この研究は、ローレンス・リバモア国立研究所LDRD 18-ERD-062(CRへ)から資金提供を受けました。John Muschler氏には、DgKO細胞とDgKI細胞を寄贈していただき、感謝します。カリフォルニア大学バークレー校電子顕微鏡研究所のスタッフの方々には、電子顕微鏡によるサンプルの調製とデータ収集に関するアドバイスと支援をしていただき、ありがとうございました。

1.解凍して分注するlaminin-111
注:精製されたラミニン-111は市販されていますが(通常、マトリゲルとして販売されているEHSマトリックスから精製されています)、すべての供給源が無傷の非分解タンパク質を提供するわけではありません。
<ol><li>Ln1を調達し、流戮サイズ排除クロマトグラフィー26 またはポリアクリルアミドゲル電気泳動26を行うことにより、Ln1が分解されていないことを確認する。変性Ln1は337 kDa、197 kDa、177 kDaのバンドを持ち、ネイティブLn1は711 kDaのシングルバンドを持つ必要があります。</li>
	<li>Ln1を冷蔵庫の氷上で一晩解凍し、低タンパク質結合チップと滅菌技術を使用して、低タンパク質吸着性微量遠心チューブ(通常、培養オーバーレイの場合は100 μg/チューブ、コーティングの場合は10 μgで分注)に分注します27。アリコートを凍結し、-80°Cで保存してください。</li>
</ol>2. 低pH緩衝液を用いたラミニン重合

