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1。 キイロタマホコリカビの走コンピテントセルの調製<ol><li>化学誘引物質cAMPに走ているD. discoideumの細胞、22で振とう培養からD3 - Tリッチメディアに成長して収穫の細胞を生成するには℃、 </li><li> （DBバッファは5mMのNa 2 HPO 4、5mMのKH 2 PO 4、2mMのMgCl 2、及び0.1mMのCaCl 2を含む）非栄養発達バッファーで細胞を2回洗浄する。 </li><li> 2 × 10 7細胞/ mlの密度でDBのバッファーで細胞を再懸濁する。 </li><li>一時間では22で100回転℃で250mlのフラスコに10ミリリットルのセルを横に振る。 </li><li> 75 nMのcAMPの最終濃度、cAMPの脈打つ治療として指定されたプロセスを達成するために6時間以上6分毎に10ミリリットルの細胞を7.5μMのcAMPの株式の100μlを提供します。 cAMPのパルスの治療、D.の5-6時間後にdiscoideumの細胞は、cAMPの勾配に向かって有能な走化性となる。 </li><li> 5分の200 gで遠心分離により細胞を収集し、DBバッファと再懸濁し、細胞は、2.5 mMのカフェインを含む、22で200 rpmで振とう℃で20分間走の状況にセルをbasolateする。 </li></ol> 2。イメージングが表示され、操作可能な化学誘引物質の勾配で細胞をchemotaxing <ol><li>埋め戻しDBのバッファー中で1μMのcAMPおよび0.1μg/μlでアレクサ594、新たに調製した30μlの溶液によるマイクロピペット。 </li><li>マイクロピペットホルダーにFemtotipを取り付け、圧力供給装置、エッペンドルフFemtoJetシステムにチューブを接続します。 </li><li>安定勾配を確立するために、安定した圧力を提供するために、マイクロマニピュレーターを電動マイクロマニピュレータ（エッペンドルフTransferMan NK2）にマイクロピペットのアセンブリを取り付けます。 </li><li>共焦点顕微鏡で40倍の油レンズを介してDBバッファー6mlを充填した1ウェルLabTekチャンバーをマウントし、明視野光学系を使用して、中心視野にFemtotip。 </li><li>圧力を投入し、cAMPを/アレクサ594混合物の勾配を確立するために70 hPaのための補償の圧力（PC）を設定します。 </li><li> 543 nmのレーザラインで励起を用いてcAMPおよびアレクサ594蛍光の所望の濃度の混合物を監視することでのcAMPの勾配を可視化する。 </li><li>セルにさらさ​​れている勾配を操作するために、目的の位置にマイクロピペットを置くと位置1、位置2のようにそれらを設定するには、マイクロマニピュレータの自動位置決め機能を使用して、ポジション3。 </li></ol> 3。不動の非極性細胞システムは、cAMP勾配感知に関与してイベントをシグナル伝達のイメージングを容易に<ol><li>カフェイン処理した後、3分間500グラムで細胞と遠心のアリコートを削除します。 </li><li>バッファを削除し、2.5mMのカフェインを含む新鮮なDB用緩衝液で5 × 10 5細胞/ mlに細胞を希釈する。 </li><li>四ウェルチャンバーの各ウェルに単一のウェルチャンバーに細胞懸濁液の1 mlあるいは0.4 mlを適用します。 </li><li> 10分間、注意深くアタッチされていないセルを削除し、同じボリュームに置き換えるためのバッファをオフにピペッティング細胞が付着することができます。 </li><li>顕微鏡下で所望の細胞を見つけて、イメージングを起動します。 </li><li>安定した勾配にさらす細胞におけるシグナル伝達成分の動態を監視するために設計された実験では、実験前に10分は、5.0μM（最終濃度）Latrunculin Bで細胞を扱う。 </li></ol> 4。同時モニタリングのヘテロ三量体Gタンパク質活性化およびPIP 3生産<ol><li>細胞を共発現GαCFPとYFPGβ（G細胞）とPIP 3インジケータPH - GFP（PH細胞）7を発現する細胞を開発する。パルス状のcAMP </li><li> 1:1の比率とプレートそれらを一つウェルまたは4ウェルチャンバー内での細胞のこれらの2つのタイプを混在させること。 </li><li> CFP、YFPとGFPが10nm幅の464から624 nmの波長範囲内のラムダスタックアクイジションモードを使用して排出指紋の基準曲線を作成して保存します。 </li><li>同時にG細胞内の画像Gタンパク質の活性化と10 nm刻みで464から544 nmの波長範囲内で同一のラムダスタックアクイジションモードで使用してPH細胞におけるPIP 3生産。 </li><li> G.における個々のチャネルにCFPとYFPの強度を分離するために数学的にラムダのスタック内の各蛍光体の寄与を計算するために保存CFPand YFPと放出された指紋を使用してツァイス510METAソフトウェアの線形アンミキシング機能を適用する</li><li>同戦略では、数学的にPHの細胞でGFPの強度を計算するために保存されているGFPと背景放射の指紋を使用して線形アンミキシング機能を適用します。 </li></ol> 5。代表的な結果： <ol><li> D.の優れたモデルシステムGPCRを介した走化性のためのカビ 。ソーシャルアメーバ、D. discoideumのは、ライフサイクルの間に顕著な走化性を示す。その遺伝的および生化学的な利点のために、D. dは強力なのが用意されています走化性を研究するystem。 </li><li> 目に見えると操作可能な化学的に誘引するフィールドの下で細胞の走化性。