著者らは、プロトコルを開発し、するための試薬を提供するための再利用可能なフローチャンバーを設計·製造​​を支援するため、FI Ataullakhanov、N. Dashkevich、N.​​ GudimchukとA. Korbalev数字のために画像を提供するため、N. GudimchukとP.ザハロフに感謝したいと思いますテキスト編集やヒントや議論のためのGrishchukラボの他のメンバーと助けをジゴキシゲニン標識微小管の種、A.ポタペンコを準備します。この作品は、によって部分的にサポートされていましたNIH付与GM R01-098389とキンメル学者でELGにペンシルベニア州マッスル研究所からのパイロット助成金、RFBRは、12 04から00111 - 、13 - 04から40190-Hを付与し、13で-04-40188-H、FI Ataullakhanovに科学幹部会補助金（分子システムインテグレーションおよび分子機序および分子細胞生物学プログラム）のロシア科学アカデミー、NIHは、JRマッキントッシュにGM R01 GM033787を付与し、ドミトリーZimin王朝財団ポスドクフェローシップへVAV

著者らは、開示することは何もありません。

多くの単一分子アッセイは、最近日常大幅に非特異的なタンパク質の付着を低減するために、特別に処理カバーグラスを使用しています。我々はここで説明する手順では、ハワード·ラボ32で開発されたオリジナルのプロトコルの変形例であり、我々はカバースリップをシラン化することであっても蛍光を使用していないDICベースのビーズアッセイ、と努力の価値であることがわかり?...