<ol><li>polyLM重合バッファーの作製:1 mM CaCl2 と20 mM酢酸ナトリウムを超純水で秤量し、混合します。次に、HClまたは酢酸を使用してpHを4.0に調整します。0.2μmフィルターに通した滅菌フィルターを使用し、4°Cで保存します。</li>
	<li>コントロール重合バッファーの作製:必要に応じて、1 mM CalCl2 と 20 mM Tris を混合し、10 N HCl または 12 N NaOH を使用して pH を 7.0 に調整します。0.2μmフィルターに通した滅菌フィルターを使用し、4°Cで保存します。 注: プロトコルはここで一時停止できます。</li>
	<li>必要な数のLn-1アリコートを冷蔵庫で一晩解凍します。</li>
	<li>滅菌細胞培養技術を用いて、適切なバッファー(ステップ2.1のpolyLM重合バッファーまたはステップ2.2のコントロール重合バッファーのいずれか)を最終濃度100 μg/mLのラミニンアリコートに添加し(すなわち、0.5 mLを50 μgアリコートに添加)、ピペッティングで穏やかに混合します。</li>
	<li>蛍光標識ラミニンを可視化に使用する場合は、再溶解し、1:50 wt/wt比(例:2 μg/100 μg)で添加し、ピペッティングで穏やかに混合します。 注:細胞接着の基質としてラミニンを使用する場合は、手順2.6〜2.8に従います。</li>
	<li>適切な緩衝液で希釈した直後に、ラミニンを培養プラスチックまたはガラス器具に転写し、37°Cの細胞培養インキュベーターで一晩インキュベートします。 カバーガラスをコーティングする場合は、平衡液滴が得られる程度に十分な容量を添加し(通常、13 mmの丸いカバーガラスには50 μLを使用)、加湿したチャンバーで37°Cで一晩保管してください。</li>
	<li>滅菌技術27を使用して、1xリン酸緩衝生理食塩水(PBS)で洗浄します。</li>
	<li>液体を慎重に取り除きます。表面を完全に乾かさないでください。 注:乳タンパク質誘導のために細胞を覆うためにラミニンを使用する場合は、手順2.9-2.12に従います。</li>
	<li>乳腺上皮細胞を培養します。2% FBS、0.01 mg/mLインスリン、0.005 μg/mL EGF、0.05 mg/mLゲンタマイシン、および0.05 g/mLノルモシンを添加したDMEM-F12で、MepGジストログリカンノックイン(DgKI)またはMepGジストログリカンノックアウト細胞(DgKO)を培養します。</li>
	<li>ラミニン刺激用のDgKOおよびDgKI細胞を10,500細胞/cm2 でウェルプレートまたはカバーガラス底皿に播種します</li>
	<li>希釈したラミニンを細胞培養インキュベーターで37°Cで30分間インキュベートします。</li>
	<li>polyLM アリコートは 2,000 x g で 20 分間遠心分離しますが、タンパク質実体のサイズが小さく、効果的に回収するにはより高速な速度が必要であるため、LM は中性 pH 20,000 x g で 20 分間遠心分離します。</li>
	<li>滅菌技術27を用いて、上清を穏やかに除去し、3 μg/mLのプロラクチン、2.8 μMのヒドロコルチゾン、および5 μg/mLのインスリンを添加したDMEM-F12の適切な量の誘導培地に再懸濁する</li>
	<li>細胞培養培地にラミニンを直ちに移し、細胞を刺激します。50-800 μg/mL Ln1で細胞を刺激することに成功しました</li>
</ol>3. 単離糖蛋白質を用いたラミニン重合
<ol><li>組換えニドゲン-1とLn1を冷蔵庫の氷上で一晩静かに解凍します。</li>
	<li>細胞培養培地中でLn1とニドゲン-1を1:1 wt/wtの比率で混合し、タンパク質結合の少ないチップとチューブを使用し、滅菌法を使用します。細胞培養培地中の純粋なLn1をコントロールとして使用します。</li>
	<li>可視化に蛍光標識ラミニンを使用する場合は、再溶解し、1:50 wt/wt 比(例:2 μg/100 μg)で添加します。ピペッティングで静かに混合します。</li>
	<li>Ln1-ニドゲンをインキュベートするか、Ln1を細胞培養インキュベーターで37°Cで30分間インキュベートします。</li>
	<li>Ln1-ニドゲン共重合体を2,000 x g で30分間遠心分離し、コントロールLn1を20,000 x g で30分間遠心分離します。</li>
	<li>上清を静かに除去し、細胞培養培地に最終濃度100〜800μgの総タンパク質/mLになるまで再懸濁します。</li>
	<li>直ちに細胞を刺激するために使用するか、カバーガラスに移し、加湿した細胞培養インキュベーターで37°Cで一晩接着させます。</li>
	<li>カバーガラスから培地を取り出し、カバーガラスを1x PBSで洗浄した後、10%ホルマリン(PBS中の4%ホルムアルデヒド)で室温で15分間固定します。</li>
</ol>4. ジストログリカン発現細胞を用いたラミニン重合
<ol><li>乳腺上皮細胞を培養します。2% FBS、0.01 mg/mLインスリン、0.005 μg/mL EGF、0.05 mg/mLゲンタマイシン、および0.05 g/mLノルモシンを添加したDMEM-F12で、MepGジストログリカンノックイン(DgKI)またはMepGジストログリカンノックアウト細胞(DgKO)を培養します。</li>
	<li>増殖速度の違いにより、DgKI細胞は5,000細胞/cm2、DgKO細胞は8,000細胞/cm2 で播種します。加湿インキュベーターで37°Cで培養し、培地を2〜3日ごとに交換し、厳格な滅菌技術を使用して4〜5日ごとに継代します。適切な増殖速度を確保するために、血球計算盤または自動セルカウンター27 で細胞を注意深くカウントします。</li>
	<li>10,500細胞/cm2 のラミニン刺激用播種細胞をウェルプレートまたはカバーガラス底皿に入れ、2日間培養し、DgKI細胞を用いてフラクタルLn1細胞およびDgKO細胞をネガティブコントロールとして作製した。Ln-1を冷蔵庫の氷上で穏やかに一晩解凍し、3 μg/mLのプロラクチン、2.8 μMのヒドロコルチゾン、および5 μg/mLのインスリンを添加したDMEM-F12の誘導培地と混合します。35 mmのディッシュには、50〜800 μgのLn1を含む1 mLの誘導媒体を使用します。</li>
	<li>蛍光Ln1を使用する場合は、50:1のwt/wt比で混合します。</li>
	<li>細胞から培地を取り除き、3 μg/mLのプロラクチン、2.8 μMのヒドロコルチゾン、および5 μg/mLのインスリンを添加したDMEM-F12のラミニンおよび誘導培地で覆います</li>
	<li>細胞を2日間培養した後、10%ホルマリンで固定します。</li>
</ol>5. 光学顕微鏡によるラミニン微細構造の解析
<ol><li>固定サンプルを採取し、1x PBSで室温で5分間3回洗浄します。</li>
