We like to thank the members of the Dogterom lab for valuable discussions. We acknowledge Stefan Diez and Marcel Janson for reagents, and Samara Reck-Peterson for help with the purification of dynein. This work was supported by funding from the European Research Council (ERC Synergy grant: MODEL-CELL).

<ol>
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The authors declare that they have no competing financial interests.

方法は、ここで油中水型エマルジョンの液滴中の基本的な紡錘体を再構成することが、本の最近の取り組みを説明しました。幾何学的境界内にスピンドル・アセンブリを研究することは、多数の利点を提供します。重要なフィードバック機構は、有糸分裂紡錘体の微小管と、それらが組み立てられた幾何学的制約の間に存在します。微小管は、それらが、紡錘体の変位をもたらすことができる幾何学的境界に成長時に力を押し生成することができます。また、物理的な障壁に成長する微小管災害を誘発することができます。また、制限された形状内のスピンドル・アセンブリは、順番に直接微小管の成長率36に影響を与えるようなチューブリンなどの個々のコンポーネント、の漸進的な枯渇につながることができます。これらは、ここに記載のアッセイを用いて研究することができるスピンドル・アセンブリの全ての必須の決定要因です。 記載のアッセイは、低濃度のdiffereに非常に敏感です液滴コンポーネントのNCES。これは、マイナーな濃度差がいずれか遅すぎるか、あまりにも急速な微小管の成長をもたらすことができるチューブリン、の場合です。この場合には、微小管の成長率は、多くの場合、依然として実験の最初の約30分の間に温度を調整することによって補正することができます。有糸分裂紡錘体の組み立て及び位置合わせは、（皮質）力発生の濃度の変化に非常に敏感です。これは、精製された成分の濃度、純度および活性に依存する可能性があり、すべての新たに精製されたタンパク質のために慎重に滴定される必要があります。 また、特定のトレードオフは、アッセイの性質に応じて、撮像設定で行われなければなりません。最初は、水溶性の蛍光チューブリン二量体の高レベルは、画像の個々の微小管にそれを困難にします。微小管が長く成長し、可溶性チューブリンを徐々に枯渇しているように、信号対雑音比が徐々に高くなり、individuのイメージングを可能にしますアル微小管。オープンシステムでセットアップとは対照的に、幾何学的な閉じ込めは重要漂白が得られ、バルクリザーバ内の蛍光分子及び酸素スカベンジャー系の成分の交換を防止します。特に経時スピンドルアセンブリと位置を監視するために、それがあっても強力な酸素スカベンジャー系の存在下で、低い光強度で画像に必要とされます。 これらの方法には、in vitroでの簡素化紡錘状構造のボトムアップ再構成を可能にします。ここで紹介成分に加えて、他の多くの要因は、双極紡錘体形成、位置決め、方向付け、成形、 等 3に影響を与えることが記載されています。このシステムは、さらに、追加の力発生器の個々の及び組み合わせの効果を研究するために拡張することができます。このシステムから、現在存在しない紡錘体形成に重要な貢献者は、有糸分裂染色体です。有糸分裂クロマチンに示されていますいわゆる「極性吐出軍3 &#39; を生成することができます （1）chromokinesins、クロマチン結合RanGEF RCC1 38によってRanGTPと勾配の（2）の形成、（脱重合と相互作用することができる（3）動原体による直接紡錘体形成）微小管39及び（4）の対向する微小管40との間における機械的バネとして機能することができるクロマチン自体。 最後に、記載されたシステムは、現在、限られた時間的・​​空間的な活動を制御し、導入力発電機の局在を提供します。細胞では、多数のスピンドル・アセンブリの要素は、特定のコンパートメントに局在化または制限された時間ウィンドウ内で活性です。顕著な例は、具体的にはCの後側に濃縮されるダイニンの活動であり、非対称の主軸位置と細胞分裂を促進する1細胞期胚をエレガンス 。このシステムでは、我々は現在、トンを模倣する可能性を模索しています光遺伝学的手法から借用した方法を用いてemporalと非対称活動。また、Cの場合と同様エレガンス胚と剛性細胞壁を有する細胞は、多くの細胞が有糸分裂に切り上げていません。幾何学的な閉じ込めの形状は、スピンドル41に作用する力の基礎影響を与えることになります。したがって、潜在的に整形し、エマルジ ​​ョン液滴42,43の記憶許可より複雑なマイクロ流体システムを使用して、同様に非球形の小滴にスピンドル・アセンブリと位置を再構成することが重要であろう。最後に、組織において、細胞 - 細胞および細胞 - 基質シグナリングおよび付着は、多くの場合、上皮細胞において観察されるように、指向軸方向に変換することができる外部の手がかりを提供し、幹細胞。これらの特殊な側面のすべては、この現在の研究では考慮されておらず、紡錘体assembのin vivo様より再構成は向かって構築するための今後の課題を提供するかもしれませんLYとポジショニング。

