パトリックカフェルティは、カナダの多発性硬化症協会からのポスドクでサポートされています。

パート1：プレ実験のセットアップ。 <ol><li>事前に一週間は、メイトはグリア特異的プロモーターの制御下にGFPを発現するその幼虫を生成するために飛ぶ。私たちの実験のために我々は、 逆極性（レポ）プロモーター（3,4）を用いてグリア細胞に発現し核局在配列でタグ付けされたGFPを可視化。 </li><li>百分の一晩乾燥したポリ- L -リジン溶液と空気中で10分間18 mmの円形のカバースリップを浸漬することによって、事前に少なくとも一日のスリップをカバーする準備。 </li><li>実験の前日に、70％エタノールで一晩成分を浸漬することによってChamlide磁気培養チャンバーをクリーニング。 </li><li>実験日に、無菌テクニックを使用してシュナイダー昆虫培地10mlにウシ胎児血清、ペニシリン - ストレプトマイシン溶液、およびインスリンを追加することにより、培養液を準備する。使用濃度は以下のとおりです。1Xウシ胎児血清、100U/mlペニシリン、0.1mg/mlストレプトマイシン、およびシュナイ​​ダー昆虫培地に0.2 mg / mlのインスリン（（5）から変更）。 </li><li>培養チャンバーは細胞培養のフードで風乾します。準備培養液で洗浄することにより培養チャンバーから残留エタノールを洗い落とす。 </li></ol>パート2： ショウジョウバエの眼と脳の複合体の解剖。 <ol><li>数分間氷上でペトリ皿の上に冷やした培地のドロップにハエのバイアルと場所の側から3齢放浪幼虫]を選択します。幼虫をなごむこと解剖を容易にするために蠕動体の収縮を減速することをお勧めします。 </li><li>解剖顕微鏡下Sylgardコートしたディッシュ上で培養液のドロップチルド幼虫を置きます。 </li><li>解剖顕微鏡下で、しっかりと後端からの方法の約3分の1を幼虫を把握するデュモン細かいピンセットを使用してください。幼虫は前端から口のフックを押し出すまで待ち​​ます。フル拡張時に鉗子の2番目のペアと口のフックをつかみます。幼虫を解剖するために、ゆっくりと反対方向に鉗子の2つのペアを引き出します。眼と脳の複合体は、唾液腺、脂肪体、そして成虫組織と一緒に、幼虫の体から離れて引っ張ってくる。口は、彼ら​​が目の脳の複雑な構成ティアオフなるそうでなければ早すぎるとフックを引っ張らないでください。 </li><li>唾液腺、脂肪体、及び超微細クリッパーのはさみを使用して成虫ディスクを切り落とす。脳と目のディスクの二つの半球は、腹側神経索と口のフックに接続されます。 </li><li>スライドグラス上に18 mmのカバースリップを配置。後カバーガラスをピックアップするために、スライドの端からぶら下がってカバースリップの片方のエッジを残す。 18 mmのカバースリップ上に培養液のドロップを追加。鉗子で口のフックを把握し、培養培地中に目と脳の複雑なを転送します。鉗子や組織が損傷されると直接眼と脳の複雑なを把握しないでください。 </li><li>細かい解剖鋏を使用して、目と脳の複雑なと廃棄から口のフックを切り落とす。口のフックの除去は、口のフックは、組織の顕微鏡検査中に移動させる培養液中の契約を継続するとして、ライブイメージングのために重要です。 </li><li>目成虫原基内にグリアの移行を可視化するために、注意深く眼茎、脳と目のディスクを接続する細い組織をカットし（図1Aを参照）脳を離れて押したり、廃棄してください。眼茎内グリア細胞を可視化するために、（図1Eを参照）、脳、眼茎、そして目成虫原基はそのままにしておく。 </li><li>鉗子を用いてカバースリップをピックアップし、永久的なマーカーとの利益の組織の周りの下に円を描く。このサークルでは、パート4で顕微鏡のための組織を見つけるのに役立ちます。 </li></ol>第3部：磁気培養チャンバーに目成虫原基をマウント。 <ol><li>鉗子を使用して、カバースリップの上盤の端をつかんで。文化の中で組織を乱すことなくChamlide磁気チェンバーの底板にカバースリップを転送します。 </li><li> Chamlideチャンバーの本体の上にシリコンO -リングを置きます。底板の上に本体を取り付けます。 </li><li>徐々にチャンバーに培養液を追加します。速すぎるまたは利益の組織が妨害される培養液を追加しないでください。完全にカバーの下にガス交換を可能にするためにチャンバーを埋めるしないでください。 </li><li>ゆっくりとChamlideチャンバーの上に目に見えないカバーを置きます。 </li></ol>パート4：目のディスクにグリア細胞の移行の可視化。 <ol><li>共焦点または蛍光顕微鏡のステージ上に培養チャンバーを配置。低倍率での参考資料としてカバースリップ上に円を使用してサンプルを探します。 </li><li> 40倍レンズを使用してサンプルに焦点を当てる。組織はカバースリップの上に定着することができます。 3-4時間以上、10〜15分目のディスクまたは光茎の画像をキャプチャ。組織は、ピクッと動くとピントが合って移動することがあります。手動で画像をキャプチャする前に組織を中心とする目を軽減します問題があります。 </li></ol>パート5：代表的な結果： 正しく実行される、我々のプロトコルは、私たちが目の成虫原基（図1 BD）に眼茎から移行したGFP -タグ付きグリア細胞の一連の画像を収集することができました。 60分間のライブイメージングは、 野生型の目成虫原基内のグリア細胞核の位置の変化を観察するのに十分でしたが、グリア細胞の遊走に必要な遺伝子の変異体におけるグリアの核は完全に光ファイバー茎（矢印を終了に失敗しました図1 FH）。 我々は、培養組織の劣化を観察する前に240分限り長期間培養眼と脳の複合体を持っている。 240分の時間ポイントに続いて、培養眼脳複合体（図の矢印Aを2 C）を囲む培養培地中でGFP陽性細胞を観察し始める。加えてのGFPで組織の完全性の崩壊を示唆して組織全体にびまん性斑に蓄積されることになります。 <img src="/files/ftp_upload/1155/figure1.png" alt="" width="589" height="241" /> 図1：開発野生型と変異型視覚システムにおけるGFP -タグ付きグリア細胞核のライブイメージング。 、E）画像を培養し、野生型と変異体目成虫ディスク（ED）の微分干渉コントラスト（DIC）顕微鏡を用いて撮影。 野生型では、グリア細胞はで生まれていると目のディスクに眼茎（OS）から移行する。脳は、平坦化し、目成虫原基のイメージングを容易にするために削除されています。 BD）（A）0、30、および60分の時点ではGFP -タグ付きグリア細胞核が目のディスク内での移行が明らかに野生型培養目成虫原基の蛍光顕微鏡。 の変異体目の脳の複雑なのFH）蛍光顕微鏡（E）0、30℃、および60分の時点では、眼茎内でGFP -タグ付きグリア細胞核（矢印）の失速を示しています。脳（BR）は、茎内グリア細胞のイメージングを容易にするために眼茎に接続されて残されている。 <img src="/files/ftp_upload/1155/Figure_2.png" alt="" width="600" height="156" /> 図2： 野生型の目成虫原基におけるGFP -タグ付きグリア膜のライブイメージング。我々は成功し、240分の期間のための目と脳の複合体を培養している。組織培養よりも長い240分では分解し始めます。 300分の時間点（C）に比べて目成虫原基の周囲の培地（ED）、眼茎（OS）、および0（A）と150分（B）で脳の前頭葉には、（BR）、、無料です。矢印で示されているGFP陽性の細胞は、。

