המחברים מודים סופיה פריד לקבלת סיוע טכני. MBS הסוכנויות מודה הבאות על תמיכת המחקר שלהם: הקרן הלאומית למדע למענקי no.1659/08, 971/08, 1503-1508 ו491 / 10, משרדי בריאות ומדע למענקים אין. 3-4114 ו3-6540, סרטן אגודת ישראל באמצעות עזבון אלכסנדר Smidoda מאוחר, וקרן משפחת Taubenblatt למענק ההצטיינות ביו הרפואה.

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+actin+cytoskeleton+in+T+cell+activation.">The actin cytoskeleton in T cell activation.</a> Annu. Rev. Immunol. 26, 233-259 (2008).</li><li>Reicher, B., Barda-Saad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+pathways+leading+from+the+T-cell+antigen+receptor+to+the+actin+cytoskeleton+network.">Multiple pathways leading from the T-cell antigen receptor to the actin cytoskeleton network.</a> FEBS Lett. 584, 4858-4864 (2010).</li><li>Barda-Saad, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+molecular+interactions+linking+the+T+cell+antigen+receptor+to+the+actin+cytoskeleton.">Dynamic molecular interactions linking the T cell antigen receptor to the actin cytoskeleton.</a> Nat. Immunol. 6, 80-89 (2005).</li><li>Bunnell, S. C., Kapoor, V., Trible, R. P., Zhang, W., Samelson, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+actin+polymerization+drives+T+cell+receptor-induced+spreading:+a+role+for+the+signal+transduction+adaptor+LAT.">Dynamic actin polymerization drives T cell receptor-induced spreading: a role for the signal transduction adaptor LAT.</a> Immunity. 14, 315-329 (2001).</li><li>Balagopalan, L., Sherman, E., Barr, V. A., Samelson, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+techniques+for+assaying+lymphocyte+activation+in+action.">Imaging techniques for assaying lymphocyte activation in action.</a> Nat. Rev. Immunol. 11, 21-33 (2011).</li><li>Zacharias, D. A., Violin, J. D., Newton, A. C., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partitioning+of+lipid-modified+monomeric+GFPs+into+membrane+microdomains+of+live+cells.">Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells.</a> Science. 296, 913-916 (2002).</li><li>Adler, J., Parmryd, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+colocalization+by+correlation:+the+Pearson+correlation+coefficient+is+superior+to+the+Mander's+overlap+coefficient.">Quantifying colocalization by correlation: the Pearson correlation coefficient is superior to the Mander's overlap coefficient.</a> Cytometry A. 77, 733-742 (2010).</li><li>Costes, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+and+quantitative+measurement+of+protein-protein+colocalization+in+live+cells.">Automatic and quantitative measurement of protein-protein colocalization in live cells.</a> Biophys. J. 86, 3993-4003 (2004).</li><li>Fang, N., Motto, D. G., Ross, S. E., Koretzky, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyrosines+113,+128,+and+145+of+SLP-76+are+required+for+optimal+augmentation+of+NFAT+promoter+activity.">Tyrosines 113, 128, and 145 of SLP-76 are required for optimal augmentation of NFAT promoter activity.</a> J. Immunol. 157, 3769-3773 (1996).</li><li>Wunderlich, L., Farag&#243;, A., Downward, J., Buday, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+Nck+with+tyrosine-phosphorylated+SLP-76+in+activated+T+lymphocytes.">Association of Nck with tyrosine-phosphorylated SLP-76 in activated T lymphocytes.</a> Eur. J. Immunol. 29, 1068-1075 (1999).</li><li>Pauker, M. H., Reicher, B., Fried, S., Perl, O., Barda-Saad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+cooperation+between+the+proteins+Nck+and+ADAP+is+fundamental+for+actin+reorganization.">Functional cooperation between the proteins Nck and ADAP is fundamental for actin reorganization.</a> Mol. Cell Biol. 31, 2653-2666 (2011).</li><li>Barda-Saad, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+interactions+at+the+SLP-76+complex+are+critical+for+actin+polymerization.">Cooperative interactions at the SLP-76 complex are critical for actin polymerization.</a> EMBO J. 29, 2315-2328 (2010).</li><li>Pauker, M. H., Hassan, N., Noy, E., Reicher, B., Barda-Saad, 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> Studying the Dynamics of SLP-76, Nck, and Vav1 Multimolecular Complex Formation in Live Human Cells with Triple-Color. 5, rs3 (2012).</li></ol>

אין לנו ניגודי עניינים לחשוף.

