Fluorescens Biomembrane Kraft Probe: Samtidig Kvantificering af receptor-ligand Kinetics og Binding-induceret intracellulær signalering på en enkelt celle
Summary
We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.
Abstract
Membran receptor-ligand-interaktioner medierer mange cellulære funktioner. Bindende kinetik og nedstrøms signalering udløses af disse molekylære interaktioner er sandsynligvis påvirkes af den mekaniske miljø, hvor binding og signalering finde sted. En nylig undersøgelse viste, at mekanisk kraft kan regulere antigen-genkendelse ved og udløsning af T-celle receptor (TCR). Dette blev muliggjort af en ny teknologi, vi udviklet og betegnet fluorescens biomembran force sonde (fBFP), som kombinerer enkelt molekyle force spektroskopi med fluorescens mikroskopi. Ved hjælp af en ultra-bløde menneskelige røde blodlegemer som følsomme force sensor, en high-speed kamera og real-time imaging sporing teknikker, den fBFP er ~ 1 pN (10 -12 N), ~ 3 nm og ~ 0,5 ms i kraft, rumlig og tidsmæssig opløsning. Med fBFP, kan man præcist måle enkelt receptor-ligand bindende kinetik under force regulering og samtidig billede binding-udløst intracellulær calcium signalering på en enkelt levende celle. Denne nye teknologi kan anvendes til at studere andre membran-receptor-ligand-interaktion og signalering i andre celler under mekanisk regulering.
Introduction
Celle-til-celle og celle-til-ekstracellulære matrix (ECM) adhæsion medieres af binding mellem celleoverfladereceptorer, ECM-proteiner og / eller lipider 1. Binding tillader celler at danne funktionelle strukturer 1, samt erkende, kommunikere og reagerer på miljøet 1-3. Modsætning opløselige proteiner (f.eks cytokiner og vækstfaktorer), der binder fra en tredimensional (3D) væskefase onto celleoverfladereceptorer, celleadhæsionsreceptorer danne bindinger med deres ligander på tværs af en smal forbindelsesepitop kløft at bygge bro to modstående overflader, der begrænser molekylære diffusion i en todimensionel (2D) grænseflade 4-7. I modsætning til 3D-kinetik, der almindeligvis måles på traditionel bindingsanalyser (f.eks overfladeplasmonresonans eller SPR), at 2D kinetik har kvantificeres med specialiserede teknikker såsom atomic force mikroskopi (AFM) 8-10, flow kammer 11,12, mikropipette 13,14, optiskpincet 15 og biomembran force probe (BFP) 16-21.
Mere end blot at give fysisk kobling til cellulær samhørighed, adhæsionsmolekyler er en vigtig del af signaleringen maskiner til cellen til at kommunikere med sine omgivelser. Der har været stigende interesse for at forstå, hvordan ligand-engagementet af adhæsionsmolekyler initierer intracellulær signalering og hvordan det første signal transduceres inde i cellen. Intuitivt egenskaber receptor-ligand-binding kan påvirke de signaler den fremkalder. Det er imidlertid vanskeligt at dissekere mekanistiske relationer mellem den ekstracellulære interaktion og intracellulære signalsystemer begivenheder ved hjælp af traditionel ensemble af biokemiske analyser på grund af deres mange begrænsninger, f.eks en dårlig tidsmæssig opløsning og den fuldstændige mangel på rumlig opløsning. Eksisterende metoder, der tillader både biofysiske (2D-receptor-ligand binding kinetik) og biokemiske (signalanlæg) bemærkninger om levendeceller omfatter substrater af justerbar stivhed 22, elastomer søjle arrays 23 og flow kammer / mikrofluidenheder indarbejdet med fluorescens kapacitet 24-26. , Udlæsninger af signalering og receptor-ligand binding har dog skal indhentes separat (oftest ved forskellige metoder), hvilket gør det vanskeligt at dissekere tidsmæssige og rumlige relationer obligations- karakteristika med signalering begivenheder.
