私たちの仕事は、NIH / NIGMS、NSF、USDA / NIFA、とBARDからVCへの補助金によってサポートされています。

背景植物細胞間の接続を介して高分子のSymplastic輸送、原形質連絡は、多くの植物病理学者と生物学者のためのものです。例えば、いくつかのウイルスタンパク質は、ウイルスの動き1-3を有効にするplasmodesmalサイズ排除限界を調節することが知られている。また、それらの重要な発達の規制当局の間でいくつかの内在性タンパク質は、非細胞自律的に4を機能させるには、おそらく原形質連絡を介して、セルからセルに移動すると仮定されています。従って、植物細胞の間に高分子輸送を特定し、視覚化する信頼性の高い方法論は非常に需要があります。 1）対象となる植物を育てる高い形質転換効率については、健康な、堅牢な植物を使用する必要があります。 （1） シロイヌナズナの植物短い光周期（130から150μEM -2 s -1の光の8時間、23℃での環境制御室内にポットでプロミックスBXの1つのシロイヌナズナの植物を（10センチ× 10センチメートル× 10 cm）に成長する40から65 C/16時間20時暗い° C）、6〜8週間5％の相対湿度。 5を説明するように、市販製品と時折それらを受精させる。 15ミリメートル× 35 mm（長さの測定は葉柄を含む）より大きいサイズを持つ葉は実験のために選択されています。 2。N. benthamianaとN.アナタバカム 長い光周期（23 ° Cで130から150μEM -2 s -1の光の16時間と環境制御室内のポット（20センチ× 20センチメートル× 20センチメートル）でプロミックスBXの1つの植物を育てる/ 8 20時暗い時間​​° C）および7〜10週間の40から65パーセントの相対湿度。記述として市販の製品と時折それらを受精させる。 Nの50ミリメートル× 70 mmを超えるサイズの葉Nのbenthamiana、または100ミリメートル× 125ミリメートルアナタバカムは、（これらの長さの測定は、葉柄が含まれていない）実験のために選択されています。 DNAでコーティングされた金微粒子による遺伝子銃のカートリッジの2）の調製この実験的なステップのためのプロトコルは、以前は次の6詳細に記載されている。それは高濃度（〜の1μg/μl）の実験の後の段階での共焦点顕微鏡分析を容易にするために最も高い形質転換効率を得るためによく精製されたプラスミドDNAを使用することが非常に重要です。 蛍光シグナルのクラスタと関連付ける細胞の数がsymplastic輸送（すなわちの程度の指標として使用されるので、このアッセイの場合、それは、準備カートリッジは高い周波数で同時に二つ以上の隣接するセルを変換しないことを確認することが重要です。マルチセル信号のクラスタは、動きを示すのに対し、信号を含む単一セル）は、何の動きがないことを示します。各カートリッジの品質は、タンパク質16〜20時間の後の砲撃の発現を分析することによって確認することができます。我々のプロトコル6はこの実験のため、適切なこの時間枠のすべての式のイベントの&lt;3％、でマルチセル式のクラスタを生成カートリッジを生成します。 DNAでコーティングされた微粒子の3）バイオリスティックデリバリー<ol><li>鋭いカミソリの刃と同じ発達段階（つまり、同じサイズと同じ年齢で、ステップ1を参照）の葉を取り除くとすぐに平らな発泡スチロールの表面に上向きに背軸側面に配置してください。葉の背軸側は、その下のトリコーム密度と薄くキューティクルによる爆撃のためのより良い基質を表します。 シロイヌナズナの葉は、それらの比較的小さいサイズの、ウィンドウのスクリーンメッシュの部分で覆われているはず、とメッシュがその後画鋲で固定するため発泡スチロールの表面に。葉が平らな維持は、粒子のデリバリーの効率が向上し、microbombardment中の組織への損傷を最小限に抑えます。 </li><li>銃へのDNA -被覆微粒子（ステップ1で調製した）でカートリッジを挿入します。 シロイヌナズナの場合、撮影は1μmの微粒子のための80から110 psiの圧力、および0.6μmの微粒子のための140から160 psiで実行されます。 Nのbenthamianaとタバコは、撮影は1μmの微粒子のための100から120 psiの圧力、および0.6μmの微粒子のための160から180 psiで実行されます。