This work is supported by the intramural fund from NIAID, NIH.

1. Preparation of chemotactic competent cells of Dictyostelium discoideum
<ol>
<li>To generate D. discoideum cells that are chemotactic to the chemoattractant cAMP, harvest cells growing in D3-T rich media from a shaking culture at 22&deg;C.</li>
<li>Wash the cells twice in non-nutrient developmental buffer (DB buffer containing 5 mM Na2HPO4, 5 mM KH2PO4, 2 mM MgCl2, and 0.1 mM CaCl2).</li>
<li>Re-suspend cells in DB buffer at a density of 2x10...

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No conflicts of interest declared.

The processes of reaching chemotactic competent stage of cells
For wild type D. discoideum cells, it takes about 5 &tilde; 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 stag...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>D3-T Growth Media</td>
			<td>KD Medical</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Caffeine</td>
			<td>Sigma</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Latrunculin B</td>
			<td>Molecular Probes</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa 594</td>
			<td>Molecular Probes</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>cAMP</td>
			<td>Sigma</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ChronTrol XT programmable timer</td>
			<td>ChronTrol Corp</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Miniplus 3 peristaltic pump</td>
			<td>Gilbson</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Platform rotary shaker</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FemtoJet microcapillary pressure supply</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Single- and four-well Lab-Tek II coverglass chambers</td>
			<td>Nalge Nunc International</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LSM 510 META or equivalent fluorescent microscope</td>
			<td>Zeiss</td>
			<td>&nbsp;</td>
			<td>a 40X 1.3 NA or 60X 1.4 NA oil DIC Plan-Neofluar objective lens</td>
		</tr>
		<tr>
			<td>Olympus X81 or equivalent</td>
			<td>Olympus</td>
			<td>&nbsp;</td>
			<td>Requires a 100X 1.47 NA TIRF objective lens</td>
		</tr>
	</tbody>
</table>

imaging g-protein coupled receptor (gpcr)-mediated signaling events that control chemotaxis of dictyostelium discoideum

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 G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; 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.

Watch this Scientific Journal Video about Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum at JoVE.com

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

imaging-g-protein-coupled-receptor-gpcr-mediated-signaling-events

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Biology

17.9K visualizações.  National Institute of Allergy and Infectious Diseases, National Institutes of Health. Aqui, descrevemos os métodos detalhados de células vivas de imagem para investigar quimiotaxia. Apresentamos fluorescência métodos microscópicos para monitorar a dinâmica espaço-temporal dos eventos de sinalização em células migratórias. Medição dos eventos de sinalização nos permite entender melhor como uma rede de sinalização GPCR-alcança gradiente de detecção de quimioatrativos e controles de migração direcional das células eucarióticas.