Konventionel BFP er en ultrasensitiv kraft spektroskopi med høj opløsning Spatiotemporal 17. Det bruger en fleksibel røde blodlegemer (RBC) som en kraft sensor, hvilket muliggør måling af enkelt-molekyle 2D kinetik, mekaniske egenskaber og konformationelle ændringer 14,16,19-21,27-29. En fluorescerende billeddannelse baseret BFP (fBFP) korrelerer de receptor-ligand bindingskinetik med bindingen-udløste cellesignalering ved enkelt-molekyle skala. Med denne opsætning in situ cellesignalering aktiviteter i forbindelse med overfladen generelt mekaniskcal-stimulering blev observeret i T-celler 27. Den fBFP er alsidig og kan bruges til undersøgelser af celleadhæsion og signalering medieret af andre molekyler i andre celler.
Protocol
Representative Results
Discussion
En vellykket fBFP eksperiment medfører nogle kritiske overvejelser. Først for den kraft beregning for at være pålidelige, mikropipetten, RBC, og sonden perle bør tilpasses så tæt til koaksial som muligt. Projektionen af RBC inde pipetten skal være omkring én probe pipette diameter, således at friktionen mellem RBC og pipetten er ubetydelig. For en typisk human RBC, den optimale pipette diameter er 2,0-2,4 um, som giver en bedste tilpasning af ligning 1 17,30. For det andet at sikre målinger i…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Research related to this paper and the development of the fBFP technology in the Zhu lab were supported by NIH grants AI044902, AI077343, AI038282, HL093723, HL091020, GM096187, and TW008753. We thank Evan Evans for inventing this empowering experimental tool, and members of the Evans lab, Andrew Leung, Koji Kinoshita, Wesley Wong, and Ken Halvorsen, for helping us to build the BFP. We also thank other Zhu lab members, Fang Kong, Chenghao Ge and Kaitao Li, for their helps in the instrumentation development.
Materials
| Table 1: Reagents/Equipment | |||
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Sodium Phosphate Monobasic Monohydrate (NaH2PO4•H2O) | Sigma-Aldrich | S9638 | Phosphate buffer preparation |
| Anhy. Sodium Phosphate Dibasic (Na2HPO4) | Sigma-Aldrich | S7907 | Phosphate buffer preparation |
| Sodium Carbonate (Na2CO3) | Sigma-Aldrich | S2127 | Carbonate/bicarbonate buffer preparation |
| Sodium Bicarbonate (NaHCO3) | Sigma-Aldrich | S5761 | Carbonate/bicarbonate buffer preparation |
| Sodium chloride (NaCl) | Sigma-Aldrich | S7653 | N2-5% buffer preparation |
| Potassium chloride (KCl) | Sigma-Aldrich | P9541 | N2-5% buffer preparation |
| Potassium phosphate monobasic (KH2PO4) | Sigma-Aldrich | P5655 | N2-5% buffer preparation |
| Sucrose | Sigma-Aldrich | S0389 | N2-5% buffer preparation |
| MAL-PEG3500-NHS | JenKem | A5002-1 | Bead functionalization |
| Biotin-PEG3500-NHS | JenKem | A5026-1 | RBC biotinylation |
| Nystatin | Sigma-Aldrich | N6261 | RBC osmolarity adjustment |
| Ammonium Hydroxide (NH4OH) | Sigma-Aldrich | A-6899 | Glass bead silanization |
| Methanol | BDH | 67-56-1 | Glass bead silanization |
| 30% Hydrogen Peroxide (H2O2) | J. T. Barker | Jan-86 | Glass bead silanization |
| Acetic Acid (Glacial) | Sigma-Aldrich | ARK2183 | Glass bead silanization |
| 3-MERCAPTOPROPYLTRIMETHOXYSILANE(MPTMS) | Uct Specialties, llc | 4420-74-0 | Glass bead functionalization |