それぞれの葉だけ直径10〜12 mmの領域の上に微粒子を広げる1回の撮影で十分な表面積を持っているのでシロイヌナズナの場合、我々は通常、葉ごとに一つのカートリッジを利用する。大きいNとbenthamianaとタバコの葉は、同じ葉の複数の爆撃が可能な、しかし、彼らは（説明を参照）と同じ発生段階で葉の領域をターゲットに半ばリブの両側にある左右対称の位置に行う必要があります。 </li><li>発泡スチロールの表面から葉を取り出して、それらに私を置いてウェットワットマンろ紙の3層ではなくペトリ皿は、パラフィルムでシャーレをシールし、そのタンパク質産物の納入導入遺伝子と細胞間移行の可能性の発現を可能にするために36〜48時間室温で振とうする。 </li></ol> 4）タンパク質発現のイメージングは​​、 一過性に発現タグタンパク質の蛍光シグナルは、共焦点顕微鏡により視覚化です。ケアは、各試験蛋白質の検出に最適な顕微鏡の設定を見つけるに注意する必要があります。たとえば、タバコモザイクウイルスの移行タンパク質（TMV MP）1-3,6のplasmodesmal局在のような弱い信号強度、と限定された細胞内蓄積を示すタンパク質は、高い解像度とに支払う40X対物レンズ、下に観察する必要があります感度は、そのような自由なYFPなどの強いシグナル強度と細胞質の分布を示すタンパク質のに対し、（図1参照）より高速なイメージングのための共焦点ズーム機能で10倍の対物レンズ下で可視化することができます。 Symplastic輸送は、蛍光シグナルを含む、マルチセルクラスタの外観から推測されます。各クラスター内のクラスターやセルの数の数は、細胞間輸送の程度の指標である。信頼性の高いデータを取得するには、少なくとも100の発現のクラスターは、各実験系ごとに記録されるべきである。例えば、タンパク質の細胞間移行が合計200の発現のクラスターの二つの異なる遺伝的背景（例えば、野生型およびトランスジェニック植物）、で比較されている場合は、記録すべきである。重要なのは、実験、相互に比較されることになっているの結果は、同時に行われるべきである。 5。代表的な結果下の図は、蛍光タグタンパク質のsymplastic輸送を検出するための代表的な実験を示しています。パネルAおよびBは、Nの次のようmicrobombardmentを得ている典型的な共焦点画像を表示TMV MP - YFPを発現するとbenthamianaの葉は、構築する。パネルではTMV MP - YFPの、symplastic動きはYFPのシグナルのマルチセルクラスタの外観に基づいて観測される。すべて一過性に発現しないTMV MP - YFPは、いくつかのmicrobombardmentsの単一セルの信号（パネルB）によって証明されるように移動することができます。統計的に、で&lt;カウント信号クラスタの40％は、TMV MP - YFPは、一方のセル間を移動することができないで&gt;クラスタの60％、タンパク質は2つのセルの拡散が最も頻繁にされて、2-5細胞間を移動（約〜50％の症例）（図1C）。 生来の運動活動せず、比較的小さな分子サイズを持つタンパク質は、細胞間を移動するためにPDを通して拡散することができます。例えば、フリーYFP、または1xYFPは（約27 kDaの、パネルD）、カウントのクラスタ（パネルC）の30％で、いくつかの細胞間に広がっています。この非特異的な拡散は、TMV MP - YFP融合タンパク質（57 kDa）のためにサイズに匹敵する、と完全に細胞自律的である並進YFPの調光器、または2xYFP（54 kDaの、パネルE）、は発生しません（図1C）。 <img src="/files/ftp_upload/2208/2208fig1.jpg" alt="図1" /> 図1。N.でsymplastic輸送アッセイから得られた典型的な結果benthamiana葉組織。 TMV MP - YFPの（A、B）可視化。信号クラスターの（C）定量。 1xYFPと2xYFP、それぞれ自由なYFPと並進YFPダイマー、。 1xYFPの（D）可視化。 2xYFPの（E）可視化。顕微鏡写真で、左パネルはYFPシグナルを示し、右側のパネルには、マージされたYFPの画像（緑の）と葉緑体の自家蛍光（白の）信号を示します。画像は、単一の焦点セクションです。顕微鏡写真の中のアスタリスクは、YFPのシグナルを示す表皮細胞を示す。バー= 50μmの。