	<li>0.5% Triton X-100、3% ウシ血清アルブミン、10% ヤギ血清、40 μg/mL 抗マウスブロッキング抗体フラグメントを含む 1x PBS でサンプルを室温で 1 時間ブロッキングし、透過処理します。 注意: Triton X-100は危険です。手袋と目の保護具を着用して取り扱い、適切に廃棄してください。</li>
	<li>抗ラミニン抗体を希釈率1:100で1x PBSと0.5% Triton X-100および3% BSAを混合し、加湿チャンバー内で室温で2時間、または4°Cで一晩サンプルに添加します(通常は、底部にウェットラボワイプを入れたチップボックスを使用します)。</li>
	<li>一次抗体を除去し、0.5% Triton X-100 および 3% BSA を含む 1x PBS で 3 回洗浄します。</li>
	<li>0.5% Triton X-100 および 3% BSA を含む 1x PBS のサンプルに適切な二次抗体を 1:500 に希釈して添加し、加湿チャンバー内で暗所の室温でインキュベートします。</li>
	<li>二次抗体を除去し、0.5% Triton X-100および3% BSAを含む1x PBSで3回洗浄します。</li>
	<li>細胞を含むサンプルの場合は、6 μM のファロイジンを添加して 45 分間添加した後、0.5% Triton X-100 と 3% BSA を含む PBS で 3 回洗浄し、続いて PBS で 3 回洗浄します。その後、0.1 μg/mL DAPI in PBSで5分間染色します。</li>
	<li>必要に応じてサンプルを取り付けます。</li>
	<li>高分解能共焦点顕微鏡を用いたln-1構造の画像化液浸対物レンズ(水浸プランアポクロマート40X/1.1NAまたは油浸プランアポクロマート63X/1.4NA)を備えたレーザー走査型顕微鏡を使用してください。</li>
</ol>6. 箱の計数次元によるlamininの構造物の性格描写
<ol><li>Matlab ツールボックスまたは ImageJ で使用可能なボックス計数次元アルゴリズムを使用して、フラクタル特性を解析します。これらの手法は、ラミニンのイメージを閾値とし、Ln1 を含むボックスの数とボックスのサイズを比較した対数対数プロットにプロットします。これらのパラメータ間の関係の傾きはボックスカウント次元であり、非フラクタルジオメトリの次元は0、1、または2になり、フラクタルジオメトリは非整数次元28になります。 注:酸処理、糖タンパク質処理、およびDgKI細胞付着Ln1はすべて、ボックスカウントフラクタル次元が~1.7であるのに対し、コントロールLn1のフラクタル次元は2.0です。</li>
</ol>7. 電子顕微鏡によるラミニンコンストラクトのキャラクタリゼーション
<ol><li>ラミニンを細胞培養培地に再懸濁し、湿度の高い37°Cの環境で90分間シリカウエハーに吸着させます。</li>
	<li>マトリックスを pH 7.2 の 0.1 M カコジル酸ナトリウム緩衝液中の 2.5% グルタルアルデヒドで固定します。 注意: グルタルアルデヒドは危険であり、手袋と目の保護具を着用してドラフトで取り扱い、有害廃棄物として処分する必要があります。</li>
	<li>pH 7.2 の 0.1 M カコジル酸ナトリウム緩衝液中の 1% 四酸化オスミウム中の後置マトリックス。 注意: 四酸化オスミウムは危険であり、手袋と目の保護具を使用してドラフトで取り扱い、適切に廃棄する必要があります。カコジル酸は危険であり、手袋と目の保護具を使用してドラフトで取り扱い、適切に廃棄する必要があります。.</li>
	<li>pH 7.2のカコジル酸ナトリウム緩衝液で3回洗浄します。</li>
	<li>エタノールの濃度を上げて脱水します。</li>
	<li>スパッタリングコートマトリックスは、金の薄い層でコーティングされます。</li>
	<li>走査型電子顕微鏡を用いた画像。</li>
</ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microstructure+of+laminin-111+compensates+for+dystroglycan+loss+in+mammary+epithelial+cells+in+downstream+expression+of+milk+proteins.">The microstructure of laminin-111 compensates for dystroglycan loss in mammary epithelial cells in downstream expression of milk proteins.</a> Biomaterials. 218, 119337 (2019).</li><li>Weir, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dystroglycan+loss+disrupts+polarity+and+beta-casein+induction+in+mammary+epithelial+cells+by+perturbing+laminin+anchoring.">Dystroglycan loss disrupts polarity and beta-casein induction in mammary epithelial cells by perturbing laminin anchoring.</a> Journal of Cell Science. 119, 4047-4058 (2006).</li><li>Hughes, C. S., Postovit, L. M., Lajoie, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+a+complex+protein+mixture+required+for+optimal+growth+of+cell+culture.">Matrigel: a complex protein mixture required for optimal growth of cell culture.</a> Proteomics. 10 (9), 1886-1890 (2010).</li><li>Palmero, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+follicular+thyroid+cell+line+PCCL3+responds+differently+to+laminin+and+to+polylaminin,+a+polymer+of+laminin+assembled+in+acidic+pH.">The follicular thyroid cell line PCCL3 responds differently to laminin and to polylaminin, a polymer of laminin assembled in acidic pH.