הרגולציה והפונקציה של תהליכים תאיים רבים תלויה בהיווצרות וסיום האינטראקציות בין חלבונים. הדמיה מיקרוסקופית מאפשרת מעקב בזמן אמת של חלבונים מתויגים fluorescently בתאים חיים. Colocalization של חלבונים מתויגים יכול להציע אינטראקציה ישירה או עקיפה בין החלבונים, וניתן להשתמש בו כדי...

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagent/Material&lt;/strong&gt;</td></tr><tr><td>Amaxa human T Cell nucleofector kit</td><td>Lonza</td><td>VCA-1002</td><td></td></tr><tr><td>Anti CD3 (UCHT1 clone) </td><td>BioLegend</td><td>300432</td><td></td></tr><tr><td>Falcon FACS Tubes</td><td>Becton Dickinson</td><td>352058</td><td></td></tr><tr><td>FCS</td><td>HyClone</td><td>SV30160.03</td><td></td></tr><tr><td>G418</td><td>Calbiochem</td><td>345810</td><td></td></tr><tr><td>German coverglass system 4 chamber slides </td><td>Lab-Tek II</td><td>155382</td><td></td></tr><tr><td>HCl</td><td>Bio Lab</td><td>8410501</td><td></td></tr><tr><td>Hepes </td><td>Biological Industries</td><td>03-025-1B</td><td></td></tr><tr><td>Hygomycin</td><td>Enzo</td><td>ALX-380-306</td><td></td></tr><tr><td>L-glutamine </td><td>Sigma</td><td>G7513</td><td></td></tr><tr><td>PBS (10X)</td><td>Sigma</td><td>D1408</td><td></td></tr><tr><td>Penicillin-Streptomycin </td><td>Sigma</td><td>P0781</td><td></td></tr><tr><td>Poly-L-lysine 0.1 % (w/v) in H2O</td><td>Sigma</td><td>P8920</td><td></td></tr><tr><td>RPMI</td><td>Sigma</td><td>R87589</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma</td><td>S2002</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt; </td></tr><tr><td>Accublock digital dry bath</td><td>Labnet</td><td>D1105A</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>Centrifuge 5810 R</td><td></td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td>LSM 510 Meta</td><td></td></tr><tr><td>Heatable mounting frame - Heating Insert P S </td><td>PeCon</td><td>130-800-031</td><td></td></tr><tr><td>TempModule S (required for the heatable mounting frame)</td><td>Zeiss</td><td>411860-9010-000</td><td></td></tr><tr><td>Electroporation device</td><td>Amaxa</td><td>Nucleofector I</td><td></td></tr><tr><td>Fluorescence activated cell sorter</td><td>Becton Dickinson</td><td>FACSVantage SE</td><td></td></tr><tr><td>Image analysis software</td><td>Bitplane</td><td>7.0.0</td><td></td></tr></tbody></table>

