Name: protrusion force microscopy: a method to quantify forces developed by cell protrusions
Uploaded: 2018-06-16T14:00:00.000+00:00
Duration: 6 min 37 s
Description: Watch this Scientific Journal Video about Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions at JoVE.com

著者は、ビデオ撮影と編集の初期活動をこの仕事とマチュー ・ サンチェスとフランソワーズ Viala アンナ Labernadie、ギヨーム ・ Charrière、パトリック ・ Delobelle に感謝しています。この作業は、l ' アジャンス ナシオナル デ ラ凝った (ANR14-CE11-0020-02)、ラ財団注ぐラ凝った Médicale (FRM DEQ2016 0334894)、INSERM 計画がん、財団トゥールーズがんや人間科学フロンティア (RGP0035/2016) によってサポートされています。

1. ホルムバール コーティング グリッドの準備
<ol><li>純粋なアセトンで電子顕微鏡グリッドをきれいしてろ紙に乾燥させる.純粋なエタノールと顕微鏡スライドをきれい、レンズ ペーパーで拭くし、ブロワーでほこりを取り除きます。</li>
	<li>ホルムバールの下の部分でのソリューションを含むフィルム キャスト装置の漏斗にエタノール洗浄ガラス スライドを垂直方向に配置します。漏斗の上をカバーしてください。</li>
	<li>レベルがスライドの 3 分の 2 に達するまでホルムバール溶液 (二塩化エチレンは 0.5%) 100 mL アトマイザー電球が付いているポンプします。</li>
	<li>1 分のためのソリューションにスライドをしてください。</li>
	<li>安定パラレルテク ホルムバール ソリューションを排出する装置のバルブを開きます。10 15 mL/秒の流量が 30-80 nm のホルムバール フィルムを得られます。フィルムの厚さは、ホルムバール溶液の濃度によって、排水速度によって決まります。 注: バルブをゆっくり開いて、圧力を介して空気弁で排水率を制御できます。排水をより速くより厚いフィルム。実習でホルムバール流量から膜厚を予測することは困難だから我々 ホルムバール映画のいくつかのバッチを準備し、AFM による厚さを制御 (手順 2 を参照)。</li>
	<li>商工会議所からスライドを削除し、繊細な任意の液体ホルムバールを削除するろ紙上で乾燥させます。</li>
	<li>かみそりの刃を持つホルムバール フィルムから 20 mm × 50 mm の四角形をカットします。</li>
	<li>クリーンの蒸留水をビーカーを記入し、ゆっくりと映画をオフにフロートするために水に垂直方向にスライドを突入: フィルムは水面に浮かぶべきであります。</li>
	<li>光沢のあるフィルム上のグリッドを場所乾燥アセトン洗浄が立ち向かいます。</li>
	<li>グリッドのいくつかの上に丸いガラス coverslip (Ø 12 mm) を配置します。この coverslip の膜厚を測定するのに役立つでしょう (手順 2 を参照)。</li>
	<li>それに合うようにサイズ変更された白いステッカーで顕微鏡スライドをカバーします。映画全体がスライドに準拠してまでフローティング ホルムバール フィルムの境界線に垂直方向にスライドを浸しなさい。ろ紙をスライドから余分な水分を削除して、それは室温で一晩乾燥させてください。</li>
</ol>2. 膜厚の測定
<ol><li>ピンセットでスライドからそれをデタッチする、coverslip の周りホルムバールをカットします。</li>
	<li>ペンで、coverslip の下のグリッドの位置をマークします。</li>
	<li>Coverslip からデタッチするマーク グリッドの周りホルムバールをカットします。したがって、穴残りホルムバール シートで、膜厚を測定することができます。PBS の coverslip をカバーし、顕微鏡のスライド ガラスの上に置きます。</li>