</a> Molecular and Cellular Endocrinology. 376 (1), 12-22 (2013).</li><li>Hochman-Mendez, C., Lacerda de Menezes, J. R., Sholl-Franco, A., Coelho-Sampaio, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polylaminin+recognition+by+retinal+cells.">Polylaminin recognition by retinal cells.</a> Journal of Neuroscience Research. 92 (1), 24-34 (2014).</li><li>Leal-Lopes, C., Grazioli, G., Mares-Guia, T. R., Coelho-Sampaio, T., Sogayar, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerized+laminin+incorporation+into+alginate-based+microcapsules+reduces+pericapsular+overgrowth+and+inflammation.">Polymerized laminin incorporation into alginate-based microcapsules reduces pericapsular overgrowth and inflammation.</a> Journal of Tissue Engineering and Regenerative Medicine. 13 (10), 1912-1922 (2019).</li><li>Paccola Mesquita, F. C., Hochman-Mendez, C., Morrissey, J., Sampaio, L. C., Taylor, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+as+a+Potent+Substrate+for+Large-Scale+Expansion+of+Human+Induced+Pluripotent+Stem+Cells+in+a+Closed+Cell+Expansion+System.">Laminin as a Potent Substrate for Large-Scale Expansion of Human Induced Pluripotent Stem Cells in a Closed Cell Expansion System.</a> Stem Cells International. 2019, 9 (2019).</li><li>Walker, J. M., Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Protein Protocols Handbook. , 61-67 (2002).</li><li>Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culture of Animal Cells: A Manual of Basic Techniques. 7th edition. , (2016).</li><li>Mandelbrot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Fractal Geometry of Nature. , (1982).</li><li>Schittny, J. C., Yurchenco, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terminal+short+arm+domains+of+basement+membrane+laminin+are+critical+for+its+self-assembly.">Terminal short arm domains of basement membrane laminin are critical for its self-assembly.</a> Journal of Cell Biology. 110 (3), 825-832 (1990).</li><li>Schuger, L., Yurchenco, P., Relan, N. K., Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+fragment+E4+inhibition+studies:+basement+membrane+assembly+and+embryonic+lung+epithelial+cell+polarization+requires+laminin+polymerization.">Laminin fragment E4 inhibition studies: basement membrane assembly and embryonic lung epithelial cell polarization requires laminin polymerization.</a> International Journal of Developmental Biology. 42 (2), 217-220 (1998).</li><li>Colognato, H., Yurchenco, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Form+and+function:+the+laminin+family+of+heterotrimers.">Form and function: the laminin family of heterotrimers.</a> Developmental Dynamics. 218 (2), 213-234 (2000).</li><li>Kabaeva, Z., Meekhof, K. E., Michele, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sarcolemma+instability+during+mechanical+activity+in+Large+myd+cardiac+myocytes+with+loss+of+dystroglycan+extracellular+matrix+receptor+function.">Sarcolemma instability during mechanical activity in Large myd cardiac myocytes with loss of dystroglycan extracellular matrix receptor function.</a> Human Molecular Genetics. 20 (17), 3346-3355 (2011).</li></ol>