ここでは、我々は最初の10μg/ mLのと混合2μMのcAMPの希釈系列によるcAMPの濃度と蛍光染料アレクサ594強の間に直線関係を得るために、単純な方法論を示すアレクサ594（図2A）。次に、我々はAlexa 594（図図2B）の強度が勾配のcAMP濃度の定量測定を確立するために、また、勾配を可視化する簡単な方法を提供しています。 </li><li> 細胞運動は細胞の極性と方向感知と非結合されています。アクチンの重合阻害剤は既存の形態学的極性を排除し、方向感知の細胞の機能（図3A）を維持しながらも、細胞の動きを防ぐことができます。目に見えると操作可能な cAMP刺激の雇用は、入力、たとえば、一律に適用される刺激や勾配を保証します。このメソッドは、グラデーション感知機械の重要なシグナル伝達成分のcAMP誘発性再分布の定量的な分析を行うことができます。これらのシグナル伝達成分の測定時空間ダイナミクスは、私たちは勾配（図3B）に偏光生化学的応答の生成中にシグナル伝達ネットワークが均一な刺激への適応を実現する方法を理解することができます。 </li><li> 安定した勾配に曝されるとシグナル伝達ネットワークを化学的感覚の動態の全身測定。これは、細胞は最初のグラデーションを経験するときに細胞内の偏光の設立に各成分が寄与する方法を理解するためにコンポーネントに信号を送る方向を感知するの力学/動力学を測定することが重要です。ハイテンポ-空間分解能でのライブセルイメージングのアプリケーションは、まず安定したcAMPの勾配（図4A - C）にさらされているセルの相性PIP3産生を示す。生細胞イメージングを適用して、体系的にcAMP刺激によるPIP3産生（図4D、E）への方向を感知する特異的シグナル伝達ネットワークのダイナミクスを測定した。 </li><li> 一様に適用されるcAMP刺激時に ​​同時モニタリングヘテロ三量体Gタンパク質活性化およびPIP 3生産。フェルスター共鳴エネルギー移動（省略形はFRET）のcAMP刺激によりヘテロ三量体Gタンパク質の活性化（解離）を監視するための効率的なアプローチを提供します。ここで、我々は、FRETが変化し、それぞれPIP 3プローブ、GとPHの細胞内PH - GFP、（図5の膜輸送を監視することにより、ヘテロ三量体Gタンパク質活性化およびPIP 3生産の同時測定のために便利な簡単な導入システムを説明）。これは一時的なPIP 3の生産をトリガしながら均一に適用されるcAMP刺激は、永続的なGタンパク質の活性化をトリガーします。 </li></ol><img src="/files/ftp_upload/3128/3128fig1.jpg" alt="図1"/> 図1：D.の優れたモデルシステムGPCRを介した走化性のためのカビ 。 A.スキームは、方向感知の簡単なシグナル伝達経路を示しています。B. cAMPの勾配は、D.の急速な走化性を誘導するdiscoideumの細胞 。細胞は、PIP 3プローブ、PH - GFP（緑）を発現。グラデーション（赤）がアレクサ594で可視化。スケールバー=50μmの。 <img src="/files/ftp_upload/3128/3128fig2.jpg" alt="図2"/> 図2：目に見えると操作可能な化学的に誘引するフィールドの下で細胞の走化。 A.グラフは、10μg/ mLのアレクサ594と混合2μMのcAMPの希釈系列によるcAMPの濃度と蛍光染料アレクサ594強の間に直線関係を示しています。線形関係で勾配のcAMP濃度のB.定量的測定は、 Aの蛍光色素アレクサ594 cAMP濃度と強度の<img src="/files/ftp_upload/3128/3128fig3.jpg" alt="図3"/> 図3：細胞運動は細胞の極性と方向感知と切り離されている。 A.イメージは、アクチン重合阻害剤Latrunculin Bの治療によって不動の細胞が方向感知の能力を維持することを示しています。細胞は、PIP 3プローブ、PH - GFP（緑）を発現。グラデーション（赤）がアレクサ594で可視化される。B.操作可能なcAMP刺激や不動電池システムは、方向感知の重要な問題に対処することができます。スケールバー=10μmの。 <img src="/files/ftp_upload/3128/3128fig4.jpg" alt="図4"/> 図4：安定した勾配に曝されるとシグナル伝達ネットワークを化学的感覚の動態の全身測定。 A.モンタージュは、安定したcAMPの勾配（レッド）にさらされているセルの相性のPIP 3生産（グリーン）。B.画像がC. Cで提示さPIP 3生産の動態の測定のための利益の領域（ROI）を、それぞれ示す。キネティクスO安定した勾配に曝露した細胞におけるfのPIP 3生産。D.スキームでは、PIP 3生産へのcAMPの刺激から方向センシングのシグナル伝達ネットワークを示します。安定した勾配に曝されると、その反応速度はEで同じ色の実線で表示されます。 <img src="/files/ftp_upload/3128/3128fig5.jpg" alt="図5"/> 図5：GPCRシグナル伝達ネットワークの同時監視、マルチイベント。 A.スキームはそれぞれ、変更およびPIP3プローブ、GとPHの細胞内PH - GFPの細胞膜の転座をFRETが。Gの虹のイメージとPHの細胞のショーのB.モンタージュ監視することにより、ヘテロ三量体Gタンパク質活性化およびPIP 3生産の同時測定を示しています。これは一時的なPIP3産生をトリガしながら均一に適用されるcAMP刺激は、末梢細胞での永続的なGタンパク質の活性化をトリガーすること。タイムポイントは、4.9s、10.2sと20.4sのために前に（0）と刺激後です。Gタンパク質の活性化と一様に適用されるcAMPの刺激によりPIP3産のC.速度論は。

