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% エタノールと紫外線照射による殺菌) カバー スリップと DMEM 培地 2 mL 6 ウェル プレートのウェルに。37 ° C、5% CO2で 24 時間加湿のインキュベーターで細胞を成長します。</li>
	<li>1stチミジン ブロック: 2 日目、徹底的に新鮮な DMEM メディア (100 mM 在庫、2 mM 最終濃度) のチミジンの必要量を混ぜます。</li>
	<li>チミジンを含む 2 mL を追加メディア 6 ウェルの細胞をプレートし、18 時間孵化させなさい。</li>
	<li>3 日目、培地を吸引し、2 mL の 1x PBS で 2 回細胞と新鮮な prewarmed DMEM; に一度洗う9 h ブロックから細胞を解放する新鮮な prewarmed DMEM 培地で細胞を育ちます。</li>
	<li>2ndチミジン ブロック: チミジンを含む 2 mL を再び追加メディア (最終濃度が 2 mM) 6 ウェル プレートの各ウェル内のセルにします。</li>
	<li>さらに 18 時間孵化させなさい。</li>
	<li>4 日目、培地を吸引し、2 mL の 1x PBS で 2 回細胞と新鮮な prewarmed DMEM を洗ってください。</li>
	<li>一緒にそれらをミックスし、室温 20 分間インキュベート希薄 Sirna (制御と Cdt1) と血清無料で 10 分間個別に培地にトランスフェクション試薬各トランスフェクションに使用する siRNA の最終濃度が 100 nM。チミジンから洗浄されたセルに反応混合物を追加します。</li>
	<li>インキュベート細胞ブロックから解放し、4% とカバー スリップ上のセルを修復する 9-10 h の 37 ° C で室温に 20 分間 PFA 注意: PFA は有毒である、適切な保護具を着用します。</li>
	<li>
Immunostain セル、常温の 10 分の 0.5% (v/v) 洗剤イオンと 5 分の 1 × PBS で 2 回細胞を洗浄後。
	<ol>
<li>1% (w/v) で一次抗体 (マウス抗 α-チューブリン抗体希釈 1:1, 000、1: 400、1: 300 で希釈した抗リン ɣ H2AX マウスで希釈ウサギ抗 Zwint1 抗体) の 50 μ L とセルを扱う続く RT で 1 h の 1x PBS で 1 %bsa (w/v) で細胞をブロックします。37 oc 以上で 1 h の 1x PBS で BSA</li>
		<li>1x PBS で細胞を 3 回洗浄した後室温 1 時間各カバー ガラス (アレクサ 488 と 1: 250 の 1% (w/v) BSA 1x PBS で希釈でローダミン レッド) 二次抗体の 50 μ L で細胞を治療します。</li>
		<li>2 回各 5 分の 1 × PBS のセルを洗浄した後セル DAPI を扱う (0.1µg/mL の 1x PBS で) 右で 5 分間</li>
		<li>2 回各 5 分の 1 × PBS のセルを洗浄した後クリア顕微鏡スライド上の適切なマウント メディアにセルに直面してカバー スリップを配置します。</li>
	</ol>
</li>
	<li>60 X 100 X 1.4 NA 計画アポクロマート DIC 油浸対物レンズと immunostained タンパク質の細胞が必要なイメージの品質に応じて適切なカメラを装備して逆の高分解能共焦点顕微鏡にマウントされているイメージ。</li>