<ol>
	<li>Waigmann, E., Ueki, S., Trutnyeva, K., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ins+and+outs+of+non-destructive+cell-to-cell+and+systemic+movement+of+plant+viruses.+.">The ins and outs of non-destructive cell-to-cell and systemic movement of plant viruses. .</a> Crit Rev Plant Sci. 23, 195-250 (2004).</li><li>Lucas, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+viral+movement+proteins:+agents+for+cell-to-cell+trafficking+of+viral+genomes.">Plant viral movement proteins: agents for cell-to-cell trafficking of viral genomes.</a> Virology. 344, 169-184 (2006).</li><li>Epel, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+viruses+spread+by+diffusion+on+ER-associated+movement-protein-rafts+through+plasmodesmata+gated+by+viral+induced+host+beta-1,3-glucanases.">Plant viruses spread by diffusion on ER-associated movement-protein-rafts through plasmodesmata gated by viral induced host beta-1,3-glucanases.</a> Semin Cell Dev Biol. 20, 1074-1081 (2009).</li><li>Zambryski, P. C., Crawford, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodesmata:+gatekeepers+for+cell-to-cell+transport+of+developmental+signals+in+plants.">Plasmodesmata: gatekeepers for cell-to-cell transport of developmental signals in plants.</a> Annu Rev Cell Dev Biol. 16, 393-421 (2000).</li><li>Rivero-Lepinckas, L., Crist, D., Scholl, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+plants+and+presercation+of+seeds.">Growth of plants and presercation of seeds.</a> Methods Mol Biol. 323, 3-12 (2006).</li><li>Ueki, S., Lacroix, B., Krichevsky, A., Lazarowitz, S. G., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+transient+genetic+transformation+of+Arabidopsis+leaves+by+biolistic+bombardment.">Functional transient genetic transformation of Arabidopsis leaves by biolistic bombardment.</a> Nat Protoc. 4, 71-77 (2009).</li><li>Oparka, K. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+spatial+regulation+of+symplastic+trafficking+during+development+in+Arabidopsis+thaliana+apices.">Temporal and spatial regulation of symplastic trafficking during development in Arabidopsis thaliana apices.</a> Development. 126, 1879-1889 (1999).</li><li>Kim, I., Cho, E., Crawford, K. M., Hempel, F. D., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-to-cell+movement+of+GFP+during+embryogenesis+and+early+seedling+development+in+Arabidopsis.">Cell-to-cell movement of GFP during embryogenesis and early seedling development in Arabidopsis.</a> Proc Natl Acad Sci USA. 102, 2227-2231 (2005).</li><li>Crawford, K. M., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-targeted+and+targeted+protein+movement+through+plasmodesmata+in+leaves+in+different+developmental+and+physiological+states.">Non-targeted and targeted protein movement through plasmodesmata in leaves in different developmental and physiological states.</a> Plant Physiol. 125, 1802-1812 (2001).</li><li>Roberts, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phloem+unloading+in+sink+leaves+of+Nicotiana+benthamiana:+comparison+of+a+fluorescent+solute+with+a+fluorescent+virus.">Phloem unloading in sink leaves of Nicotiana benthamiana: comparison of a fluorescent solute with a fluorescent virus.</a> Plant Cell. 9, 1381-1396 (1997).</li><li>Guenoune-Gelbart, D., Elbaum, M., Sagi, G., Levy, A., Epel, B. 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利害の衝突は宣言されません。

symplastic輸送アッセイの成功の鍵は、統計的に有意かつ容易に検出可能信号クラスターの生産を可能にする高い形質転換効率を、得ることである。これは健康な、堅牢な植物から収穫された葉を使用して、そして純粋な、そして濃縮したDNAの調製により被覆された金粒子を準備達成することができます。 同じ成長段階で葉を使用しても、アッセイの信頼性にとって不可欠...

