We thank the Dictyostelium Stock Center for strains and reagents. This study was financed by grants from the Swiss National Science Foundation and the Canton of Basel.

<ol><li>タイムラプス顕微鏡による細胞性粘菌の早期の飢餓応答を観察します。 <ol><li> 5グラムプロテオースペプトン、5グラムthiotone Eペプトン、10gのグルコース、5gの酵母エキス、0.35グラムHPO 4 2のNa：DH1.10細胞またはHL-5培地（1L用を含むエルレンマイヤーフラスコ中のコーラ欠損細胞成長* 7H 2 O、0.35グラムのKH 160回転の回転に振盪インキュベーターで22℃で2 PO 4、0.05グラムジヒドロ-硫酸塩、pHは6.6）。 0.01×10 6細胞/ mlおよび2×10 6細胞/ mlの密度で細胞を保管してください。 </li><li>収穫対数期に成長しているDH1.10セル21または22℃でのHL-5培地で振とう培養で増殖させたコーラ欠損細胞DH1.10のバックグラウンドで生成された10、によって、細胞凝集を調べます。これを行うには、400×gで3分間、細胞（通常10〜50 ml）を、遠心分離の適切な量を取り、BSS（10mMの塩化ナトリウム、10mMのKClの中で細胞を2回洗浄、2.5 mMのCaCl 2を、pHは6.5）。 </li><li>血球計数器を用いて細胞を数えます。続いて、（5、10、20、または40）の密度でプレートの細胞を24ウェルプレートに10 4細胞/ cm×2。彼らはBSSに22℃で1時間接着させます。 </li><li> 10前に説明したように、画像ごとに135秒を取って、タイムラプス顕微鏡により凝集を視覚化します。設定5Xの客観的かつ適切なソフトウェアによって自動化された電子増倍電荷結合素子カメラを搭載したライブセルイメージングを使用して（ 材料表を参照してください）。 </li></ol></li><li>飢餓時の細胞性粘菌細胞のcAMPのパルス化。 <ol><li>外部から細胞を採取することにより、DH1.10細胞21またはDH1.10 コーラ欠損細胞10の開発にcAMPのパルスを適用の効果を調べます。これを行うには、400×gで3分間、適切な細胞の量（通常10〜50ミリリットル）、遠心分離機を取り、BSSで二回洗います。 </li><li> COUNT血球計数器を用いて細胞やBSSで1×10 7細胞/ mlの密度に細胞を再懸濁します。パルスを印加する前に、2時間22℃で培養（160 rpm）を振ります。タイマー制御蠕動ポンプを使用したcAMPのパルスを追加します。 5時間かけて5秒パルスに50 nMでのcAMP（最終濃度）の15μlのあらゆる6.5分を提供するためにポンプをプログラムします。 </li><li>血球計数器を用いて細胞を数えます。続いて、（5、10、20、または40）の密度で細胞をプレート24ウェルプレートに10 4細胞/ cm×2。 BSSで1時間接着させます。 </li><li> 5Xの対物レンズを使用して明視野顕微鏡による22℃で16時間後に凝集を視覚化します。 </li></ol></li><li>馴化培地への暴露による細胞性粘菌開発の誘導。 <ol><li> 22に記載されているように 、新鮮な馴化培地を準備します。カルトを振ってから、対数期DH1.10細胞21またはDH1.10 コーラ欠損細胞10を収集400×gで3分間、ピペット、遠心分離器を用いて22℃でHL-5培地でURESとPBMで細胞を3回洗浄し（0.02 Mリン酸カリウム、10μMのCaCl 2、1mMのMgCl 2、pHは6.1）。 </li><li>血球計数器を用いて細胞をカウントし、mlの1×10 7細胞/の密度でPBMでこれらを再懸濁し、110回転数/ 22℃で20時間振とうします。 </li><li> 3分間400×gで遠心分離後、馴化培地を収集し、4℃で15分間8000×gで遠心分離することにより明らかにしました。 </li><li> 0.45μmフィルターを通してフィルター条件培地は、（ 材料表を参照）、PBMで3倍に希釈します。 </li><li> （5、10、20、または40）の密度で血球計およびプレートを用いて細胞をカウントし、24ウェルプレートに10 4細胞/ cm×2。彼らは22℃で1時間接着させます。 </li><li>先に調製した馴化培地を用いた細胞の交換上清。 </li><li>集約aを可視化5Xの対物レンズを使用して明視野顕微鏡によるFTER 16時間。 </li></ol></li></ol>

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No conflicts of interest declared.

coroninタンパク質は、真核生物クレードのほとんどの分類群で発見されています。coronin欠損細胞は初期発生時に集計中心を形成することができないので、 細胞性粘菌の coroninのA、哺乳類coronin 1のホモログは、早期の飢餓応答に関与していますサイクル10。定量的かつ正確に系統間で開発の遅れを評価することができるようにするには、自動化されたステージコントローラと顕?...

