The authors would like to thank Drs. C. Szalinski, H. Teng, and M. Bruchez for help with Imaris, R. Vistein, and D. Shiwarski for technical help and advice, and Dr. M von Zastrow, Dr. T Kirchhausen, Dr. D Drubin, and Dr. W Almers for reagents and helpful discussion. Funding provided by T32 grant NS007433 to SLB and NIH DA024698 and DA036086 to MAP.

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The authors have nothing to disclose.

Hier beschreiben wir den Einsatz von TIRFM zu Clathrin-vermittelte Endozytose (CME) auf der Ebene der einzelnen CCPs in lebenden Zellen in Echtzeit zu visualisieren. CME ist eine schnelle und hochdynamische Veranstaltung durch die kumulative Wirkung vieler räumlich und zeitlich getrennte Einzelveranstaltungen vermittelt. Die meisten Assays, die gegenwärtig verwendet werden, wie zum Beispiel biochemische Messungen der Internalisierung mit Oberflächen Biotinylierung oder Liganden-Bindung, Durchflusszytometrie oder fixi...

Der Prozess der Clathrin-vermittelte Endozytose (CME) ist abhängig von der rechtzeitigen Ankunft der vielen Komponenten der Clathrin-vermittelten endozytischen Maschinen, um Fracht in die Vesikel 1-3 sammeln und die Plasmamembran zu manipulieren. CME ist durch Membran Verformung und Fracht-Adapterproteine, die an werdende Stellen der Endozytose 1 zusammen kommen eingeleitet. Diese Proteine ​​rekrutieren das Hüllprotein Clathrin, die in eine käfigartige Struktur, die die Clathrin-beschichteten Nadel (KPCh) 4 bildet versammelt. Nachdem die KPCh ist vollständig in eine Kugelform, Membranspaltung, vor allem durch Maßnahmen der großen GTPase, Dynamin montiert, erzeugt freie Clathrin-umhüllten Vesikeln (CCV) 5,6. Diese Internalisierung triggert schnelle Demontage der Clathrin-Mantel, so dass für mehrere Runden von CME Komponenten wiederverwendet werden. Die Entdeckung und Charakterisierung der Proteine ​​in CME beteiligt wurde in der traditionellen Bioch verwurzeltemical, genetischen und mikroskopischen Techniken 4-6,8. Diese Tests haben die Rollen und Interaktionspunkte dieser endozytischen Komponenten erläutert. Obwohl sehr nützlich zur Definition von wesentlichen Komponenten Handel Maschinen sind diese Tests sehr bei der Erfassung des dynamischen Verhaltens von CME Komponenten oder Ladungskonzentration begrenzt. Dies ist eine kritische Einschränkung, da CME wird durch die Anordnung choreographiert von Sätzen von Protein-Module in definierten Schritten angetrieben wird, und da kleine Veränderungen in der Dynamik der einzelnen endocytic Ereignisse große kumulativen Auswirkungen auf die Endozytose haben. Ferner bisherigen Daten zeigen, dass einzelne CCP kann sowohl in ihrer Zusammensetzung und in ihrem Verhalten unterscheiden, was darauf hindeutet, dass die physiologische Regulation dieses Verfahren ist sehr räumlich und zeitlich eingeschränkt 9-14. Visualisierung einzelner endozytischen Ereignisse ist daher wichtig zu verstehen, warum es mehrere redundante Proteine ​​in CME beteiligt und wie diese Proteine ​​könnte gesteuert werden by physiologische Signale, um Fracht Internalisierung regulieren. Hier beschreiben wir den Einsatz von Total Internal Reflection Fluorescence Microscopy (TIRFM) CME auf der Ebene der Dynamik der einzelnen CCPs in lebenden Zellen zu beobachten. TIRFM beruht auf dem Unterschied im Brechungsindex zwischen dem Deckglas und der Fluidumgebung der Zellen 15,16. Wenn das Anregungslicht auf die Zellen zu mehr als dem kritischen Winkel gerichtet ist, wird intern reflektiert, wodurch eine abklingende Welle, die einen dünnen Bereich der Beleuchtung, der sich ungefähr 100 nm oberhalb des Deckglases beibehält. Dies stellt sicher, dass nur die Fluoreszenzmoleküle in diesem engen Bereich angeregt werden. Praktisch kann dies die Anregung von Fluoreszenzmolekülen auf oder in der Nähe der Plasmamembran, und minimiert die Fluoreszenz von den inneren Teilen der Zelle. Dies stellt einen wesentlich höheren Signal-zu-Rausch-Verhältnis und z-Achsenauflösung auf Ereignisse in der Plasmamembran, CO visualisierenzu häufig verwendeten Modi wie herkömmliche Epifluoreszenz oder konfokaler Fluoreszenzmikroskopie mpared. Wir beschreiben auch, zu einem Einführungs und praktischen Ebene, um die Verwendung einer gängigen Bildanalyse-Plattform zu analysieren und zu quantifizieren einfache morphologische Merkmale und Dynamik der einzelnen Lade endozytischen Veranstaltungen.