この研究は、米国エネルギー省の後援の下、ローレンス・リバモア国立研究所が契約DE-AC52-07NA27344の下で実施しました。IMリリースLLNL-JRNL-799443。

ラミニンは、上皮系、筋肉系、神経系におけるECMの重要な要素であり、in vitroでは、細胞の機能分化の調節に重要な役割を果たすことが示されています。細胞に示されるラミニンの構造が、そのシグナル伝達と結果として生じる細胞表現型を調節することを示す証拠が増えており15,16、したがって、Ln1微細構造を制御する方法は、これらの分野で働?...

成長因子やサイトカインとは異なり、細胞外マトリックス(ECM)タンパク質は構造ネットワークに集合することができます。ECM機能障害は多くの疾患の重要な要素であるため、細胞がECM組成、微細構造、および生体力学からのシグナルを感知および伝達するメカニズムは、治療法開発の魅力的な標的です。これらのネットワークの微細構造が、これらのタンパク質が細胞にシグナルを伝達する仕組みに大きな役割を果たしていることを示唆する証拠が増えています。現在までに、ECMの微細構造と細胞機能とのこの連関は、コラーゲンI1、フィブロネクチン2、およびフィブリン3について示されています。
ラミニン-111(Ln1)は、上皮細胞4、筋肉細胞5、神経細胞6に接触する細胞外マトリックスの重要な構造成分である三量体の十字型タンパク質です。乳腺では、Ln1は乳腺上皮細胞から乳汁産生細胞への機能分化に必要であり7,8,9、Ln1は腫瘍復帰の誘導に不可欠である10,11。Ln1と他のラミニンは、筋肉における皮質アクチンネットワークの発達と筋膜の組織化12,13、および神経細胞の移動と神経突起伸長13に必要である。このように、ラミニンが細胞にシグナルを送るメカニズムを理解することは、活発な研究分野です。
Ln1には、インテグリン、シンデカン、ジストログリカン、LBP-110、LamRなど、幅広い受容体に対する複数の接着ドメインが含まれています(図1A)4,14。ラミニンα1のc末端球状ドメインを含むE8断片は、ジストログリカンとインテグリリンα6β1およびα3β1の結合に必要であり15、上皮細胞の機能分化に必要である15,16。対照的に、短腕は異なる生物学的活性を示し17、ネットワーク形成後にこれらの異なるLn1ドメインを認識できることは、細胞の挙動を変化させる可能性があることを意味する。
我々は最近、高分子ネットワークに配列されたLn1がフラクタル特性(すなわち、複数の長さスケールにわたって自己相似な構造)を示すことを実証した18。フラクタルネットワークは、この配列を欠いているLn1と比較して、上皮細胞に対して異なるシグナルを伝達する19。このフラクタルネットワークは、特定のアセンブリ構造を示しており、ここで、長腕は、細胞18に優先的に曝露される。この異なる長腕表示の結果として、上皮細胞は、よく組織化されたタイトジャンクションとアクチンストレス線維の抑制を有する乳生産細胞に機能分化する19。
本稿では、Ln1長腕の高い表示を示す短腕-短腕結合相互作用からLn1ネットワークを生成する3つの方法、すなわち、1)酸性緩衝液による無細胞集合、2)糖タンパク質との共インキュベーション、または3)細胞表面ジストログリカンとの相互作用について述べる。これら3つの方法は、3つの非常に異なるメカニズムによって類似したラミニンの微細構造を生成するため、実験計画に柔軟性を持たせることができます。以前の研究では、これら3つの方法が生物学的頑健性を表す可能性があることが示唆されています:乳腺上皮では、細胞表面のジストログリカン、糖タンパク質、または人工的に構造化されたラミニンのいずれかが機能分化を誘導することができます19。したがって、研究者は研究対象に基づいてこれらの方法から選択することができます:細胞膜中のジストログリカンがフラクタルネットワークに正しい重合を誘導するため、ジストログリカン発現細胞はLn-1微細構造に対する感受性を示さない19,20。ラミニンと糖タンパク質の混合物であるマトリゲル21は、Ln-1の最も経済的な供給源であり、ジストログリカンノックアウト乳腺細胞を分化させるのに必要な微細構造を提供する20が、ロットごとにばらつきのあるタンパク質の複雑な混合物を含んでいる。PolyLMは、この機能を提供する最もクリーンで還元主義的なシステムですが、マトリゲル19と比較して、乳腺細胞の分化を誘導するコストが高く、有効性が低くなります。
これら3つの方法は、拡散制限凝集18,19に特徴的なフラクタルネットワーク構造を有し、複数の実験系22,23,24,25において凝集したラミニンと比較して生物活性の変化を示す。これらの方法を用いた今後の研究により、上皮分化に必要な受容体とシグナル伝達を同定し、腫瘍などの異常な微小環境20の細胞において正常な細胞-マトリックス相互作用を回復させることが決定される可能性がある。

