Name: characterization of cell membrane extensions and studying their roles in cancer cell adhesion dynamics
Uploaded: 2018-03-26T14:30:00.000+00:00
Duration: 8 min 11 s
Description: Watch this Scientific Journal Video about Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics at JoVE.com

大瀬助成財団の資金この作品。

1. セル拡張機能特性
<ol><li>SW 13、SW-ネオと SW DKK3 標準加湿のインキュベーターでは、37.0 ° C、5% 10% 牛胎児血清と 10,000 U/mL ペニシリンとストレプトマイシン ('成長培地' として指定) 補われる CO2ダルベッコ変更イーグル培地で細胞を維持します。<ol>
<li>診断、セルをカウントし、別 6 ウェル プレートに SW 13、SW-ネオ、SW DKK3 ラインにウェルあたり 5,000 の細胞をプレート培地に無菌ガラス coverslips に一晩成長します。</li>
	</ol>
</li>
	<li>各井戸のための一晩の成長は、成長培地を吸引後、1 ml の温かいリン酸緩衝生理食塩水 (PBS) の細胞を洗浄し、1 ml の 10 分の 3.7% のホルムアルデヒドのセルを修復します。</li>
	<li>30 分洗って余分な 0.05% クリスタル バイオレットの 1 mL の各ウェルの吸引ホルムアルデヒドと染色細胞を染める 1 mL の脱イオン水の 3 つの洗浄を使用しています。 注: 染料の通風管のレベルに応じて細胞の可視化を促進するために染色の背景を削除するいくつかの追加の洗浄は必要かもしれない。<ol>
<li>微細先端の鉗子を使用して、coverslips を取得し、クリア マウント試薬のドロップでラベル付きガラス スライドに顔ダウン配置します。</li>
	</ol>
</li>
	<li>光学顕微鏡の下で細胞延長の試金のため各 coverslip から非重複の細胞の 20 件がランダムに選択します。<ol>
<li>各視野の 400 倍の倍率での顕微鏡写真を取る。直接各 20 の代表的な細胞の拡張の各種類の数をカウントします。 注: など、20 の隔離されたセルを信頼性の高い結果を保証するテスト条件ごとに検討すべき。糸状は 5 ミクロン以下のベースと 1 つの頂点の細胞拡張機能によって定義されました。Lobopodia は、複数の頂点を持つ拡張機能を含む長さの 5 に 20 ミクロンのベース携帯電話の拡張機能によって定義されました。ケラトサイトは 20 ミクロンの長さと頂の有無よりも大きいセル拡張によって定義されました。テスト細胞の種類に応じてセル拡張のサイズは異なる場合があります、パラメーターの特定の洗練さがあります。</li>
	</ol>
</li>
	<li>検査 6 井戸全体平均 (すなわちSW-13、SW ネオ、SW DDK) にそれぞれの細胞の種類に各セルの拡張数を決定します。</li>
	<li>一方向の分散分析 (ANOVA) の検定を使用して、別のテスト細胞のタイプ間の細胞膜拡張の割合の平均値の違いを比較します。</li>
</ol>2. 接着アッセイ
<ol><li>SW 13、SW-ネオと SW DKK3 細胞培地に CO2を 37.0 ° C、5% で標準加湿インキュベーターで維持します。<ol>
<li>診断を使用して、カウントし別 6 ウェル プレートによくあたり SW 13、SW-ネオ、SW DKK3 の 100,000 細胞をプレートし、成長培地で一晩培養します。</li>
		<li>セル タイプごとにシード 6 ウェル プレート テスト、下テスト 6 の指定された時間のポイントごとに 1 つのプレートを使用します。 メモ: 異なる種類の細胞の時間ポイントが異なる場合があります。</li>
	</ol>
</li>
	<li>一晩インキュベートした後一度暖かい PBS のセルを洗浄し、各ウェルに非酵素的細胞解離液 x 1 の 0.5 mL を追加します。<ol>
<li>細胞解離ソリューションを普及するプレートを旋回します。定義された時点 (つまり1、2、3、5、10、および 15 分) で指定されたプレートを吸引し、3 回 1 mL 温 PBS 溶液で洗浄します。</li>
		<li>洗浄の間緩く接続されたセルをデタッチするプレートを軽くたたきます。</li>
	</ol>
</li>
	<li>残りのすべての PBS と修正 1 ml 10 分の 3.7% のホルムアルデヒドのプレートに接続されている残りの細胞を吸引します。</li>
	<li>30 分 0.05% クリスタル バイオレットの 1 ml の細胞を染色し、1 mL の脱イオン水で余分な染料を洗浄します。細胞の可視化を促進するために洗浄を滴定しなさい。 注: 染料の通風管のレベルに応じていくつかの追加の洗浄は細胞の可視化を促進するために必要かもしれない。</li>
	<li>光学顕微鏡を使用すると、100 倍の倍率で、各プレートの各ウェルに接続されている残りのセルをカウントします。 注: 透明な定規またはマーキング プレートの下側の先の細いペン ガイドの使用は同じ細胞の繰り返しカウントを避けるために役立ちます。</li>
	<li>各プレートのウェルあたり添付の細胞の平均の数を決定します。</li>
	<li>双方向分散統計的検定を用いて指定された期間にわたって SW 13、SW-ネオと SW DDK3 細胞の間細胞の剥離率を比較します。</li>
</ol>