real-time live imaging of t-cell signaling complex formation

הגנה מפני מחלות זיהומיות מתווכת על ידי 1,2 המערכת החיסונית. לימפוציטים מסוג T הם רכזי האב של מערכת החיסון, המסדיר את ההפעלה ותגובות של תאים חיסוניים 3,4 מרובים. הפעלת תא T תלויה בהכרה של אנטיגנים ספציפיים המוצגים על ידי תאי מציגי אנטיגן (נגמ&quot;שים). קולט אנטיגן T-cell (TCR) ספציפי לכל שיבוט תאי T וקובע סגוליות אנטיגן 5. הכריכה של TCR לאנטיגן גורמת לזירחון של מרכיבי קומפלקס TCR. על מנת לקדם את הפעלת תא T, אות זה חייב להיות transduced מן הקרום אל הציטופלסמה והגרעין, ייזום תגובות שונות חיוניות כגון גיוס של חלבוני איתות לTCR; אתר APC (סינפסה החיסונית), ההפעלה המולקולרית שלהם, סידור מחדש cytoskeletal, גובה של ריכוז סידן תוך תאי, ושינויים בביטוי גני 6,7. הנכוןitiation וסיום של אותות המפעילים הוא קריטי לתגובות תא T המתאימות. הפעילות של חלבוני איתות תלויה בהיווצרות וסיום האינטראקציות בין חלבונים, לפרסם שינויי translational כגון זרחון חלבון, היווצרות של קומפלקסי חלבונים, ubiquitylation החלבון וגיוס של חלבונים לאתרים שונים 8 סלולריים. הבנת הפעולה הפנימית של תהליך הפעלת תא T היא קריטית עבור מחקר חיסוני ויישומים קליניים. מבחני שונים פותחו כדי לחקור אינטראקציות בין חלבונים, עם זאת, מבחני ביוכימיים, כגון שיטת שיתוף immunoprecipitation בשימוש נרחב, אינם מאפשרים מיקום חלבון להיה להבחין, ובכך נמנע אפשרות התצפית של תובנות רבות ערך לדינמיקה של הסלולר מנגנונים. בנוסף, מבחני אלה בדרך כלל בתפזורת לשלב חלבונים מהתאים רבים ושונים שעשויות להיות בשלבים שונים של t שוניםהוא חקר את התהליך סלולרי. זה יכול להיות השפעה מזיקה על החלטה זמנית. השימוש בהדמיה בזמן אמת של תאי חיים מאפשר גם מעקב המרחבי של חלבונים ואת היכולת להבחין בין temporally אירועי איתות, ובכך שופך אור על הדינמיקה של תהליך 9,10. אנו מציגים שיטה של ​​הדמיה בזמן אמת של היווצרות איתות-מורכבת במהלך הפעלת תא T. T-cell קווים, כגון Jurkat T-תאים ראשוניים או, הם transfected עם פלסמידים קידוד לחלבונים של עניין התמזגו לחלבוני ניאון monomeric, מניעת oligomerization הלא פיסיולוגי 11. חיים תאי T הם ירדו מעל coverslip מראש מצופה עם נוגדן תא T הפעלת 8,9, אשר נקשר למתחם CD3/TCR, גרימת הפעלת תאי T תוך התגברות על הצורך בהפעלת אנטיגנים ספציפיים. תאים הופעל הם צילמו כל הזמן עם השימוש במיקרוסקופיה confocal. נתוני הדמיה מנותחים להניב תוצאות כמותיות, כגון colמקדם ocalization של חלבוני האיתות.

אנו מציגים דוגמה של הדמיה T-תא חי וניתוח. לפני ניסוי ההדמיה, SLP76 תאי T לקויים (J14) נותחו עם השימוש בFACS על מנת לקבוע את הביטוי של חלבוני הניאון (איור 1). תאי T transfected עם איתות חלבונים המתויגים mCFP-NCK וSLP76-mYFP הופקדו על שקופיות הפעלה וצלמו במהלך T-תא הפצה (איור 2). ?...

Watch this Scientific Journal Video about Real-time Live Imaging of T-cell Signaling Complex Formation at JoVE.com

הדמיה חיה בזמן אמת של היווצרות מורכבות איתות של תאי T | אימונולוגיה וזיהום | וִידֵאוֹ

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

אנו מתארים שיטת הדמיה לחיות תאים המספקת תובנה דינמיקת חלבון במהלך תהליך תא T ההפעלה. אנו מדגימים את השימוש בשילוב של הפצת assay תא T, מיקרוסקופיה confocal וניתוח הדמיה להניב תוצאות כמותיות לעקוב איתות היווצרות מורכבת לאורך כל הפעלת תא T.

הדמית חיה בזמן אמת של T-תא איתות גיבוש קומפלקס

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

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13.6K Views.  Bar-Ilan University. אנו מתארים שיטת הדמיה לחיות תאים המספקת תובנה דינמיקת חלבון במהלך תהליך תא T ההפעלה. אנו מדגימים את השימוש בשילוב של הפצת assay תא T, מיקרוסקופיה confocal וניתוח הדמיה להניב תוצאות כמותיות לעקוב איתות היווצרות מורכבת לאורך כל הפעלת תא T.

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

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.