<ol>
	<li>Aigouy, B., Lepelletier, L., Giangrande, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+chain+migration+requires+pioneer+cells.">Glial chain migration requires pioneer cells.</a> J. Neurosci. 28, 11635-11641 (2008).</li><li>Silies, M., Yuva, Y., Engelen, D., Aho, A., Stork, T., Klambt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cell+migration+in+the+eye+disc.">Glial cell migration in the eye disc.</a> J. Neurosci. 27, 13130-13139 (2007).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Sepp, K. J., Auld, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+lacZ+enhancer+trap+lines+to+GAL4+lines+using+targeted+transposition+in+Drosophila+melanogaster.">Conversion of lacZ enhancer trap lines to GAL4 lines using targeted transposition in Drosophila melanogaster.</a> Genetics. 151, 1093-1101 (1999).</li><li>Gibson, M. C., Patel, A. B., Nagpal, R., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emergence+of+geometric+order+in+proliferating+metazoan+epitheia.">The emergence of geometric order in proliferating metazoan epitheia.</a> Nature. 442, 1038-1041 (2006).</li><li>Lee, T., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+with+a+repressible+cell+marker+(MARCM)+for+Drosophila+neural+development.">Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development.</a> Trends Neurosci. 24, 251-254 (2001).</li></ol>

The authors have nothing to disclose.

このプロトコルでは、ライブ顕微鏡を使用して目成虫原基にグリア細胞の遊走の観察を説明します。私たちの野生型の例（図1 AD）においては、我々は1時間かけ目のディスクにグリア細胞の動きを観察するために核GFPマーカーを使用。現在我々の研究室で研究されてグリア細胞の移行に必要な候補遺伝子の変異では、我々は、1時間の期間（図1 EH）の間に眼茎内のグリア細胞核の失速を...