<ol>
	<li>Lijima, M., Huang, Y. E., 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=Temporal+and+spatial+regulation+of+chemotaxis.">Temporal and spatial regulation of chemotaxis.</a> Dev Cell. 3, 469-469 (2002).</li><li>Murphy, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+biology+of+leukocyte+chemoattractant+receptors.">The molecular biology of leukocyte chemoattractant receptors.</a> Annu Rev Immunol. 12, 593-593 (1994).</li><li>Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+protein-linked+signaling+pathways+control+the+developmental+program+of+Dictyostelium.">G protein-linked signaling pathways control the developmental program of Dictyostelium.</a> Neuron. 12, 235-235 (1994).</li><li>Jin, T., Xu, X., Hereld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis,+chemokine+receptors+and+human+disease.">Chemotaxis, chemokine receptors and human disease.</a> Cytokine. 44, 1-1 (2008).</li><li>Meier-Schellersheim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Key+role+of+local+regulation+in+chemosensing+revealed+by+a+new+molecular+interaction-based+modeling+method.">Key role of local regulation in chemosensing revealed by a new molecular interaction-based modeling method.</a> PLoS Comput Biol. 2, e82-e82 (2006).</li><li>Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+mechanism+of+a+GPCR+and+a+heterotrimeric+G+protein+during+chemoattractant+gradient+sensing+in+Dictyostelium.">Coupling mechanism of a GPCR and a heterotrimeric G protein during chemoattractant gradient sensing in Dictyostelium.</a> Sci Signal. 3, 71-71 (2010).</li><li>Xu, X., Meier-Schellersheim, M., Jiao, X., Nelson, L. E., Jin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+imaging+of+single+live+cells+reveals+spatiotemporal+dynamics+of+multistep+signaling+events+of+chemoattractant+gradient+sensing+in+Dictyostelium.">Quantitative imaging of single live cells reveals spatiotemporal dynamics of multistep signaling events of chemoattractant gradient sensing in Dictyostelium.</a> Mol Biol Cell. 16, ra71-ra71 (2005).</li><li>Xu, X., Meier-Schellersheim, M., Yan, J., Jin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locally+controlled+inhibitory+mechanisms+are+involved+in+eukaryotic+GPCR-mediated+chemosensing.">Locally controlled inhibitory mechanisms are involved in eukaryotic GPCR-mediated chemosensing.</a> J. Cell Biol. 178, 141-141 (2007).</li><li>Jin, T., Zhang, N., Long, Y., Parent, C. A., Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+the+G+protein+betagamma+complex+in+living+cells+during+chemotaxis.">Localization of the G protein betagamma complex in living cells during chemotaxis.</a> Science. 287, 1034-1034 (2000).</li><li>Janetopoulos, C., Jin, T., 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=Receptor-mediated+activation+of+heterotrimeric+G-proteins+in+living+cells.">Receptor-mediated activation of heterotrimeric G-proteins in living cells.</a> Science. 291, 2408-2408 (2001).</li><li>Funamoto, S., Milan, K., Meili, 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=Role+of+phosphatidylinositol+3'+kinase+and+a+downstream+pleckstrin+homology+domain-containing+protein+in+controlling+chemotaxis+in+dictyostelium.">Role of phosphatidylinositol 3' kinase and a downstream pleckstrin homology domain-containing protein in controlling chemotaxis in dictyostelium.</a> J. Cell Biol. 153, 795-795 (2001).</li><li>Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+PLC-beta2+and+-beta3+and+PI3Kgamma+in+chemoattractant-mediated+signal+transduction.">Roles of PLC-beta2 and -beta3 and PI3Kgamma in chemoattractant-mediated signal transduction.</a> Science. 287, 1046-1046 (2000).</li><li>Sasaki, A. T., Chun, C., Takeda, 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=Localized+Ras+signaling+at+the+leading+edge+regulates+PI3K,+cell+polarity,+and+directional+cell+movement.">Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement.</a> J. Cell Biol. 167, 505-505 (2004).</li><li>Meili, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoattractant-mediated+transient+activation+and+membrane+localization+of+Akt/PKB+is+required+for+efficient+chemotaxis+to+cAMP+in+Dictyostelium.">Chemoattractant-mediated transient activation and membrane localization of Akt/PKB is required for efficient chemotaxis to cAMP in Dictyostelium.</a> EMBO J. 18, 2092-2092 .</li><li>Parent, C. A., Blacklock, B. J., Froehlich, W. M., Murphy, D. B., Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+protein+signaling+events+are+activated+at+the+leading+edge+of+chemotactic+cells.">G protein signaling events are activated at the leading edge of chemotactic cells.</a> Cell. 95, 81-81 (1998).</li><li>Funamoto, S., Meili, R., Lee, S., Parry, L., 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=Spatial+and+temporal+regulation+of+3-phosphoinositides+by+PI+3-kinase+and+PTEN+mediates+chemotaxis.">Spatial and temporal regulation of 3-phosphoinositides by PI 3-kinase and PTEN mediates chemotaxis.</a> Cell. 109, 611-611 (2002).</li><li>Iijima, M., 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=Tumor+suppressor+PTEN+mediates+sensing+of+chemoattractant+gradients.">Tumor suppressor PTEN mediates sensing of chemoattractant gradients.</a> Cell. 109, 599-599 (2002).</li><li>Haastert, P. J. V. a. n., Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis:+signalling+the+way+forward.">Chemotaxis: signalling the way forward.</a> Nat Rev Mol Cell Biol. 5, 626-626 (2004).</li></ol>

