Chemotaxis Signal Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health
This article is a part ofJoVE General. If you think this article would be useful for your research, please recommend JoVE to your institution's librarian.Recommend JoVE to Your Librarian
Current Access Through Your IP Address
Current Access Through Your Registered Email Address
Xu, X., Jin, T. Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum. J. Vis. Exp. (55), e3128, doi:10.3791/3128 (2011).
Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into Gγ and Gβγ subunits 7, 9, 10. Gβγ subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.
1. Preparation of chemotactic competent cells of Dictyostelium discoideum
2. Imaging chemotaxing cells in a visible and manipulatable chemoattractant gradient
3. Immobile nonpolarized cell system facilitates imaging signaling events involved in cAMP gradient sensing
4. Simultaneous monitoring heterotrimeric G protein activation and PIP3 production
5. Representative results:
Figure 1: An excellent model system of D. discoideum for GPCR mediated chemotaxis. A. Scheme shows a brief signaling pathway of directional sensing. B. cAMP gradient induces rapid chemotaxis of D. discoideum cells. Cells express PIP3 probe, PH-GFP (Green). Gradient (Red) is visualized by Alexa 594. Scale bar=50μm.
Figure 2: Chemotaxis of cells under a visible and manipulatable chemoattract fields. A. Graph shows a linear relationship between cAMP concentration and the intensity of a fluorescent dye Alexa 594 by a dilution series of 2 μM cAMP mixed with 10 μg/mL Alexa 594. B. Quantitative measurement of cAMP concentration of a gradient by the linear relationship of cAMP concentration and intensity of fluorescent dye Alexa 594 in A.
Figure 3: Cell motility is uncoupled with cell polarization and directional sensing. A. Image shows that immobile cells by the treatment of actin polymerization inhibitor Latrunculin B maintain the capability of directional sensing. Cells express PIP3 probe, PH-GFP (Green). Gradient (Red) is visualized by Alexa 594. B. Manipulatable cAMP stimulation and immobile cell system allows to address key questions of directional sensing. Scale bar=10μm.
Figure 4: Systemic measurements of kinetics of chemosensing signaling network upon exposure to a steady gradient. A. Montage shows a biphasic PIP3 production (Green) of cell which is exposed to a steady cAMP gradient (Red). B. Image shows the regions of interests (ROIs) for measurement of kinetics of PIP3 production presented in C. C. Kinetics of PIP3 production in the cells exposed to a steady gradient. D. Scheme shows the signaling network of directional sensing from cAMP stimulation to PIP3 production. Their kinetics upon exposure to a steady gradient is presented in the same color solid lines in E.
Figure 5: Simultaneous monitoring multi-events of GPCR signaling networks. A. Scheme shows simultaneous measurement of heterotrimeric G protein activation and PIP3 production by monitoring the FRET change and membrane translocation of PIP3 probe, PH-GFP in G and PH cells, respectively. B. Montage of rainbow images of G and PH cells shows that a uniformly applied cAMP stimulation triggers a persistent G protein activation at the cell peripheral, while which triggers a transient PIP3 production. The time points are before (0s) and after stimulation for 4.9s, 10.2s and 20.4s. C. Kinetics of G protein activation and PIP3 production upon a uniformly applied cAMP stimulation.
The processes of reaching chemotactic competent stage of cells
For wild type D. discoideum cells, it takes about 5 ˜ 6 hours pulsing development at room temperature to induce them into a well-chemotactic competent stage during which cells display a well polarized cellular morphology and rapid cell migration (Fig. 1). Several factors, such as cAMP concentration for pulsing, temperature, and different genetic backgrounds, may affect the process of reaching chemotactic competent stage. A cAMP gradient guides the cells to move toward the source of cAMP, a directed cell migration designated as chemotaxis. However, cells that have not reached the chemotactic competent stage due to insufficient development or their genetic background may not show two typical features: polarized morphology and rapid cell movement. On the other hand, cells that have passed the chemotactic competent stage because of over-development often form clumps of the cells, which are very difficult to separate into individual cells for imaging experiments..