	<li>ゴールデン ストライプ春の先端に表示されないかどうかを確かめて、原子間力顕微鏡ガラス ブロックのシリコン窒化カンチレバーをマウントします。 注: 感度とカンチレバーのばね定数する必要があります以前較正されて PBS で。</li>
	<li>AFM モジュール上にガラス ブロックをマウントし、顕微鏡の上にモジュールを配置します。</li>
	<li>観測室ホルムバール コーティング coverslip を置き、500 μ L の PBS でそれをカバーします。</li>
	<li>コンタクト モードと 0.5 nN の力セット ポイントで原子間力顕微鏡を用いたホルムバール シートの穴の境界線をスキャンします。</li>
	<li>高さ測定の参照としてガラス coverslip 表面を使用し、断面の高さとホルムバール厚を評価 (実行ホルムバール バッチごとの少なくとも 10 測定)。</li>
</ol>3. グリッド上のセルのシード
<ol><li>無菌フードの下で上、両面粘着テープのストリップ (12 mm × 3 mm) を配置します。</li>
	<li>ホルムバール コーティング グリッドをピンセットで切り取って逆さまに接着剤ストリップ (グリッドの縁のみがテープに接続する必要があります)。ホルムバール フィルムは、coverslip に直面する必要があります。</li>
	<li>まあ文化にこのデバイスを置きます。</li>
	<li>ひと単球16から区別される 10 の4大食細胞を含む FCS なし RPMI の 10 μ L の液滴を配置します。 注: を確認ホルムバール コーティングの損傷を防ぐためにピペット チップでグリッドに触れないようにします。</li>
	<li>(37 ° C、5% CO2) 細胞付着させる 30 分間インキュベートします。</li>
	<li>2 ml の 10% の FCS を含む RPMI の井戸を埋めます。</li>
	<li>細胞の AFM 観察する前に付着する CO2を 5% と 37 ° C で 2 時間デバイスを孵化させなさい。</li>
</ol>4 Podosome 誘起変形の地形計測
<ol><li>ガラス底ペトリ皿に倍増した両面粘着テープの 2 つのストリップを固執します。 2 つのストリップ間の距離はグリッドの直径よりも小さくなる必要があります。</li>
	<li>慎重に粘着テープからマクロファージとメッキ グリッドをデタッチします。</li>
	<li>裏返して、グリッド セルをガラスに直面するために、2 つの接着剤のストリップ間グリッドを固執します。</li>
	<li>2 つのグリッドは、新しい倍増両面接着剤のストリップ修正: AFM チップにアクセス可能なグリッドの中央の領域を残して 2 つの接着剤ストリップしたがってサンドイッチされ、グリッド。 グリッドが固定もあり、ねじれていない; 注: を確認します。そうでなければ AFM カンチレバーはホルムバール表面に近づくことができるかもしれない。</li>
	<li>10 mM HEPES (pH 7.4) を添加した予熱の 37 ° C 培養液 (10 %fcs と RPMI) 2 mL をシャーレをご記入ください。</li>
	<li>37 ° C 皿ヒーターに培養皿を置きます。</li>
	<li>原子間力顕微鏡ステージ上皿ヒーターをインストールします。</li>
	<li>37 ° C で安定化システムします。</li>
	<li>0.5 nN の力で接触モード エキスタチンを検索する最初のグローバル イメージを作成し、ホルムバール地形 3 Hz で約 20 nm 大ピクセルします。 メモ: は、グリッドの端近くをスキャンしないでください。</li>
</ol>

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利害の対立が宣言されていません。

材料特性
ホルムバール、私たちのケースで、変形可能な膜材料の選択は、いくつかの要件を満たす必要があります。材料必要がある可視光線を通す、明視野と蛍光顕微鏡で観察できるように限られた自己蛍光を展示します。薄膜の粗さは、10 をかなり下回っている必要があります nm 細胞接着に対する任意の地形の影響を避けるために、AFM イメージングによ?...