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細胞の走化性有能な段階に到達するプロセス野生型のためのD. discoideumの細胞 、それは細胞がうまく偏光細胞形態と迅速な細胞の遊走（図1）が表示される間も、走化性有能な段階にそれらを誘導するために、室温で開発をパルス〜5約6時間かかります。このようなcAMPのパルスの濃度、温度、および異なる遺伝的背景など、いくつかの要因は、走化性有能なステージに...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント</th></tr><tr><td> D3 - Tの増殖培地</td><td> KDメディカル</td><td></td><td></td></tr><tr><td>カフェイン</td><td>シグマ</td><td></td><td></td></tr><tr><td> Latrunculin B </td><td>分子プローブ</td><td></td><td></td></tr><tr><td>アレクサ594 </td><td>分子プローブ</td><td></td><td></td></tr><tr><td> cAMPの</td><td>シグマ</td><td></td><td></td></tr><tr><td> ChronTrol XTプログラマブルタイマ</td><td> ChronTrolコーポレーション</td><td></td><td></td></tr><tr><td> Miniplus 3蠕動ポンプ</td><td> Gilbson </td><td></td><td></td></tr><tr><td>プラットフォームロータリーシェーカー</td><td></td><td></td><td></td></tr><tr><td> FemtoJetマイクロキャピラリー圧力供給</td><td>エッペンドルフ</td><td></td><td></td></tr><tr><td>シングル四ウェルのLab - Tek社IIカバーガラスチャンバー</td><td> Nalgeヌンクインターナショナル</td><td></td><td></td></tr><tr><td> LSM 510 METAまたは同等の蛍光顕微鏡</td><td>ツァイス</td><td></td><td> 40X 1.3 NAまたは60X 1.4 NA油DICプラン- Neofluarの対物レンズ</td></tr><tr><td>オリンパスX81または同等の</td><td>オリンポス</td><td></td><td> 100X 1.47 NA TIRF対物レンズが必要です。 </td></tr></tbody></table>