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		<th>Comment</th>
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<tr>
<td>Poly-L-lysine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P8920</td>
<td>&nbsp;</td>
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<td>Schneider's Insect Media</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S0146</td>
<td>&nbsp;</td>
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<td>Penicillin-Streptomycin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P4458</td>
<td>&nbsp;</td>
<tr>
<td>Insulin solution from bovine pancreas</td>
<td>Reagent</td>
<td>Sigma</td>
<td>I0516</td>
<td>&nbsp;</td>
<tr>
<td>Chamlide Magnetic chamber</td>
<td>Tool</td>
<td>Live cell Instrument</td>
<td>CM-R-10</td>
<td>35 mm dish type chamber for 18 mm coverslip</td>
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<td>Ultra fine clipper scissors</td>
<td>Tool</td>
<td>Fine scientific tools</td>
<td>15200-00</td>
<td>&nbsp;</td>
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<td>Dumont #5 forceps</td>
<td>Tool</td>
<td>Fine scientific tools</td>
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<td>Fluorescent microscope</td>
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<td>Zeiss</td>
<td>&nbsp;</td>
<td>Any fluorescent imaging system that has the necessary filters and excitation for GFP can be used. </td>
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live imaging of glial cell migration in the drosophila eye imaginal disc

脊椎動物と無脊椎動物の生物の両方のグリア細胞はensheathするために、最終的な標的領域へ​​の移行と関連する神経細胞をサポートする必要があります。最近の進展は、開発途上蛹の翼（1）、の研究ではグリア細胞のライブマイグレーションを記述するためになされている一方で<em&gt;ショウジョウバエ</em&gt;グリア細胞の遊走は、通常、固定した組織の検査を含んでいた。運動性細胞のライブ顕微鏡分析は、速度と成長の方向の変化を決定するを含む渡り鳥プロセス、全体の細胞挙動を検査する能力を提供しています。遺伝学的ツールの使用と対に、ライブイメージングは​​、開発の過程で特定の遺伝子のためのより正確な役割を決定するために使用することができます。 Siliesで前の仕事<em&gt;ら。</em&gt;（2）固定組織における眼成虫原基に、眼茎、開発の目と脳を結ぶ構造に由来するグリア細胞の移行を説明しています。ここでは、中にグリア細胞のライブマイグレーションを検討するためのプロトコルの概要を説明<em&gt;ショウジョウバエ</em&gt;目の成虫原基。我々は、野生型および変異動物におけるグリア細胞の進行に従うことを開発するグリア細胞でGFPを発現し、ショウジョウバエのラインを活用する。

Watch this Scientific Journal Video about Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc at JoVE.com

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

ここでは、GFPタグ付きのグリア細胞とペアライブ顕微鏡分析を使用して開発をショウジョウバエの眼にグリア細胞の遊走を調べるためのプロトコルについて説明します。

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 ...

live-imaging-glial-cell-migration-drosophila-eye-imaginal

Research

JoVE Journal

Biology

10.7K ビュー.  University of British Columbia - UBC. ここでは、GFPタグ付きのグリア細胞とペアライブ顕微鏡分析を使用して開発をショウジョウバエの眼にグリア細胞の遊走を調べるためのプロトコルについて説明します。

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

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

3齢幼虫ショウジョウバエの幼虫から成虫ディスクの解剖

生物学

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

ライブの視覚化ショウジョウバエ</em蛍光色素で>グリア - 神経筋接合部

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries made using this system have taken advantage of the spatiotemporal nature of photoreceptor differentiation in the developing eye imaginal disc. To use this system it is first necessary for the researcher to learn to identify and dissect the eye disc. We describe a novel RFP reporter to aid in the identification of the eye disc and the visualization of specific cell types in the developing eye. We detail a methodology for dissection of the eye imaginal disc from third instar larvae and describe how the eye-RFP reporter can aid in this dissection. This eye-RFP reporter is only expressed in the eye and can be visualized using fluorescence microscopy either in live tissue or after fixation without the need for signal amplification. We also show how this reporter can be used to identify specific cells types within the eye disc. This protocol and the use of the eye-RFP reporter will aid researchers using the Drosophila eye to address fundamentally important biological questions.

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

We describe a novel red fluorescent protein (RFP) reporter that is expressed specifically in the Drosophila eye. We detail a methodology for dissection of the eye imaginal disc and how this reporter can be used to aid in the dissection and identification of specific cell types in the developing eye.

のアイ成虫原基の可視化を支援するために新しいRFPレポーターショウジョウバエ</em

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries ...

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 ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

リビングでのイメージング、細胞の形状の変更ショウジョウバエ胚

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

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 ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

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 Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

初期のオートファジーイベントの細胞イメージングライブOmegasomesを以降

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

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, ...