微小管は高度に細胞構造、細胞運動性、細胞分裂、細胞内輸送および1のために重要である細胞骨格構造が保存されている。これらの動的なポリマーは、GTPの存在下でチューブから組み立て、そして、彼らは自然に成長と2の短縮を切り替える。微小管は、従って、コントラストを高めるために特殊な光学技術は、光学顕微鏡で微小管を観察するために使用されるべきである（直径がわずか25 nm）の非常に薄い。これらのポリマーを用いた以前の研究は、微分干渉コントラスト（DIC）3を使用して動的挙動を調べた。 試験管内でこれと同様の研究は、一般的な実験条件の下で、微小管は、まれにしか解重合に破局し、スイッチを受けていないことを明らかにした一回5〜15分間（この周波数は、28〜32℃で調べた7月15日ミリモル、可溶性チューブリン濃度である）4。異なる技術は、このようにインダクタに提案されている制御された方法でのE微小管脱重合。微小管の短縮は、ここで説明するように、離れて可溶性チューブ5,6を洗浄するレーザ光​​7と微小管を切断するか、またはセグメント化された微小管8を用いてトリガすることができる。セグメント化された微小管だけでなく、確率的に切り替えるポリマーを使用してこれまでの研究は、このような染色体、小胞、およびタンパク質をコーティングしたビーズのような小さな細胞内貨物は、短縮微小管9月13日の端に移動させることを見出した。この現象は、有糸分裂細胞における染色体の動きのための直接含意を持っていると考えられており、基本的なメカニズムは、アクティブな調査14〜16の下に現在されている。 最近では、全反射蛍光（TIRF）顕微鏡などの蛍光ベースの技術は、動的微小管と運動性を研究するために使用されてきたが17-24を終了します。このアプローチの利点は、相互作用の試験を可能にすることである異なる蛍光で標識したタンパク質を用いて、リアルタイムで微小管と微小管結合タンパク質間のS。いくつかのタンパク質複合体は、微小管末端を伸長及び/又は短縮してprocessively移動することが見出された。彼らは、微小管結合タンパク質DAM1 10,12,18、Ska1 19、およびXMAP215 20だけでなく、キネシンモーターKif18A 21,22、MCAK 23およびCENP-Eの24が含まれています。これらのタンパク質は、EB1 25のような古典的な先端追跡タンパク質のそれとは根本的に異なるセッ先端追跡を呈する。 EB1分子および関連パートナーは、安定的に、動的微小管に結合したままでいるように見えるものの、終了、個々の分子は急速に可溶性のプール26を交換するだけ〜0.8秒間、微小管の先端に結合したまま。これとは対照的に、進行的先端トラッカーは、DAM1のように、微小管との旅行が多くミクロンのために終了し、微小管の先端との関連は、男のために最後にすることができます任意の秒数。先端会合時間だけでなく、追跡の結果率は、先端追跡複雑な27を形成する分子の数に強く依存する。より大きなタンパク質のアンサンブルは、通常、はるかに優れた先端トラッカーです。例えば、単離された酵母動原体のような複雑なアセンブリは、微小管に結合されたままでは時間28で終了。いくつかの微小管結合タンパク質、 例えば Ndc80の動原体タンパク質複合体、微小管は、単一分子レベルで終了し、まだNdc80ビード貨物19,29-31の動きを結合する非常に効率的であるとともに追跡することができないことが見出されている。したがって、異なるタンパク質複合体、ならびにそれらの生物学的役割によって先端追尾のメカニズムを理解するためには、先端追跡複合体中の分子の数の関数としての先端トラッキングを調べるために、ならびに決定することが重要であるビーズ貨物の表面に集合的な運動性を発揮するこれらの複合体の能力。 図1A）を用いた実験を準備し、実施するための詳細なプロトコルを提供下。まず、市販のガラススライドは、短いポリエチレンチューブ（プロトコル1）を取り付けるために修飾される。再使用可能な顕微鏡流チャンバは、その後、スライドおよび化学的またはプラズマ洗浄、シラン化カバースリップ（プロトコル2）32-34から組み立てられる。結果として生じるチャンバ容積は、入口管の体積を含むのみ20-25液（又は15μlのできるだけ小さい、プロトコル1の注3を参照）である。市販のフローチャンバーを使用することもできるが、その体積は、タンパク質の不必要な浪費をもたらす、通常は大きくなる。大きなチャンバーを採用した場合は、以下のプロトコルのすべてのソリューションの量が比例して拡大縮小する必要があります。微小管の種子は、その後徐々に加水分解性GTP類似体、GMPCPP（グアノシン-5&#39;-[（α、β）-methyleno]三リン酸）（プロトコル3を使用して調製される、ハイマン&lt;も参照してください。EM&gt;ら35）。種子を洗浄し、カバースリップ上に固定され、表面は、その後、他のタンパク質の非特異的吸収を防ぐためにブロックされている32（プロトコル4を用いてジゴキシゲニン種子の固定化を記載）。セグメント化された微小管は、次いで、プロトコル5を用いて調製することができる。このアプローチの主な根拠は、GTPの存在下で形成動的微小管ポリマーは、GMPCPPを含有する安定なチューブセグメントの短い「キャップ」を付加することによって、一時的に安定させることができることである。これらのキャップはまた、ローダミン標識したチューブリンを含有するので、530〜550 nmのレーザーまたは水銀アークランプ（プロトコル6）36で視野を照明することによって簡単に除去することができる。先端トラッキング信号の蛍光強度は、次いで考慮顕微鏡視野照明（プロトコル7）のばらつきを考慮して、分解、微小管の端部に移動する分子の数を推定するために使用することができる。同様のアプローチを使用することができる微小管脱重合および27に記載したように調製したタンパク質被覆ビーズ（プロトコール8）との間の相互作用を研究する。いくつかのタンパク質は容易にセグメント化された微小管の壁に結合するが、レーザーピンセットはまた、それによって、その結合を促進し、微小管の壁の近くにビードを保持するために使用することができる。