有糸分裂の間、複製されたゲノムの染色体は新たに形成された娘細胞の上に姉妹染色分の平等な分配を確保するために、セル赤道で編成されています。染色体の位置および分離は、二つの対向の中心体から発するスピンドル微小管への結合によって媒介されます。忠実な染色体分配を確保することに加えて、有糸分裂紡錘体の配向は、細胞分裂面1を決定します 。細胞分裂の協調姿勢は、開発の多くの段階中および組織の恒常性2に必須です。具体的には、規制紡錘体の位置は、細胞内容の非対称分布が生じ、あるいは異なるサイズ2の娘細胞を生成することができます。紡錘体アセンブリおよび向きは、前期に開始し、協力し力発電機3,4に拮抗するとの間の複雑な相互作用によって編成されています。発生する力は、Bから構成されOTH引っ張り、押す力。これらの力は、細胞の皮質とスピンドルの連絡先からだけでなく、スピンドル内の両方から発信され、微小管ダイナミクスによってだけでなく、分子モーターとクロスリンカー（ 図1）によって生成することができます。 微小管は、細胞皮質のような剛性の構造に対して成長時押圧力を発生させることができます。システム内で発生する総抵抗力に応じて、これは、有糸分裂紡錘体（低力）の再配置をもたらすか、微小管の座屈や大惨事（強い力）5-8をトリガすることができます 。長さは、微小管座屈9の強力な決定因子であるので、押すことによって発生させることができる力の量は、微小管の長さに依存します。さらに、1つの中心体に由来微小管は、他の中心体に対して早期前期3,10に中心体分離を駆動するために提案された機構を押すことができます。 広告で成長している微小管によって発生した力を押す、微小管が接触し末端細胞表層へのditionも力11を引っ張っ媒介することができます。複数の研究室からの研究は、皮質力発電機の制御された活性は、インビボ 1 で紡錘体の非対称の位置決めのために必要であることを示しています。これらの引っ張り力にエッセンシャル（以下「ダイニン」という）皮質局在細胞質ダイニン、マイナスエンド向かう微小管モーター蛋白質8,12,13です。例えば、酵母、皮質14に沿ってスライドモーター依存微小管における微小管の格子結果に皮質ダイニンの横方向の相互作用を出芽インチしかし、皮質引っ張り力はまた、微小管8を解重合する末端にアタッチメントを形成するダイニンの機能によって生成することができます。微小管収縮によって発生するエネルギーは、一般的により高い一桁力につながる可能性があり（〜50 PN（15を参照</s個々のモータータンパク質（〜7-8 PN（参考文献16））によって生成される力より））&gt;アップ。脱重合微小管への皮質ダイニンのエンド上の結合は出芽酵母17およびCで主軸の位置決めを促進しますエレガンス 13。モーター依存スライドおよび微小管脱重合駆動の両方皮質引っ張る力が協力またはin vivoで相互に排他的であることができるかどうかを現在不明です。 微小管押圧力とダイニン媒介皮質引っ張り力に加えて、中心体の位置決めは、多くの他のタンパク質によって有糸分裂紡錘体の中から制御されます。キネシン-5モータは、例えば、外側にスライド力18-20の生成をもたらす、逆平行極間微小管を架橋することができる四量体として存在します。キネシン-5モーターファミリーのメンバーは、Cの例外を除いて、研究した全ての真核生物における中心体分離およびバイポーラスピンドルアセンブリのために必要とされますエレガンス<html> （参照21に概説されています）。 また、ASE1 / PRC1ファミリーの拡散性架橋剤はまた、 インビボおよびインビトロ 22-27 に極間微小管の微小管重複領域に局在することが知られています。重複微小管領域のASE1主導の展開によって発生する力は、in vitro 28,29 にキネシン-14媒介スライディング力を相殺するのに十分な大きさです。細胞では、ASE1 / PRC1は、スピンドル中間帯25-27,30で微小管の重複領域を拡散性架橋剤によって発生する力が大幅にスピンドル組織に貢献できることを示唆しているの安定形成に必要です。最後に、有糸分裂クロマチンからの力および動原体は、種々の経路3を介して紡錘体のアセンブリおよび位置決めに影響を与えます。これらのコンポーネントは、ここで説明する再構成アッセイの一部ではないので、詳細には説明しません。 jove_content &quot;&gt;別の理論は、異なる分子成分と力がスピンドルアセンブリと位置決めに協力、私たちはまだ遠い定量的理解からである上記の方法に存在する。生細胞での実験に加えて、精製された成分を用いたin vitroの実験の強力なを提供しますこの目標を達成するのに役立つルート。ここでは、最近公開された方法（ロスら 31）の適応と拡張版の視覚ガイドを提示し、基本的なスピンドルが始まる、油中水型エマルジ ​​ョン液滴中で再構成されていますマイクロ流体技術を使用してコンポーネントの最小数は、球状液滴が分裂細胞に匹敵するサイズで生成される。これらの液滴内で、精製された中心体およびチューブリンは、微小管アスター動態を研究発生及びアスター・アスター相互作用を強制するために組み合わせることができる。で皮質（例えば、ダイニンなど）およびインター極性（例えば、キネシン5 / ASE1）力発電機を導入し、我々in vivoの状況に似ていると起動し、ますます複雑紡錘状の構造を再構成します。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1. Preparation of microfluidic chips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Photomask (Photomask on film substrate)</td>
			<td>Selba S.A. Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 3025</td>
			<td>MicroChem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafer (4 inch silicon wafer p/Boron &#60;1-0-0&#62; 10-20 &#937;-cm, 500-550&#956;m, SSP, w/2 flats)</td>
			<td>WRS Materials</td>
			<td>4P0SSP-005</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater&#160;</td>
			<td>Polos</td>
			<td>SPIN150I</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS pre-polymer RVT615 A+B</td>
			<td>Lubribond</td>
			<td>9481</td>
			<td></td>
		</tr>
		<tr>
			<td>Corona treater</td>
			<td>Electro-technic products</td>
			<td>BD-20ACV</td>
			<td></td>
		</tr>
		<tr>
			<td>2. Microfluidic setup</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>DOPS (1,2-Dioleoyl-sn-glycero-3-phospho-L-serine)</td>
			<td>Avanti Polar Lipids</td>
			<td>840035</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinyl PE (1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl))</td>
			<td>Avanti Polar Lipids</td>
			<td>870285</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>650498</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass pipets</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Laboport</td>