<table border="0" cellpadding="0" cellspacing="0" style="width:665px;" width="665">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10% formalin</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">HT501128</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Laminin primary antibody</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">L9393</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-Rabbit Alexafluor 488 secondary antibody</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">A32731</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">C4901 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell Culture Incubator</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">Heracell 150 or similar</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Centrifuge for microcentrifuge tubes</td>
			<td style="width:121px;">Eppendorf</td>
			<td style="width:161px;">5418 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Confocal Microscope</td>
			<td style="width:121px;">Zeiss</td>
			<td style="width:161px;">LSM 710 or equivalent</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Coverslips</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">25CIR-1 or similar</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">DMEM-F12, Liquid, High Glucose, +HEPES, L-glutamine, +Phenol red</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">11330107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EGF (Epidermal Growth Factor)</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">11376454001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluorescently Labeled Laminin</td>
			<td style="width:121px;">Cytoskeleton Inc</td>
			<td style="width:161px;">LMN02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gentamicin</td>
			<td style="width:121px;">VWR</td>
			<td style="width:161px;">VWRV0304-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutaraldehyde</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">G5882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HCl</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">320331 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hydrocortisone</td>
			<td style="width:121px;">Sigma-Aldrich</td>
			<td style="width:161px;">H0888-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Insulin</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">16634-50mg</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MepG Dystroglycan Knockout Cells</td>
			<td style="width:121px;">LBNL</td>
			<td style="width:161px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaOH</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">S8045 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Normocin</td>
			<td style="width:121px;">Invivogen</td>
			<td style="width:161px;">ant-nr-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Osmium Tetroxide</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">75633</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Ovine Prolactin</td>
			<td style="width:121px;">Los Angeles Biomedical Research Institute</td>
			<td style="width:161px;">Ovine Pituitary Prolactin</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phalloidin</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">A22287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phosphate Buffered Saline</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">10010023 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Purified Laminin-111</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">L2020-1MG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Recombinant human Nidogen-1, carrier free</td>
			<td style="width:121px;">R&#38;D systems</td>
			<td style="width:161px;">2570-ND-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Acetate</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">S2889 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Cacodylate</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">C0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile filters</td>
			<td style="width:121px;">Millipore</td>
			<td style="width:161px;">SLGP033RS or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">252859 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:121px;">Sigma-Aldrich</td>
			<td style="width:161px;">X100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultra-low protein binding tubes</td>
			<td style="width:121px;">VWR</td>
			<td style="width:161px;">76322-516, 76322-522 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultrapure water</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">15230253 or similar</td>
			<td></td>
		</tr>
	</tbody>
</table>