<ol>
	<li>Libe, R., Fratticci, A., Bertherat, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenocortical+cancer:+pathophysiology+and+clinical+management.">Adrenocortical cancer: pathophysiology and clinical management.</a> Endocrine-related cancer. 14 (1), 13-28 (2007).</li><li>Veeck, J., Dahl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+Wnt+pathway+in+cancer:+the+emerging+role+of+Dickkopf-3.">Targeting the Wnt pathway in cancer: the emerging role of Dickkopf-3.</a> Biochimica et biophysica acta. 1825 (1), 18-28 (2012).</li><li>Friedl, P., Gilmour, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+cell+migration+in+morphogenesis,+regeneration+and+cancer.">Collective cell migration in morphogenesis, regeneration and cancer.</a> Nat Rev Mol Cell Biol. 10 (7), 445-457 (2009).</li><li>Jacquemet, G., 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=L-type+calcium+channels+regulate+filopodia+stability+and+cancer+cell+invasion+downstream+of+integrin+signalling.">L-type calcium channels regulate filopodia stability and cancer cell invasion downstream of integrin signalling.</a> Nat Commun. 7, 13297 (2016).</li><li>Machesky, L. M., Li, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fascin:+Invasive+filopodia+promoting+metastasis.">Fascin: Invasive filopodia promoting metastasis.</a> Commun Integr Biol. 3 (3), 263-270 (2010).</li><li>Petrie, R. J., Harlin, H. M., Korsak, L. I., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activating+the+nuclear+piston+mechanism+of+3D+migration+in+tumor+cells.">Activating the nuclear piston mechanism of 3D migration in tumor cells.</a> J Cell Biol. 216 (1), 93-100 (2017).</li><li>Korah, R., 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=Epigenetic+silencing+of+RASSF1A+deregulates+cytoskeleton+and+promotes+malignant+behavior+of+adrenocortical+carcinoma.">Epigenetic silencing of RASSF1A deregulates cytoskeleton and promotes malignant behavior of adrenocortical carcinoma.</a> Molecular cancer. 12, 87 (2013).</li><li>Cheng, J. 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=A+novel+FOXO1-mediated+dedifferentiation+blocking+role+for+DKK3+in+adrenocortical+carcinogenesis.">A novel FOXO1-mediated dedifferentiation blocking role for DKK3 in adrenocortical carcinogenesis.</a> BMC cancer. 17 (1), 164 (2017).</li><li>Barry, D. J., Durkin, C. H., Abella, J. V., Way, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+source+software+for+quantification+of+cell+migration,+protrusions,+and+fluorescence+intensities.">Open source software for quantification of cell migration, protrusions, and fluorescence intensities.</a> J Cell Biol. 209 (1), 163-180 (2015).</li><li>Cliffe, A., 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=Quantitative+3D+analysis+of+complex+single+border+cell+behaviors+in+coordinated+collective+cell+migration.">Quantitative 3D analysis of complex single border cell behaviors in coordinated collective cell migration.</a> Nat Commun. 8, 14905 (2017).</li></ol>

著者が明らかに何もありません。

ここで簡単に、いくつかの落とし穴と各種のテスト条件に適用することができます信頼性の高い再現性とセル拡張機能を特徴付けるための体外定量的手法について述べる。また、細胞膜拡張機能の観察された変化の潜在的な機能的意義を関連付けるためシンプルで定量化可能な接着や運動アッセイを同時に実行できます。
しかし、これらのメソッドは、いくつか?...

