Name: investigating the function of coronin a in the early starvation response of dictyostelium discoideum by aggregation assays
Uploaded: 2016-06-18T15:00:00.000+00:00
Duration: 7 min 48 s
Description: 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

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>

<ol>
	<li>Weijer, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenetic+cell+movement+in+Dictyostelium.">Morphogenetic cell movement in Dictyostelium.</a> Semin Cell Dev Biol. 10, 609-619 (1999).</li><li>Devreotes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dictyostelium+discoideum:+a+model+system+for+cell-cell+interactions+in+development.">Dictyostelium discoideum: a model system for cell-cell interactions in development.</a> Science. 245, 1054-1058 (1989).</li><li>Kimmel, A. R., Firtel, 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=cAMP+signal+transduction+pathways+regulating+development+of+Dictyostelium+discoideum.">cAMP signal transduction pathways regulating development of Dictyostelium discoideum.</a> Curr Opin Genet Dev. 1, 383-390 (1991).</li><li>Clarke, M., Gomer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PSF+and+CMF,+autocrine+factors+that+regulate+gene+expression+during+growth+and+early+development+of+Dictyostelium.">PSF and CMF, autocrine factors that regulate gene expression during growth and early development of Dictyostelium.</a> Experientia. 51, 1124-1134 (1995).</li><li>Robertson, A., Drage, D. J., Cohen, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Aggregation+in+Dictyostelium+discoideum+by+an+External+Periodic+Pulse+of+Cyclic+Adenosine+Monophosphate.">Control of Aggregation in Dictyostelium discoideum by an External Periodic Pulse of Cyclic Adenosine Monophosphate.</a> Science. 175, 333-335 (1972).</li><li>Konijn, T. M., Chang, Y. Y., Bonner, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+cyclic+AMP+in+Dictyostelium+discoideum+and+Polysphondylium+pallidum.">Synthesis of cyclic AMP in Dictyostelium discoideum and Polysphondylium pallidum.</a> Nature. 224, 1211-1212 (1969).</li><li>Iranfar, N., Fuller, D., Loomis, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+expression+analyses+of+gene+regulation+during+early+development+of+Dictyostelium+discoideum.">Genome-wide expression analyses of gene regulation during early development of Dictyostelium discoideum.</a> Eukaryot Cell. 2, 664-670 (2003).</li><li>Darmon, M., Brachet, P., Da Silva, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotactic+signals+induce+cell+differentiation+in+Dictyostelium+discoideum.">Chemotactic signals induce cell differentiation in Dictyostelium discoideum.</a> Proc Natl Acad Sci U S A. 72, 3163-3166 (1975).</li><li>Mann, S. K., Firtel, 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=Cyclic+AMP+regulation+of+early+gene+expression+in+Dictyostelium+discoideum:+mediation+via+the+cell+surface+cyclic+AMP+receptor.">Cyclic AMP regulation of early gene expression in Dictyostelium discoideum: mediation via the cell surface cyclic AMP receptor.</a> Mol Cell Biol. 7, 458-469 (1987).</li><li>Vinet, A. F., 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=Initiation+of+multicellular+differentiation+in+Dictyostelium+discoideum+is+regulated+by+coronin+A.">Initiation of multicellular differentiation in Dictyostelium discoideum is regulated by coronin A.</a> Mol Biol Cell. 25, 688-701 (2014).</li><li>Eckert, C., Hammesfahr, B., Kollmar, 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+holistic+phylogeny+of+the+coronin+gene+family+reveals+an+ancient+origin+of+the+tandem-coronin,+defines+a+new+subfamily,+and+predicts+protein+function.">A holistic phylogeny of the coronin gene family reveals an ancient origin of the tandem-coronin, defines a new subfamily, and predicts protein function.</a> BMC Evol Biol. 11, 268 (2011).</li><li>Pieters, J., Muller, P., Jayachandran, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+guard:+coronin+proteins+in+innate+and+adaptive+immunity.">On guard: coronin proteins in innate and adaptive immunity.</a> Nat Rev Immunol. 13, 510-518 (2013).</li><li>Gatfield, J., Albrecht, I., Zanolari, B., Steinmetz, M. O., Pieters, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+the+Leukocyte+Plasma+Membrane+with+the+Actin+Cytoskeleton+through+Coiled+Coil-mediated+Trimeric+Coronin+1+Molecules.">Association of the Leukocyte Plasma Membrane with the Actin Cytoskeleton through Coiled Coil-mediated Trimeric Coronin 1 Molecules.