			<td>KNF</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum chamber</td>
			<td>Kartell</td>
			<td>Polypropylene Vacuum Desiccator</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma-Aldrich</td>
			<td>M5904</td>
			<td></td>
		</tr>
		<tr>
			<td>Surfactant (Span80)</td>
			<td>Sigma-Aldrich</td>
			<td>85548</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Branson</td>
			<td>M2800H</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td></td>
			<td></td>
			<td>24x60mm, thickness 1.5</td>
		</tr>
		<tr>
			<td>Glass-slides</td>
			<td></td>
			<td></td>
			<td>26x76mm</td>
		</tr>
		<tr>
			<td>Puncher</td>
			<td>Harris Uni-Core</td>
			<td>135840/135841/135843</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory sealing film</td>
			<td>Parafilm</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Valap (Vaseline, lanolin, paraffin wax melted at equal concentrations)</td>
			<td></td>
			<td></td>
			<td>Home made</td>
		</tr>
		<tr>
			<td>Brightfield Microscope</td>
			<td>Leica</td>
			<td>PMIRB&#160;</td>
			<td>Any inverted brightfield would suffice</td>
		</tr>
		<tr>
			<td>Pressure controler</td>
			<td>Fluigent</td>
			<td>MFCS-FLEX-4C-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>PEEK Tubing</td>
			<td>Cluzeau Info. Labo, France</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microfluidics vials (Tubes Micrew 0.5 ml and 1 ml)</td>
			<td>VWR, The Netherlands</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3. Reconstituting spindle formation and positioning</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Dextran-647nm (Dextran, Alexa Fluor 647, 10,000 MW, Anionic, Fixable)</td>
			<td>Life Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA - Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose (D-(+)-Glucose)</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose-oxidase (Glucose oxidase from Aspergillus niger)</td>
			<td>Sigma-Aldrich</td>
			<td>G6125</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT (DL-dithioltheitol)</td>
			<td>Sigma-Aldrich</td>
			<td>646563</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase (Catalase form bovine liver)</td>
			<td>Sigma-Aldrich</td>
			<td>C9322</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubulin (Tubulin from bovine brain)</td>
			<td>Cytoskeleton Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tubulin-488/561 (HiLyte-488/Rhodamine-labeled tubulin from porcine brain)</td>
			<td>Cytoskeleton Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GTP (Guanosine-&#39;5-triphosphate, sodium salt hydrate)</td>
			<td>Sigma-Aldrich</td>
			<td>51120</td>
			<td></td>
		</tr>
		<tr>
			<td>&#954;-casein (&#954;-casein from bovine serum milk)</td>
			<td>Sigma-Aldrich</td>
			<td>C0406</td>
			<td></td>
		</tr>
		<tr>
			<td>Airfuge (Air-driven ultracentrifuge)</td>
			<td>Beckman-Coulter</td>
			<td>CLS</td>
			<td></td>
		</tr>
		<tr>
			<td>4. Introducing spindle-assembly factors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Neutravidin</td>
			<td>Sigma-Aldrich</td>
			<td>A2666</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP (Adenosine-&#39;5-triphosphate, disodium salt hydrate)</td>
			<td>Sigma-Aldrich</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr>
			<td>PEP (Phospho(enol)pyruvic acid monosodium salt hydrate &#8805; 97% (enzymatic))</td>
			<td>Sigma-Aldrich</td>
			<td>P0564</td>
			<td></td>
		</tr>
		<tr>
			<td>PK/LDH -(Pyruvate kinase/lactic dehydrogenase enzymes from rabbit muscle)</td>
			<td>Sigma-Aldrich</td>
			<td>P0294</td>
			<td></td>
		</tr>
	</tbody>
</table>

