この作品は、マリーキュリー社会復帰グラント（IRG249084）によってサポートされていました。私たちは、Kurant研究所のすべてのメンバーに感謝します。

食細胞とアポトーシス細胞：細胞の2つの集団が標識されている必要があり、生体内でアポトーシス細胞クリアランスの力学に従ってください。 マクロファージ、グリア及び外胚葉8：貪食細胞集団をマークするために我々は胚で排他的に食細胞を標識シミュレーションcytGFPマーカーを含むショウジョウバエラインを使用。一つは、血球固有Gal4のドライバ（CRQ-Gal4の ）含む行またはグリア固有Gal4のドライバ（ レポGal4の ）とUAS制御（UAS-GFP）の下で遺伝的に符号化された蛍光レポーターを含む食細胞に対して異なるマーカーを使用することができます。 アポトーシス細胞のクリアランスの間にアポトーシス細胞を監視するために我々は異なったアポトーシス/貪食マーカーを使用。アネキシンV（Molecular Probes社）は10アポトーシス細胞の早期マーカーとして機能します。Phiphilux（OncoImmunin）は後でapoptoとして使用されている蛍光カスパーゼ-3基板であるSISマーカー、及びLysoTracker（Molecular Probes社）はファゴソームマーカーとして機能します。我々はマイクロインジェクションシステムPicoPump PV 820を使用して、これらの試薬を注入する。 1。準備中<ol><li>ヘプタンのり： 両面テープをアンロールし、シンチレーションバイアル中で、24時間のためにパラフィルムでバイアル、およびロックを封印、ヘプタンで埋めることができる限り置く。接着剤は厚すぎるヘプタン追加。 </li><li>我々は2時間ペースト+酵母ハエは予め温めグレープジュース寒天プレート上に置くことができ、その後°C 10-12時（所望の段階に依存します）のための事前のマイクロインジェクションと時間経過の録音を25にプレートを転送注入された胚の。そこで我々は、ステージ15や開発の16から胚を提供し、25℃に維持寒天プレートから胚を集める。 </li><li>針の調製： 注射用の針を準備するには、我々は &#39;サター楽器&#39;ニードルプラーと薄肉ガラスフィラメント（FHCキャピラリーチューブ）を使用します。プールされたキャピラリーの先端を破っている0.5ミクロンの直径からn。 </li><li>接着剤でカバースリップ： ピペットチップを使用すると、カバースリップの途中で一行でヘプタン接着剤のドロップを分散。数秒のためにそれが乾燥してみましょう。これらのカバースリップのいくつかを準備します。 </li></ol> 2。胚の準備<ol><li>寒天プレートから胚を集め、きれいなペイントブラシと流水を使ってセルストレーナー（SPLライフサイエンス）に転送します。ストレーナーは、廃水を収集するようにビーカーの上に保持されている。 </li><li>すべての酵母ペーストが除去されるまで水を使用してセルストレーナーで胚を洗浄します。 </li><li>キムワイプを用いたストレーナの外側を拭いて余分な水分を乾かします。 </li><li>きれいなペトリ皿内のセルストレーナーを置き、セルストレーナーで胚をカバーするのに十分な50％の漂白剤を追加します。時折2分（2-3回）、それらを攪拌するための胚をDechorionate。 </li><li>胚漂白剤の臭い緩いまで広範囲に水で洗い流してください。 </li><li>きれいにセルストレーナーを置きペトリ皿に、最大乾燥を防ぐために水で胚をカバーしています。 </li><li>胚の設定を開始する前に、2本の針（時々針が目詰まりすることができます）に必要な試薬1μlのをロードします。これは、針の先端に到達するための液体のため、約5分かかります。 </li><li>グレープジュース寒天の長方形の部分を切り取り、顕微鏡スライド上に置きます。ペトリ皿からきれいなペイントブラシを使って寒天ピースにdechorionated胚を転送します。 </li><li>蛍光解剖顕微鏡下で適切に選択し、ウェットペイントブラシを使用したGFPマーカーを発現胚を上演し、近くに別の（ 図1A）の後行1で寒天片の端に各胚を配置。胚は、中枢神経系（CNS）におけるグリアによるイメージング食作用のための彼らの腹側にまで配置する必要があります。 </li><li> 1行目には約10胚を設定します。 </li><li>ヘプタン接着剤のストリップ（ 図1B、Cで途中で被覆されたカバースリップに胚を取り付け</strong&gt;）。 </li><li>約5分（これは部屋の湿度と注入される量に依存します）のために脱水チャンバー内にカバースリップを置きます。 </li><li>さらに脱水症状を避けるためにハロカーボン油700（Sigma）で胚をカバーするまで乾燥した後。 </li><li>上向きに胚と顕微鏡スライド上にカバースリップを置きます。あなたは、カバースリップの移動を防止するために、スライド上に水滴を置いてもよい。胚は今注射のための準備が整いました。噴射位置は、胚の途中で側方に典型的である。 </li></ol> 3。胚インジェクション<ol><li>マイクロマニピュレータに針を取り付けます。 </li><li>針先を破るためには視野内にカバースリップの端を入れて、同じ焦点面に針を調整します。それは針の先端に当たって、それを壊すまで、非常に慎重にカバースリップを移動します。 </li><li>胚に着目し、油に針を配置し、針から液滴を得ることを確認してください。これは、針の先端がされていることを意味しよく壊れた。 </li><li>ニードルホルダーに触れることなく針に胚を移動し、胚にドロップを注入。離れて胚を移動し、すべての胚が注入されるまで、次のいずれかに進みます。 </li></ol> 4。イメージング 40Xまたは100Xを目的とした倒立共焦点顕微鏡で我々は画像胚を。我々は強力なGFPの発現と注射後アポトーシス細胞の良いラベルを示し途中でCNSとうまく配置胚、探します。 ライブ胚の時間経過の録音のために我々は通常、5〜6共焦点スライス（厚さ2μmのそれぞれ）を選択します。その後、我々は全体の細胞を観察するために3スライス（6ミクロン厚）の投影を行う。 我々は安定しており、簡単に漂白しないアポトーシス細胞のマーカーを使用しています。我々は60秒の間隔で録音を行う漂白GFPを避けるために。 場合によっては、胚は圧延開始することができる録画の時間の間。したがって、我々は、停止し、必要に応じて、最初からやり直すために、記録でたまに見てみましょう。

