Name: spatio-temporal manipulation of small gtpase activity at subcellular level and on timescale of seconds in living cells
Uploaded: 2012-03-09T13:00:00
Description: Watch this Scientific Journal Video about Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells at JoVE.com

Denne undersøgelse blev støttet af NIH finansiering af forskning (DK090868 og GM092930 til TI). Der er en verserende patent på et indhegnet rapamycin analog.

1. Transfektion af plasmid DNA <ol><li> Tilsættes plasmid-DNA&#39;er, 0,5 ug membran-bundne FRB (herefter benævnt LDR) og 0,5 ug FKBP-Tiam1 (T-cellelymfom invasion og metastase-inducerende protein 1: Rac aktivator) mærket med fluorescerende protein til 37,5 gl dH 2 O. Tilsæt 1 gl FuGENE HD. </li><li> Vortex, og der inkuberes ved stuetemperatur i 20 minutter. I mellemtiden, skal du fortsætte til trin 1,3-1,8. </li><li> Vaske hver brønd i en 8-brønds kammer med 50 ul poly-D-lysin. </li><li> Vask i midt...

<ol>
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Beskrev vi en teknik, der anvender en hidtil ukendt bur forbindelse sammen med FKBP-FRB heterodimerisering systemet for at manipulere Rho GTPase-aktivitet ved en præcis subcellulære lokalisering på en tidsskala sekunder. Der er tre begrænsninger af den foreliggende fremgangsmåde. For det første fordi metoden er baseret på at forhindre eller tillade diffusion af dimerizer over plasmamembranen, målet cellulære placering skal være plasmamembranen eller dens nærhed. Yderligere optimer...

<table border="1"><tbody><tr><td> Navn på reagenset / udstyr </td><td> Firma </td><td> Katalog nummer </td></tr><tr><td> Rapamycin </td><td> Tecoland </td><td> RAPA99 </td></tr><tr><td> FuGENE HD </td><td> Roche </td><td> 04709691001 </td></tr><tr><td> Avidin </td><td> Sigma </td><td> A9275-10mg </td></tr><tr><td> DMSO </td><td> Sigma </td><td> D2650-5X5ML </td></tr><tr><td> Størrelse udelukkelse kolonne </td><td> GE Healthcare </td><td> Spin Trap G-25 </td></tr><tr><td> Glas bund 8-godt kammer </td><td> Thermo Scientific </td><td> 12-565-47 </td></tr><tr><td> Inverted Fluorescensmikroskop </td><td> Zeiss </td><td> Axiovert200M </td></tr><tr><td> UV-LED-lyskilde </td><td> Rapp Opto Elektronik </td><td> UVILED </td></tr><tr><td> Konfokal roterende skive </td><td> Yokogawa Electric Corp </td><td> CSU10 </td></tr><tr><td> CCD-kamera </td><td> Hamamatsu Photonics </td><td> ORCA-ER </td></tr><tr><td> 100x Objektivlinsen </td><td> Zeiss </td><td> Plan Apochromat </td></tr></tbody></table> Syntetisk ordning CRb (Copyright ACS2011) 6 <a href="/files/ftp_upload/3794/3794scheme1_lg.jpg"><img src="/files/ftp_upload/3794/3794scheme1.jpg" alt="Skema 1" /></a> Syntetiske betingelser: (a) BnBr, K2CO 3, DMF (b) f.HNO 3, AcOH (c) TFA (d) propargyl-Br, K2CO 3, DMF, (e) glycerol, kat. PTSA, toluen (f) NaBH3CN, TiCl4, MeCN (g) Tf H2O, pyridin (h) methanol HCI, (i) LiAIH4, THF (j) TsCI, pyridin, CH 2Cl 2 (k) NaN 3, DMF (l) 8 2,6-di-t-butylpyridin, CH2 Cl2 (m) 13, CuSO4, ascorbat, 2-propanol, H2O, CH2 Cl2.

spatio-temporal manipulation of small gtpase activity at subcellular level and on timescale of seconds in living cells

Dynamisk regulering af Rho familie af små guanosin triphosphatases (GTPaser) med stor spatiotemporal præcision er nødvendig for forskellige cellulære funktioner og arrangementer 1, 2. Deres spatiotemporally dynamiske karakter er blevet afsløret ved hjælp af visualisering af deres aktivitet og lokalisering i realtid 3. For at opnå en dybere forståelse af deres roller i forskellige cellulære funktioner på det molekylære niveau, bør næste skridt være forstyrrelse af protein aktiviteter på en præcis subcellulære placering og timing. For at nå dette mål, har vi udviklet en fremgangsmåde til lysinduceret, spatio-tidsmæssigt styret aktivering af små GTPaser ved at kombinere to teknikker: (1) rapamycin-induceret FKBP-FRB heterodimerisering og (2) en foto-bure metode rapamycin. Med anvendelse af rapamycin-medieret FKBP-FRB heterodimerisering, har vi udviklet en fremgangsmåde til hurtig inducerbare aktivering eller inaktivering af små GTPaser including Rac 4, Cdc42 4, RhoA 4 og Ras 5, hvor rapamycin inducerer translokation af FKBP-kondenserede GTPaser, eller deres aktivatorer, til plasmamembranen, hvor FRB er forankret. Til kobling med dette heterodimerisering system, har vi også udviklet en foto-bure system rapamycin analoger. Et foto-bur forbindelsen er et lille molekyle, hvis aktivitet er undertrykt med en fotospaltelig beskyttende gruppe kendt som en anbringelse i bur gruppe. At undertrykke heterodimerisering aktivitet fuldstændigt vi udviklet en bur rapamycin, som er bundet til et makromolekyle, således at den resulterende store komplekset ikke kan krydse plasmamembranen, hvilket fører til praktisk talt ingen baggrund aktivitet som en kemisk dimerizer i celler 6. Figur 1 illustrerer et skema i vores system. Med kombinationen af ​​disse to systemer, lokalt har vi ansat en Rac aktivator til plasmamembranen på en tidsskala sekunder og opnåede lys-induceret Rac aktivering på det subcellulære Level 6.

Watch this Scientific Journal Video about Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells at JoVE.com

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

En fremgangsmåde til spatio-temporal kontrol lille GTPase-aktivitet af lys, er beskrevet. Denne metode er baseret på rapamycin-induceret FKBP-FRB heterodimerisering og foto-bure systemer. Optimering af lys-bestråling muliggør spatio-tidsmæssigt styret aktivering af små GTPaser på det subcellulære niveau.

Spatio-tidsmæssige Manipulation af Lille GTPase aktivitet på Subcellulær niveau og på Tidsplan for Seconds i levende celler

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

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