reconstitution of basic mitotic spindles in spherical emulsion droplets

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and cross-linkers. Growing microtubules can generate pushing forces, while depolymerizing microtubules can convert the energy from microtubule shrinkage into pulling forces, when attached, for example, to cortical dynein or chromosomes. In addition, motor proteins and diffusible cross-linkers within the spindle contribute to spindle architecture by connecting and sliding anti-parallel microtubules. In vivo, it has proven difficult to unravel the relative contribution of individual players to the overall balance of forces. Here we present the methods that we recently developed in our efforts to reconstitute basic mitotic spindles bottom-up in vitro. Using microfluidic techniques, centrosomes and tubulin are encapsulated in water-in-oil emulsion droplets, leading to the formation of geometrically confined (double) microtubule asters. By additionally introducing cortically anchored dynein, plus-end directed microtubule motors and diffusible cross-linkers, this system is used to reconstitute spindle-like structures. The methods presented here provide a starting point for reconstitution of more complete mitotic spindles, allowing for a detailed study of the contribution of each individual component, and for obtaining an integrated quantitative view of the force-balance within the mitotic spindle.

前のセクションで提示される方法は、油中水型エマルジョン液滴の幾何学的な閉じ込めを使用して複雑さを増加させると、スピンドル状構造の再構成を可能にします。このセクションでは、質的にこれらのアッセイの能力を実証する代表的な結果を説明しています。 バイポーラスピンドルが組み立てられたとき、有糸分裂の間、細胞はヒト細胞のために測定されるように、約15ミクロンの直径を有する球を形成するために切り上げます。この特性有糸分裂細胞の形状が制限され、両方がスピンドルサイズや向き35,36を指示する幾何学的境界を提供します。マイクロ流体技術は、正確に生きた細胞で観察された状況に似ているため、in vitroでの紡錘体のボトムアップ再構成を可能にする幾何学的封じ込めのレベルを提供します。 <p class="jove_content" fo:keep-together.within-微小管の成長は、幾何学的境界によって制限されているときにページ= &quot;1&quot;&gt;微小管アスターは既にそれ自体で複雑な動作が可能です。微小管が成長するにつれ、新たなチューブリン二量体を組み込むことにより生成された押し込み力はエマルジョン液滴の両側に2中心体を駆動します。最初は、中心体が自由に閉じ込められたボリューム（ 図4a、左パネル）内に拡散します。