Name: combining mitotic cell synchronization and high resolution confocal microscopy to study the role of multifunctional cell cycle proteins during mitosis
Uploaded: 2017-12-05T14:00:00
Description: Watch this Scientific Journal Video about Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis at JoVE

MCherry ヒストン H2B と GFP-α-チューブリンを安定発現 HeLa 細胞を共有するため東北大学の田中幸三に感謝しております。この作品は、DV (R00CA178188) に NCI の助成金、ノースウェスタン大学からスタートアップ資金にサポートされていました。

1. 二重チミジン ブロックおよび解放: 試薬の準備
<ol><li>ダルベッコ修飾イーグル培地 (DMEM) 培 10 %fbs、ペニシリン、ストレプトマイシンと 500 mL を作る。</li>
	<li>チミジンの 100 mM の在庫は、滅菌水と因数-80 ° C でストア</li>
</ol>2 細胞分裂の進行 (図 1 a) の固定セルイメージ投射のためのプロトコル
<ol><li>日 1、5 HeLa 細胞の 10 倍 ~ 2 種 (70% エタ...

<ol>
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Chem. 285 (10), 7197-7207 (2010).</li><li>Rosner, M., Schipany, K., Hengstschl&#228;ger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Merging+high-quality+biochemical+fractionation+with+a+refined+flow+cytometry+approach+to+monitor+nucleocytoplasmic+protein+expression+throughout+the+unperturbed+mammalian+cell+cycle.">Merging high-quality biochemical fractionation with a refined flow cytometry approach to monitor nucleocytoplasmic protein expression throughout the unperturbed mammalian cell cycle.</a> Nature Protocols. 8 (3), 602-626 (2013).</li><li>Coquelle, 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=Enrichment+of+non-synchronized+cells+in+the+G1,+S+and+G2+phases+of+the+cell+cycle+for+the+study+of+apoptosis.">Enrichment of non-synchronized cells in the G1, S and G2 phases of the cell cycle for the study of apoptosis.</a> Biochem. Pharmacol. 72 (11), 1396-1404 (2006).</li><li>Amon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+procedures.">Synchronization procedures.</a> Methods Enzymol. 351, 457-467 (2002).</li><li>Cooper, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rethinking+synchronization+of+mammalian+cells+for+cell+cycle+analysis.">Rethinking synchronization of mammalian cells for cell cycle analysis.</a> Cell Mol. Life Sci. 60 (6), 1099-1106 (2003).</li><li>Whitfield, M. L., Sherlock, G., Saldanha, A. J., Murray, J. I., Ball, C. A., Alexander, K. E., Matese, J. C., Perou, C. M., Hurt, M. M., Brown, P. O., Botstein, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+genes+periodically+expressed+in+the+human+cell+cycle+and+their+expression+in+tumors.">Identification of genes periodically expressed in the human cell cycle and their expression in tumors.</a> Mol. Biol. Cell. 13 (6), 1977-2000 (2002).</li><li>Vassilev, L. T., 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=Selective+small-molecule+inhibitor+reveals+critical+mitotic+functions+of+human+CDK1.">Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1.</a> Proc Natl Acad Sci U S A. 103 (28), 10660-10665 (2006).</li><li>Varma, D., Chandrasekaran, S., Sundin, L. J., Reidy, K. T., Wan, X., Chasse, D. A., Nevis, K. R., DeLuca, J. G., Salmon, E. D., Cook, J. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+the+double+thymidine+block+for+synchronizing+mammalian+cells+at+the+G1-S+border.">An evaluation of the double thymidine block for synchronizing mammalian cells at the G1-S border.</a> Exp. Cell Res. 68 (1), 163-168 (1971).</li><li>Martin-Lluesma, S., Stucke, V. M., Nigg, E. 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+Hec1+in+spindle+checkpoint+signaling+and+kinetochore+recruitment+of+Mad1/Mad2.">Role of Hec1 in spindle checkpoint signaling and kinetochore recruitment of Mad1/Mad2.</a> Science. 297, 2267-2270 (2002).</li><li>Brouwers, N., Mallol Martinez, N., Vernos, I. <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+Kif15+and+its+novel+mitotic+partner+KBP+in+K-fiber+dynamics+and+chromosome+alignment.">Role of Kif15 and its novel mitotic partner KBP in K-fiber dynamics and chromosome alignment.</a> PLoS One. 12 (4), e0174819 (2017).</li><li>Dou, Z., 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=Dynamic+localization+of+Mps1+kinase+to+kinetochores+is+essential+for+accurate+spindle+microtubule+attachment.">Dynamic localization of Mps1 kinase to kinetochores is essential for accurate spindle microtubule attachment.</a> Proc Natl Acad Sci USA. 112 (33), E4546-E4555 (2015).</li><li>Chan, Y. 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二重チミジン同期の最も重要な利点は、染色体の整列、バイポーラの解析もでき、一斉に有糸分裂を入力これらの細胞の多くは短時間の窓の分裂期細胞の増加し、サンプル サイズを提供すること主軸形成と染色体分配のエネルギー効率の向上。
多くの規定する蛋白質複合体とシグナル伝達経路有糸分裂を通して通常進行を確保するために専念しているし、このプロセス?...

