This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.
G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of β-arrestins3. β-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with β-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and β-arrestin1 tagged with green fluorescent protein (β-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.
Methodology
Description of Microscope
Live cell imaging experiments performed using TE-FM Epi-Fluorescence system attached to Nikon Inverted Microscope Eclipse TE300. The microscope equipped with heating stage. A cool snap HQ digital B/W CCD (Roper Scientific) camera and LAMDA 10-2 optical filter changer (Sutter instrument company) is attached to microscope. Excitation and emission wavelengths are controlled with filter wheels and controlled by Lamba 10-2 filter wheel controller, Sutter Instruments Co. Exposure time 500 ms should be enough to view RFP or GFP in live cells. Hardware control and acquisition of images are controlled by Metamorph software. The choice of the filters for the present study are, filter sets S480/20x, S525/40m and S565/25x, S620/60m for GFP and RFP, respectively; EGFP/DsRed dual dichroic, Chroma Technology. This filter wheel can accommodate up to six filter sets. Microscope is attached with Prior Proscan stage controller with Joy stick. All these hardware attachments can be controlled by Metamorph software. The microscope is also attached with Micromanipulators to hold the micropipettes.
A. Measuring Leukotriene B4 Directed Dendritic Cell Migration
Using the above described microscope setting, we developed methods for following ligand directed dendritic cell migration in real time. Two micromanipulators (Narishige) are attached to the microscope. The chemotaxis of mouse bone marrow derived dendritic cells (BMDCs) towards LTB4 gradient was recorded. Here, we describe the methods to prepare the micro-pipettes using micro-pipette puller, loading the ligand into micro-pipette and setting up the chemotaxis experiment on the heated stage of the microscope to monitor directional migration of cells. This method can be applied to test the efficacy of distinct chemoattractants in inducing migration as well as effect of inhibitors of chemotaxis in live cells. The isolation of BMDCs21 from mouse has been described in several publications including JoVE22.
1. Preparation of BMDCs
Preparation of ligand loaded micro pipette:
B. Measuring GPCR (BLT1) and Cytosolic Protein (β-arrestin) Interactions, Translocation in Live Cells
1. Preparation of Plasmid DNA Constructs
The details of DNA plasmid constructs were described in Jala et al. (2005)18.
2. Transfection of Human BLT1-RFP and β-Arrestin-GFP into RBL-2H3 Cells
3. Live Cell Imaging
Acquisition of images
Process of images and quantification of fluorescence of proteins and localization
Representative Results
A. Measuring Leukotriene B4 Directed Dendritic Cell Migration
The method described in this protocol was used to determine the dendritic cell migration towards leukotriene B4 (Figure 1 and attached video 1)21. We can apply similar methodology to determine the chemotactic capacity of cells to a particular ligand in live cells.
Figure 1. Mouse bone marrow derived dendritic cell migration towards 100 nM LTB4.
Video 1. Live cell migration of BMDCs towards LTB4. Click here to see video.
B. Measuring GPCR (BLT1) and Cytosolic Protein (β-arrestin) Interactions, Translocation in Live Cells
Upon completion of this procedure, one can obtain the following information in GPCR signaling events.
Figure 2. Expression of BLT1-RFP (A), β-arrestin-GFP (B), pNuc-CFP (C) into RBL-2H3 cells. Color combined image shown in panel D.
Figure 3. Ligand induced translocation of receptor and β-arrestin. Line scan of fluorescent intensities in the cells are shown.
Figure 4. Kinetics of receptor internalization and β-arrestin translocation upon addition of 1 μM LTB4. The fluorescence intensities were measured as a function of time at membrane and cytosolic locations of the cell.
Video 2 Live imaging of cells expressing BLT1-RFP and β-arrestin-GFP upon the addition of 1 μM LTB4. Click here to watch video
Live cell imaging is a powerful tool to demonstrate the function and interactions of specific proteins as they occur in real-time. The methods described in this manuscript clearly show that LTB4 can induce rapid migration of dendritic cells. These methods not only expand the aspects of LTB4 function to diverse cell types, they allow similar methods to be applied to a variety of other chemokines and testing their efficacy as chemotactic agents on different leukocyte sub populations. Fluorescence imaging in this system allows for monitoring signaling events in real-time. Furthermore, these methods may also be extended to examine the tight association of molecules in vivo by following fluorescence resonance energy transfer (FRET) using appropriate flurochromes tagged molecules.
The authors have nothing to disclose.
The research is supported by National Institutes of Health grants AI-52381, CA138623 and Kentucky Lung Cancer Research Board and institutional support from James Graham Brown Cancer Center.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Cell lines: | ||||
Rat Basophilic Leukomia Cell line (RBL-2H3) or HEK293 cells. | ATCC | CRL-2256 | ||
Media: | ||||
Delbecco’s modified Eagle’s Medium (DMEM) | Invitrogen | 11995 | ||
Phenol red free RPMI or DMEM | Invitrogen | 11835-030 | ||
Fetal Bovine Serum | Invitrogen | 16000-044 | ||
L-Glutamine (200 mM) | Invitrogen | 25030 | ||
Penicillin-streptomycin (10000 U/mL) | Invitrogen | 15140 | ||
Trypsin, 0.05% (1X) with EDTA 4Na, liquid | Invitrogen | 25300 | ||
HEPES (1M) | Invitrogen | 15630 | ||
Others: | ||||
35 mm sterile glass coverslip-bottomed Fluoro dishes (0.17 mm thick) (WillCo-dish) | WPI | FD35-100 | ||
Sterile Gene Pulser Cuvette (0.4 cm electrode gap) (Bio-Rad) | Bio-Rad | 16552088 | ||
Instruments/software: | ||||
Gene Pulser II electroporater | Bio-Rad | |||
TE-FM Epi-Fluorescence system attached to Nikon Inverted Microscope Eclipse TE300 | Nikon | |||
Metamorph Software | Universal Imaging | |||
Vertical Micro-pipette puller | Narishige International | |||
Micro-Forge M-900 | Narishige International | |||
Hadraulic Micromanipulator MO-188NE | Narishige International | |||
Coarse Manual Manipulator, MN-188NE | Narishige International | |||
cDNA constructs: | ||||
cDNA of G-Protein coupled receptor tagged with red fluorescence protein at C-terminus (hBLT1-RFP) | Jala et al 2005 | |||
cDNA of cytosolic protein tagged with GFP (β-arrestin1-GFP in present study). | Jala et al 2005 |