Name: generating a fractal microstructure of laminin-111 to signal to cells
Uploaded: 2020-09-28T13:00:00.000+00:00
Duration: 6 min 56 s
Description: Watch this Scientific Journal Video about Generating a Fractal Microstructure of Laminin-111 to Signal to Cells at JoVE.com

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

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JoVE Journal

Bioengineering

924 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

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

タンパク質、細胞閉じ込めのための二次元加工基板を作成する

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

生物工学

Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and how the domains are oriented/positioned. Here, a protocol with the potential for elucidating which specific domains mediate interactions in multicomponent system through ab initio modeling is described. A method for calculating solution structures of macromolecules and their assemblies is provided that involves integrating data from small angle X-ray scattering (SAXS), chromatography, and atomic resolution structures together in a hybrid approach. A specific example is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins and forms an extended, curved nanostructure. One of its globular domains attaches to laminin &#947;-1, which structures the basement membrane. This provides a basis for determining accurate structures of flexible multidomain protein complexes and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows rapid analysis in which multiple oligomeric states are separated just prior to SAXS data collection. The analysis yields information on the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high-resolution structures of the component proteins is also given.

Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution.

小角 x 線散乱による溶液中の高分子の構造研究

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and ...

生化学

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

表面開始アセンブリの使用ECMタンパク質のナノファイバーおよびナノ構造エンジニア

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

サンドイッチ状の微小環境は、Cell /材料の相互作用を活用します

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D相互作用の研究のための3Dコラーゲンゲルおよびマイクロチャネルの作製<em&gt;インビボ</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

細胞外マトリックス内に埋め込まれたエンジニアリング三次元上皮組織

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

サポートされている脂質二重層中のリガンドナノクラスター配列

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

マイクロパターントラクション顕微鏡のパターン生成

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the cells themselves. Here, we present the method of Light-Induced Molecular Adsorption of Proteins (LIMAP) using the PRIMO system as a patterning technique to produce micro-patterned extracellular matrix (ECM) substrates using a single or combination of proteins. The method enables printing of ECM patterns in micron resolution with excellent reproducibility. We provide a step-by-step protocol and demonstrate how this can be applied to study the processes of neuronal pathfinding. LIMAP has significant advantages over existing micro-printing methods in terms of the ease of patterning more than one component and the ability to generate a pattern with any geometry or gradient. The protocol can easily be adapted to study the contribution of almost any chemical component towards cell fate and cell behavior. Finally, we discuss common issues that can arise and how these can be avoided.

Our overall aim is to understand how cells sense extracellular cues that lead to directed axonal growth. Here, we describe the methodology of Light-Induced Molecular Adsorption of Proteins, used to produce defined micro-patterns of extracellular matrix components in order to study specific events that govern axon outgrowth and pathfinding.

細胞外マトリックスタンパク質に対する細胞応答を研究するためのマイクロパターニングのためのPRIMOシステムを用いてタンパク質の光誘導分子吸着

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the ...

神経科学

Fabricating Engineered Nanofibrillar Collagen with Directed Patterning

Production of Nanofibrillar Patterned Collagen for Tissue Engineering

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are designed to emulate the biophysical properties of native tissue, providing cellular phenotypic and morphological guidance that contributes to the restoration of the regenerative tissue niche. Collagen, a prevalent extracellular matrix protein, is a common component of these regenerative biomaterials due to its biocompatibility and other favorable properties. The current study describes a novel and straightforward method for the fabrication of engineered nanofibrillar collagen with directed fibril patterning. Through the manipulation of shear stress, temperature, and pH, collagen fibrillogenesis and alignment are precisely controlled without requiring specialized equipment. This approach allows for the creation of nanofibrillar collagen biomaterials that mimic the native structure of tissues exhibiting either anisotropic or isotropic characteristics. The flexibility in collagen nanofibril patterning not only facilitates the study of nanoscale patterning on cell behavior but also offers diverse possibilities for patterned tissue engineering applications.

The following protocol presents a shear-based extrusion method for the fabrication of collagen hydrogels with nanoscale patterned fibrils, which can be applied to a broad range of tissue engineering applications.

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are ...