細胞周期細胞は彼らのゲノムと増殖の正確な重複規制の厳しいと一時的制御イベントのシリーズを受けます。哺乳類細胞周期の間期と M 期ので構成されます。中間期の 3 つの段階 - G1、S から成っている、セル G2 のゲノムを複製し、正常な細胞周期進行1,2のために必要な成長を経る。(前期 prometaphase、中期、後期、終期) 有糸分裂と細胞質分裂から成る M 相親細胞は遺伝的に同一の 2 つの娘細胞を生成します。続いて中期プレート (中期) の整列の有糸分裂、姉妹染色分け体重複ゲノムの凝縮 (前期)、組み立て有糸分裂紡錘体 (prometaphase) の微小管による自分の体に取得されたドライブの姉妹染色分け体のに向かって分割し、反対側のスピンドルに運ばれる極 (後期) とき偏析と等しい。2 つの娘細胞は、アクチンに基づく収縮環 (終期と細胞質分裂) の活動によって物理的に分かれています。動原体染色分け体のセントロメア領域で組み立てて特殊なタンパク質の構造は、紡錘体微小管の添付ファイルをサイトとして.その主な機能は、染色体キャプチャ、配置を駆動し、染色体分離3,4の忠実性を維持するために紡錘アセンブリ チェックポイントを仲介しながら、不適切なスピンドル微小管の添付ファイルを修正することを支援することです。
セル同期の手法は、細胞周期の進行に関与する分子と構造のイベントを理解するための理想的なツールとして機能します。このアプローチは、さまざまな種類の解析、遺伝子発現のプロファイリングを含む細胞の生化学的なプロセスの解析とタンパク質の細胞内局在性の検出の特定のフェーズでセル人口を豊かに使用されています。個々 の遺伝子産物の研究のみならず遺伝子式5、miRNA 発現パターン6のマイクロ アレイ解析を含む全体のゲノムの解析を伴うアプローチ同期の哺乳類細胞を使用できます。翻訳制御7、および蛋白質の変更8のプロテオーム解析。同期は、細胞周期の進行に及ぼす影響の遺伝子発現やタンパク質ノックダウンまたはノックアウト、または化学物質の研究にも使用できます。
細胞は、細胞周期の異なった段階で同期できます。物理的、化学的、広くセル同期に使用されます。セル同期の最も重要な基準は、同期する必要があります noncytotoxic と可逆であること。潜在的な不利な細胞結果の薬理学的エージェントによってセルを同期する、ため化学依存メソッドはキー細胞周期イベントの勉強のために有利にできます。たとえば、ヒドロキシウレア、amphidicolin、ミモシン、ロバスタチン、G1/S 期の細胞の同期に使用できるが、彼らを阻害する生化学的経路の効果のために彼らは細胞周期チェックポイント機構をアクティブにし、重要なを殺すセル9,10の割合です。その一方で、「チミジン ブロック」として知られている成長媒体にチミジンを追加して DNA 複製のフィードバック阻害は特定ポイント11,12,13で細胞周期を挙げることが出来る。セルは、ノコダゾールと RO 33069,14と扱うことによって G2/m 期で同期できます。微小管重合を防ぐノコダゾールが比較的高い細胞毒性です。また、ノコダゾール逮捕細胞分裂すべりによって中間期の早期に返すことができます。二重チミジン ブロック逮捕セル G1/S 段階およびブロックからの解放後、細胞はある G2 から、有糸分裂に同期的に続行します。細胞周期細胞チミジン ブロックから解放のための通常の進行は、セル固定またはライブ イメージングによる高分解能共焦点顕微鏡の下で観察できます。セルを入力し、二重チミジン ブロックからリリース後有糸分裂に進むときに具体的に、有糸分裂蛋白質の摂動の効果を学ぶことができます。Cdt1、多機能性蛋白質、DNA 複製が G1 期でライセンスを起源に関与する有糸分裂15の中にも動原体微小管の添付ファイル必要です。有糸分裂 Cdt1 の機能を研究、レプリケーション ライセンス G1 フェーズ G2/M フェーズのみで具体的にその枯渇に影響を与えて同時に一方で、枯渇の影響を回避する手法を採用する必要があります。ここでは、固定と生細胞イメージングによる蛋白質の細胞周期の異なった段階の間に複数の機能を実行するの分裂の役割を研究する二重チミジン ブロックに基づく詳細なプロトコルを提案する.