タンパク質のcoroninファミリは、高度真核生物を通じて保存されています。これらのタンパク質は、カルボキシ末端のコイルドコイルドメイン13,14（ 図1）に接続されたユニークな領域が続くアミノ末端トリプトファン、アスパラギン酸（WD）リピート含有領域が存在することを特徴とします。 Coroninsは、細胞骨格調節およびシグナル伝達12を含む、種々の細胞機能に関与しています。哺乳類では、最大6つの短いcoronin分子（coronin 1-6）と同様に7をcoronin「タンデム」は、12,15を共発現させることができます。 Coronin 1は、最​​も広く研究ファミリーのメンバーであり、病原体の破壊、T細胞の生存および神経伝達に関与することが示されました。どのように、正確に、coronin 1は、これらの活動は不明なままで行います。 coroninながら1は、CAを調節することが示さ2+およびcAMP依存性シグナル伝達だけでなく、F-アクチン細胞骨格の調節16-18、潜在的な共同ました哺乳類で最大7家族の-expressionは、それが困難なこれらのシステムにcoroninsの分子機能を研究するために潜在的な冗長性のために作られました。哺乳類の生物とは異なり、下等真核生物細胞性粘菌は明らかに非冗長機能15,19,20と2つだけcoronin家族（coroninのA、哺乳類coronin 1とcoronin Bのオルソログ、哺乳類coronin 7のオルソログ）を表します。この事実は、 細胞性粘菌に coroninsの機能を研究するための強力なモデルになります。 細胞性粘菌でcoronin Aの役割を研究するために、我々は、10 coronin欠く野生型細胞または細胞のいずれかを用いて平衡塩類溶液（BSS）緩衝液を含む組織培養プレート中で飢餓によって発達サイクルを誘導しました。私たちはcoronin Aを欠く細胞が飢餓時に多細胞凝集体を形成することができなかったことがわかりました。この表現型の正確な定量的評価のためのこのプロトコールに記載の自動化生細胞イメージングは​​不可欠なツールです。 coronin欠く細胞における早期の飢餓応答の開始における欠陥は、cAMPカスケードの上流の行為をcoroninことを示唆し、cAMPのパルスを供給することによって救出することができます。開発の開始をシミュレートするためのcAMPパルスの外因性の適用は、過去8,9にいくつかの研究室で利用されてきました。しかしながら、この手順は、細胞密度及びタイミングに大きく依存することが知られています。したがって、ここで説明するプロトコルは、再現性の高い度合いを保証するために、これらの変動性を低減することを目的とします。まとめると、これらの研究に利用技術は、 細胞性粘菌の発達サイクルの初期段階でのタンパク質の機能を調査するために強力なツールを提供し、アップ識別だけでなく、機能をcoroninの下流エフェクター可能性を秘めています。

<table>
	<tbody>
		<tr>
			<td>HL-5 media (for 1L: 5 g proteose peptone, 5 g thiotone E peptone, 10 g glucose, 5 g yeast extract, 0.35 g Na2HPO4&#8727;2H2O, 0.35 g KH2PO4, 0.05 g dihydrostreptomycin-sulfate, pH 6.6)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Proteose peptone</td>
			<td>BD Bioscience</td>
			<td>211693</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiotone E peptone</td>
			<td>BD Bioscience</td>
			<td>211684</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>BD Bioscience</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>AppliChem</td>
			<td>A3666</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4&#8727;2H2O</td>
			<td>Fluka</td>
			<td>71643</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>AppliChem</td>
			<td>A1043</td>
			<td></td>
		</tr>
		<tr>
			<td>dihydrostreptomycin-sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>D1954000</td>
			<td></td>
		</tr>
		<tr>
			<td>PBM (0.02 M potassium phosphate, 10 &#956;M CaCl2, and l mM MgCl2, pH 6.1)</td>
			<td></td>
			<td></td>
			<td>self made</td>
		</tr>
		<tr>
			<td>BSS (10 mM NaCl, 10 mM KCl, 2.5 mM CaCl2, pH 6.5)</td>
			<td></td>
			<td></td>
			<td>self made</td>
		</tr>
		<tr>
			<td>0.45-&#956;m Filtropure S filter</td>
			<td>Sarstedt</td>
			<td>83.1826</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 24-well Tissue culture plate</td>
			<td>Fisher Scientific</td>
			<td><a href="https://www.fishersci.com/shop/products/falcon-tissue-culture-plates-23/087721h">08-772-1H</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Cellobserver microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>custom built</td>
		</tr>
		<tr>
			<td>AxioVision software</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IPC Microprocessor&#8211;controlled dispensing pump</td>
			<td>ISMATEC</td>
			<td>ISM 931</td>
			<td></td>
		</tr>
		<tr>
			<td>Axiovert 135M microscope</td>
			<td>Zeiss</td>
			<td>491237-0001-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubation Shaker</td>
			<td>Inforst HT Minitron</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

investigating the function of coronin a in the early starvation response of dictyostelium discoideum by aggregation assays