<table>
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td></td>
<td></td>
<td></td>
<td>Table 1: Microscopy and other equipment.</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss 
			Nikon</td>
			<td>Axio Imager 2 
			Eclipse Ti</td>
			<td>other microscope models capable of DIC and epi-fluorescence-imaging can be used</td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>Zeiss 
			Nikon</td>
			<td>420490-9900-000 
			CFI Apo 100x Oil 1.49</td>
			<td>100X, DIC, 1.3-1.49 NA</td>
		</tr>
		<tr>
			<td>Objective heater</td>
			<td>Bioptechs</td>
			<td>150803, 150819-19</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent filter cube</td>
			<td>Chroma</td>
			<td>49004 or 49008 
			41017 or 49020</td>
			<td>optimized for Rhodamine fluorescence&#160; 
			optimized for GFP fluorescence</td>
		</tr>
		<tr>
			<td>Acquisition software</td>
			<td>freeware MicroManager 
			Molecular Devices</td>
			<td>not applicable 
			MetaMorph 7.5</td>
			<td>http://valelab.ucsf.edu/~MM/MMwiki/ 
			other software can be used to acquire images and for a particle tracking</td>
		</tr>
		<tr>
			<td>EMCCD camera</td>
			<td>Andor</td>
			<td>iXon3, DU-897E-cs0-#BV</td>
			<td>Highly sensitive EMCCD camera</td>
		</tr>
		<tr>
			<td>Trapping laser</td>
			<td>IPG Photonics</td>
			<td>YLR-10-1064-LP</td>
			<td>1064 nm laser, 10 W</td>
		</tr>
		<tr>
			<td>Fluorescence excitation lasers</td>
			<td>Coherent, Inc. 
			Coherent, Inc.</td>
			<td>Sapphire 488 LP 
			Sapphire 552 LP</td>
			<td>excitation of green fluorophores 
			excitation of red fluorophores</td>
		</tr>
		<tr>
			<td>Plasma Cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial flow chambers</td>
			<td>Warner Instruments</td>
			<td>RC-20 or RC-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion pump</td>
			<td>Cole Palmer 
			Harvard Apparatus</td>
			<td>Masterflex 77120-52 
			Pico Plus</td>
			<td>Both pumps provide the required rate of liquid flow but a peristaltic pump may pulse at very slow speed. The flow with a syringe pump is more consistent for a wide range of rates but this pump has inertia.&#160;</td>
		</tr>
	</tbody>
</table>
<table>
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td></td>
<td></td>
<td></td>
<td>Table 2: Microscopy chamber preparation.</td>
		</tr>
		<tr>
			<td>Modified microscope slides for reusable chambers</td>
			<td>Precision Glassblowing of Colorado</td>
			<td>Custom order www.precisionglassblowing.com</td>
			<td>Sonic slots in slides using schematics in Figure 1</td>
		</tr>
		<tr>
			<td>Polyethylene tubing</td>
			<td>Intramedic</td>
			<td>427410</td>
			<td>I.D. 0.58 mm, O.D. 0.965 mm; use these tubes to connect assembled chamber to the pump and waste container</td>
		</tr>
		<tr>
			<td>Polyethylene tubing</td>
			<td>Intramedic</td>
			<td>427400</td>
			<td>I.D. 0.28 mm, O.D. 0.61 mm; use these tubes to make the reusable chamber</td>
		</tr>
		<tr>
			<td>Regular microscope slides</td>
			<td>VWR</td>
			<td>48312-003</td>
			<td>Other similar slides can be used</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>VWR</td>
			<td>48393-150, 48366-067</td>
			<td>Other similar coverslips can be used</td>
		</tr>
		<tr>
			<td>Silicon sealant</td>
			<td>World Precision Instruments</td>
			<td>KIT, SILICON SEALANT 5 MIN CURE</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy glue</td>
			<td>Loctite</td>
			<td>83082</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate adhesive</td>
			<td>Scotch 3M</td>
			<td>AD114</td>
			<td>Or cyanoacrylate adhesive from other manufacturers</td>
		</tr>
	</tbody>
</table>
<table>
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td></td>
<td></td>
<td></td>
<td>Table 3: Coverslips cleaning and coating.</td>
		</tr>
		<tr>
			<td>Molecular Sieves, Grade 564</td>
			<td>Macron</td>
			<td>4490-04</td>
			<td width="200px"></td>
		</tr>
		<tr>
			<td>Coverglass Staining Jar</td>
			<td>Ted Pella, Inc.</td>
			<td>21036</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip Ceramic Holder</td>
			<td>Thomas Scientific</td>
			<td>8542e40</td>
			<td></td>
		</tr>
		<tr>
			<td>PlusOne Repel Silane</td>
			<td>GE Healthcare Biosciences</td>
			<td>17-1332-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma-Aldrich</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-digoxigenin AB</td>
			<td>Roche applied science</td>
			<td>11093274910</td>
			<td></td>
		</tr>
	</tbody>
</table>
<table>
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td></td>
<td></td>
<td></td>
<td>Table 4: Preparation of seeds and segmented microtubules.</td>
		</tr>
		<tr>
			<td>Tubulin</td>
			<td>purified from cow brains 
			Cytoskeleton, Inc</td>
			<td>&#160; 
			T238P</td>
			<td>For purification protocols see 49&#8211;51 
			Unlabeled porcine tubulin</td>
		</tr>
		<tr>
			<td>Labeled tubulin</td>
			<td>Cytoskeleton, Inc 
			Invitrogen 
			Invitrogen</td>
			<td>TL590M 
			C1171 (Rhodamine) 
			A-2952 (Digoxigenin)</td>
			<td>Rhodamine-labeled porcine tubulin 
			Tubulin can be labeled with any amine-reactive dye as in reference52.</td>
		</tr>
		<tr>
			<td>GMPCPP</td>
			<td>Jena Biosciences</td>
			<td>NU-405</td>
			<td>Aliquot and store at -70 &#176;C</td>
		</tr>
		<tr>
			<td>VALAP</td>
			<td>vaseline, lanolin, and paraffin at 1:1:2 by mass</td>
			<td></td>
			<td>see ref. 9</td>
		</tr>
	</tbody>
</table>

preparation of segmented microtubules to study motions driven by the disassembling microtubule ends