generating a fractal microstructure of laminin-111 to signal to cells

ラミニン-111(Ln1)は、上皮系、筋肉系、神経系の細胞外マトリックスの重要な部分です。我々は以前、Ln1の微細構造が細胞へのシグナル伝達の仕方を変化させることを実証したが、これはおそらくLn1がネットワークに集合すると異なる接着ドメインが露出するためであろう。このプロトコルでは、重合されたLn1を発生させる3つの方法を記述する。

ラミニン-111は、短腕の末端に3つの自己組織化ドメインを持つ三量体タンパク質です(図1A)。適切に処理すると、短いアームは六方格子に重合し(図1B、1C)、より大きな空間スケールで拡散限界凝集フラクタル特性を示します(図2)。カルシウム含有中性pH緩衝液中でインキュベートしたラミニンは、抗Ln1抗体(<strong cla...

Watch this Scientific Journal Video about Generating a Fractal Microstructure of Laminin-111 to Signal to Cells at JoVE.com

Generating a Fractal Microstructure of Laminin-111 to Signal to Cells

重合されていないLn1とは異なる方法で細胞にシグナルを送るフラクタル特性を持つLn1ポリマーを生成する3つの方法について説明します。

細胞にシグナルを送るためのラミニン-111のフラクタル微細構造の生成

Laminin-111 (Ln1) is an essential part of the extracellular matrix in epithelia, muscle and neural systems. We have previously demonstrated that the ...

generating-a-fractal-microstructure-of-laminin-111-to-signal-to-cells

Research

JoVE Journal

Bioengineering

914 ビュー.  Texas Heart Institute. 重合されていないLn1とは異なる方法で細胞にシグナルを送るフラクタル特性を持つLn1ポリマーを生成する3つの方法について説明します。

ビデオ: Generating a Fractal Microstructure of Laminin-111 to Signal to Cells

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

のための周期的圧力のバイオリアクターの設計生体外で</em大動脈心臓弁の>研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

生物工学

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

生体材料の血管新生を評価するために、完全な皮膚欠損モデル<em&gt;インビボ</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

キナーゼのアップコン バージョンのナノ粒子を介した光活性化によって細胞内シグナル伝達をリモートでトリガーする統合システム

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the microscale&#8213;to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert (&#39;caged&#39;) by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

微生物の挙動を研究するための制御された動的化学景観の生成

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...