	<li>顕微鏡用ソフトウェアを用いた 0.2 μ m 厚の z スタックが適切なは、室温で画像を取得します。</li>
</ol>3. 細胞分裂の進行 (図 3 a) の生きているセルイメージ投射のためのプロトコル
<ol><li>1 日に 1.5 mL DMEM 培地で 35 mm ガラス底培養皿に mCherry H2B と GFP-α-チューブリン (少しセルの種類および蛋白質の表現に基づいて変数) を安定発現約 0.5-1 x 105 HeLa 細胞を播くし、加湿のそれらを育てる37 oC と 5% CO2で 24 h のためのインキュベーター。</li>
	<li>1stチミジン ブロック: 2 日目、チミジンを含む 1.5 mL を加える料理 (100 mM チミジン、2 mM 最終濃度) のセルにメディア。</li>
	<li>18 時間孵化させなさい。</li>
	<li>3 日目、チミジンを含む培地を吸引し、2 mL の 1x PBS で 2 回と新鮮な prewarmed DMEM 三度、セルを洗浄します。</li>
	<li>一緒にそれらをミックスし、室温 20 分間インキュベート希薄 Sirna (制御と Hec1) と 10 分ごとに個別に血清フリー培地でのトランスフェクション試薬各トランスフェクションに使用する siRNA の最終濃度が 100 nM。チミジンから洗浄されたセルに反応混合物を追加します。</li>
	<li>1.5 mL 8 h ブロックから細胞を解放する新鮮な prewarmed DMEM 培地で細胞を成長します。</li>
	<li>2ndチミジン ブロック: チミジンを含む 1.5 mL を追加料理のセルにメディア (2 mM 最終濃度)。</li>
	<li>さらに 18 時間孵化させなさい。</li>
	<li>4 日目、培地を吸引し、2 mL の 1x PBS で 2 回と新鮮な prewarmed DMEM 三度、セルを洗浄します。細胞を成長し、新鮮な prewarmed ・ リーボヴィッツの (L-15) 媒体補われる 10 %fbs と 20 mM HEPES pH 7.0 8 h ブロックから細胞を解放します。</li>
	<li>既にステージと実験の温度を安定させるためにイメージングを開始する前に少なくとも 30 分間点灯する高分解能共焦点顕微鏡で設定温度制御チャンバーに皿を置きます。</li>
	<li>セルが表示される、まで 60 × 対物と明るいフィールドにフォーカスし、手動で選択した領域に、ステージ上でふるいにかけます。</li>
	<li>光とレーザー パワー/露出、画像集録パラメーターと選択の顕微鏡画像集録ソフトウェアを使用してテスト期間を設定します。GFP (488 励起; 385 nm 発光) と mCherry (561 nm 励起と 385 nm 発光) 画像を取得するためのフィルターを使用します。</li>
	<li>タイムラプスの実験を実行するには、別に 16 h までの期間にパルス送信される光と蛍光画像 10 分ごとを取得します。</li>
	<li>12 1.0 μ m で区切られたソフトウェアを使用して二重チミジン ブロックからのリリース後 9 h z-飛行機の顕微鏡に適切なは、画像を取得します。</li>
	<li>細胞分裂の進行を追跡画像を解析し、最終的にソース ソフトウェア フィジー/ImageJ で対応する映画を組み立てます。</li>
</ol>