<ol>
	<li>Kinchen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+to+die+for:+signaling+to+apoptotic+cell+removal+in+worm,+fly+and+mouse.">A model to die for: signaling to apoptotic cell removal in worm, fly and mouse.</a> Apoptosis. 15, 998-1006 (2010).</li><li>Kinchen, J. M., Ravichandran, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagosome+maturation:+going+through+the+acid+test.">Phagosome maturation: going through the acid test.</a> Nat. Rev. Mol. Cell Biol. 9, 781-795 (2008).</li><li>Kinchen, J. M., Ravichandran, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytic+signaling:+you+can+touch,+but+you+can't+eat.">Phagocytic signaling: you can touch, but you can't eat.</a> Curr. Biol. 18, 521-524 (2008).</li><li>Stuart, L. M., Ezekowitz, 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=Phagocytosis:+elegant+complexity.">Phagocytosis: elegant complexity.</a> Immunity. 22, 539-550 (2005).</li><li>Lauber, K., Blumenthal, S. G., Waibel, M., Wesselborg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clearance+of+apoptotic+cells:+getting+rid+of+the+corpses.">Clearance of apoptotic cells: getting rid of the corpses.</a> Mol. Cell. 14, 277-287 (2004).</li><li>Henson, P. M., Hume, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+cell+removal+in+development+and+tissue+homeostasis.">Apoptotic cell removal in development and tissue homeostasis.</a> Trends Immunol. 27, 244-250 (2006).</li><li>Kurant, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+the+CNS+clear:+glial+phagocytic+functions+in+Drosophila.">Keeping the CNS clear: glial phagocytic functions in Drosophila.</a> Glia. 59, 1304-1311 (2011).</li><li>Kurant, E., Axelrod, S., Leaman, D., Gaul, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Six-microns-under+acts+upstream+of+Draper+in+the+glial+phagocytosis+of+apoptotic+neurons.">Six-microns-under acts upstream of Draper in the glial phagocytosis of apoptotic neurons.</a> Cell. 133, 498-509 (2008).</li><li>Mergliano, J., Minden, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase-independent+cell+engulfment+mirrors+cell+death+pattern+in+Drosophila+embryos.">Caspase-independent cell engulfment mirrors cell death pattern in Drosophila embryos.</a> Development. 130, 5779-5789 (2003).</li><li>van den Eijnde, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+exposure+of+phosphatidylserine+during+apoptosis+is+phylogenetically+conserved.">Cell surface exposure of phosphatidylserine during apoptosis is phylogenetically conserved.</a> Apoptosis. 3, 9-16 (1998).</li></ol>

著者は、彼らが競合する経済的利益を持っていないことを宣言します。

アポトーシス細胞のクリアランスは非常に動的であるアポトーシスの重要な最後の段階である。したがって、プロセスの実時間研究は極めて重要である。ここでは、開発してショウジョウバエの胚を生体内アポトーシス細胞のクリアランスの監視を可能にするプロトコルを記述します。アポトーシス細胞と食細胞：この手順では、細胞の2つの集団は、特異的なマーカーを使用してマー...