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		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Name of Material/ Equipment</td>
			<td style="width:180px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:207px;">Comments/Description</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">DMEM/High Glucose with L-glutamine and sodium pyruvate</td>
			<td style="width:180px;">Fisher Scientific&#160;</td>
			<td>SH3024301</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Dulbecco&#39;s Phosphate Buffered Saline (DPBS), no calcium, no magnesium</td>
			<td style="width:180px;">Gibco, by Life Technologies</td>
			<td>21600-010</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">EDTA Free Acid</td>
			<td style="width:180px;">Amresco</td>
			<td style="width:157px;">0322-500G</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Fetal Bovine Serum</td>
			<td style="width:180px;">Gibco, by Life Technologies</td>
			<td style="width:157px;">10437-028</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Leibovitz&#39;s L-15 Medium, no phenol red</td>
			<td style="width:180px;">Gibco, by Life Technologies</td>
			<td>21083-027</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Opti-MEM</td>
			<td style="width:180px;">Gibco, by Life Technologies</td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">HEPES</td>
			<td style="width:180px;">CellPURE by Fisher Scientific&#160;</td>
			<td>BP2937-100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Effectene&#160;</td>
			<td style="width:180px;">Qiagen</td>
			<td style="width:157px;">301425</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">25 millimeter coverglass</td>
			<td style="width:180px;">Fisher Scientific&#160;</td>
			<td>12-545-86&#160;</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Corning cell culture treated flasks, 25cm2</td>
			<td style="width:180px;">Fisher Scientific&#160;</td>
			<td>10-126-28</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Cell culture 6-well plate</td>
			<td style="width:180px;">Greiner Bio-One, by VWR</td>
			<td>82050-896</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Monoclonal ANTI-FLAG M1</td>
			<td style="width:180px;">Sigma Aldrich</td>
			<td>F3040-5MG</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">[D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt (DAMGO)</td>
			<td style="width:180px;">Sigma Aldrich</td>
			<td>E7384-5MG</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Alexa Fluor 647 Protein Labeling Kit</td>
			<td style="width:180px;">Life Technologies</td>
			<td style="width:157px;">A20173</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">ImageJ</td>
			<td style="width:180px;">NIH</td>
			<td style="width:157px;"></td>
			<td>http://rsb.info.nih.gov/ij/</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Imaris Image analysis software</td>
			<td style="width:180px;">BitPLane</td>
			<td style="width:157px;"></td>
			<td>http://www.bitplane.com/imaris/imaris, for automated analysis</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Nikon Eclipse Ti inverted microscope and required accessories including filter cubes and filters</td>
			<td style="width:180px;">Nikon</td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Nikon TIRF arm with required adapters for Nikon Eclipse Ti</td>
			<td style="width:180px;">Nikon</td>
			<td style="width:157px;"></td>
			<td>For adjusting angle of incidence</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">iXon+ EMCCD camera and adapters</td>
			<td style="width:180px;">Andor</td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Verwendung lebender Zellen TIRF Mikroskopie wir die endozytischen Dynamik des μ-Opioid-Rezeptor (MOR) aufgezeichnet ist, ein G-Protein-gekoppelten Rezeptor (GPCR) und deren endocytic Adapterprotein β-Arrestin. Die β-Arrestin-Konstrukt wurde vorübergehend in einem stabil exprimieren MOR-Zelllinie unter Verwendung des Protokolls in Abbildung 1 skizziert transfiziert und 96 Stunden später abgebildet. Die MOR in der stabilen Zelllinie ist N-terminal mit einem pH-sensitive GFP markiert. Diese fluoreszie...

Watch this Scientific Journal Video about Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy at JoVE.com

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

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.

Visualisierung Clathrin-vermittelte Endozytose von G-Protein-gekoppelten Rezeptoren an Einzelereignis Resolution über TIRF-Mikroskopie

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

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Research

JoVE Journal

Biology

80.1K Aufrufe.  Carnegie Mellon University. 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. 

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

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43&reg;) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

Imaging Exocytose in Retinal bipolaren Zellen mit TIRF-Mikroskopie

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

Forschung

Biologie

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Visualizing Einzel-Molekül-DNA-Replikation mit Fluoreszenz-Mikroskopie

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

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.

In vivo Quantifizierung der G Protein-gekoppelten Rezeptor-Wechselwirkungen mit spektral aufgelöste Zwei-Photonen-Mikroskopie

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

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

In-vivo-Imaging von Intact Drosophila-Larven auf subzellulärer Auflösung

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

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.

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-gekoppelten Rezeptor (GPCR)-vermittelten Signaltransduktion, die Steuerung der Chemotaxis Dictyostelium discoideum</em

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

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.

Die Quantifizierung der Wirkung als Agonist am G-Protein-gekoppelte Rezeptoren

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 ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

Visualisierung Bakterien in Nematoden durch Fluoreszenzmikroskopie

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

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.

Genetisch kodierte Molecular Probes, um G-Protein-gekoppelte Rezeptoren Studieren

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

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques &#8211; stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 &#181;m from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

Super-Resolution Imaging der zytokinetischen Z-Ring im Live-Bakterien mit Schnelle 3D-Structured Illumination Microscopy (F3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

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).

Charakterisierung von G-Protein gekoppelten Rezeptoren durch eine Fluoreszenz-basierte Calciummobilisierungsassay

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