טכניקה מיקרוסקופית TIRF הדמיה בזמן אמת סימולטני של TCR וחלבונים הקשורים אליו לשידור אותות

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

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

דמיין את הדינמיקה של משטח הפני-תא באמצעות מיקרוסקופ גיליון אור של סריג

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

Biochemistry

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological synapses&#39;. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of &#39;semi-professional APCs&#39;. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic &#39;planar cellular APC model&#39; for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

דגם תא האנדותל מישורי הדמיה החיסוני סינפסה Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

הדמית תא CD4 T גירת ביניים בדרמיס המוסת

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

קליפת המוח אקטין זרימה בתאי T לכמת ידי מתאם תמונת ספקטרוסקופיה מרחב ובזמן של תאורה מיקרוסקופית נתונים מובנים

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological synapse, a stereotyped cell-cell junction that regulates T cell activation and directionally targets effector responses. To study these processes effectively, an imaging approach that is tailored to capturing fast, polarized responses is necessary. This protocol describes such a system, which is based on a photoactivatable peptide-major histocompatibility complex (pMHC) that is non-stimulatory until it is exposed to ultraviolet light. Targeted decaging of this reagent during videomicroscopy experiments enables precise spatiotemporal control of TCR activation and high-resolution monitoring of subsequent cellular responses by total internal reflection (TIRF) imaging. This approach is also compatible with genetic and pharmacological perturbation strategies. This allows for the assembly of well-defined molecular pathways that link TCR signaling to the formation of the polarized cytoskeletal structures that underlie the immunological synapse.

This protocol describes an imaging-based method to activate T lymphocytes using photoactivatable peptide-MHC, enabling precise spatiotemporal control of T cell activation.

יכולות בקרה של תא T הפעלה באמצעות אגוניסט Photoactivatable

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

הערכה של הממשק סינפטית של תאי T האנושי העיקרי של דם היקפיים ורקמות הלימפה

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Imaging the Human Immunological Synapse

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90&#176; angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS.

This protocol images both the immunological synapse formation and the subsequent polarized secretory traffic towards the immunological synapse. Cellular conjugates were formed between a superantigen-pulsed Raji cell (acting as an antigen-presenting cell) and a Jurkat clone (acting as an effector helper T lymphocyte).

הדמיה של הסינפסה החיסונית של האדם

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) ...

<meta charset="utf8"></head><body>טכניקת הדמיה ברזולוציה גבוהה לאיתור מיקרו-דומיינים Ca2+ בתאי T

Imaging Initial Ca2+ Microdomains in Primary T Cells

Local, sub-second Ca2+ signals, termed Ca2+ microdomains, are highly dynamic and short-lived Ca2+ signals, which result in a global [Ca2+]i elevation and might already determine the fate of a T cell. Upon T cell receptor activation, NAADP is formed rapidly, binding to NAADP binding proteins (HN1L/JPT2, LSM12) and their respective receptors (RyR1, TPC2) sitting on intracellular Ca2+ stores, like the ER and lysosomes, and leading to subsequent release and elevation of [Ca2+]i. To capture these fast and dynamically occurring Ca2+ signals, we developed a high-resolution imaging technique using a combination of two Ca2+ indicators, Fluo-4 AM and FuraRed AM. For postprocessing, an open-source, semi-automated Ca2+ microdomain detection approach was developed based on the programming language Python. Using this workflow, we are able to reliably detect Ca2+ microdomains on a subcellular level in primary murine and human T cells in high temporal and spatial resolution fluorescence videos. This method can also be applied to other cell types, like NK cells and murine neuronal cell lines.

<meta charset="utf8"></head><body>מחבר זרקור: חשיפת ההפעלה הראשונית של התגובה החיסונית הנרכשת להתערבות טיפולית

Here, we provide a comprehensive protocol to resolve initial local Ca2+ signals, known as Ca2+ microdomains, in primary murine and human T cells using fluorescence microscopy. This protocol serves as a valuable resource for researchers examining Ca2+ signaling pathways within immune cells and to further unravel their function.

הדמיה ראשונית של מיקרודומיינים Ca2+ בתאי T ראשוניים

Local, sub-second Ca2+ signals, termed Ca2+ microdomains, are highly dynamic and short-lived Ca2+ signals, which result in a global [Ca2+]i elevation and ...