調節不全 WNT シグナル伝達は、副腎悪性腫瘍1で重要な役割を果たしています。本研究で使用される方法を調査かどうか DKK3 のサイレンシング、wnt シグナル伝達の負の調節因子副腎皮質における脱分化イベントを表しますセル拡張レパートリー変更のコンテキストで腫瘍形成を促進します。DKK3 は 38 kDa 分泌される糖タンパク質 N 末端のシグナルペプチドとその強制式は積極的な悪性の挙動を抑制、逆に上皮間葉こと、細胞周期の停止で起因した前の調査は示した遷移2。
副腎皮質癌 (ACC) および他の癌の悪性の挙動も含む腫瘍細胞浸潤を円滑に細胞外のマトリックスでは、周囲の表面とのインタ フェースする腫瘍細胞の機能に影響し、移行3。癌の進行の特定細胞膜拡張機能の役割はますます糸状の形成を主に、さまざまなコンテキストで例示されています。たとえば、L 型カルシウム チャネルの過剰発現は、糸状の形成を誘導し、腫瘍細胞浸潤4を促進する発見されています。同様に、ファシン、アクチン結合タンパク質の最小正常組織単位も発現がん細胞突起の形成5に。Lobopodia 形成により細胞外のマトリックスを通って効果的に移行する非悪性線維芽細胞、しかしでは線維肉腫細胞が細胞の移動を容易にする lobopodia の代わりにマトリックスメタロプロテアーゼ活性に頼ることを示されていると侵略6。我々 はその腫瘍を示されている抑制、Ras 協会ドメイン家族 1 アイソ フォーム (RASSF1A) を含む、DKK3、骨格要素を変えるとケラトサイト形成を促進し、侵襲プロパティ7、8を阻むことができます。
など、発がん、細胞膜拡張子変更、特に糸状、lobopodia、およびケラトサイト形成試験条件下での評価との関係に関与する遺伝子の効果を特徴付けるため重要です。現在最新の技術は、データ収集と解釈のますます洗練された顕微鏡検査方法、蛍光標識および複雑なコンピューターのアルゴリズムの使用を含んでいます。これらのメソッドは、新しい強力な分析ツールを提供する、その複雑さは、彼らの広範な使用と細胞生物学実験の適応性を制限します。さらに、正確な定量化とセル拡張形態変化の観察は通常測定9,10ではありません。対照的に、ここで標準的な顕微鏡技術と容易に適応できる体外メソッドを使用してセルの拡張子変更を正確に定量化する手法を紹介します。これらのメソッドはまた同時に分析の各セルの各セル拡張機能の種類を定量化し、細胞膜の拡張レパートリーで全体的な変更を確認します。また、どのようにこれらの変更は、細胞接着特性を関連付けることができます.
実験例として、SW-13 SW-DDK3、pCMV6-エントリ/DKK3 プラスミッドのベクトル安定発現されている、構成 DKK3、副腎における差別化要因を染色を指定の以前に作成された細胞株を使用します。非-南西 13 細胞と SW 13 細胞安定発現する空のベクター (pCMV6 エントリ)、SW-ネオを指定は、実験コントロールとして機能します。

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>ThermoFisher</td><td>11965-092</td><td>Supplemented with fetal bovine serum and penicllin and streptomycin</td></tr><tr><td>Deulbeco&#039;s Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>D8537</td><td>1X solution, used directly</td></tr><tr><td>3.7% formaldehyde&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F8775</td><td>Dilute stock solution</td></tr><tr><td>0.05% crystal violet&amp;nbsp;</td><td>Harleco</td><td>192-12</td><td>Dilute stock solution</td></tr><tr><td>De-ionized water</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>Microscope with 400X magnification&amp;nbsp;</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>6-well plates</td><td>Corning</td><td>353046</td><td>NA</td></tr><tr><td>Cell dissociation solution non-enzymatic 1X</td><td>Sigma-Aldrich</td><td>C5914&amp;nbsp;</td><td>1X solution, used directly</td></tr></tbody></table>

characterization of cell membrane extensions and studying their roles in cancer cell adhesion dynamics