<table>
	<tbody>
		<tr>
			<td>DMEM (1X)</td>
			<td>Life Technologies</td>
			<td>11965-092</td>
			<td>Store at 4 degree C</td>
		</tr>
		<tr>
			<td>DPBS (1X)</td>
			<td>Life Technologies</td>
			<td>14190-144</td>
			<td>Store at 4 degree C</td>
		</tr>
		<tr>
			<td>Leibovitz&#8217;s (1X) L-15 medium</td>
			<td>Life Technologies</td>
			<td>21083-027</td>
			<td>Store at 4 degree C</td>
		</tr>
		<tr>
			<td>Serum reduced medium (Opti-MEM)</td>
			<td>Life Technologies</td>
			<td>319-85-070</td>
			<td>Store at 4 degree C</td>
		</tr>
		<tr>
			<td>Penicillin and streptomycin</td>
			<td>Life Technologies</td>
			<td>15070-063 (Pen Strep)</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Dharmafect2</td>
			<td>GE Dharmacon</td>
			<td>T-2002-02</td>
			<td>Store at 4 degree C</td>
		</tr>
		<tr>
			<td>Thymidine</td>
			<td>MP Biomedicals LLC</td>
			<td>103056</td>
			<td>Dissolved in sterile distiled water</td>
		</tr>
		<tr>
			<td>Cdt1 siRNA</td>
			<td>Life Technologies</td>
			<td></td>
			<td>Ref 10</td>
		</tr>
		<tr>
			<td>Hec1 siRNA</td>
			<td>Life Technologies</td>
			<td></td>
			<td>Ref 13</td>
		</tr>
		<tr>
			<td>HeLa cells expressing GFP-H2B</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa cells expressing GFP-&#945;-tubulin and mCherry H2B</td>
			<td>Generous gift from Dr. Kozo Tanaka of Tohoku university, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Sigma-Aldrich Corporation</td>
			<td>F8775</td>
			<td>Toxic, needs caution</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich Corporation</td>
			<td>D9542</td>
			<td>Toxic, needs caution</td>
		</tr>
		<tr>
			<td>Mouse anti-&#945;-tubulin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>Sc32293</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Rabbit ant-Zwint1</td>
			<td>Bethyl</td>
			<td>A300-781A</td>
			<td>1:400 dilution</td>
		</tr>
		<tr>
			<td>Mouse anti-phospho-&#947;H2AX (Ser139)</td>
			<td>Upstate Biotechnology</td>
			<td>05-626, clone JBW301</td>
			<td>1:300 dilution</td>
		</tr>
		<tr>
			<td>Alexa 488</td>
			<td>Jackson ImmunoResearch</td>
			<td></td>
			<td>1:250 dilution</td>
		</tr>
		<tr>
			<td>Rodamine Red-X</td>
			<td>Jackson ImmunoResearch</td>
			<td></td>
			<td>1:250 dilution</td>
		</tr>
		<tr>
			<td>BioLite 6 well multidish</td>
			<td>Thermo Fisher Scientific</td>
			<td>130184</td>
			<td></td>
		</tr>
		<tr>
			<td>35MM Glass bottom dish</td>
			<td>MatTek Corporation</td>
			<td>P35GCOL-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse TiE inverted microscope</td>
			<td>Nikon Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning disc for confocal</td>
			<td>Yokagawa</td>
			<td>CSU-X1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra 888 EM-CCD Camera</td>
			<td>Andor</td>
			<td>iXon Ultra EMCCD</td>
			<td></td>
		</tr>
		<tr>
			<td>4 wave length laser</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubation System for Microscopes</td>
			<td>Tokai Hit</td>
			<td>TIZB</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-elements software</td>
			<td>Nikon Instruments</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

combining mitotic cell synchronization and high resolution confocal microscopy to study the role of multifunctional cell cycle proteins during mitosis