</a> Mol Biol Cell. 16, 2786-2798 (2005).</li><li>Kammerer, R. A., 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=A+conserved+trimerization+motif+controls+the+topology+of+short+coiled+coils.">A conserved trimerization motif controls the topology of short coiled coils.</a> Proc Natl Acad Sci U S A. 102, 13891-13896 (2005).</li><li>Uetrecht, A. C., Bear, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronins:+the+return+of+the+crown.">Coronins: the return of the crown.</a> Trends Cell Biol. 16, 421-426 (2006).</li><li>Jayachandran, R., 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=Coronin+1+Regulates+Cognition+and+Behavior+through+Modulation+of+cAMP/Protein+Kinase+A+Signaling.">Coronin 1 Regulates Cognition and Behavior through Modulation of cAMP/Protein Kinase A Signaling.</a> PLoS Biol. 12, e1001820 (2014).</li><li>Suo, D., 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=Coronin-1+is+a+neurotrophin+endosomal+effector+that+is+required+for+developmental+competition+for+survival.">Coronin-1 is a neurotrophin endosomal effector that is required for developmental competition for survival.</a> Nat Neurosci. 17, 36-45 (2014).</li><li>Mueller, P., 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=Regulation+of+T+cell+survival+through+coronin-1-mediated+generation+of+inositol-1,4,5-trisphosphate+and+calcium+mobilization+after+T+cell+receptor+triggering.">Regulation of T cell survival through coronin-1-mediated generation of inositol-1,4,5-trisphosphate and calcium mobilization after T cell receptor triggering.</a> Nat Immunol. 9, 424-431 (2008).</li><li>de Hostos, E. L., Bradtke, B., Lottspeich, F., Guggenheim, R., Gerisch, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronin,+an+actin+binding+protein+of+Dictyostelium+discoideum+localized+to+cell+surface+projections,+has+sequence+similarities+to+G+protein+beta+subunits.">Coronin, an actin binding protein of Dictyostelium discoideum localized to cell surface projections, has sequence similarities to G protein beta subunits.</a> EMBO J. 10, 4097-4104 (1991).</li><li>Shina, M. C., 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=Redundant+and+unique+roles+of+coronin+proteins+in+Dictyostelium.">Redundant and unique roles of coronin proteins in Dictyostelium.</a> Cell Mol Life Sci. 68, 303-313 (2011).</li><li>Cornillon, S., 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=Phg1p+is+a+nine-transmembrane+protein+superfamily+member+involved+in+dictyostelium+adhesion+and+phagocytosis.">Phg1p is a nine-transmembrane protein superfamily member involved in dictyostelium adhesion and phagocytosis.</a> J Biol Chem. 275, 34287-34292 (2000).</li><li>Gomer, R. H., Yuen, I. S., Firtel, 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=A+secreted+80+x+10(3)+Mr+protein+mediates+sensing+of+cell+density+and+the+onset+of+development+in+Dictyostelium.">A secreted 80 x 10(3) Mr protein mediates sensing of cell density and the onset of development in Dictyostelium.</a> Development. (112), 269-278 (1991).</li><li>Iijima, N., Takagi, T., Maeda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+proteinous+factor+mediating+intercellular+communication+during+the+transition+of+Dictyostelium+cells+from+growth+to+differentiation.">A proteinous factor mediating intercellular communication during the transition of Dictyostelium cells from growth to differentiation.</a> Zoological science. 12, 61-69 (1995).</li><li>Mehdy, M. C., Firtel, 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=A+secreted+factor+and+cyclic+AMP+jointly+regulate+cell-type-specific+gene+expression+in+Dictyostelium+discoideum.">A secreted factor and cyclic AMP jointly regulate cell-type-specific gene expression in Dictyostelium discoideum.</a> Mol Cell Biol. 5, 705-713 (1985).</li><li>Jain, R., Yuen, I. S., Taphouse, C. R., Gomer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+density-sensing+factor+controls+development+in+Dictyostelium.">A density-sensing factor controls development in Dictyostelium.</a> Genes Dev. 6, 390-400 (1992).</li><li>Loomis, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+signaling+during+development+of+Dictyostelium.">Cell signaling during development of Dictyostelium.</a> Dev Biol. 391, 1-16 (2014).</li></ol>

