Farging fosfor-epitoper i Cilierte Organs av Whole Mount Sebrafisk embryo
Summary
Teknikker er beskrevet i immunfarging av fosfo-epitoper i hele sebrafisk embryo og deretter gjennomføre to-farge fluorescerende confocal lokalisering i cellulære strukturer så små som primær flimmerhårene. Teknikkene for reparasjon og avbildning kan definere plasseringen og kinetikken til forekomsten eller aktivering av spesifikke proteiner.
Abstract
Den raske proliferasjon av celler, vev-spesifikk ekspresjon av genene og fremveksten av signaleringsnettverk karakter tidlig embryoutvikling av alle virveldyr. De kinetikk og plassering av signaler – også innenfor enkeltceller – i utvikling av embryo utfyller identifisering av viktige utviklings gener. Immunfarging teknikker er beskrevet som har vist seg å definere kinetikken av intracellulære og hele dyre signaler i konstruksjoner så små som primær cilia. Teknikkene for feste, bilde- og prosesserings bilder ved hjelp av en laser-scanning confocal sammensatt mikroskop kan gjennomføres på så få som 36 timer.
Sebrafisk (Danio rerio) er en ønskelig organisme for etterforskere som ønsker å gjennomføre studier i en virveldyrarter som er rimelig og relevant for sykdom hos mennesker. Genetiske knockouts eller knockdowns må bekreftes av tapet av selve proteinprodukt. En slik bekreftelse av proteintapkan oppnås ved hjelp av teknikker som er beskrevet her. Nøkkelen til signalveier kan også påvist ved hjelp av antistoffer som er reaktive med proteiner som er post-translatorisk modifisert ved fosforylering. Bevare og optimalisere den fosforylerte tilstanden til en epitop er derfor kritisk for denne bestemmelse, og oppnås ved denne protokollen.
Denne studien beskrives teknikker for å løse embryoene i løpet av første 72 timer for utvikling og samtidig lokalisere en rekke relevante epitoper med cilier i Kupffer s Vesikkel (KV), nyrene og det indre øret. Disse teknikkene er enkel, krever ikke disseksjon og kan gjennomføres på en relativt kort tidsperiode. Prosjektering konfokal bildestakker inn et enkelt bilde er et nyttig middel til å presentere disse dataene.
Introduction
The techniques described here are the outcome of studies that have sought to define downstream targets of Ca2+ signals during events that occur during early development, including fertilization, gastrulation, somitogenesis and trunk, eye, brain and organ formation.1-3 The original discoveries of embryonic Ca2+ signaling were dependent on the use of natural and engineered Ca2+ indicators, such as aequorin4 and fura-2.5 Even with current technology, the detection of transient elevations of Ca2+ requires cumbersome analytical tools and does not reveal the targets of such Ca2+ signals.
This laboratory investigates Ca2+ signals that act through the Ca2+/calmodulin-dependent (multifunctional) protein kinase known as CaMK-II, an enzyme that is enriched in the central nervous system and originally identified as a regulator of long-term potentiation.6 CaMK-II is not brain-specific, is widely expressed and highly conserved throughout the entire lifespan and bodies of species throughout the animal kingdom, including invertebrates.7,8 CaMK-II has the unique capability of sustaining its own activity even after Ca2+ levels have diminished due to its ability to autophosphorylate at Thr287. In this autophosphorylated state, CaMK-II remains active in a Ca2+/CaM-independent manner, until dephosphorylated.6 Thus, the localization of phosphorylated CaMK-II (Thr287) can identify cells in which natural, relevant Ca2+ elevations have occurred.