位相依存的に細胞周期の様々 な規制のイベントの研究は、細胞増殖と分裂についての明確な理解を提供します。細胞周期の特定の段階のセル人口の同期は、このような実験的努力で非常に役に立つ発見されています。比較的より少なく有毒な化学薬品処理による細胞の同期は、結果として細胞周期イベントの選択した分裂段階の特定の濃縮を得ようと研究の薬理学的阻害薬の使用上有利なすることができます。ここで、セルのさまざまな段階で同期のひと細胞周期 S 期と M 段階二重チミジン ブロックの両方を含むと染色体の整列の有糸分裂蛋白質の機能を研究するための手順をリリース プロトコルを説明します。分離。このプロトコルは、確立された界面の機能を持つ多機能タンパク質の細胞分裂の役割を研究するため非常に便利になっています。私たちのケースでは Cdt1、レプリケーション元ライセンス G1 期での重要な蛋白質の細胞分裂の役割を効果的に学ぶことができます G2/M 固有 Cdt1 が枯渇することができる場合にのみ。二重チミジンの同期を使用して特定の G2/M Cdt1 の枯渇のための詳しいプロトコルについて述べる。また、セル固定のプロトコルを説明し、チミジン リリース後高分解能共焦点顕微鏡を用いた細胞イメージングを住んでいます。メソッドは Hec1、複雑な Ndc80 のコンポーネントのように生理と摂動条件下で細胞分裂蛋白質の機能を分析し固定と生きている細胞の分裂期の細胞の大規模なサンプル サイズを取得することができるため便利分析も紹介します。

有糸分裂の進行と細胞内微小管の安定性に関する研究は二重チミジン ブロックからリリース後固定 Cdt1 は G1 期に DNA の複製の起源のライセンスに関与しています。それは S 期は劣化が、G2/m 期で再蓄積します。有糸分裂におけるその役割を研究、内因性 Cdt1 を使用して最も適したセル同期方式の二重チミジン ブロック15G/M 段階で具体?...

Watch this Scientific Journal Video about Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis at JoVE

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

高分解能共焦点顕微鏡を用いた解析に続いて HeLa 細胞の二重チミジン同期するためのプロトコルを提案します。このメソッドは、多数の有糸分裂、また界面機能を有する多機能性蛋白質の細胞分裂の役割の解明を可能にする S 期から同期的に進めるセルを取得するキーです。

有糸分裂の多機能の細胞周期蛋白質の役割を研究する有糸分裂細胞同期と高分解能共焦点顕微鏡を組み合わせた

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...