微小管の解重合は、 インビトロで異なるタンパク質複合体およびタンパク質被覆ビーズを輸送する力を提供することができる。基本的なメカニズムは、細胞分裂時の微小管依存染色体の運動に重要な役割を果たしていると考えられているが、関連するタンパク質およびそれらの正確な役割は明確に定義されています。したがって、精製された成分と定義され、生化学的環境を用いて、インビトロでそのような運動性を研究するためのものでアッセイを開発する必要性が高まっている。微小管は、しかしながら、本質的に不安定なポリマーであり、成長との間の短縮それらの切り替えは、確率的と制御が困難である。我々はここで説明するプロトコルはphotoablatableがキャップを安定化して作られてセグメント化された微小管を利用しています。そのようなセグメント化された微小管の脱重合することにより、分解、微小管の端部に運動性の研究を補助する、高い時間および空間分解能でトリガすることができる。この技術は、約するために使用することができる動的な微小管が終了するとprocessively移動する蛍光標識されたタンパク質複合体中の分子数の定量分析を行っRRY。このにおける信号対雑音比、および他の定量的蛍光アッセイを最適化するために、カバーガラスを可溶性蛍光標識されたタンパク質の非特異的吸収を減少させるために処理されるべきである。詳細なプロトコルを考慮に蛍光照明ムラを取ると等距離ガウスフィットを使用して、単一のフルオロフォアの強度を決定するために提供される。最後に、進行的貨物の動きに結合する微小管脱重合に異なるモーターと非運動タンパク質の能力への洞察を提供し、タンパク質でコーティングされたマイクロビーズの微小管依存の動きを研究するセグメント化された微小管の使用を記載している。

微小管脱重合を追跡するタンパク質が終了する。酵母動原コンポーネントDAM1が圧倒的に脱重合微小管のベストのヒント-トラッカーである14を終了します。 GFPで標識されたこの10サブユニット複合体は、容易に発現し、細菌細胞18,38から精製し、私たちは、先端追跡アッセイのポジティブコントロールとして、それを使用することをお勧めすることができます。微...

Watch this Scientific Journal Video about Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends at JoVE.com

Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends

微小管は本質的に不安定なポリマーであり、成長との間の短縮その切り替えは、確率および制御することは困難である。ここでは、photoablatable安定キャップでセグメント化された微小管を使用してプロトコルを記述します。セグメント化された微小管の脱重合することにより、分解、微小管末端を有する運動の分析を支援する、高い時間および空間分解能でトリガすることができる。

逆アセンブル微小管端部によって駆動される運動を研究するセグメント化微小管の調製

Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are ...

preparation-segmented-microtubules-to-study-motions-driven

Research

JoVE Journal

Biology

14.3K ビュー.  Russian Academy of Sciences. 微小管は本質的に不安定なポリマーであり、成長との間の短縮その切り替えは、確率および制御することは困難である。ここでは、photoablatable安定キャップでセグメント化された微小管を使用してプロトコルを記述します。セグメント化された微小管の脱重合することにより、分解、微小管末端を有する運動の分析を支援する、高い時間および空間分解能...

ビデオ: Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

トカゲの運動の速度と方向の視覚感度のテスト

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

生物学

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

コンピュータによって生成された動物モデルの刺激

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

の単離と精製ショウジョウバエ</em磁気ビーズソート別>末梢ニューロン

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

の生産<em&gt;アフリカツメガエルトロピカ</em微小管関連RNAを識別するために、&gt;卵抽出

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the Mgm101 protein is involved in the recombinational repair of mitochondrial DNA. Recent investigations have indicated that Mgm101 is related to the Rad52-type recombination protein family. These proteins form large oligomeric rings and promote the annealing of homologous single stranded DNA molecules. However, the characterization of Mgm101 has been hindered by the difficulty in producing the recombinant protein. Here, a reliable procedure for the preparation of recombinant Mgm101 is described. Maltose Binding Protein (MBP)-tagged Mgm101 is first expressed in Escherichia coli. The fusion protein is initially purified by amylose affinity chromatography. After being released by proteolytic cleavage, Mgm101 is separated from MBP by cationic exchange chromatography. Monodispersed Mgm101 is then obtained by size exclusion chromatography. A yield of ~0.87 mg of Mgm101 per liter of bacterial culture can be routinely obtained. The recombinant Mgm101 has minimal contamination of DNA. The prepared samples are successfully used for biochemical, structural and single particle image analyses of Mgm101. This protocol may also be used for the preparation of other large oligomeric DNA-binding proteins that may be misfolded and toxic to bacterial cells.