| Borosilicate Glass beads | Distrilab Particle Technology | 9002 | Glass bead functionalization |
| Streptavidin−Maleimide | Sigma-Aldrich | S9415 | Glass bead functionalization |
| BSA | Sigma-Aldrich | A0336 | Ligand functionalizing |
| Fura2-AM | Life Technologies | F-1201 | Intracellular calcium fluorescence dye loading |
| Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D2650 | Intracellular calcium fluorescence dye loading |
| Quantibrite PE Beads | BD Biosciences | 340495 | Density quantification |
| Flow Cytometer | BD Biosciences | BD LSR II | Density quantification |
| Capillary Tube 0.7-1.0mm x 30" | Kimble Chase | 46485-1 | Micropipette making |
| Flaming/Brown Micropipette Puller | sutter instrument | P-97 | Micropipette making |
| Pipette microforce | Narishige | MF-900 | Micropipette making |
| Mineral Oil | Fisher Scientific | BP2629-1 | Chamber assembly |
| Microscope Cover Glass | Fisher Scientific | 12-544-G | Chamber assembly |
| Micro-injector | World Precision Instruments | MF34G-5 | Chamber assembly |
| 1ml Syringe | BD | 309602 | Chamber assembly |
| Micropipette holder | Narishige | HI-7 | Chamber assembly |
| Home-designed mechanical parts and adaptors fabrications using CNC machining. | Biophysics Instrument | All parts are customized according to the CAD designs. | BFP system |
| Microscope (TiE inverted) | Nikon | MEA53100 | BFP system |
| Objective CFI Plan Fluor 40x (NA 0.75, WD 0.72mm, Spg) | Nikon | MRH00401 | BFP system |
| Camera, GE680, 640×480, GigE, 1/3" CCD, mono | Graftek Imaging | 02-2020C | BFP system |
| Prosilica GC1290 – ICX445, 1/3", C-Mount, 1280×960, Mono., CCD, 12 Bit ADC | Graftek Imaging | 02-2185A | BFP system |
| Manual submicron probehead with high resolution remote control | Karl Suss | PH400 | BFP system |
| Anti-vibration table (5’ x 3’) | TMC | 77049089 | BFP system |
| 3D manual translational stage | Newport | 462-XYZ-M | |
| SolidWorks 3D CAD software | SOLIDWORKS Corp. | Version 2012 SP5 | BFP system |
| LabVIEW software | National Instruments | Version 2009 | BFP system, BFP program |
| 3D piezo translational stage | Physik Instrumente | M-105.3P | BFP system |
| Linear piezo accuator | Physik Instrumente | P-753.1CD | BFP system |
| Micromanager software | Version 1.4 | fBFP system, fluorescence imaging program | |
| Dual Cam (DC-2) | Photometrics | 77054724 | fBFP system |
| Dual Cam emission filter (T565LPXR) | Photometrics | 77054725 | fBFP system |
| Fluorescence Camera | Hamamatsu | ORCA-R2 C10600-10B | fBFP system |
| Plastic paraffin film (Parafilm) | Bemis Company, Inc | PM996 | bottle sealing |
| Table 2: Buffer solutions | |||
| Carbonate/bicarbonate buffer (pH 8.5) | |||
| Sodium Carbonate (Na2CO3) | 8.4g/L | ||
| Sodium Bicarbonate (NaHCO3) | 10.6g/L | ||
| Phosphate buffer (pH 6.5-6.8) | |||
| NaPhosphate monobasic NaH2PO4•H2O | 27.6g/L | ||
| Anhy. NaPhosphate dibasic Na2HPO4 | 28.4g/L | ||
| N2-5% buffer (pH 7.2) | |||
| Potassium chloride (KCl) | 20.77g/L | ||
| Sodium chloride (NaCl) | 2.38g/L | ||
| Potassium phosphate monobasic (KH2PO4) | 0.13g/L | ||
| Anhy. Sodium Phosphate Dibasic (Na2HPO4) | 0.71g/L | ||
| Sucrose | 9.70g/L | ||
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