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著者は、彼らが競合する財務持分を有してないことを宣言します。

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 &amp;deg;C</td></tr><tr><td>DPBS (1x)</td><td>Life Technologies</td><td>14190-144</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Leibovitz&amp;rsquo;s (1x) L-15 medium</td><td>Life Technologies</td><td>21083-027</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Serum reduced medium (Opti-MEM)</td><td>Life Technologies</td><td>319-85-070</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Penicillin and streptomycin</td><td>Life Technologies</td><td>15070-063 (Pen Strep)</td><td>1:1,000 dilution</td></tr><tr><td>Dharmafect2</td><td>GE Dharmacon</td><td>T-2002-02</td><td>Store at 4 &amp;deg;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-&amp;alpha;-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-&amp;alpha;-tubulin</td><td>Santa Cruz Biotechnology</td><td>Sc32293</td><td>1:1,000 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-&amp;gamma;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>35 mm 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 ...

combining-mitotic-cell-synchronization-high-resolution-confocal

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

14.2K ビュー.  Northwestern University. 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。

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

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

2つの相補的細胞同期プロトコルによる細胞周期制御遺伝子発現の研究

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

遺伝学

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

細胞分裂時の染色体分配の生きているセルイメージ投射

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

発生生物学

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

siRNAトランスフェクション後の有糸分裂のタイムラプスイメージング

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

生物学

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

哺乳類の細胞分裂量的共焦点反射顕微鏡を介して 3 D 行列

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

生物工学

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

リビングでの動的動原体タンパク質の挙動と染色体の動きを図解して有糸分裂チェックポイントを勉強<em&gt;ショウジョウバエ</em&gt;合胞体胚

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

全細胞集団の単一細胞レベルでサイクリンBの分解を勉強

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and development. Cytokinesis involves a series of events that are well coordinated in time and space. Cytokinesis involves the formation of an actomyosin ring at the division site, followed by ring constriction, membrane furrow formation and extra cellular matrix remodeling. The fission yeast, Schizosaccharomyces pombe (S.&#160;pombe)&#160;is a well-studied model system that has revealed with substantial clarity the initial events in cytokinesis. However, we do not understand clearly how different cytokinetic events are coordinated spatiotemporally. To determine this, one needs to analyze the different cytokinetic events in great details in both time and in space. Here we describe a microscopy approach to examine different cytokinetic events in live cells. With this approach it is possible to time different cytokinetic events and determine the time of recruitment of different proteins during cytokinesis. In addition, we describe protocols to compare protein localization, and distribution at the site of cell division. This is a basic protocol to study cytokinesis in fission yeast and can also be used for other yeasts and fungal systems.

The fission yeast, Schizosaccharomyces pombe&#160;is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

分裂酵母における細胞質分裂のイベントの時空間解析

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

蛍光生細胞顕微鏡によるミトティック・マイオシス酵母核ダイナミクスの検討

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, misunderstood, or misinterpreted by the fixed-cell analysis. While the fixed cell imaging and analysis is robust and sufficient to observe cellular steady-state, it can be limited in defining a temporal order of events at the cellular level and is ill-equipped to assess the transient nature of dynamic processes including mitotic progression. In contrast, live cell imaging is an eloquent tool that can be used to observe cellular processes at the single-cell level over time and has the capacity to capture the dynamics of processes that would otherwise be poorly represented in fixed cell imaging. Here we describe an approach to generate cells carrying fluorescently labeled markers of chromatin and microtubules and their use in live cell imaging approaches to monitor metaphase chromosome alignment and mitotic exit. We describe imaging-based techniques to assess the dynamics of spindle formation and mitotic progression, including the identification of cells at various stages in mitosis, identification&#160;and tracking of mitotic defects, and analysis of spindle dynamics and mitotic cell fate following the treatment with mitotic inhibitors.

Here we present a protocol to assess the dynamics of spindle formation and mitotic progression. Our application of time-lapse imaging enables the user to identify cells at various stages of mitosis, track and identify mitotic defects, and analyze spindle dynamics and mitotic cell fate upon exposure to anti-mitotic drugs.

水動紡錘摂動後のメタフェイズタイミングと細胞運命のダイナミクスを評価するライブ細胞イメージング

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, ...

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae

Time-lapse fluorescence microscopy has revolutionized the understanding of meiotic cell-cycle events by providing temporal and spatial data that is often not seen by imaging fixed cells. Budding yeast has proved to be an important model organism to study meiotic chromosome segregation because many meiotic genes are highly conserved. Time-lapse microscopy of meiosis in budding yeast allows the monitoring of different meiotic mutants to show how the mutation disrupts meiotic processes. However, many proteins function at multiple points in meiosis. The use of loss-of-function or meiotic null mutants can therefore disrupt an early process, blocking or disturbing the later process and making it difficult to determine the phenotypes associated with each individual role. To circumvent this challenge, this protocol describes how the proteins can be conditionally depleted from the nucleus at specific stages of meiosis while monitoring meiotic events using time-lapse microscopy. Specifically, this protocol describes how the cells are synchronized in prophase I, how the anchor away technique is used to deplete proteins from the nucleus at specific meiotic stages, and how time-lapse imaging is used to monitor meiotic chromosome segregation. As an example of the usefulness of the technique, the kinetochore protein Ctf19 was depleted from the nucleus at different time points during meiosis, and the number of chromatin masses was analyzed at the end of meiosis II. Overall, this protocol can be adapted to deplete different nuclear proteins from the nucleus while monitoring the meiotic divisions.

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae

Time-lapse microscopy is a valuable tool for studying meiosis in budding yeast. This protocol describes a method that combines cell-cycle synchronization, time-lapse microscopy, and conditional depletion of a target protein to demonstrate how to study the function of a specific protein during meiotic chromosome segregation.

S. cerevisiaeの減数分裂を研究するためのタイムラプス顕微鏡とタンパク質のステージ特異的核枯渇の使用

Time-lapse fluorescence microscopy has revolutionized the understanding of meiotic cell-cycle events by providing temporal and spatial data that is often ...