Name: imaging g-protein coupled receptor (gpcr)-mediated signaling events that control chemotaxis of dictyostelium discoideum
Uploaded: 2011-09-20T12:00:00
Description: Watch this Scientific Journal Video about Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum at JoVE.com

Detta arbete stöds av intramural fonden NIAID, NIH.

1. Beredning av kemotaxi behöriga celler Dictyostelium discoideum <ol><li> För att generera D. discoideum celler som kemotaxi till chemoattractant cAMP, skörd celler växer i D3-T-rik media från en skakning kultur vid 22 ° C. </li><li> Tvätta cellerna två gånger i icke-näringsämnen utvecklings-buffert (DB buffert innehållande 5 mM Na 2 HPO 4, 5 mm KH 2 PO 4, 2 mm MgCl 2 och 0,1 mM CaCl 2). </li><li> Återsuspendera cellerna i BF buff...

<ol>
	<li>Lijima, M., Huang, Y. E., Devreotes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+spatial+regulation+of+chemotaxis.">Temporal and spatial regulation of chemotaxis.</a> Dev Cell. 3, 469-469 (2002).</li><li>Murphy, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+biology+of+leukocyte+chemoattractant+receptors.">The molecular biology of leukocyte chemoattractant receptors.</a> Annu Rev Immunol. 12, 593-593 (1994).</li><li>Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+protein-linked+signaling+pathways+control+the+developmental+program+of+Dictyostelium.">G protein-linked signaling pathways control the developmental program of Dictyostelium.</a> Neuron. 12, 235-235 (1994).</li><li>Jin, T., Xu, X., Hereld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis,+chemokine+receptors+and+human+disease.">Chemotaxis, chemokine receptors and human disease.</a> Cytokine. 44, 1-1 (2008).</li><li>Meier-Schellersheim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Key+role+of+local+regulation+in+chemosensing+revealed+by+a+new+molecular+interaction-based+modeling+method.">Key role of local regulation in chemosensing revealed by a new molecular interaction-based modeling method.</a> PLoS Comput Biol. 2, e82-e82 (2006).</li><li>Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+mechanism+of+a+GPCR+and+a+heterotrimeric+G+protein+during+chemoattractant+gradient+sensing+in+Dictyostelium.">Coupling mechanism of a GPCR and a heterotrimeric G protein during chemoattractant gradient sensing in Dictyostelium.</a> Sci Signal. 3, 71-71 (2010).</li><li>Xu, X., Meier-Schellersheim, M., Jiao, X., Nelson, L. E., Jin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+imaging+of+single+live+cells+reveals+spatiotemporal+dynamics+of+multistep+signaling+events+of+chemoattractant+gradient+sensing+in+Dictyostelium.">Quantitative imaging of single live cells reveals spatiotemporal dynamics of multistep signaling events of chemoattractant gradient sensing in Dictyostelium.</a> Mol Biol Cell. 16, ra71-ra71 (2005).</li><li>Xu, X., Meier-Schellersheim, M., Yan, J., Jin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locally+controlled+inhibitory+mechanisms+are+involved+in+eukaryotic+GPCR-mediated+chemosensing.">Locally controlled inhibitory mechanisms are involved in eukaryotic GPCR-mediated chemosensing.</a> J. Cell Biol. 178, 141-141 (2007).</li><li>Jin, T., Zhang, N., Long, Y., Parent, C. A., Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+the+G+protein+betagamma+complex+in+living+cells+during+chemotaxis.">Localization of the G protein betagamma complex in living cells during chemotaxis.</a> Science. 287, 1034-1034 (2000).</li><li>Janetopoulos, C., Jin, T., Devreotes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor-mediated+activation+of+heterotrimeric+G-proteins+in+living+cells.">Receptor-mediated activation of heterotrimeric G-proteins in living cells.</a> Science. 291, 2408-2408 (2001).</li><li>Funamoto, S., Milan, K., Meili, R., Firtel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+phosphatidylinositol+3'+kinase+and+a+downstream+pleckstrin+homology+domain-containing+protein+in+controlling+chemotaxis+in+dictyostelium.">Role of phosphatidylinositol 3' kinase and a downstream pleckstrin homology domain-containing protein in controlling chemotaxis in dictyostelium.</a> J. Cell Biol. 153, 795-795 (2001).</li><li>Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+PLC-beta2+and+-beta3+and+PI3Kgamma+in+chemoattractant-mediated+signal+transduction.">Roles of PLC-beta2 and -beta3 and PI3Kgamma in chemoattractant-mediated signal transduction.</a> Science. 287, 1046-1046 (2000).</li><li>Sasaki, A. T., Chun, C., Takeda, K., Firtel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+Ras+signaling+at+the+leading+edge+regulates+PI3K,+cell+polarity,+and+directional+cell+movement.">Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement.</a> J. Cell Biol. 167, 505-505 (2004).</li><li>Meili, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoattractant-mediated+transient+activation+and+membrane+localization+of+Akt/PKB+is+required+for+efficient+chemotaxis+to+cAMP+in+Dictyostelium.">Chemoattractant-mediated transient activation and membrane localization of Akt/PKB is required for efficient chemotaxis to cAMP in Dictyostelium.</a> EMBO J. 18, 2092-2092 .</li><li>Parent, C. A., Blacklock, B. J., Froehlich, W. M., Murphy, D. B., Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+protein+signaling+events+are+activated+at+the+leading+edge+of+chemotactic+cells.">G protein signaling events are activated at the leading edge of chemotactic cells.</a> Cell. 95, 81-81 (1998).</li><li>Funamoto, S., Meili, R., Lee, S., Parry, L., Firtel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+regulation+of+3-phosphoinositides+by+PI+3-kinase+and+PTEN+mediates+chemotaxis.">Spatial and temporal regulation of 3-phosphoinositides by PI 3-kinase and PTEN mediates chemotaxis.</a> Cell. 109, 611-611 (2002).</li><li>Iijima, M., Devreotes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+suppressor+PTEN+mediates+sensing+of+chemoattractant+gradients.">Tumor suppressor PTEN mediates sensing of chemoattractant gradients.</a> Cell. 109, 599-599 (2002).</li><li>Haastert, P. J. V. a. n., Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis:+signalling+the+way+forward.">Chemotaxis: signalling the way forward.</a> Nat Rev Mol Cell Biol. 5, 626-626 (2004).</li></ol>

