Summary

ホールマウントゼブラフィッシュ胚の繊毛器官におけるリン酸化エピトープを免疫染色

Published: February 19, 2016
doi:

Summary

技術は全体のゼブラフィッシュ胚におけるホスホ – エピトープを免疫染色した後、一次繊毛のように小さな細胞構造の二色の蛍光共焦点の局在化を行うことが記載されています。固定およびイメージングのための技術は、特定のタンパク質の出現または活性化の位置と速度を定義することができます。

Abstract

細胞の急速な増殖は、遺伝子の組織特異的発現およびシグナル伝達ネットワークの出現は、すべての脊椎動物の初期胚発生を特徴付けます。動態および信号の場所 – であっても、単一の細胞内 – 胚では、重要な発達遺伝子の同定を補完します。免疫染色技術は、一次繊毛のように小さな構造に細胞内および動物全体の信号の速度を定義することが示されているが記載されています。レーザー走査型共焦点複合顕微鏡を用いた定着、撮像処理画像のための技術は、わずか36のような時間で完了することができます。

ゼブラフィッシュ( ゼブラフィッシュ)は、手頃な価格とヒト疾患に関連する脊椎動物種の研究を行うことを求める研究者のための望ましい生物です。遺伝的ノックアウトまたはノックダウンは、実際のタンパク質産物の損失によって確認されなければなりません。タンパク質損失のような確認ここに記載された技術を用いて達成することができます。シグナル伝達経路への手がかりはまた、翻訳後リン酸化によって修飾されたタンパク質と反応する抗体を使用して解読することができます。エピトープのリン酸化状態を維持し、最適化することは、この決意することが重要であり、このプロトコルによって達成されます。

本研究では、開発の最初の72時間と共局在クッパーの小胞(KV)における繊毛と関連する様々なエピトープ、腎臓および内耳中に胚を固定するための手法について説明します。これらの技術は、切開を必要としない、単純であり、比較的短時間で完了することができます。単一の画像に共焦点画像スタックを投写することは、これらのデータを提示する有用な手段です。

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

このプロトコルでのゼブラフィッシュの手順は、バージニア・コモンウェルス大学の施設内動物管理使用委員会(IACUC)によって承認されています。 試薬の調製 4%PFA / PBS。ドラフト内でパラホルムアルデヒドの8グラム(PFA)を秤量します。まだドラフト中ながら、50℃まで撹拌しながら加熱し、H 2 Oを蒸留〜80ミリリットルで乾燥PFAを溶解します。 PFAが?…

Representative Results

ホスホ-エピトープを可視化するための最適な条件 ゼブラフィッシュ胚におけるタンパク質エピトープの免疫学的局在を記述する方法は、in situハイブリダイゼーションを介してmRNAをローカライズするためのものに比べて比較的疎でした。ゼブラフィッシュ胚の繊毛細胞におけるタンパク質のエピトー?…

Discussion

PFA /メタノール法は、ゼブラフィッシュの開発中に、ホスホT 287 -CaMK-IIエピトープの免疫局在を最適化する第一の目的と本研究室で開発されました。このメソッドは、正常にゼブラフィッシュKV、12内耳14と腎臓を含むいくつかの繊毛器官の形成時にP-のCaMK-IIをローカライズ。13、特にKV段階で、この技術が必要でした。この方法の成功は、b)は、リン酸化のCaMK…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

この作品は、国立科学財団の助成金IOS-0817658によってサポートされていました。

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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Citazione di questo articolo
Rothschild, S. C., Francescatto, L., Tombes, R. M. Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos. J. Vis. Exp. (108), e53747, doi:10.3791/53747 (2016).

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