動物細胞は、物理的に行列とその環境1を構成する他の細胞と対話します。これは、移行、体を内面化、外部情報を取得を区別するために必要です。このようなプロセスでセルは機械力を生成する必要がある、その生物学的挙動、例えば増殖の監督と近年多くの研究が示している、力を生成し、その環境をプローブする細胞の能力の影響か分化2,3。ターンでは、細胞の力の測定は力生成の規制を勉強し、細胞行動と組織の運命4,5にその意義を理解する主要な援助です。
近年では、その環境では6セルを出せる力を測定する多数の技術の開発を目撃しました。これらの大半は牽引力細胞出す携帯電話プローブまたは変形可能な基板をはめると、明らかに尽力されています。しかし、細胞外の環境に突起に関連する機械的な力測定技術の不足に苦しむ、日でなく特徴にしています。
この制限を克服するためには基板に直交に加わる力を測定する手法を提案します。それは細胞細胞基質の変形を測定し、軍の関与を推測することが可能となって、直交方向に変形することができます薄い弾性シートをめっきで構成されます。基板形状、原子間力顕微鏡によるナノスケールの分解能で測定し、変形から力の評価は、前方の細胞構造7,8,の幾何学の知識に依存しています9。
ここでは、セットアップとエキスタチン、三次元環境10、11、間葉系移行の大食細胞によって形成される前方接着構造によって生成された力を測定への応用について述べる 12,13,14,15,16,17。この手法が力の発生とその突起を含む多くの細胞プロセス制御の理解を進めると考えています。

<table><tbody><tr><td>200 mesh nickel grids</td><td>Electron Microscopy Sciences</td><td>G200-Ni</td><td></td></tr><tr><td>Filter paper</td><td>Sigma-Aldrich</td><td>1001-055</td><td></td></tr><tr><td>Microscope slides</td><td>Fisher Scientific</td><td>10235612</td><td></td></tr><tr><td>White stickers 26 x 70 mm</td><td>Avery</td><td>DP033-100</td><td></td></tr><tr><td>Film casting device with valve in its outlet</td><td>Electron Microscopy Sciences</td><td>71305-01</td><td></td></tr><tr><td>Razorblades</td><td>Electron Microscopy Sciences</td><td>72000</td><td></td></tr><tr><td>Ethanol</td><td>VWR</td><td>1.08543.0250</td><td></td></tr><tr><td>Acetone</td><td>VWR</td><td>20066.321</td><td></td></tr><tr><td>Formvar 0.5% solution in ethylene dichloride</td><td>Electron Microscopy Sciences</td><td>15820</td><td></td></tr><tr><td>12 mm coverslips</td><td>VWR</td><td>631-0666</td><td></td></tr><tr><td>Inverted microscope</td><td>Carl Zeiss</td><td>Axiovert 200</td><td></td></tr><tr><td>Atomic Force Microscope</td><td>JPK Instruments</td><td>NanoWizard III</td><td></td></tr><tr><td>Temperature-controlled sample holder&amp;nbsp;</td><td>JPK Instruments</td><td>BioCell</td><td></td></tr><tr><td>Silicon nitride cantilever with a nominal spring constant of 0.01 N/m</td><td>Veeco Instruments</td><td>MLCT-AUHW</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>14190-094</td><td></td></tr><tr><td>Double-sided adhesive tape</td><td>APLI AGIPA</td><td>118100</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>31870-025</td><td></td></tr><tr><td>FCS</td><td>Sigma-Aldrich</td><td>F7524</td><td></td></tr><tr><td>HEPES&amp;nbsp;</td><td>Sigma-Aldrich</td><td>H0887</td><td></td></tr><tr><td>35 mm glass-bottom Petri dishes</td><td>WPI</td><td>FD35-100</td><td></td></tr></tbody></table>

protrusion force microscopy: a method to quantify forces developed by cell protrusions