imaging g-protein coupled receptor (gpcr)-mediated signaling events that control chemotaxis of dictyostelium discoideum

多くの真核細胞は、その環境での化学信号の勾配を検出し、それに応じて1を移行することができます。このガイド付きの細胞遊走は、免疫細胞や神経細胞2、3のパターニングの人身売買など、その機能を遂行するために様々な細胞に不可欠な走化性、と呼ばれます。 G -タンパク質共役受容体（GPCR）の大家族は、in vivoで 4 の細胞移動を指示するために、ケモカインとして知られている変数の小さなペプチドを、検出する。走化性研究の最終目標は、GPCRの機械感覚のケモカインの勾配とコントロールが走化性につながるイベントを通知する方法を理解することです。この目的のために、我々は、リアルタイムで、化学誘引物質の勾配に誘引物質、細胞運動の時空間濃度を監視するためのイメージング技術を使って、GPCRは三量体G -タンパク質の活性化を仲介し、5月8日真核細胞の走化性に関与する細胞内シグナル伝達イベント。単純な真核生物、 キイロタマホコリカビは 、白血球のに似ているchemotaxic動作を表示し、D.カビは真核生物の化学走性を研究するための重要なモデル系である。自由生活アメーバ、D.などdiscoideumの細胞が豊富な培地で割ります。飢餓時には、細胞は、multicullular構造を形成するためにcAMPを介した走化性を介して集約した発生プログラムを入力してください。 cAMPに走化性に関与する多くのコンポーネントは、Dで確認されているカビ 。 GPCR（CAR1）へのcAMPの結合はGγとGβγサブユニット7、9、10にヘテロ三量体G -タンパク質の解離を誘導する。 Gβγサブユニットは、順番に細胞膜11-13 PIP 3にPIP 2を変換し、PI3Kを活性化Rasを活性化する。 PIP 3はこのように膜14、15にこれらのタンパク質を募集し、プレクストリン相同（PH）ドメインを持つタンパク質の結合部位として機能する。 CAR1受容体の活性化はまた、PIP 2 16、17にPIP 3を脱リン酸化PTEN、の膜の関連付けを制御します。分子メカニズムは、進化的にそのような好中球18のようなヒト細胞のケモカインGPCRを介した走化性で保存されている。我々は、D.の走化性を研究するための次の方法を提示discoideumの細胞 。 1。走化性成分の細胞の調製。 2。 cAMPの勾配における細胞のイメージングは​​、走化性。 3。単一の生細胞におけるGタンパク質ヘテロのGPCR誘導活性を監視する。 4。リアルタイムで単一の生細胞のイメージングは、化学誘引物質トリガダイナミックPIP 3の応答。私たちの開発した撮像法は、ヒト白血球の走化性を研究するために適用することができます。