細胞膜の拡張レパートリーは、がん細胞の接着と渡り鳥の電位を含む様々 な悪性挙動を変調します。正確に分類細胞延長の定量化し、細胞の接着能に及ぼす影響を測定する能力はどのように細胞シグナル伝達イベント衝撃がん細胞挙動と積極性を決定する重要です。ここでは、体外でデザインと、セル拡張メソッドの使用数量と共に設立の in vitroモデルの粘着能力試験と副腎皮質癌 (ACC) について述べる。具体的には、DKK3 の効果推定腫瘍抑制因子とプロの分化抑制因子 SW 13 ACC 細胞株の膜の拡張表現型をテストします。比較的単純な信頼性の高いを提供するためにこれらのアッセイを提案し、簡単に解釈できるメトリック測定様々 な実験条件下でのこれらの特性。

上記の試金を使用して、細胞の拡張の形態と細胞接着特性に及ぼす DKK3 の過剰発現は、確立された ACC セルライン SW-13、体外でテストされました。DKK3 を過剰発現細胞は安定株 Myc DDK タグ pCMV6-エントリ/DKK3 プラスミッドのベクトルし、SW DKK3 として指定で SW 13 細胞によって生成されました。同様に、空のベクター pCMV6 エントリ安定発現細胞は、トランスフェク?...

Watch this Scientific Journal Video about Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics at JoVE.com

Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics

本研究は、ユーティリティおよび定量的細胞膜拡張測定と細胞の接着能の相関性を示します。代表的な例として、Dickkopf 関連蛋白質 3 を紹介して (DKK3) が増加 lobopodia 形成を促進し、副腎皮質癌における細胞接着細胞の in vitro。

細胞膜の拡張機能と癌細胞の接着の役割を勉強のキャラクタリゼーション

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ...

characterization-cell-membrane-extensions-studying-their-roles-cancer

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

7.5K ビュー.  Yale University School of Medicine. 本研究は、ユーティリティおよび定量的細胞膜拡張測定と細胞の接着能の相関性を示します。代表的な例として、Dickkopf 関連蛋白質 3 を紹介して (DKK3) が増加 lobopodia 形成を促進し、副腎皮質癌における細胞接着細胞の in vitro。

ビデオ: Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

モデリングとイメージング、3次元の集団細胞浸潤

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

医学

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. While 75% of bladder cancers are non-invasive and unlikely to metastasize, about 25% progress to an invasive growth pattern. Up to half of the patients with invasive cancers will develop lethal metastatic relapse. Thus, understanding the mechanism of invasive progression in bladder cancer is crucial to predict patient outcomes and prevent lethal metastases. In this article, we present a three-dimensional cancer invasion model which allows incorporation of tumor cells and stromal components to mimic in vivo conditions occurring in the bladder tumor microenvironment. This model provides the opportunity to observe the invasive process in real time using time-lapse imaging, interrogate the molecular pathways involved using confocal immunofluorescent imaging and screen compounds with the potential to block invasion. While this protocol focuses on bladder cancer, it is likely that similar methods could be used to examine invasion and motility in other tumor types as well.

The processes governing bladder cancer invasion represent opportunities for biomarker and therapeutic development. Here we present a bladder cancer invasion model which incorporates 3-D culture of tumor spheroids, time-lapse imaging and confocal microscopy. This technique is useful for defining the features of the invasive process and for screening therapeutic agents.

侵略を勉強し、膀胱癌の治療を評価するための 3次元細胞培養システム

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. ...