約20〜30分後、最初の微小管は微小管がすべての方向に皮質に対して成長するにつれて拡散が制限となり、可視および中心体になります。中間の長さ（液滴直径の約50％）の微小管とアスターは、（立体的に）、他の中心体および皮質互いに押し微小管と、それぞれ別のを撃退することができます。これは、互いに（ 図4a、中央のパネル）二つの対向中心体を有する典型的な「バイポーラ」紡錘状の配置になります。微小管は、液滴直径の〜50％以上成長するとメーターは、中心体を液滴皮質（ 図4a、右パネル）に沿って成長している微小管と、液滴の対向する境界にさらにプッシュされます。これらのアッセイにおける微小管の成長率は温度とチューブリン濃度の両方に非常に敏感であることに注意することが重要です。これらのパラメータは、したがって、強く核生成が最初に観察され、定常状態のアスター位置に達したときの時間に影響します。 細胞では、皮質引っ張る力が微小管プラス端と皮質関連ダイニンとの間に耐荷重添付ファイルの形成を介して生成されます。動物細胞では、この関連はのGαi1のN末端 ​​ミリストイル化を経由して、原形質膜を標的とするのGαi/ LGN / NuMAの複合体に依存します。これらの再構成アッセイでは、のGαi/ LGN /のNuMA複合体の要件を直接介して、ビオチン化脂質とダイニンを結合させることによりバイパスされますビオチン - ストレプトアビジン - ビオチン複合体の形成。これらの結合は比較的安定（K D〜10 -14 M）37であり、急速に形成する（通常は微小管の核形成が明らかになる前に、エマルション滴形成後10分以内）（ 図4b）。ダイニンの非存在下で、中心体を液滴皮質（ 図4c）の反対側に押され、一方の皮質ダイニンの存在下では、中心体は、典型的には、複数の中央位置を保持します。私たちは、これを防止し、微小管押す力に対抗する二つの効果の結果である理由。 （1）ダイニンは直接それによって微小管の長さを制限し、過度の微小管の座屈を防止し、微小管の大災害を促進し、（2）皮質引っ張る力は、アスター、アスター反発力に対抗個々アスター8、上の正味のセンタリング力につながります。 拡散性架橋剤ASE1がfを誘導します逆平行微小管29の重なり領域を増大させる傾向があるorces。これと一致し、ASE1の存在（とダイニンの不在）で中心体は、ASE1がバンドルさ極間微小管（ 図4d）にローカライズと密接一緒に発見されました。キネシン-5ファミリーのメンバーは、（導入を参照）スピンドル内から押し出す力を提供することにより、中心体の分離を駆動します。 Cut7（S.ポンベキネシン5オルソログ）の存在下では、中心体であってもASE1（ 図4e）の存在下で、エマルション液滴の反対側に押し出されます。皮質およびインター極性力発生器を組み合わせることによって、複雑さのレベルは、さらに最終的には双極性有糸分裂紡錘体の包括的な理解をもたらす、これらの実験で増加させることができます。これらの結果の詳細な定量的記述は、将来的に利用できるようになります（ロスら、原稿準備中）。 <p class="jove_content" fo:keeP-together.withinページ= &quot;1&quot;&gt;時点で固定瞬間中心体の位置を観察し、そして微小管の観察された長さに彼らの行動に関連することに加えて、有益な情報が渡って測位処理を以下により得ることができます時間。露光時間及びレーザ強度​​を低減することにより、それだけで約30％の漂白剤を有する少なくとも1時間、2分間隔でアスター位置に追従することが可能です。これは、集中介して自由に拡散する中心体からアスターアセンブリおよび位置決めの監視（0分）ことができます（12分）分散型アスター（36分、さらに）（ 図5c）に。これは、両方の定常状態の挙動とスピンドルアセンブリ動態の研究を促進します。同一試料中の異なる内容の小滴を組み合わせることによって、そのままでおよび皮質力発生器（ 図5b）なしで小滴に中心体の位置決め及びスピンドルアセンブリ動態を比較するために、例えば、可能です。 図1： 紡錘体に作用する力 、いくつかの力を発生する分子は紡錘体の形成および位置決めを促進するために、紡錘体の微小管に作用します。細胞表層に成長する微小管は中心体に押圧力を発生させます。 （紫）皮質ダイニンは、脱重合微小管をキャプチャし、中心体に引っ張り力を発生させます。 PRC1 / ASE1ファミリーのクロスリンカーは、対向する外側にスライドする力を発生に対し、スピンドル内では、キネシン-5 / Cut7モータータンパク質は、反平行微小管を外側にスライドする力を与える。 <a href="https://www.jove.com/files/ftp_upload/54278/54278fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。 。</a> <img alt="図2" src="/files/ftp_upload/54278/54278fig2.jpg" /> 図2：4マイクロ流体チップを含む4インチのフォトマスクのマイクロ流体チップの設計 （A）設計。 （B）シングルチップの詳細な表現。チップは、1つの出口チャネルとダストフィルタが続く3の入口チャネルが含まれています。入口チャネル1が水相を含有し、脂質/油相とチャネル3 2チャネルは、追加の脂質/油相で形成された液滴を希釈するために使用することができます。 （C）（上下から来る）脂質/油相（左から来る）水相に会う接合部の詳細な表現。ジャンクショ​​ンで、液滴が形成され、右側の出口流路に向かって流れます。 （D）2ミクロンチャネルを有する防塵フィルタの詳細な表現。 <a href="https://www.jove.com/files/ftp_upload/54278/54278fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/54278/54278fig3.jpg" /> 図 3： 油中水滴型エマルジ ​​ョン液滴形成のための方法論 （A）明視野顕微鏡上のマイクロ流体チップとマイクロ流体チューブ。水及び脂質/油相の管の両方が、それぞれチップ口1,2に接続されています。 （B）水と脂質/油相が満たすマイクロ流体チップの制限。圧力を変化させることにより、液滴サイズを制御することができます。 PDMSコーティングされたフローセルの（C）設計。フローセル内への液滴をロードした後、開放端部には、追加Valapで閉鎖されています。 （D）液滴形成の概略図。液滴は、中心体、チューブリンおよび紡錘体形成のために必要な追加の構成要素を封入するために使用されます。皮質ダイニンターゲティングの（E）の回路図。ビオチン化（バイオ）ダイニンは、ストレプトアビジン（STRP）を介してビオチン化脂質を標的とします。 <ahref = &quot;https://www.jove.com/files/ftp_upload/54278/54278fig3large.jpg&quot;ターゲット= &quot;_空白&quot;&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。 <img alt="図4" src="/files/ftp_upload/54278/54278fig4.jpg" /> 図4： 複雑さの増加に伴ってスピンドル・再構成はの代表的な結果 （A）、短い、中間、及び長い微小管の一例を示す2中心体を含む液滴の最大強度投影。中心体と微小管10％HiLyte-488チューブリン標識の添加により可視化されます。スケールバー=10μmです。 （B）非存在下でのGFP-ビオチンダイニン-TMRのローカライズ（ - 、左）とストレプトアビジンの存在（+、右）（STRP）。皮質GFP-ビオチンダイニン-TMRのまたは存在（+、右） - （C）微小管アスター不在の位置決め（左）。 （D）微小管アスター（左パネル、GRASE1の存在下でEEN）ポジショニング（中央のパネル、赤）。 ASE1とCut7（キネシン-5）（中央のパネル、赤）の存在下で、（E）微小管アスター（左パネル、緑）ポジショニング。 <a href="https://www.jove.com/files/ftp_upload/54278/54278fig4large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図5" src="/files/ftp_upload/54278/54278fig5.jpg" /> 図 5：in vitroでの イメージングアスターポジショニングダイナミクス （A）、数時間までのために液滴イメージングを可能にするPDMSカップのデザイン。異なる内容を含む液滴の（B）の生成、ストレージおよびイメージング（送信）は、不在蛍光デキストランの（左）インクルージョン（右）（アレクサ647）によって示されています。広告のzスタック（2μmの距離）から取られた60分のタイムラプスの（C）シングル平面画像（2分間隔）単一の中心体を含むroplet。 <a href="https://www.jove.com/files/ftp_upload/54278/54278fig5large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets at JoVE.com

Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets

The assembly and positioning of the mitotic spindle depend on the combined forces generated by microtubule dynamics, motor proteins and cross-linkers. Here we present our recently developed methods in which the geometrical confinement of spherical emulsion droplets is used for the bottom-up reconstitution of basic mitotic spindles.

球状​​エマルジョン液滴での基本的な紡錘体の再構成

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and ...

reconstitution-basic-mitotic-spindles-spherical-emulsion

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9.7K ビュー.  Delft University of Technology. The assembly and positioning of the mitotic spindle depend on the combined forces generated by microtubule dynamics, motor proteins and cross-linkers. Here we present our recently developed methods in which the geometrical confinement of spherical emulsion droplets is used for the bottom-up reconstitution of basic mitotic spindles.

ビデオ: Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets

Title Cell Encapsulation by Droplets

滴タイトルセルのカプセル化

生物学

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the richness of biological information it provides. MR does not require any ionizing radiation and can noninvasively provide higher resolution and better signal-to-noise ratio in comparison to other tomographic or spectroscopic modalities. In this protocol, we will focus on small animal MR imaging and MR spectroscopy (MRI/MRS) to noninvasively acquire relaxation weighted 1H images of mouse and to obtain 31P spectra of mouse muscle. This work does not attempt to cover every aspect of small animal MRI/MRS but rather introduces basic procedures of mouse MRI/MRS experiments. The main goal of this work is to inform researchers of the basic procedures for in vivo MR experiments on small animals. The goal is to provide a better understanding of basic experimental procedures to allow researchers new to the MR field to better plan for non-MR components of their studies so that both MR and non-MR procedures are seamlessly integrated.