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		<th>Catalogue Number</th>
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<td>Gold microparticles, 1.0 &mu;m in diameter</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2262</td>
<td>&nbsp;</td>
<tr>
<td>Gold microparticles, 0.6 &mu;m in diameter</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2263</td>
<td>&nbsp;</td>
<tr>
<td>Spermidine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S0266-1G</td>
<td>&nbsp;</td>
<tr>
<td>Tefzel tubing</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2441</td>
<td>&nbsp;</td>
<tr>
<td>Helios cartridge preparatory station</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2420</td>
<td>&nbsp;</td>
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<td>Tubing cutter</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2422</td>
<td>&nbsp;</td>
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<td>Helios gene gun</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2432</td>
<td>&nbsp;</td>
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<td>Helium gas regulator</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2413</td>
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a cell-to-cell macromolecular transport assay in planta utilizing biolistic bombardment

ここで、我々は、 植物体中の細胞間の高分子輸送の範囲を検出し、評価するために簡単かつ迅速にプロトコルを提示する。このプロトコルでは、関心の蛍光標識タンパクが一時的にそのエンコーディングのDNA構築物の遺伝子銃送達後の植物組織に発現しています。タグタンパク質の内と細胞間の配布は、共焦点顕微鏡で分析される。我々は、アッセイにステップバイステップのプロトコルを提供する、詳細にこの技術を説明し三植物種のsymplasticタンパク質輸送の範囲、 シロイヌナズナ 、 タバコbenthamianaとNを評価するアナタバカム （タバコ）。

Watch this Scientific Journal Video about A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment at JoVE.com

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

植物細胞の間に高分子の人身売買は、一過性の関心の蛍光タグ融合タンパク質を発現し、共焦点顕微鏡によるその内および細胞間の分布を分析することによって評価することができます。

細胞間の高分子輸送アッセイプランタで微粒子銃砲撃を活用

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

a-cell-to-cell-macromolecular-transport-assay-planta-utilizing

Research

JoVE Journal

Biology

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

11.8K ビュー.  State University of New York at Stony Brook. 植物細胞の間に高分子の人身売買は、一過性の関心の蛍光タグ融合タンパク質を発現し、共焦点顕微鏡によるその内および細胞間の分布を分析することによって評価することができます。

ビデオ: A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

トランスジェニックの世代 C.エレガンスバイオリスティック変換による

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

生物学

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

高分子結晶学による構造決定のための脂質メソフェーズで栽培膜タンパク質の収穫とクライオ冷却結晶

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

PDZ-依存性のCFTRの高分子シグナル伝達複合体のin vitroでの解析で

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

培養<em&gt;線虫（Caenorhabditis elegans）</em &gt;微小粒子衝撃による無菌液体培地およびトランスジェニックワームの作成に

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

自動化された蛍光寿命イメージング顕微鏡を利用してオープンソースのハイコンテンツな解析

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Optimization of Crystal Growth for Neutron Macromolecular Crystallography