多数の生物学的文脈で動物細胞を機械的な力を開発することによって、環境物理的にやり取りする必要があります。これらのうち、牽引力はよ特徴付けられた、されているが、基板に直交細胞突起力測定技術の不足があります。付着性のセルの基板上で突出力を測定する実験装置を考案しました。準拠ホルムバール シート メッキ細胞変形この基板、ナノメートル スケールでの原子間力顕微鏡 (AFM) によって生成された地形にマップされます。フォース値は、前方の細胞構造の幾何学に基づく変形分布の解析から、抽出されます。したがって、時間の経過とともに細胞の個々 の突出の単位によって力を測定できます。この手法は、力の発生とその突起を含む多くの細胞プロセス制御の研究を有効になります。ここでは、その人間の大食細胞によって形成されるエキスタチンによって生成された前方力を測定への応用について述べる.

上記のプロトコルでは、マクロファージ エキスタチン ホルムバール基板上で前突荷重を定量化する実験のセットアップを準備する方法について説明します。これは原子間力顕微鏡を使って実現されますが、図 1に示します。
エキスタチン JPK データ処理ソフトウェアを使用しての下の膨らみの地形?...

Watch this Scientific Journal Video about Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions at JoVE.com

Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

ここでは、エキスタチン適用対応フィルム地形画像の自動解析するフィルムの調製から突出力を評価するために使用する実験手法を詳しく説明します。

突起力顕微鏡: 細胞突起が開発した力を定量化する方法

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction ...

protrusion-force-microscopy-method-to-quantify-forces-developed-cell

Research

JoVE Journal

Biology

5.5K ビュー.  IPBS, Université de Toulouse, CNRS, UPS. ここでは、エキスタチン適用対応フィルム地形画像の自動解析するフィルムの調製から突出力を評価するために使用する実験手法を詳しく説明します。

ビデオ: Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton

The mechanical properties of the nucleus determine its response to mechanical forces generated in cells. Because the nucleus is molecularly continuous with the cytoskeleton, methods are needed to probe its mechanical behavior in adherent cells. Here, we discuss the direct force probe (DFP) as a tool to apply force directly to the nucleus in a living adherent cell. We attach a narrow micropipette to the nuclear surface with suction. The micropipette is translated away from the nucleus, which causes the nucleus to deform and translate. When the restoring force is equal to the suction force, the nucleus detaches and elastically relaxes. Because the suction pressure is precisely known, the force on the nuclear surface is known. This method has revealed that nano-scale forces are sufficient to deform and translate the nucleus in adherent cells, and identified cytoskeletal elements that enable the nucleus to resist forces. The DFP can be used to dissect the contributions of cellular and nuclear components to nuclear mechanical properties in living cells.

In this protocol, we describe a micropipette method to directly apply a controlled force to the nucleus in a living cell. This assay allows interrogation of nuclear mechanical properties in the living, adherent cell.

核と細胞骨格との間の機械的統合を測定するための直接力プローブ

The mechanical properties of the nucleus determine its response to mechanical forces generated in cells. Because the nucleus is molecularly continuous ...

生化学

Perturbing Endothelial Biomechanics via Connexin 43 Structural Disruption

Endothelial cells have been established to generate intercellular stresses and tractions, but the role gap junctions play in endothelial intercellular stress and traction generation is currently unknown. Therefore, we present here a mechanics-based protocol to probe the influence of gap junction connexin 43 (Cx43) has on endothelial biomechanics by exposing confluent endothelial monolayers to a known Cx43 inhibitor 2,5-dihydroxychalcone (chalcone) and measuring the impact this inhibitor has on tractions and intercellular stresses. We present representative results, which show a decrease in both tractions and intercellular stresses under a high chalcone dosage (2 &#181;g/mL) when compared to control. This protocol can be applied to not just Cx43, but also other gap junctions as well, assuming the appropriate inhibitor is used. We believe this protocol will be useful in the fields of cardiovascular and mechanobiology research.