An antibody against autophosphorylated (P-Thr287) mammalian CaMK-II has been well-characterized and was initially used to localize activated CaMK-II in brain tissue.9 Zebrafish (Danio rerio) have seven CaMK-II genes10,11 whose protein products contain a sequence of MHRQE[pT287]VECLK in this region.10,11 This sequence is very similar to the phosphopeptide antigen used to create this rabbit polyclonal antibody (MHRQE[pT]VDCLK; Upstate/Millipore) and therefore it was not a complete surprise that this antibody cross-reacted with zebrafish CaMK-II. This laboratory showed that this antibody reacts with zebrafish CaMK-II in proportion to autophosphorylation and Ca2+/CaM-independent activity.12 Additional pan-specific CaMK-II antibodies have also been shown to cross-react with zebrafish CaMK-II.13
This antibody has been used to demonstrate that zebrafish CaMK-II is preferentially activated in cells on one side of the zebrafish Kupffer’s Vesicle (KV), the ciliated organ necessary for establishment of left/right asymmetry.12 This antibody was used to demonstrate that CaMK-II is transiently activated in four adjacent cells on the left side of the KV during the exact same developmental phase that organ positioning is determined.12 In addition to the Kupffer’s Vesicle (KV), autophosphorylated (P-T287) was also located in specific intracellular sites in other ciliated tissues including the kidney, neuromasts, and inner ear.12,13 In the zebrafish kidney, P-T287-CaMK-II is enriched along the apical border of ciliated ductal cells and within cloacal cilia where it influences their assembly.13 Finally, in the developing inner ear, P-T287-CaMK-II is intensely concentrated at the base of cilia and influences cell differentiation through the Delta-Notch signal pathway.14 In summary, the detection of activated CaMK-II has pinpointed sites of intracellular Ca2+ release and illuminated potential new signaling pathways.
These discoveries were completely dependent on developing a sensitive and accurate method to localize activated (P-T287-autophosphorylated) CaMK-II. The methods to fix and immunostain the zebrafish KV, kidney and inner ear are described. The limitations of this technique are also described. These techniques should be useful to any investigator who seeks to obtain high-resolution images in two fluorescent channels of not just phospho-epitopes, but any epitope, during early vertebrate development.
Protocol
Representative Results
Discussion
PFA / metanol-metoden ble utviklet i dette laboratoriet med hovedmål å optimalisere immunolocalization av fosfor-T 287 -CaMK-II epitop i sebrafisk utvikling. Denne metoden med hell lokaliserte P-CaMK-II under dannelsen av flere cilierte organer, inkludert sebrafisk KV, 12 indre øret 14 og nyre. 13 Spesielt ved KV stadium, denne teknikken var nødvendig. Suksessen med denne metoden er sannsynligvis på grunn av en kombinasjon av a) minimalisering av autofluorescens, b) bevar…
Declarações
The authors have nothing to disclose.
Acknowledgements
Dette arbeidet ble støttet av National Science Foundation tilskudd IOS-0817658.
Materials
| 1-phenyl-2-thiourea (PTU) | Sigma | P-7629 | 0.12% Stock solution. Dilute 1:40 in system water |
| Alexa488 anti-mouse IgG | Life Technologies | A11001 | Goat polyclonal, use at 1:500 |
| Alexa488 anti-rabbit IgG | Life Technologies | A11008 | Goat polyclonal, use at 1:500 |
| Alexa488 phalloidin | Life Technologies | A12379 | Preferentially binds to F-actin |
| Alexa568 anti-mouse IgG | Life Technologies | A11004 | Goat polyclonal, use at 1:500 |
| Alexa568 anti-rabbit IgG | Life Technologies | A11011 | Goat polyclonal, use at 1:500 |
| anti-acetylated a-tubulin | Sigma | T7451 | Mouse monoclonal, use at 1:500 |
| anti-phospho-T287 CaMK-II | EMD Millipore | 06-881 | Rabbit polyclonal, use at 1:20 |
| anti-total CaMK-II | BD Biosciences | 611292 | Mouse monoclonal, use at 1:20 |
| Ethanol | Fisher | S96857 | Lab grade, 95% denatured |
| Forceps | Fine Science Tools | 11252-20 | Dumont #5 |
| Glass coverslips | VWR | 16004-330 | #1 thickness |
| Glass microscope slides | Fisher | 12-550-15 | Standard glass slides |
| Methanol | Fisher | A411 | Store in freezer |
| Microcentrifuge tubes | VWR | 20170-038 | capped tubes, not sterile |
| Normal goat serum | Life Technologies | 16210-064 | Aliquot 1ml tubes, store in freezer |
| Paraformaldehyde | Sigma | P-6148 | Reagent grade, crystalline |
| Phosphate buffered saline (PBS) | Quality Biological | 119-069-131 | 10X stock solution or made in lab |
| Triton X-100 | Sigma | BP-151 | 10% solution in water, store at room temp |
| Tween-20 | Life Technologies | 85113 | 10% solution in water, store at room temp |
| Compound microscope | Nikon | E-600 | Mount on vibration-free table |
| C1 Plus two-laser scanning confocal | Nikon | C1 Plus | Run by EZ-C1 program, but upgrades use "Elements" |
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