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 1 L: 5 g proteose peptone, 5 g thiotone E peptone, 10 g glucose, 5 g yeast extract, 0.35 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;*2H&lt;sub&gt;2&lt;/sub&gt;O, 0.35 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 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>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;*2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fluka</td><td>71643</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</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 &amp;mu;M CaCl&lt;sub&gt;2&lt;/sub&gt;, and l mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 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 CaCl&lt;sub&gt;2&lt;/sub&gt;, pH 6.5)</td><td></td><td></td><td>self made</td></tr><tr><td>0.45-&amp;mu;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>08-772-1H</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&amp;ndash;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

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

キイロタマホコリカビの選択に基づいて細胞と細菌の成長の遺伝子工学

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

遺伝学

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis elegans is a well-established model organism that has been widely used in the field to study protein aggregation and toxicity. Its optical transparency enables the direct visualization of protein aggregation by fluorescence microscopy. Moreover, the fast reproductive cycle and short lifespan make the nematode a suitable model to screen for genes and molecules that modulate this process.
However, the quantification of aggregate load in living animals is poorly standardized, typically performed by manual inclusion counting under a fluorescence dissection microscope at a single time point. This approach can result in high variability between observers and limits the understanding of the aggregation process. In contrast, amyloid-like protein aggregation in vitro is routinely monitored by thioflavin T fluorescence in a highly quantitative and time-resolved fashion.
Here, an analogous method is presented for the unbiased analysis of aggregation kinetics in living C. elegans, using a high-throughput confocal microscope combined with custom-made image analysis and data fitting. The applicability of this method is demonstrated by monitoring inclusion formation of a fluorescently labeled polyglutamine (polyQ) protein in the body wall muscle cells. The image analysis workflow allows the determination of the numbers of inclusions at different timepoints, which are fitted to a mathematical model based on independent nucleation events in individual muscle cells. The method described here may prove useful to assess the effects of proteostasis factors and potential therapeutics for protein aggregation diseases in a living animal in a robust and quantitative manner.

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Here, a method is presented for the analysis of protein aggregation kinetics in the nematode Caenorhabditis elegans. Animals from an age-synchronized population are imaged at different time points, followed by semiautomated inclusion counting in CellProfiler and fitting to a mathematical model in AmyloFit.

Caenorhabditisエレガンスにおける自動包接計数を用いた生体内のタンパク質凝集動態のモニタリング

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis ...

生化学

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 ...

生物学

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse formation and maintenance. Many of these fundamental interactions occur in trans and are typically induced by heterophilic or homophilic interactions between cells expressing membrane anchored binding pairs. Elucidating how disease relevant mutations disrupt these fundamental protein interactions can provide insight into a myriad of cell biology fields. Many protein-protein interaction assays do not typically disambiguate between cis and trans interactions, which potentially leads to an overestimation of the extent of binding that is occurring in vivo and involve labor intensive purification of protein and/or specialized monitoring equipment. Here, we present an optimized simple protocol that allows for the observation and quantification of only trans interactions without the need for lengthy protein purifications or specialized equipment. The HEK cell aggregation assay involves the mixing of two independent populations of HEK cells, each expressing membrane-bound cognate ligands. After a short incubation period, samples are imaged and the resulting aggregates are quantified.

Here, we present an optimized protocol to rapidly and semiquantitatively measure ligand-receptor interactions in trans in a heterologous cell system using fluorescence microscopy.

異種細胞系におけるタンパク質凝集による細胞間相互作用の測定

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse ...

神経科学

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

タンパク質の品質管理システムを学びます<em&gt; D。 discoideumの</em&gt;温度制御された生細胞イメージングを用い

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

キイロタマホコリカビ急性の機械的刺激に対する応答の評価

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

線虫の加齢とともに固有の蛋白質の集合の変化を研究するためのメソッド

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

フローサイトメトリーによる細胞性粘菌キイロタマホコリカビマクロピノサイトーシスの高スループット測定

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.

We here introduce a procedure to measure protein oligomers and aggregation in cell lysate and live cells using fluorescence correlation spectroscopy.

蛍光相関分光法を用いたタンパク質凝集の検出

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's ...

初期の飢餓応答でCoronin Aの機能を調査

要約

さらに動画を探す

この動画の章

キイロタマホコリカビの選択に基づいて細胞と細菌の成長の遺伝子工学

Caenorhabditisエレガンスにおける自動包接計数を用いた生体内のタンパク質凝集動態のモニタリング

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

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

異種細胞系におけるタンパク質凝集による細胞間相互作用の測定

タンパク質の品質管理システムを学びます

キイロタマホコリカビ急性の機械的刺激に対する応答の評価

線虫の加齢とともに固有の蛋白質の集合の変化を研究するためのメソッド

フローサイトメトリーによる細胞性粘菌キイロタマホコリカビマクロピノサイトーシスの高スループット測定

蛍光相関分光法を用いたタンパク質凝集の検出