1. Imaging chemotaxing cells in a visible and manipulatable chemoattractant gradient
It is a technical advance to apply fluorescently labeled and manipulatable chemoattractant stimulation into an experimental system. Historically, we had applied either homogenously applied stimulation (also called uniform stimulation) or gradient stimulation to observe cell morphology and behaviors. However, an "blind" stimulation shows no tempo-spatial information on how the stimuli reaches cells, therefore casts doubts on any "abnormal" observations of cell responses. Here, we show an application of fluorescent dye (Alexa594, Molecular Probe) with chemoattractant to establish a linear relationship between the concentration of chemoattractant and intensity of monitored fluorescent day and (Fig. 2A, B). An acquisition combination of green fluorescent protein (GFP) and a red emission of fluorescent dye (Alexa594) which allows us to monitor both a stimulation and cell responses upon this stimulation (Fig. 2C).
According to the experimental requirement, there are more combinations available. While considering a new fluorescent dye, it is critical to confirm the fluorescent is suitable for your experiment, especially for a gradient experiment, it is important to confirm the diffusion co-efficiency of your stimuli and fluorescent dye. Also, in order to obtain a linear relationship of stimuli concentration and intensity of the mixed fluorescent dye, it is necessary to acquire the maximum concentration of stimuli without saturation of intensity.
2. Immobile nonpolarized cell system facilitates live cell imaging
Chemotaxis is a complicated cellular process; however, it can be dissected into three basic aspects: cellular polarization, motility, and directional sensing. Morphologically, the polarization of the a is referred to a clearly defined leading front and trailing end of a cell. Many signaling components involved in chemotaxis localize in either the fornt or the back in a polarized cells. Directional sensing is the capability of a cell to translate an extracellular gradient into a polarized intracellular biochemical polarization. As shown in Fig. 3A, the treatment of cells with actin polymerization inhibitor (Latrunculin B) rapidly eliminates cell motility and its original polarization. Strikingly, cells are still able to establish an intracellular polarization, such as an accumulation of PIP3 in the front of the cells facing the gradient (shown as a crescent by PIP3biosensor, PH-GFP), indicating that directional sensing of the cells can be uncoupled from cell polarization and cell motility. This non-polarized cell system allows us to determine the kinetics of signaling components essential for chemosensing without the complication of cell motility and previous polarization. Pro and con, the treatment of actin polymerization inhibitor abolishes all the dynamics that dependents on actin-cytoskeleton.
3. Monitoring signaling network by multi-spectral live cell imaging
Engagement of cAMP to its receptor triggers activation of heterotrimeric G protein. As a subsequently, Gβγ subunit dissociates from Gα subunit and activates downstream effectors to induce cell responses such as PIP3 production. Technical advances on tempo-spatial resolution of fluorescent microscopy allow a simultaneous imaging of several signaling events at subcellular level in live cells. In this section, we first show a simultaneous imaging of two signaling molecules along with monitoring cAMP gradient (Fig. 4A). Next, we present spectral imaging of G activation and its downstream cell response of PIP3 production (Fig. 4B). Förster resonance energy transfer (abbreviated FRET), also known as fluorescence resonance energy transfer, resonance energy transfer (RET) or electronic energy transfer (EET), is a mechanism describing energy transfer between two chromophores. There are three criteria for occurrence of FRET: overlap of donor emission and acceptor excitation, appropriate orientation, and short distance (<10 nm). The distance/orientation requirement of two molecules facilitates FRET to be an efficient way to detect protein-protein interaction or intromolecular conformation change of a protein. Here, we used the common used CFP/YFP FRET pair (Gα-CFP/Gβ-YFP) to monitor the dynamic dissociation of G protein α/βγ subunits in live cells. As a clever experimental design we described here is to mix two different types of cells in order to simultaneously monitor both G protein activation and PIP3 production when it is not convenient to measure all dynamics in one cell.
No conflicts of interest declared.
This work is supported by the intramural fund from NIAID, NIH.
|D3-T Growth Media||KD Medical|
|Latrunculin B||Molecular Probes, Life Technologies|
|Alexa 594||Molecular Probes, Life Technologies|
|ChronTrol XT programmable timer||ChronTrol Corp|
|Miniplus 3 peristaltic pump||Gilbson|
|Platform rotary shaker|
|FemtoJet microcapillary pressure supply||Eppendorf|
|Single- and four-well Lab-Tek II coverglass chambers||Nalge Nunc international|
|LSM 510 META or equivalent fluorescent microscope||Carl Zeiss, Inc.||a 40X 1.3 NA or 60X 1.4 NA oil DIC Plan-Neofluar objective lens|
|Olympus X81 or equivalent||Olympus Corporation||Requires a 100X 1.47 NA TIRF objective lens|