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 2x107 cells/ml. </li>
<li>Shake 10 ml cells in a 250 ml flask at 100 rpm at 22&deg;C for one hour. </li>
<li>Deliver 100 &mu;l of 7.5 &mu;M cAMP stock to the 10 ml cells every six minutes over 6 hours to achieve a final concentration of 75 nM cAMP, a process designated as cAMP pulsing treatment. After 5-6 hours of cAMP pulsing treatment, D. discoideum cells become chemotactic competent toward cAMP gradient. </li>
<li>Collect cells by centrifugation at 200 g for 5 min and then resuspend cells with DB buffer containing 2.5 mM caffeine, and shake at 200 rpm at 22&deg;C for 20 min to basolate cell to a chemotactic situation.</li>
</ol>
2. Imaging chemotaxing cells in a visible and manipulatable chemoattractant gradient
<ol>
<li>Backfill a micropipette with a freshly prepared 30 &mu;l solution of 1 &mu;M cAMP and Alexa 594 at 0.1&mu;g/&mu;l in DB buffer.</li>
<li>Attach the Femtotip to a micropipette holder and connect the tubing to a pressure supply apparatus, Eppendorf FemtoJet system. </li>
<li>Attach the micropipette assembly to a micromanipulator (Eppendorf TransferMan NK2) motorized micromanipulator to provide a steady pressure in order to establish a stable gradient.</li>
<li>Mount a one-well LabTek chamber filled with 6 ml of DB buffer over a 40X oil lens on a confocal microscope and use bright-field optics, center the Femtotip into the field of view. </li>
<li>Turn on the pressure supply and set the compensation pressure (Pc) for 70 hPa to establish a gradient of the cAMP/Alexa 594 mixture. </li>
<li>Visualize cAMP gradient by monitoring the mixture of desired concentration of cAMP and Alexa 594 fluorescence using excitation with a 543 nm laser line. </li>
<li>Use auto-positioning function of the micromanipulator to put micropipette to the desired positions and set them as Position 1, Position 2, and Position 3 to manipulate the gradient to which cell are exposed to.</li>
</ol>
3. Immobile nonpolarized cell system facilitates imaging signaling events involved in cAMP gradient sensing
<ol>
<li>After caffeine treatment, remove an aliquot of cells and centrifuge at 500g for 3 min.</li>
<li>Remove buffer and dilute cells to 5x105 cells/ml with fresh DB buffer containing 2.5 mM caffeine.</li> 
<li>Apply 1 ml of cell suspension to a single-well chamber or 0.4 ml to each well of a four-well chamber. </li>
<li>Allow cells to adhere for 10 min, carefully pipette off the buffer to remove unattached cells and replace with the same volume.</li>
<li>Locate the desired cells under microscope and start imaging. </li>
<li>For an experiment designed to monitor dynamics of signaling components in the cells exposing to a steady gradient, treat the cells with 5.0 &mu;M (final concentration) Latrunculin B for 10 min prior to the experiments. </li>
</ol>
4. Simultaneous monitoring heterotrimeric G protein activation and PIP3 production
<ol>
<li>cAMP pulsing develop cells co-expressing G&alpha;CFP and YFPG&beta; (G cells) and cells expressing PIP3 indicator PH-GFP (PH cells) 7.</li> 
<li>Mix these two types of cells with 1:1 ratio and plate them in one-well or 4-well chambers.</li>
<li>Create and save the emission fingerprint reference curve CFP, YFP and GFP using Lambda Stack Acquisition mode within the spectral range from 464 to 624 nm with a 10 nm width.</li>
<li>Simultaneously image G protein activation in G cells and PIP3 production in PH cells using with same Lambda Stack Acquisition mode within the spectral range from 464 to 544 nm with a 10 nm increments.</li>
<li>Apply Linear Unmixing function of Zeiss 510META software using saved CFPand YFP and emission fingerprints to mathematically calculate the contribution of each fluorophore in the Lambda Stack to separate the CFP and YFP intensity into individual channels in G.</li>
<li>With the same strategy, apply Linear Unmixing function using saved GFP and background emission fingerprints to mathematically calculate GFP intensity in PH cells.</li>
</ol>
5. Representative results:
<ol>
<li>An excellent model system of D. discoideum for GPCR mediated chemotaxis. A social amoeba, D. discoideum exhibits a striking chemotaxis during the life cycle. Due to its genetic and biochemical advantages, D. d provides a powerful system to study chemotaxis.</li>
<li>Chemotaxis of cells under a visible and manipulatable chemoattract fields. Here, we first show a simple methodology to obtain a linear relationship between cAMP concentration and the intensity of a fluorescent dye Alexa 594 by a dilution series of 2 &mu;M cAMP mixed with 10 &mu;g/mL Alexa 594 (Fig. 2A). Next, we provide an easy way to visualize the gradient, moreover, to establish a quantitative measurement of cAMP concentration of a gradient by the intensity of Alexa 594 (Fig. Fig. 2B).</li>
<li>Cell motility is uncoupled with cell polarization and directional sensing. An actin polymerization inhibitor eliminates pre-existing morphological polarity and also prevents cell movement while maintaining the cells' capability of directional sensing (Fig. 3A). Employment of visible and manipulatable cAMP stimulation guarantees the input, for example uniformly applied stimulations or a gradient. This method allows a quantitative analysis of cAMP-induced redistribution of key signaling components in the gradient sensing machinery. Measured spatiotemporal dynamics of these signaling components allow us to understand how the signaling network achieves adaptation to uniform stimulations while generating polarized biochemical responses to gradients (Fig. 3B).</li> 
<li>Systemic measurements of kinetics of chemosensing signaling network upon exposure to a steady gradient. It is critical to measure the dynamics/kinetics of directional-sensing signaling components to understand how each component contributes to the establishment of intracellular polarization when cells experience first gradient. Application of live cell imaging with a high tempo-spatial resolution, we first show a biphasic PIP3 production of cell which is exposed to a steady cAMP gradient (Fig. 4A-C). Applying live cell imaging, we have systematically measured dynamics of directional-sensing specific signaling network from cAMP stimulation to PIP3 production (Fig. 4D, E).</li>
<li>Simultaneous monitoring heterotrimeric G protein activation and PIP3 production upon uniformly applied cAMP stimulation. F&ouml;rster resonance energy transfer (abbreviated FRET) provides an efficient approach to monitor heterotrimeric G protein activation (dissociation) upon cAMP stimulation. Here, we described an convenient easy-adopt system for a 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 (Fig. 5). A uniformly applied cAMP stimulation triggers a persistent G protein activation while which triggers a transient PIP3 production.</li>
</ol>
<img src="/files/ftp_upload/3128/3128fig1.jpg" alt="Figure 1"/> 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=50&mu;m.
<img src="/files/ftp_upload/3128/3128fig2.jpg" alt="Figure 2"/> 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 &mu;M cAMP mixed with 10 &mu;g/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. 
<img src="/files/ftp_upload/3128/3128fig3.jpg" alt="Figure 3"/> 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=10&mu;m.
<img src="/files/ftp_upload/3128/3128fig4.jpg" alt="Figure 4"/> 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.
<img src="/files/ftp_upload/3128/3128fig5.jpg" alt="Figure 5"/> 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.