Biomimetic Replication of Root Surface Microstructure using Alteration of Soft Lithography

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, biomimetic surfaces mimicking the microstructure of leaf surface, were used to study the effects of leaf microstructure on leaf-environment interactions. However, no such tool exists for roots. We developed a tool allowing the synthetic mimicry of the root surface microstructure into an artificial surface. We relied on the soft lithography method, known for leaf surface microstructure replication, using a two-step process. The first step is the more challenging one as it involves the biological tissue. Here, we used a different polymer and curing strategy, relying on the strong, rigid, polyurethane, cured by UV for the root molding. This allowed us to achieve a reliable negative image of the root surface microstructure including the delicate, challenging features such as root hairs. We then used this negative image as a template to achieve the root surface microstructure replication using both the well-established polydimethyl siloxane (PDMS) as well as a cellulose derivative, ethyl cellulose, which represents a closer mimic of the root and which can also be degraded by cellulase enzymes secreted by microorganisms. This newly formed platform can be used to study the microstructural effects of the surface in root-microorganism interactions in a similar manner to what has previously been shown in leaves. Additionally, the system enables us to track the microorganism&#8217;s locations, relative to surface features, and in the future its activity, in the form of cellulase secretion.

Biomimetics has been previously used as a tool to study leaf-microorganism interactions. However, no such tool exists for roots. Here, we develop a protocol to form synthetic surfaces mimicking root surface microstructure for the study of root-environment interactions.

軟リソグラフィの改変を用いた根表面微細構造の生物模倣複製

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

ヒト人工多能性幹細胞由来心筋細胞における単細胞光学作用電位測定

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

静止ヒト造血幹細胞を維持する培養法

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.

This protocol describes the assembly and operation of a low-cost acoustofluidic device for rapid molecular delivery to cells via sonoporation induced by ultrasound contrast agents.

細胞への分子化合物の送達を強化するためのアキュトー流体デバイスの組立と操作

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

スクリーニング用途のための線維芽細胞由来ヒト工学的結合組織

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often detrimental to protein folding and, in particular, chromophore maturation in green fluorescent proteins and related variants. A reasonable alternative is to manipulate the translation of the protein so that all Pro residues are replaced residue-specifically by analogs, a method known as selective pressure incorporation (SPI). The built-in chemical modifications can be used as a kind of &#34;molecular surgery&#34; to finely dissect measurable changes or even rationally manipulate different protein properties. Here, the study demonstrates the usefulness of the SPI method to study the role of prolines in the organization of the typical &#946;-barrel structure of spectral variants of the green fluorescent protein (GFP) with 10-15 prolines in their sequence: enhanced green fluorescent protein (EGFP), NowGFP, and KillerOrange. Pro residues are present in connecting sections between individual &#946;-strands and constitute the closing lids of the barrel scaffold, thus being responsible for insulation of the chromophore from water, i.e., fluorescence properties. Selective pressure incorporation experiments with (4R)-fluoroproline (R-Flp), (4S)-fluoroproline (S-Flp), 4,4-difluoroproline (Dfp), and 3,4-dehydroproline (Dhp) were performed using a proline-auxotrophic E. coli strain as expression host. We found that fluorescent proteins with S-Flp and Dhp are active (i.e., fluorescent), while the other two analogs (Dfp and R-Flp) produced dysfunctional, misfolded proteins. Inspection of UV-Vis absorption and fluorescence emission profiles showed few characteristic alterations in the proteins containing Pro analogs. Examination of the folding kinetic profiles in EGFP variants showed an accelerated refolding process in the presence of S-Flp, while the process was similar to wild-type in the protein containing Dhp. This study showcases the capacity of the SPI method to produce subtle modifications of protein residues at an atomic level (&#34;molecular surgery&#34;), which can be adopted for the study of other proteins of interest. It illustrates the outcomes of proline replacements with close chemical analogs on the folding and spectroscopic properties in the class of &#946;-barrel fluorescent proteins.

To overcome the limitations of classical site-directed mutagenesis, proline analogs with specific modifications were incorporated into several fluorescent proteins. We show how the replacement of hydrogen by fluorine or of the single by double bonds in proline residues ("molecular surgery") affects fundamental protein properties, including their folding and interaction with light.

蛍光タンパク質中のプロリン類似体によるプロリンの残基特異的交換:骨格の「分子手術」が折り畳みと安定性にどのように影響するか

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often ...