アポトーシスとその後の食作用を通じて不要なまたは異常な細胞の適切な除去は、胚発生のためだけでなく、成人で組織の恒常性のために重要です。アポトーシス細胞やアポトーシス細胞の貪食クリアランスは4段階で進行する非常にダイナミックなプロセスである：アポトーシス細胞の貪食細胞の（1） 募集 （ &#39;見つける-私&#39;）、食作用の標的として細胞の（2） 認識 （ &#39; -私を食べて&#39;）、（3）の貪食 、アポトーシス粒子1-5の（4） ファゴソーム成熟と劣化が続く。 &#39;プロ&#39;マクロファージや未熟樹状細胞、および&#39;非専門家&#39;組織常駐隣接セル、後生動物の開発6,7時のアポトーシス細胞のクリアランスのために重要である：食細胞の2つのタイプがあります。 ショウジョウバエの開発では、アポトーシスを通して余分細胞の除去は、3メートルで発生後期胚の半ばで初めて、その後中旬蛹で、再び初期の成人におけるAINフェーズ、。胚発生の間にアポトーシス粒子は&#39;プロ&#39;食細胞、マクロファージ、および&#39;非専門家&#39;外胚葉およびグリア8,9によって除去される。 我々の以前の研究では、胚発生の間に食細胞にのみ発現している、（シミュレーション）六ミクロンアンダー、貪食受容体を同定した。 シミュレーションのプロモーターを用いて、我々は、ライブ胚8で同時に外胚葉およびグリア、マクロファージを監視することを可能に貪食細胞集団（ シミュレーションcytGFP）に特異的マーカーを生成しました。さらに、アポトーシスの異なる段階においてアポトーシス細胞に特異的なマーカーを用いて、我々は、 生体内のアポトーシス細胞クリアランスのダイナミクスに従うことができる。

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>Annexin V </td>
 <td>Molecular Probes</td>
 <td>A35108</td>
 <td></td>
 </tr>
 <tr>
 <td>PhiPhiLux G2D2</td>
 <td>OncoImmunin</td>
 <td>A304R2G-5</td>
 <td></td>
 </tr>
 <tr>
 <td>LysoTracker</td>
 <td>Molecular Probes</td>
 <td>L-7528</td>
 <td></td>
 </tr>
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 <td>Halocarbon oil 700</td>
 <td>Sigma</td>
 <td>H8898</td>
 <td></td>
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 <td></td>
 <td></td>
 <td></td>
 <td>Material</td>
 </tr>
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 <td>Capillary tubing</td>
 <td>FHC </td>
 <td>30-30-0</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell Strainer</td>
 <td>SPL</td>
 <td>93100</td>
 <td></td>
 </tr>
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 <td>Paintbrush</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

live imaging of apoptotic cell clearance during drosophila embryogenesis

アポトーシスとその後の食作用を通じて不要なまたは異常な細胞の適切な除去（アポトーシス細胞のクリアランス）は、すべての後生生物の正常な発達のために重要です。アポトーシス細胞のクリアランスは密接に細胞死に関連付けられている非常にダイナミックなプロセスである; unengulfedアポトーシス細胞はほとんど通常の条件下では、生体内で見られる。アポトーシス細胞のクリアランスの異なるステップを理解し、 &#39;プロ&#39;食と比較するためには-にマクロファージや樹状細胞を&#39;非専門家&#39; -組織常駐隣接セルは、プロセスのin vivoライブイメージングでは、非常に貴重なものです。ここでは、ライブショウジョウバエ胚におけるアポトーシス細胞のクリアランスを研究するためのプロトコルを記述します。貪食に異なるステップのダイナミクスに追従するために、我々はアポトーシス細胞と食細胞に対して特異的なマーカーを使用しています。また、並行して2食のシステムを監視することができます： &#39;プロ&#39;マクロファージおよび &#39;セミプロフェッショナルエディション現像中枢神経系におけるssional &#39;グリア（CNS）。ここで説明する方法は、アポトーシス細胞クリアランスのリアルタイムの研究のための優れたモデルとしてショウジョウバエ胚を用いる。