細胞性粘菌のアメーバは細菌を餌に、土壌中に発見されています。食料源が不足になると、それらは、単一の細胞が凝集中心1-4向かっ走化その間多開発プログラムを開始する因子を分泌します。このプロセスは、環状アデノシン一リン酸（cAMP）5のリリースに依存しています。 cAMPは、アデニル酸シクラーゼ、およびホスホジエステラーゼの協調作用によって電波で産生され、Gタンパク質共役受容体のcAMP 6,7に結合されています。広く使用されているアッセイは、水没条件8,9における細胞凝集の観察に基づいている下等真核生物細胞性粘菌の発達サイクルに関与するメカニズムを分析します。このプロトコルは、平衡塩溶液（BSS）10の中に沈め組織培養プレート中で飢餓によって発達サイクルのcoronin Aの役割の分析を記載します。 Coronin Aは広く保存された広報のメンバーであります活動11,12の多種多様に関与しているcoroninsの家族otein。coronin Aを欠く細胞性粘菌の細胞は、多細胞凝集体を形成することができない、そしてこの欠陥が上流の行為をcoroninことを示唆し、cAMPのパルスを供給することによって救出することができますcAMPは10をカスケード。これらの研究に記載された技術は、cAMPカスケードの上流細胞性粘菌の発達サイクルの初期段階中にタンパク質の機能を調べるための強力なツールを提供します。したがって、この凝集アッセイを利用して機能をcoroninのさらなる研究を可能にし、coronin生物学の我々の理解を進めることができます。

ショーを開発の初期段階で欠陥をcoroninにおける欠損細胞（ 図2）。細胞をcoroninの不在下では細胞性粘菌の発達サイクル中の初期段階である多細胞凝集体を形成することができません。したがって、coronin Aは初期の飢餓応答および/またはcAMPシグナル伝達の際に役割を果たしていると思われます。実際、coronin Aの非存在下で、多細胞凝集体形成の欠?...

Watch this Scientific Journal Video about Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays at JoVE.com

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

The social amoeba Dictyostelium discoideum undergoes a developmental transition into a multicellular organism when starved. The evolutionary conserved protein coronin A plays a crucial role in the initiation of development. Using aggregation assays as our main method, we aim to elucidate the role of coronin A in early development.

初期の飢餓応答でCoronin Aの機能を調査<em&gt;細胞性粘菌</em&gt;凝集アッセイによって

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular ...

investigating-function-coronin-early-starvation-response

Research

JoVE Journal

Biology

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

7.2K ビュー.  University of Basel. The social amoeba Dictyostelium discoideum undergoes a developmental transition into a multicellular organism when starved. The evolutionary conserved protein coronin A plays a crucial role in the initiation of development. Using aggregation assays as our main method, we aim to elucidate the role of coronin A in early development.

ビデオ: Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

Investigating the Microbial Community in the Termite Hindgut - Interview

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes.     The symbiotic relationship existing between the host insect and lignocellulose-degrading gut microbes is explained, as well as the industrial uses of these microbes for degrading plant biomass and generating biofuels.

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes. The symbiotic relationship existing between the host insect and lignocellulose-degrading gut microbes is explained, as well as the industrial uses of these microbes for degrading plant biomass and generating biofuels.

インタビュー - シロアリ後腸内微生物群集の調査

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes.     The ...

生物学

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

ゼブラフィッシュの視覚機能を研究するために視運動性応答を使用して

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

電離放射線への応答でγH2AX病巣の定量化

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

イメージングG -タンパク質共役受容体（GPCR）を介するシグナル伝達事象その制御走キイロタマホコリカビ</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

初期のニワトリ胚の形態形成の組織の変形を追跡

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

のNa、KとH、K-ATPaseのインによって陽イオン輸送を測定する<em&gt;アフリカツメガエル</em原子吸光法による&gt;卵母：アイソトープアッセイの代替

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

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 ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

複屈折およびタッチ呼び起こすエスケープ応答アッセイによる幼虫のゼブラフィッシュ骨格筋の欠陥の分析

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

高リン酸化タウタンパク質を用いたインビトロ凝集アッセイにおいて

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

大人のゼブラフィッシュの腎臓におけるネフロン構成と機能の解析

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...