Processerna för att nå kemotaxi behöriga skede av celler För vilda typ D. discoideum celler, det tar ca 5 ~ 6 timmar pulserande utveckling i rumstemperatur för att förmå dem till en väl kemotaxi behöriga etapp under vilken celler som visar ett väl polariserad cellulär morfologi och snabb cell migration (Fig. 1). Flera faktorer, såsom cAMP-koncentrationen för pulserande, temperatur och olika genetiska bakgrunder, kan påverka processen att nå kemotaxi behöriga skede. Ett ...

<table border="1"><tbody><tr><th> Reagensets namn </th><th> Företag </th><th> Katalognummer </th><th> Kommentarer </th></tr><tr><td> D3-T Tillväxt Media </td><td> KD Medicinska </td><td></td><td></td></tr><tr><td> Koffein </td><td> Sigma </td><td></td><td></td></tr><tr><td> Latrunculin B </td><td> Molekylära sonder </td><td></td><td></td></tr><tr><td> Alexa 594 </td><td> Molekylära sonder </td><td></td><td></td></tr><tr><td> cAMP </td><td> Sigma </td><td></td><td></td></tr><tr><td> ChronTrol XT programmerbar timer </td><td> ChronTrol Corp </td><td></td><td></td></tr><tr><td> Miniplus 3 peristaltiska pumpen </td><td> Gilbson </td><td></td><td></td></tr><tr><td> Plattform rotationsskak </td><td></td><td></td><td></td></tr><tr><td> FemtoJet microcapillary trycket försörjning </td><td> Eppendorf </td><td></td><td></td></tr><tr><td> Enkel-och fyra-och Lab-Tek II coverglass kammare </td><td> Nalge Nunc International </td><td></td><td></td></tr><tr><td> LSM 510 META eller motsvarande fluorescerande mikroskop </td><td> Zeiss </td><td></td><td> en 40X 1,3 NA eller 60X 1,4 NA olja DIC Plan-Neofluar objektiv </td></tr><tr><td> Olympus X81 eller motsvarande </td><td> Olympus </td><td></td><td> Kräver en 100X 1,47 NA TIRF objektiv </td></tr></tbody></table>