がん研究

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

三次元コラーゲンマトリックス内の細胞移動の解析

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

生物工学

Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the homeostasis of adult tissues. Interestingly, oxidative stress can alter these processes, thus contributing to the pathophysiology of diseases such as metastatic cancer. Therefore, understanding the mechanism(s) of how cells attach and spread on the ECM during perturbations in redox status can provide insight into normal and disease states. Described below is a step-wise protocol that utilizes an immunofluorescence-based assay to specifically quantify cell adhesion and spreading of immortalized fibroblast cells on fibronectin (FN) in vitro. Briefly, anchorage-dependent cells are held in suspension and exposed to the ATM kinase inhibitor Ku55933 to induce oxidative stress. Cells are then plated on FN-coated surface and allowed to attach for predetermined periods of time. Cells that remain attached are fixed and labeled with fluorescence-based antibody markers of adhesion (e.g., paxillin) and spreading (e.g., F-actin). Data acquisition and analysis are performed using commonly available laboratory equipment, including an epifluorescence microscope and freely available Fiji software. This procedure is highly versatile and can be modified for a variety of cell lines, ECM proteins, or inhibitors in order to examine a broad range of biological questions.

This method is useful for quantifying the early dynamics of cellular adhesion and spreading of anchorage-dependent cells onto the fibronectin. Furthermore, this assay can be used to investigate the effects of altered redox homeostasis on cell spreading and/or cell adhesion-related intracellular signaling pathways.

酸化ストレス中のフィブロネクチン上皮細胞の細胞接着と広がりのダイナミクスを調べる

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the ...

生化学

Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration in more appropriate 3D environments, which better resemble the dimensionality of the physiological processes in question. Migratory cells can substantially differ in their morphology and mode of migration depending on whether they are moving on 2D or 3D substrates. Due to technical difficulties and incompatibilities with most standard protocols, structural and functional analysis of cells embedded within 3D matrices still remains uncommon. This article describes methods for preparation and imaging of 3D cancer cell cultures, either as single cells or spheroids. As an appropriate ECM substrate for cancer cell migration, we use nonpepsinized rat tail collagen I polymerized at room-temperature and fluorescently labeled to facilitate visualization using standard confocal microscopes. This work also includes a protocol for 3D immunofluorescent labeling of endogenous cell cytoskeleton. Using these protocols we hope to contribute to a better description of the molecular composition, localization, and functions of cellular structures in 3D.

This article presents a method to fluorescently label collagen that can be further used for both fix and live imaging of 3D cell cultures. We also provide an optimized protocol to visualize endogenous cytoskeletal proteins of cells cultured in 3D environments.

3Dで浸潤癌細胞の細胞骨格組織を明らかにする

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration ...

Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions

Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely F-actin-negative membrane projections in endothelial and cancer cells that can be visualized through TM4SF1 (Transmembrane-4-L-six-family-1) immunofluorescence staining. TM4SF1 clusters in 100-300 &#956;m diameter TMED (TM4SF1 enriched microdomains) containing 3 to as many as 14 individual TM4SF1 molecules. TMED are arranged intermittently along nanopodia at a regular spacing of 1 to 3 TMED per &#956;m and firmly anchor nanopodia to matrix. This enables nanopodia to extend more than 100 &#956;m from the leading front or trailing rear of polarized endothelial or tumor cells, and causes membrane residues to be left behind on matrix when the cell moves away. TMED and nanopodia have been overlooked because of their extreme fragility and sensitivity to temperature. Routine washing and fixation disrupt the structure. Nanopodia are preserved by direct fixation in paraformaldehyde (PFA) at 37 &#176;C, followed by brief exposure to 0.01% Triton X-100 before staining. Nanopodia open new vistas in cell biology: they promise to reshape our understanding of how cells sense their environment, detect and identify other cells at a distance, initiate intercellular interactions at close contact, and of the signaling mechanisms involved in movement, proliferation, and cell-cell communications. The methods that are developed for studying TM4SF1-derived nanopodia may be useful for studies of nanopodia that form in other cell types through the agency of classic tetraspanins, notably the ubiquitously expressed CD9, CD81, and CD151.

Nanopodia are thin but fragile membrane channels that extend up to 100 &#956;m from a cell's leading front or trailing rear and sense the cellular environment. Direct fixation at 37 &#176;C, gentle washing, and avoidance of organic solvents like ethanol, methanol, or acetone and of higher Triton X-100 concentrations are required to observe these cellular structures.