The use of neutron macromolecular crystallography (NMX) is expanding rapidly with most structures determined in the last decade thanks to new NMX beamlines having been built and increased availability of structure refinement software. However, the neutron sources currently available for NMX are significantly weaker than equivalent sources for X-ray crystallography. Despite advances in this field, significantly larger crystals will always be required for neutron diffraction studies, particularly with the tendency to study ever-larger macromolecules and complexes. Further improvements in methods and instrumentation suited to growing larger crystals are therefore necessary for the use of NMX to expand.
In this work, we introduce rational strategies and a crystal growth bench (OptiCrys) developed in our laboratory that combines real-time observation through a microscope-mounted video camera with precise automated control of crystallization solutions (e.g., precipitant concentration, pH, additive, temperature). We then demonstrate how this control of temperature and chemical composition facilitates the search for optimal crystallization conditions using model soluble proteins. Thorough knowledge of the crystallization phase diagram is crucial for selecting the starting position and the kinetic path for any crystallization experiment. We show how a rational approach can control the size and number of crystals generated based on knowledge of multidimensional phase diagrams.

Structural studies of biomacromolecules by crystallography require high-quality crystals. Here we demonstrate a protocol that can be used by OptiCrys (a fully automated instrument developed in our lab) and/or microdialysis buttons for growing large high-quality crystals based on knowledge of the crystallization phase diagram.

中性子高分子結晶学における結晶成長の最適化

The use of neutron macromolecular crystallography (NMX) is expanding rapidly with most structures determined in the last decade thanks to new NMX ...

Simultaneous Live Imaging of Multiple Insect Embryos in Sample Chamber-Based Light Sheet Fluorescence Microscopes

Light sheet-based fluorescence microscopy offers efficient solutions to study complex processes on multiple biologically relevant scales. Sample chamber-based setups, which are specifically designed to preserve the three-dimensional integrity of the specimen and usually feature sample rotation, are the best choice in developmental biology. For instance, they have been used to document the entire embryonic morphogenesis of the fruit fly Drosophila melanogaster and the red flour beetle Tribolium castaneum. However, many available live imaging protocols provide only experimental frameworks for single embryos. Especially for comparative studies, such approaches are inconvenient, since sequentially imaged specimens are affected by ambient variance. Further, this limits the number of specimens that can be assayed within a given time. We provide an experimental framework for simultaneous live imaging that increases the throughput in sample chamber-based setups and thus ensures&#160;similar ambient conditions for all specimens. Firstly, we provide a calibration guideline for light sheet fluorescence microscopes. Secondly, we propose a mounting method for multiple embryos that is compatible with sample rotation. Thirdly, we provide exemplary three-dimensional live imaging datasets of Drosophila, for which we juxtapose three transgenic lines with fluorescently labeled nuclei, as well as of Tribolium, for which we compare the performance of three transgenic sublines that carry the same transgene, but at different genomic locations. Our protocol is specifically designed for comparative studies as it pro-actively addresses ambient variance, which is always present in sequential live imaging. This is especially important for quantitative analyses and characterization of aberrational phenotypes, which result e.g., from knockout experiments. Further, it increases the overall throughput, which is highly convenient when access to light sheet&#160;fluorescence microscopes is limited. Finally, the proposed mounting method can be adapted for other insect species and further model organisms, e.g., zebrafish, with basically no optimization effort.

Light sheet-based fluorescence microscopy is the most valuable tool in developmental biology. A major issue in comparative studies is ambient variance. Our protocol describes an experimental framework for simultaneous live imaging of multiple specimens and, therefore, addresses this issue pro-actively.

サンプルチャンバーベースの光シート蛍光顕微鏡における複数の昆虫胚の同時ライブイメージング

Light sheet-based fluorescence microscopy offers efficient solutions to study complex processes on multiple biologically relevant scales. Sample ...

FluoZin-3アッセイによる哺乳類細胞における亜鉛輸送メカニズムの調査

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

著者スポットライト:ZnT1機能による細胞亜鉛制御の探索 

Zinc transport has proven challenging to measure due to the weak causal links to protein function and the low temporal resolution. This protocol describes a method for monitoring, with high temporal resolution, Zn2+ extrusion from living cells by utilizing a Zn2+ sensitive fluorescent dye, thus providing a direct measure of Zn2+ efflux.

In Vitro亜鉛輸送アッセイを用いた哺乳類亜鉛トランスポーターの特性評価(英語)

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...