टी सेल की वास्तविक समय लाइव इमेजिंग परिसर गठन संकेतन
Summary
हम टी सेल सक्रियण प्रक्रिया के दौरान प्रोटीन गतिशीलता में अंतर्दृष्टि प्रदान करता है कि एक जीवित कोशिका इमेजिंग विधि का वर्णन है. हम मात्रात्मक परिणाम टी सेल सक्रियण भर जटिल गठन के संकेत का पालन करने की उपज के लिए टी सेल प्रसार परख, confocal माइक्रोस्कोपी इमेजिंग और विश्लेषण के संयुक्त उपयोग को प्रदर्शित करता है.
Abstract
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.
Protocol
Representative Results
Discussion
कई सेलुलर प्रक्रियाओं का विनियमन और समारोह प्रोटीन, प्रोटीन बातचीत के गठन और समाप्ति पर निर्भर करते हैं. सूक्ष्म इमेजिंग जीवित कोशिकाओं में fluorescently टैग प्रोटीन की वास्तविक समय पर नज़र रखने की अनुमति दे?…
Declarações
The authors have nothing to disclose.
Acknowledgements
लेखकों तकनीकी सहायता के लिए सोफिया फ्राइड धन्यवाद. अनुदान no.1659/08, 971/08, 1503-1508 और 491/10, कोई अनुदान के लिए स्वास्थ्य एवं विज्ञान के मंत्रालयों के लिए इसराइल विज्ञान फाउंडेशन: एमबीएस उनके शोध के समर्थन के लिए निम्नलिखित एजेंसियों धन्यवाद. 3-4114 और 3-6540, देर अलेक्जेंडर Smidoda के एस्टेट के माध्यम से इसराइल कैंसर एसोसिएशन, और जैव चिकित्सा उत्कृष्टता अनुदान के लिए Taubenblatt परिवार फाउंडेशन.
Materials
| 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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