The overall goal of this procedure is to use double thymidine synchronization and high-resolution confocal microscopy to study the mitotic roles of multifunctional proteins that may possess critical interphase functions. This method can help answer key questions in the cell biology field, about how to delineate the normal functions of proteins involved in cell cycle and mitosis. The main advantages of this technique are that, the cells can maintain their normal physiological behavior and that this method is also very easy to perform.Demonstrating this procedure will be Dr.Mohammed Amin, a post-doc from my laboratory, who is an expert in the use of this method. For fixed cell imaging of mitotic cell progression, begin by seeding approximately two times 10 to the fifth HeLa cells into each well of a six-well plate, containing a 70%ethanol and UV sterilized cover slip and two milliliters of DMEM medium. After 24 hours in a cell culture incubator, block the cells with two milliliters of freshly diluted thymidine and fresh medium per well, and return the plate to the incubator for another 18 hours.The next day, wash the cells two times with two milliliters of PBS, and one time with fresh 37 degree Celsius medium. Return the cells to the incubator for nine hours to release the cells from the block, followed by the addition of another two milliliters of thymidine-supplemented medium per well. After the second blocking incubation, wash the cells two times with two milliliters of PBS, and one time with two milliliters of fresh 37 degree Celsius medium, and add 100 nanomolar of freshly prepared siRNA to the appropriate wells.After nine to 10 hours, aspirate the supernatant and fix the cells onto the cover slips with four percent paraformaldehyde for 20 minutes at room temperature. After washing with PBS, permeabilize the fixed cells with 0.5%detergent for 10 minutes at room temperature, followed by two five-minute washes in PBS. Block the cells with one percent BSA in PBS for one hour at room temperature.Then label the cells with 50 microliters of the primary antibodies of interest for one hour at 37 degrees Celsius. At the end of the incubation, wash the cells three times in PBS, and label them with 50 microliters of the appropriate secondary antibodies of interest. After one hour at room temperature, wash the cells two times in PBS for five minutes each wash, and label the cells with DAPI for five minutes at room temperature.Follow the DAPI staining with two five-minute washes in PBS and use the appropriate mounting medium to mount the cover slips on individual clear microscope slides. Then, using 60 or 100 X 1.4 numerical aperture plan apochromatic DIC oil immersion objectives mounted on an inverted high resolution confocal microscope equipped with an appropriate camera, acquire images of the immunostained proteins in Z stacks of 0.2 micrometer thickness. For live cell imaging of mitotic cell progression, seed approximately 0.5 to one times 10 to the fifth HeLa cells stably expressing mCherry H2B and GFP alpha-tubulin into 35-milliliter glass-bottom dishes containing 1.5 milliliters of DMEM medium.Grow the cells in a cell culture incubator for 24 hours. Then, thymidine block the cells two times as just demonstrated, this time washing the cells two times with two milliliters of PBS and one time with one milliliter of pre-warmed DMEM medium at the end of each thymidine block and treating the cells with siRNA after the first block during the first thymidine washout. Grow the cells in fresh pre-warmed Leibovitz's medium, supplemented with 10%FBS and 20 millimolar HEPES for eight hours to release the cells from the second block.Then place the first plate in the temperature-controlled chamber of a high-resolution confocal microscope, and use the 60 X objective and bright field imaging to bring the cells into focus. When the cells are visible, manually adjust the plate position to the region of choice and use the image acquisition software to set the laser power and exposure, image acquisition parameters, and experiment duration period. Select the appropriate GFP and mCherry filters and acquire pulsed transmitted light and fluorescence images every 10 minutes for up to 16 hours, to obtain time-lapse images of the mitosis process.Nine hours after release from the double thymidine block, acquire the images at 12 one-micrometer separated z-planes. Then analyze the images by tracking individual mitotic cells and use the appropriate source software to assemble a corresponding movie. Although most of the control and Cdt1 siRNA cells are at the metaphase stage at nine hours post-release from double thymidine block, fixation at 10 hours reveals that most of the Cdt1 siRNA-treated cells are still arrested in late prometaphase, while the control cells enter anaphase and segregate their chromosomes as expected.Cold treatment nine hours after release from double thymidine block facilitates the retention of stable kinetochore microtubules which are relatively less robust in Cdt1-depleted cells compared to those within control-depleted cells. DNA replication origin licensing is not perturbed in Cdt1-or control-depleted cells during the G2-M phase, as these cells were not found to induce the accumulation of phosphoralated gamma H2AX, a marker for DNA damage, during the subsequent G2 phase. Cdt1 siRNA transfection of asynchronous cultures, however, induces the accumulation of phospho gamma H2AX positive foci possibly due to the DNA damage induced by improper DNA replication licensing.After nuclear envelope breakdown, normal cells enter mitosis and undergo anaphase onset to exit from mitosis within about 30 to 60 minutes. RNAi-mediated knockdown of Hec1, a key kinetochore protein required for robust kinetochore microtubule attachment formation, however, delays the normal mitotic progression to anaphase onset or exit from mitosis even after several hours of mitosis delay. Once mastered, this technique can be completed in three to four days if it is performed properly.While attempting the procedure, it's important to remember to dissolve the thymidine completely after you thaw it out from the freezer. Following this procedure, other methods like western blotting can be performed to answer additional questions like, what are the expression patterns of proteins of interest during mitosis compared to interphase? After its development, this technique paved the way for researchers in the field of cell biology to explore cancer biogenesis in human cell culture models, and to use high-resolution confocal microscopy to identify the cell cycle related functions of proteins of interest.Don't forget that working with reagents like paraformaldehyde and DAPI can be extremely hazardous and that precautions such as wearing gloves and a lab coat, should always be taken while performing this procedure.

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