The yeast mitochondrial nucleoid protein, Mgm101, is a Rad52-type recombination protein that forms large oligomeric rings. A protocol is described to prepare soluble recombinant Mgm101 using the Maltose Binding Protein (MBP)-tagging strategy coupled with cation exchange and size exclusion chromatography.

MBPベースタギング戦略によってMgm101組換えタンパク質の調製

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

腸によって脂質輸送を研究するためのCaco-2細胞を用いました

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a genetically encoded marker for the positive ends of microtubules (EB3 labelled with tdTomato fluorescent protein) and high-speed, high-resolution, live-cell imaging using spinning disk confocal microscopy. Cell cycle synchronization and increased density of microtubules are achieved by inhibiting centrosomal separation in mitotic cells, and analysis of growth is performed using open-source U-Track software. The use of a bright and red-shifted fluorescent protein, in combination with the lower laser power and reduced exposure time required for spinning disk microscopy reduce phototoxicity and the probability of light-induced artifacts. This allows for imaging a larger number of cells in the same preparation while maintaining the cells in a growth medium under standard culture conditions. Because the analysis is performed in a supervised automatic fashion, the results are statistically robust and reproducible.

Here we present a robust and detailed method of microtubule dynamics analysis in cells synchronized in prometaphase using live-cell spinning disk confocal microscopy and MATLAB-based image processing.

単極有糸状スピンドルにおけるスピンディスク顕微鏡による微小管ダイナミクスの測定

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a ...

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, to elucidate the function and assembly of flagellar parts, and to examine and understand patterns of movement. Here we demonstrate a combination of three of these methodologies. Motility in soft agar is the oldest, offering a strong selection for isolating gain-of-function suppressor mutations in motility-impaired strains, where motility is restored through a second mutation. The cell-tethering technique, first employed to demonstrate the rotary nature of the flagellar motor, can be used to assess the impact of signaling effectors on the motor speed and its ability to switch rotational direction. The &#8220;border-crossing&#8221; assay is more recent, where swimming bacteria can be primed to transition into moving collectively as a swarm. In combination, these protocols represent a systematic and powerful approach to identifying components of the motility machinery, and to characterizing their role in different facets of swimming and swarming. They can be easily adapted to study motility in other bacterial species.

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Many bacteria use flagella-driven motility to navigate their environment and colonize favorable surroundings both individually and as a collective. Demonstrated here is the use of three established methods that exploit motility as a selection tool to identify components/pathways contributing to swimming and swarming motility.

シリーズで3つの確立された技術を適用することにより 、大腸菌 におけるフラゲラ駆動運動性の調査

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, ...

A Step-by-Step Guide to Mosquito Electroantennography

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when acquiring a blood-meal. To locate a host to feed on, mosquitoes rely on a wide range of sensory cues, including visual, mechanical, thermal, and olfactory. The study details a technique, electroantennography (EAG), that allows researchers to assess whether the mosquitoes can detect individual chemicals and blends of chemicals in a concentration-dependent manner. When coupled with gas-chromatography (GC-EAG), this technique allows to expose the antennae to a full headspace/complex mixture and determines which chemicals present in the sample of interest, the mosquito can detect. This is applicable to host body odors as well as plant floral bouquets or other ecologically relevant odors (e.g., oviposition sites odorants). Here, we described a protocol that permits long durations of preparation responsiveness time and is applicable to both female and male mosquitoes from multiple genera, including Aedes, Culex, Anopheles, and Toxorhynchites mosquitoes. As olfaction plays a major part in mosquito-host interactions and mosquito biology in general, EAGs and GC-EAG can reveal compounds of interest for the development of new disease vector control strategies (e.g., baits). Complemented with behavioral assays, the valence (e.g., attractant, repellent) of each chemical can be determined.

The present article details a step-by-step protocol for successful and low noise electroantennograms in several genera of mosquitoes, including both females and males.

蚊のエレクトロアンテノグラフィーのステップバイステップガイド

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

根の滲出液中の根茎細菌の化学誘引物質の迅速な同定のための改良された気化症アッセイ

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...