Here, we present a mechanics-based protocol to disrupt the gap junction connexin 43 and measure the subsequent impact this has on endothelial biomechanics via observation of tractions and intercellular stresses.

コネクシン43構造破壊による内皮バイオメカニクスの摂動

Endothelial cells have been established to generate intercellular stresses and tractions, but the role gap junctions play in endothelial intercellular ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

蛍光生体膜フォースプローブ：単一細胞上の受容体 - リガンド速度論の同時定量との結合によって誘導される細胞内シグナル伝達

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

生物工学

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to understand morphogenetic processes is to directly measure cellular forces and mechanical properties in vivo during embryogenesis. Here, we present a setup of optical tweezers coupled to a light sheet microscope, which allows to directly apply forces on cell-cell contacts of the early Drosophila embryo, while imaging at a speed of several frames per second. This technique has the advantage that it does not require the injection of beads into the embryo, usually used as intermediate probes on which optical forces are exerted. We detail step by step the implementation of the setup, and propose tools to extract mechanical information from the experiments. By monitoring the displacements of cell-cell contacts in real time, one can perform tension measurements and investigate cell contacts&#39; rheology.

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

We present a setup of optical tweezers coupled to a light sheet microscope, and its implementation to probe cell mechanics without beads in the Drosophila embryo.

ショウジョウバエの胚のビーズ無料光ピンセット プローブ細胞力学

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to ...

発生生物学

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue morphogenesis goes in hand with molecular and structural changes at the single cell level that result in variations of subcellular mechanical properties. As a consequence, even within the same cell, different organelles and compartments resist differently to mechanical stresses; and mechanotransduction pathways can actively regulate their mechanical properties. The ability of a cell to adapt to the microenvironment of the tissue niche thus is in part due to the ability to sense and respond to mechanical stresses. We recently proposed a new mechanosensation paradigm in which nuclear deformation and positioning enables a cell to gauge the physical 3D environment and endows the cell with a sense of proprioception to decode changes in cell shape. In this article, we describe a new method to measure the forces and material properties that shape the cell nucleus inside living cells, exemplified on adherent cells and mechanically confined cells. The measurements can be performed non-invasively with optical traps inside cells, and the forces are directly accessible through calibration-free detection of light momentum. This allows measuring the mechanics of the nucleus independently from cell surface deformations and allowing dissection of exteroceptive and interoceptive mechanotransduction pathways. Importantly, the trapping experiment can be combined with optical microscopy to investigate the cellular response and subcellular dynamics using fluorescence imaging of the cytoskeleton, calcium ions, or nuclear morphology. The presented method is straightforward to apply, compatible with commercial solutions for force measurements, and can easily be extended to investigate the mechanics of other subcellular compartments, e.g., mitochondria, stress-fibers, and endosomes.

Here, we present a protocol to investigate the intracellular mechanical properties of isolated embryonic zebrafish cells in three-dimensional confinement with direct force measurement by an optical trap.

光ピンセットを用いた閉じ込めにおける細胞下力学の直接力測定

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue ...

