Name: long-term, high-resolution confocal time lapse imaging of arabidopsis cotyledon epidermis during germination
Uploaded: 2012-12-31T14:00:00
Description: Watch this Scientific Journal Video about Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination Video at JoVE.com

Vi takker Amanda Rychel for bistand til at udvikle tidsforskydningen protokollen og Lynn Pillitteri til konstruktion POLAR :: POLAR-eGFP. Vi er også taknemmelige for ABRC til at forsyne pm-rb-konstruktion. Denne protokol blev udviklet gennem en støttegruppe fra PRESTO pris fra Japan Videnskab, Teknologi og agenturet. Forskning i POLAR blev også støttet af University of Washington Royalty Research Fund (RRF-4098) og National Science Foundation (MCB-0.855.659). KMP er en NSF Graduate Research Fellow (DGE-0.718.124), og KUT er en HHMI-GBMF investigator.

1. Seed Sterilisation <ol><li> Fremstille frø sterilisering opløsning: 33% husholdningsblegemiddel, 0,1% Triton X-100. </li><li> Sted frø bærer den ønskede fluorescerende reporterkonstruktion (er) og genotype (er) i 1,7 ml rør og anvende 1 ml sterilisering opløsning. Inkuber på nutator i 15 minutter. </li><li> I en steril hætte, anvende en pipette til at fjerne sterilisering opløsningen fra røret, forlader frø bagefter. Skyl med 1 ml sterilt vand. Gentages fire gange. </li><li> Inkuber ved 4 ° C i to dage...

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Denne time-lapse konfokal teknik tillader longitudinelle studier af fluorescens mærket protein udtryk og lokalisering i individuelle celler i Arabidopsis cotyledon epidermis, som i tilfælde af Polar og andre dynamisk skiftende proteiner er afgørende for en korrekt forståelse af deres funktion. Tidligere har forlænget tid bortfalder billeddannelse blevet anvendt til at undersøge Arabidopsis root svampeinfektion 11 og meristem vækst 5,12, men tilsætning af cotyledon epidermi...

<table border="1"><tbody><tr><td> Navn på reagens </td><td> Firma </td><td> Catalog Number </td><td> Kommentarer </td></tr><tr><td> Bacto agar </td><td> BD </td><td> 214.010 </td><td></td></tr><tr><td> One-kammer slide </td><td> Nunc (Thermo Scientific) </td><td> 155.360 </td><td> Eller to-kammer (155.379) </td></tr><tr><td> Laserscanning konfokal mikroskop </td><td> Zeiss </td><td> LSM700 </td><td> Zen 2009 software </td></tr><tr><td> 20x objektiv </td><td> Zeiss </td><td> 420650-9901 </td><td> NA 0,8, Plan-APOCHROMAT </td></tr><tr><td> Dissektionsmikroskop </td><td> Benz (National) </td><td> 431TBL </td><td> Lyser nedefra </td></tr><tr><td> # 5 pincet, biologi tip </td><td> Roboz Surgical Instrument </td><td> RS-4978 </td><td> Meget fine tipser kritiske </td></tr></tbody></table>

long-term, high-resolution confocal time lapse imaging of arabidopsis cotyledon epidermis during germination

Imaging in vivo dynamik cellulær adfærd gennem en udviklingsmæssig sekvens kan være en kraftfuld teknik til at forstå mekanikken i væv mønster. Under animalsk udvikling forekommer centrale celleproliferation og mønsterdannende begivenheder meget hurtigt. For eksempel alle de celledelinger nødvendige for larvestadiet krop planen i Caenorhabditis elegans er afsluttet inden for seks timer efter befrugtningen, med syv mitotiske cyklus 1; de seksten eller flere mitose af Drosophila embryogenese forekommer hos mindre end 24 timer 2. I modsætning hertil er celledelinger under plantens udvikling langsom, typisk i størrelsesordenen af en dag 3,4,5. Dette indebærer en enestående udfordring og et behov for langsigtet direkte billedvisning til at dokumentere dynamiske opførsler af celledeling og differentiering begivenheder i løbet plante organogenesen. Arabidopsis epidermis er en fremragende model system til undersøgelsesdommere signalering, celle skæbne og udvikling i planter. I kotyledonen består dette væv af luft-og vand-resistente fortovet celler spækket med jævnt fordelt stomata, ventiler, der åbner og lukker for at kontrollere gasudveksling og vandtab. Korrekt afstand af disse stomata er kritisk for deres funktion, og deres udvikling følger en sekvens af asymmetrisk opdeling og celledifferentiering trin til at producere den organiserede epidermis (fig. 1). Denne protokol tillader observation af celler og proteiner i epidermis over flere dage for udvikling. Denne tidsramme gør præcis dokumentation af stamcelle divisioner og differentiering af epidermale celler, herunder stomata og epidermale fortov celler. Fluorescerende proteiner kan fusioneres til proteiner af interesse at vurdere deres dynamik ved celledeling og differentieringsprocesser. Denne teknik gør det muligt for os at forstå lokalisering af et nyt protein, POLAR 6, under spredning etape af stomatal-afstamningsceller i the Arabidopsis cotyledon epidermis, når det udtrykkes i celler forud asymmetriske division events og flytter til en karakteristisk område af cellen cortex kort før opdeling finder sted. Billeder kan registreres og strømlinet video nemt produceret ved hjælp af public domain software til at visualisere dynamisk protein lokalisering og celletyper, som de ændrer sig over tid.

<html> Et sæt af informative tidspunkter opsamlet med denne fremgangsmåde er vist i figur 3.. Cellemembraner er mærket med RFP (pm-rb) og GFP er fusioneret til POLAR protein under dets native promotor (POLAR :: POLAR-GFP) 6 I 30 minutter tidsskala, ser vi celledelinger sammen med ændringer i protein lokalisering forud for disse. Asymmetriske celledelinger i stomatal afstamning formular stamcelle-lignende stomatale forstadier kaldet meristemoids, som bevarer evnen til at gennemgå ...

Watch this Scientific Journal Video about Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination Video at JoVE.com

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination Video (Video) | JoVE

Vi beskriver en protokol med kammer dias og medier til at immobilisere plante kimbladene for konfokal afbildning af epidermis over flere dages udvikling, som dokumenterer stomatal differentiering. Fluorophor-mærkede proteiner kan spores dynamisk ved ekspression og subcellulær lokalisering, øget forståelse for deres mulige roller ved celledeling og celle-type differentiering.

Langsigtet, høj opløsning Konfokal Time Lapse Imaging af<em&gt; Arabidopsis Kimblad</em&gt; Epidermis under spiring

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Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

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