Watch this Scientific Journal Video about Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum at JoVE.com

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

ここでは、走化性を調査するための詳細な生細胞イメージングの方法を説明します。我々は、遊走細胞のシグナル伝達事象の時空間ダイナミクスを監視するために蛍光顕微鏡の方法を提示する。シグナル伝達イベントの測定は、私たちはさらにGPCRシグナリングネットワークは真核細胞の化学誘引物質とコントロールの方向マイグレーションの勾配のセンシングを実現する方法を理解することが可能。

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 ...

imaging-g-protein-coupled-receptor-gpcr-mediated-signaling-events

Research

JoVE Journal

Biology

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

17.9K ビュー.  National Institute of Allergy and Infectious Diseases, National Institutes of Health. ここでは、走化性を調査するための詳細な生細胞イメージングの方法を説明します。我々は、遊走細胞のシグナル伝達事象の時空間ダイナミクスを監視するために蛍光顕微鏡の方法を提示する。シグナル伝達イベントの測定は、私たちはさらにGPCRシグナリングネットワークは真核細胞の化学誘引物質とコントロールの方向マイグレーションの勾配のセン?...

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

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

スペクトル的に解決された二光子顕微鏡を用いたGタンパク質共役受容体相互作用の in vivo定量で

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

生物学

Quantifying Agonist Activity at G Protein-coupled Receptors

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.
Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (Kb) is much greater than that for the inactive state (Ka). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (Kobs), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (&#949;) (Figure 2).
Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the Kobs and relative efficacy of an agonist 1,2. In this report, we show how to modify this analysis to estimate the agonist Kb value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate Kb in absolute units of M-1.
Our method of analyzing agonist concentration-response curves 3,4 consists of global nonlinear regression using the operational model 5. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of Kobs and a parameter proportional to efficacy (&tau;). The estimate of &tau;Kobs of one agonist, divided by that of another, is a relative measure of Kb (RAi) 6. For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (&tau;sys). In this case, the Kb value of an agonist is equivalent to &tau;Kobs/&tau;sys 3.
Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.

A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.

Gタンパク質共役受容体でアゴニスト活性を定量化

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

単分子バイオセンサーを用いた生細胞中のシグナル伝達事象をリアルタイムで監視するための顕微鏡のFRET

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

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 ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

遺伝的にコードされた分子プローブは、Gタンパク質共役受容体を研究する

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

蛍光ベースのカルシウム動員アッセイによるGタンパク質共役受容体のキャラクタリゼーション

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

TIRF顕微鏡を経由してシングルイベント解像度のGタンパク質共役型受容体のクラスリン依存性エンドサイトーシスの可視化

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

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

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular development program, during which single cells chemotax towards aggregation centers1-4. This process is dependent on the release of cyclic adenosine monophosphate (cAMP)5. cAMP is produced in waves through the concerted action of adenylate cyclase and phosphodiesterases, and binds to G protein-coupled cAMP receptors6,7. A widely used assay to analyze the mechanisms involved in the developmental cycle of the lower eukaryote Dictyostelium discoideum is based on the observation of cell aggregation in submerged conditions8,9. This protocol describes the analysis of the role of coronin A in the developmental cycle by starvation in tissue-culture plates submerged in balanced salt solution (BSS)10. Coronin A is a member of the widely conserved protein family of coronins that have been implicated in a wide variety of activities11,12. Dictyostelium cells lacking coronin A are unable to form multicellular aggregates, and this defect can be rescued by supplying pulses of cAMP, suggesting that coronin A acts upstream of the cAMP cascade10. The techniques described in these studies provide robust tools to investigate functions of proteins during the initial stages of the developmental cycle of Dictyostelium discoideum upstream of the cAMP cascade. Therefore, utilizing this aggregation assay may allow the further study of coronin A function and advance our understanding of coronin biology.

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 ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

イメージングGタンパク質共役型受容体媒介走化性及び好中球様HL60細胞におけるそのシグナル伝達のイベント

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

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 ...