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

This work describes basic procedures of noninvasive small animal MRI and MRS in vivo.

非侵襲的インビボでの小動物MRIとMRS：基本的な実験手順

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

アクティブT.の試験管内再構成でcastaneumのテロメラーゼ

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Biochemical Reconstitution of Steroid Receptor&#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, including transcription factors (e.g. nuclear receptors, p53) and protein kinases (e.g. Src, Raf, Akt kinase) involved in cell cycling, tumorigenesis, apoptosis, and multiple eukaryotic signaling pathways 1,2. Of these many 'client' proteins for hsp90, the assembly of steroid receptor&bull;hsp90 complexes is the best defined (Figure 1). We present here an adaptable glucocorticoid receptor (GR) immunoprecipitation assay and in vitro GR&bull;hsp90 reconstitution method that may be readily used to probe eukaryotic hsp90 functional activity, hsp90-mediated steroid receptor ligand binding, and molecular chaperone cofactor requirements. For example, this assay can be used to test hsp90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process. 
The GR has been a particularly useful system for studying hsp90 because the receptor must be bound to hsp90 to have an open ligand binding cleft that is accessible to steroid 3. Endogenous, unliganded GR is present in the cytoplasm of mammalian cells noncovalently bound to hsp90. As found in the endogenous GR&bull;hsp90 heterocomplex, the GR ligand binding cleft is open and capable of binding steroid. If hsp90 dissociates from the GR or if its function is inhibited, the receptor is unable to bind steroid and requires reconstitution of the GR&bull;hsp90 heterocomplex before steroid binding activity is restored 4 . GR can be immunoprecipitated from cell cytosol using a monoclonal antibody, and proteins such as hsp90 complexed to the GR can be assayed by western blot. Steroid binding activity of the immunoprecipitated GR can be determined by incubating the immunopellet with [3H]steroid. 
Previous experiments have shown hsp90-mediated opening of the GR ligand binding cleft requires hsp70, a second molecular chaperone also essential for eukaryotic cell viability. Biochemical activity of hsp90 and hsp70 are catalyzed by co-chaperone proteins Hop, hsp40, and p23 5. A multiprotein chaperone machinery containing hsp90, hsp70, Hop, and hsp40 are endogenously present in eukaryotic cell cytoplasm, and reticulocyte lysate provides a chaperone-rich protein source 6. 
In the method presented, GR is immunoadsorbed from cell cytosol and stripped of the endogenous hsp90/hsp70 chaperone machinery using mild salt conditions. The salt-stripped GR is then incubated with reticulocyte lysate, ATP, and K+, which results in the reconstitution of the GR&bull;hsp90 heterocomplex and reactivation of steroid binding activity 7. This method can be utilized to test the effects of various chaperone cofactors, novel proteins, and experimental hsp90 or GR inhibitors in order to determine their functional significance on hsp90-mediated steroid binding 8-11.

An in vitro method for preparing functional glucocorticoid receptor (GR)&#x2022;hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications.

ステロイド受容体の生化学的再構成には、•Hsp90のタンパク質複合体とリガンド結合の再活性化

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, ...

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 ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

基本的な<em&gt;線虫Caenorhabditis elegans</em&gt;方法：同期との観測

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, &beta;-catenin. Two different methods are described to assess &beta;-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess &beta;-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of &beta;-catenin degradation.

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

β-カテニンの分解にの再構成<em&gt;アフリカツメガエル</em&gt;卵抽出

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

浸透水透過係数（Pの測定<sub&gt; F</sub&gt;）球状細胞の：例として単離された植物プロトプラスト

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

植物や緑藻の光捕集複合体のin vitro再構成では、

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

球状​​エマルジョン液滴での基本的な紡錘体の再構成

球状エマルジョン液滴での基本的な紡錘体の再構成