Grow Jurkat T cells in culture medium [10% fetal calf serum (FCS), 1% Penicillin-Streptomycin solution, and 2 mM L-glutamine in RPMI]. Optimally, cells should be used 1-2 weeks after thawing. Using cell cultures in logarithmic growth is crucial for high transfection efficiency and cell.......

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We present an example of live T-cell imaging and analysis. Prior to the imaging experiment, SLP76 deficient T cells (J14) were analyzed with the use of FACS to determine the expression of the fluorescent proteins (Figure 1). T cells transfected with the tagged signaling proteins mCFP-Nck and SLP76-mYFP were deposited over activating slides and imaged during T-cell spreading (Figure 2). Collected images show the dynamics of protein localization throughout activation and spreading (

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The regulation and function of multiple cellular processes depend on the formation and termination of protein-protein interactions. Microscopic imaging allows the real-time tracking of fluorescently tagged proteins in living cells. Colocalization of tagged proteins can suggest a direct or indirect interaction between the proteins, and can be used to reinforce findings obtained by biochemical methods, such as immunoprecipitation. Unlike biochemical methods, live-cell imaging allows these inter-protein interactions to be o.......

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The authors thank Sophia Fried for technical assistance. MBS thanks the following agencies for their research support: The Israel Science Foundation for grants no.1659/08, 971/08, 1503/08 and 491/10, the Ministries of Health & Science for grants no. 3-4114 and 3-6540, the Israel Cancer Association through the Estate of the late Alexander Smidoda, and the Taubenblatt Family Foundation for the Bio-medicine excellence grant.

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Name Company Catalog Number Comments
Reagent/Material Company Catalog Number Comments
Amaxa human T Cell nucleofector kit Lonza VCA-1002  
Anti CD3 (UCHT1 clone) BioLegend 300432  
Falcon FACS Tubes Becton Dickinson 352058  
FCS HyClone SV30160.03  
G418 Calbiochem 345810  
German coverglass system 4 chamber slides Lab-Tek II 155382  
HCl Bio Lab 8410501  
Hepes Biological Industries 03-025-1B  
Hygromycin Enzo ALX-380-306  
L-glutamine Sigma G7513  
PBS (10X) Sigma D1408  
Penicillin-Streptomycin Sigma P0781  
Poly-L-lysine 0.1% (w/v) in H2O Sigma P8920  
RPMI Sigma R8758  
Sodium azide Sigma S2002  
Equipment      
Accublock digital dry bath Labnet D1105A  
Centrifuge Eppendorf Centrifuge 5810 R  
Confocal microscope Zeiss LSM 510 Meta  
Heatable mounting frame - Heating Insert P S PeCon 130-800-031  
TempModule S (required for the heatable mounting frame) Zeiss 411860-9010-000  
Electroporation device Amaxa Nucleofector I  
Fluorescence activated cell sorter Becton Dickinson FACSVantage SE  
Image analysis software Bitplane 7.0.0  

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