アポトーシス細胞は、アネキシンVで標識されており、グリア、外胚葉およびマクロファージをシミュレーションcytGFPで標識された映画からの代表フレームが図2に示されている。各フレームは3スライス2μmとそれぞれの投影である。 ステージ15の胚を適切によくラベルグリア（g）のと途中で胚性CNSを示す配置されている。 CNS外にほとんどであるマクロ?...

Watch this Scientific Journal Video about Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis at JoVE.com

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

ここでは、アポトーシス細胞のクリアランスのダイナミクスを研究するための効果的な方法を説明<em生体内で&gt;</em&gt;。この方法では、住んで採用<em&gt;ショウジョウバエ</emアポトーシス細胞と食細胞の特異的標識を用いたアポトーシス細胞の貪食を監視するための強力なモデルとして&gt;胚。

アポトーシス細胞のクリアランスのイメージング中に生きる<em&gt;ショウジョウバエ</em&gt;胚

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

live-imaging-apoptotic-cell-clearance-during-drosophila

Research

JoVE Journal

Biology

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

13.4K ビュー.  Technion-Israel Institute of Technology. ここでは、アポトーシス細胞のクリアランスのダイナミクスを研究するための効果的な方法を説明。この方法では、住んで採用ショウジョウバエ胚。

ビデオ: Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

ショウジョウバエの眼成虫ディスクに付属してグリア細胞の移行のイメージングを生きる

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

生物学

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

のライブイメージングキイロショウジョウバエ胚性血球移行

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

開発と大人の準備ショウジョウバエ</emライブイメージングのための>脳と網膜

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

胚形成の間に機能的な冗長性をテストするための逆遺伝学的アプローチ

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

個々の準備ショウジョウバエ</emライブイメージングのための>エッグチェンバース

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

でGFP標識タンパク質のイメージングを生きる<em&gt;ショウジョウバエ</em&gt;卵母細胞

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

生細胞中のタンパク質凝集の4Dイメージング

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

モザイク大人のライブイメージングのためのトマト/ GFP-FLP / FRT方法<em&gt;ショウジョウバエ</em&gt;視細胞

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

精子形成細胞学的分析：ライブとの固定の準備<em&gt;ショウジョウバエ</em&gt;精巣

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by available technologies, apoptosis is generally assumed to occur shortly thereafter. Caspases can cleave many functional and structural components to cause rapid and complete cell destruction within a few minutes. However, accumulating evidence indicates that in normal healthy cells the same caspases have other functions, presumably at lower enzymatic levels. Studies of non-apoptotic caspase activity have been hampered by difficulties with detecting low levels of caspase activity and with tracking ultimate cell fate in vivo. Here, we illustrate the use of an ultrasensitive caspase reporter, CaspaseTracker, which permanently labels cells that have experienced caspase activity in whole animals. This in vivo dual color CaspaseTracker biosensor for Drosophila melanogaster transiently expresses red fluorescent protein (RFP) to indicate recent or on-going caspase activity, and permanently expresses green fluorescent protein (GFP) in cells that have experienced caspase activity at any time in the past yet did not die. Importantly, this caspase-dependent in vivo biosensor readily reveals the presence of non-apoptotic caspase activity in the tissues of organ systems throughout the adult fly. This is demonstrated using whole mount dissections of individual flies to detect biosensor activity in healthy cells throughout the brain, gut, malpighian tubules, cardia, ovary ducts and other tissues. CaspaseTracker detects non-apoptotic caspase activity in long-lived cells, as biosensor activity is detected in adult neurons and in other tissues at least 10 days after caspase activation. This biosensor serves as an important tool to uncover the roles and molecular mechanisms of non-apoptotic caspase activity in live animals.

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

To detect healthy cells in whole animals that contain low levels of caspase activity, the highly sensitive biosensor designated CaspaseTracker was generated for Drosophila. Caspase-dependent biosensor activity is detected in long-lived healthy cells throughout the internal organs of adult animals reared under optimized conditions in the absence of death stimuli.

インビボでのバイオセンサーは、 ショウジョウバエの非アポトーシスカスパーゼ活性を追跡します

Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by ...