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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

Research

JoVE Journal

Biology

17.9K Views.  National Institute of Allergy and Infectious Diseases, National Institutes of Health. Qui, descriviamo dettagliati vivere metodi di imaging per lo studio delle cellule chemiotassi. Vi presentiamo fluorescenza metodi microscopici di monitorare le dinamiche spazio-temporali di segnalazione eventi nella migrazione delle cellule. Misurazione di eventi di segnalazione ci permette di capire meglio come un GPCR di segnalazione rete raggiunge gradiente di rilevamento di fattori chemiotattici e controlli migratori direzionale delle cellule eucariotiche.

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

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Quantifying Agonist Activity at G Protein-coupled Receptors

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.
Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (Kb) is much greater than that for the inactive state (Ka). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (Kobs), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (&#949;) (Figure 2).
Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the Kobs and relative efficacy of an agonist 1,2. In this report, we show how to modify this analysis to estimate the agonist Kb value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate Kb in absolute units of M-1.
Our method of analyzing agonist concentration-response curves 3,4 consists of global nonlinear regression using the operational model 5. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of Kobs and a parameter proportional to efficacy (&tau;). The estimate of &tau;Kobs of one agonist, divided by that of another, is a relative measure of Kb (RAi) 6. For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (&tau;sys). In this case, the Kb value of an agonist is equivalent to &tau;Kobs/&tau;sys 3.
Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.

A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular development program, during which single cells chemotax towards aggregation centers1-4. This process is dependent on the release of cyclic adenosine monophosphate (cAMP)5. cAMP is produced in waves through the concerted action of adenylate cyclase and phosphodiesterases, and binds to G protein-coupled cAMP receptors6,7. A widely used assay to analyze the mechanisms involved in the developmental cycle of the lower eukaryote Dictyostelium discoideum is based on the observation of cell aggregation in submerged conditions8,9. This protocol describes the analysis of the role of coronin A in the developmental cycle by starvation in tissue-culture plates submerged in balanced salt solution (BSS)10. Coronin A is a member of the widely conserved protein family of coronins that have been implicated in a wide variety of activities11,12. Dictyostelium cells lacking coronin A are unable to form multicellular aggregates, and this defect can be rescued by supplying pulses of cAMP, suggesting that coronin A acts upstream of the cAMP cascade10. The techniques described in these studies provide robust tools to investigate functions of proteins during the initial stages of the developmental cycle of Dictyostelium discoideum upstream of the cAMP cascade. Therefore, utilizing this aggregation assay may allow the further study of coronin A function and advance our understanding of coronin biology.

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

The social amoeba Dictyostelium discoideum undergoes a developmental transition into a multicellular organism when starved. The evolutionary conserved protein coronin A plays a crucial role in the initiation of development. Using aggregation assays as our main method, we aim to elucidate the role of coronin A in early development.

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...