Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with β-arrestin

Summary

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.

Abstract

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-proteins^1-2. 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 atherosclerosis^10-17. 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 techniques^18-19.

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 lines^18-19. 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.

Protocol

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 image....

Discussion

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 ima.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Rat Basophilic Leukomia Cell line (RBL-2H3) or HEK293 cells.	ATCC	CRL-2256
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
35 mm sterile glass coverslip-bottomed Fluoro dishes (0.17 mm thick) (WillCo-dish)	World Precision Instruments, Inc.	FD35-100
Sterile Gene Pulser Cuvette (0.4 cm electrode gap) (Bio-Rad)	Bio-Rad	16552088
Gene Pulser II electroporater	Bio-Rad
TE-FM Epi-Fluorescence system attached to Nikon Inverted Microscope Eclipse TE300	Nikon Instruments
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