Nanopodia  - 細胞運動および細胞間の相互作用において役割を有する薄い、壊れやすい膜見通し

Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely ...

生物学

Quantitative Analysis of Cell Edge Dynamics during Cell Spreading

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out shape. Following the cell-substrate attachment, the cell forms a thin sheet of lamellipodia emanating from the cell body. In the lamellipodia, globular actin (G-actin) monomers polymerize into a dense filamentous actin (F-actin) meshwork that pushes against the plasma membrane, thereby providing the mechanical forces required for the cell to spread. Notably, the molecular players that control the actin polymerization in lamellipodia are essential for many other cellular processes, such as cell migration and endocytosis. 
Since spreading cells form continuous lamellipodia that span the entire cell periphery and persistently expand outward, cell spreading assays have become an efficient tool to assess the kinetics of lamellipodial protrusions. Although several technical implementations of the cell spreading assay have been developed, a detailed description of the workflow, which would include both a step-by-step protocol and computational tools for data analysis, is currently lacking. Here, we describe the experimental procedures of the cell spreading assay and present an open-source tool for quantitative and unbiased analysis of cell edge dynamics during spreading. When combined with pharmacological manipulations and/or gene-silencing techniques, this protocol is amenable to a large-scale screen of molecular players regulating lamellipodial protrusions.

In this protocol, we present the experimental procedures of a cell spreading assay that is based on live-cell microscopy. We provide an open-source computational tool for the unbiased segmentation of fluorescently labeled cells and quantitative analysis of lamellipodia dynamics during cell spreading.

細胞拡散時の細胞エッジダイナミクスの定量的解析

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.

接着フットプリントアッセイによる細胞転動における分子接着性のイメージング

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of ...

Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the construction and regeneration of tissues, and is critical in embryonic development, immunological responses, and wound healing. Dysregulation of cell motility contributes to pathological disorders, such as chronic inflammation and cancer metastasis. Cell migration, tissue invasion, axon, and dendrite outgrowth all initiate with actin polymerization-mediated cell-edge protrusions. Here, we describe a simple, efficient, time-saving method for the imaging and quantitative analysis of cell-edge protrusion dynamics during spreading. This method measures discrete features of cell-edge membrane dynamics, such as protrusions, retractions, and ruffles, and can be used to assess how manipulations of key actin regulators impact cell-edge protrusions in diverse contexts.

This protocol aims to measure the dynamic parameters (protrusions, retractions, ruffles) of protrusions at the edge of spreading cells.

生細胞顕微鏡による拡散時の細胞端突出ダイナミクスの測定

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the ...

Cell Adhesion Molecules - Types and Functions

Cell adhesion molecules (CAMs) are pivotal to multicellularity and the coordinated functioning of tissues and organ systems. They enable physical interactions between cells and provide mechanical strength to tissues. They also function as receptors for signal transmission across the plasma membrane. The CAMs are broadly classified into four families - integrins, cadherins, selectins, and immunoglobulin-like CAMs (IgCAMs).
CAM Families
The Integrin family of proteins is primarily&#160; involved in a cell&#8217;s interaction with its surrounding matrix. However, some integrins can bind CAMs on another cell&#8217;s surface and participate in direct cell-cell interactions. For example, the integrins on immune cells bind IgCAMs expressed on the vascular endothelium.
Cadherins are a superfamily of calcium-dependent glycoproteins primarily involved in establishing strong cell adhesions. They compose the adherens junctions and desmosomes in tissues, such as the epithelium.
Selectins and IgCAMs are involved in transient cell interactions and participate in directing cells towards target sites. For example, they help in the selective recruitment of lymphocytes to the secondary lymphoid organs.
CAM and Multicellularity
CAMs are found&#160; across virtually all multicellular organisms - from sponges and simple nematodes to complex invertebrates and vertebrates. The complexity of the cellular interactions, and therefore of the CAMs, increases with the complexity of the organisms. For example,&#160; while the fruit fly D.melanogaster has approximately&#160; 500 genes involved in cell adhesion, complex vertebrates like mammals have over a thousand genes that code for different types of CAMs.

細胞膜の拡張機能と癌細胞の接着の役割を勉強のキャラクタリゼーション

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