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

Många eukaryota celler kan upptäcka gradienter av kemiska signaler i sina miljöer och migrera detta 1. Denna guidade cell migration som avses kemotaxi, vilket är viktigt för olika celler att utföra sina funktioner såsom handel med immunceller och mönstring av neuronala celler 2, 3. En stor familj av G-proteinkopplade receptorer (GPCRs) känner variabel små peptider, som kallas kemokiner, direkt cell migration in vivo 4. Det slutliga målet för kemotaxi forskning är att förstå hur en GPCR maskiner sinnen kemokinreceptorantagonist lutningar och kontroller signalering händelser som ledde till chemotaxis. För detta ändamål använder vi avbildningstekniker för att övervaka i realtid, Spatiotemporal koncentrationer av chemoattractants, cellens rörelse i en lutning på chemoattractant, medierad GPCR aktivering av heterotrimeric G-protein, och intracellulär signalering skeenden inblandade i chemotaxis av eukaryota celler 5-8 . Den enkla eukaryot organism, Dictyostelium discoideum visar chemotaxic beteenden som liknar dem av leukocyter, och D. discoideum är ett viktigt modellsystem för att studera eukaryota chemotaxis. Som fritt levande amöbor, D. discoideum celler delar sig i rika medium. Vid svält, celler ange ett utvecklings-program där de sammanlagda via cAMP-medierad chemotaxis att bilda multicullular strukturer. Många komponenter som chemotaxis till lägret har identifierats i D. discoideum. Bindningen av cAMP till en GPCR (car1) inducerar dissociation av heterotrimeric G-proteiner i Gγ och subenheter Gβγ 7, 9, 10. Gβγ subenheter aktivera Ras, som i sin tur aktiverar PI3K, konvertering PIP 2 till PIP 3 på cellmembranet 11-13. PIP-3 fungerar som bindningsställen för proteiner med pleckstrin homologi (PH) domäner, vilket rekrytera dessa proteiner till membran 14, 15. Aktivering av car1 receptorer styr också membranet sammanslutningar av PTEN som dephosphorylates PIP 3 till PIP-2 16, 17. De molekylära mekanismerna är evolutionärt bevarade i kemokinreceptorantagonist GPCR-medierad chemotaxis av mänskliga celler, såsom neutrofiler 18. Vi presenterar följande metoder för att studera chemotaxis av D. discoideum celler. 1. Beredning av kemotaxi komponent celler. 2. Imaging chemotaxis av celler i ett läger lutning. 3. Övervakning en GPCR inducerad aktivering av heterotrimeric G-protein i enstaka levande celler. 4. Imaging chemoattractant-styrd dynamisk PIP 3 svar i enskilda levande celler i realtid. Vår utvecklade imaging metoder kan tillämpas för att studera chemotaxis av humana leukocyter.

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

Här beskriver vi detaljerat leva metoder cell imaging för att undersöka chemotaxis. Vi presenterar fluorescens mikroskopiska metoder för att övervaka Spatiotemporal dynamik signalering händelser i vandrande celler. Mätning av signalering händelser som tillåter oss att ytterligare förstå hur en GPCR-signalnätet uppnår lutning avkänning av chemoattractants och kontroller riktad migration av eukaryota celler.

Imaging G-proteinkopplade receptor (GPCR)-medierad signalering Händelser som Kontroll chemotaxis av 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 ...

Research

JoVE Journal

Biology

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

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

In vivo Kvantifiering av G Tillsammans Interaktioner protein receptor med spektralt korrigerats inom två-photon Mikroskopi

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

Kvantifiera agonist vid G-protein-kopplade receptorer

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

FRET mikroskopi för realtidsövervakning av signalering händelser i levande celler Använda unimolekylära Biosensorer

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

Levande cell imaging av Early autophagy Evenemang: Omegasomes och 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 ...

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

Genetiskt kodade molekylära sonder för att studera G-proteinkopplade receptorer

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

Karaktärisering av G-proteinkopplade receptorer av en fluorescensbaserad kalciummobilisering Assay

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

Visualisera clathrin medierad endocytos av G-proteinkopplade receptorer på Single-event upplösning via TIRF Mikroskopi

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

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

Undersöker funktion Coronin A i tidig Svält svar av<em&gt; Dictyostelium discoideum</em&gt; Genom aggregering Analyser

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

Imaging G-proteinkopplad receptor-medierad kemotaxi och dess signaleringshändelser i neutrofil-liknande HL60-celler

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

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

Studera Protein system för kvalitetskontroll<em&gt; D. discoideum</em&gt; Använda Temperaturstyrd levande 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 ...