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial cells regulate permeability of the endothelium, and many studies have indicated the important contribution of the actin cytoskeleton to determining junctional integrity1-5. A cortical actin belt is thought to be important for the maintenance of stable junctions1, 2, 4, 5. In contrast, actin stress fibers are thought to generate centripetal tension within endothelial cells that weakens junctions2-5. Much of this theory has been based on studies in which endothelial cells are treated with inflammatory mediators known to increase endothelial permeability, and then fixing the cells and labeling F-actin for microscopic observation. However, these studies provide a very limited understanding of the role of the actin cytoskeleton because images of fixed cells provide only snapshots in time with no information about the dynamics of actin structures5. 
Live-cell imaging allows incorporation of the dynamic nature of the actin cytoskeleton into the studies of the mechanisms determining endothelial barrier integrity. A major advantage of this method is that the impact of various inflammatory stimuli on actin structures in endothelial cells can be assessed in the same set of living cells before and after treatment, removing potential bias that may occur when observing fixed specimens. Human umbilical vein endothelial cells (HUVEC) are transfected with a GFP-&beta;-actin plasmid and grown to confluence on glass coverslips. Time-lapse images of GFP-actin in confluent HUVEC are captured before and after the addition of inflammatory mediators that elicit time-dependent changes in endothelial barrier integrity. These studies enable visual observation of the fluid sequence of changes in the actin cytoskeleton that contribute to endothelial barrier disruption and restoration. 
Our results consistently show local, actin-rich lamellipodia formation and turnover in endothelial cells. The formation and movement of actin stress fibers can also be observed. An analysis of the frequency of formation and turnover of the local lamellipodia, before and after treatment with inflammatory stimuli can be documented by kymograph analyses. These studies provide important information on the dynamic nature of the actin cytoskeleton in endothelial cells that can used to discover previously unidentified molecular mechanisms important for the maintenance of endothelial barrier integrity.

Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.

GFP -アクチンを発現するライブ内皮細胞におけるアクチン細胞骨格の研究

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

生物学

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their microenvironment. While many approaches exist for measuring cell-induced material strain, here we provide a methodology for monitoring strain with sub-micron resolution in a reference-free manner. Using a two-photon activated photolithographic patterning process, we demonstrate how to generate mechanically and bio-actively tunable synthetic substrates containing embedded arrays of fluorescent fiducial markers to easily measure three-dimensional (3D) material deformation profiles in response to surface tractions. Using these substrates, cell tension profiles can be mapped using a single 3D image stack of a cell of interest. Our goal with this methodology is to make traction force microscopy a more accessible and easier to implement tool for researchers studying cellular mechanotransduction processes, especially newcomers to the field.

This protocol provides instructions for implementing multiphoton lithography to fabricate three-dimensional arrays of fluorescent fiducial markers embedded in poly(ethylene glycol)-based hydrogels for use as reference-free, traction force microscopy platforms. Using these instructions, measurement of 3D material strain and calculation of cellular tractions is simplified to promote high-throughput traction force measurements.

リファレンスフリートラクションフォース顕微鏡プラットフォームの製造と実装

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their ...

Traction Force Microscopy to Study B Lymphocyte Activation

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements from an experimentally observed displacement field produced by a cell pulling on an elastic substrate. Here, we adapted TFM to investigate the spatial and temporal structure of the force field exerted by B cells when activated by antigen engagement of the B cell receptor. Gel rigidity, bead density, and protein functionalization must be optimized for the study of relatively small cells (~ 6 &#181;m) that interact with, and respond specifically to ligands for cell surface receptors.

Here we present a protocol used to perform traction force microscopy experiments on B cells. We describe the preparation of soft polyacrylamide gels and their functionalization, as well as data acquisition at the microscope and a summary of data analysis.

Bリンパ球活性化を研究するための牽引力顕微鏡

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements ...

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 ...

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes depend on the advances of growth cones that located at the tips of neurites. Axonal growth cones generate driving forces by sensing their local microenvironment and modulating cytoskeletal dynamics and actin-adhesion coupling (clutch coupling). Decades of research have led to the identification of guidance molecules, their receptors, and downstream signaling cascades for regulating neuronal migration and axonal guidance; however, the molecular machineries required for generating forces to drive growth cone advance and navigation are just beginning to be elucidated. At the leading edge of neuronal growth cones, actin filaments undergo retrograde flow, which is powered by actin polymerization and actomyosin contraction. A clutch coupling between F-actin retrograde flow and adhesive substrate generates traction forces for growth cone advance. The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging. Importantly, when combined with an F-actin marker Lifeact, this technique can quantify 1) the F-actin polymerization rate and 2) the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate. Both are critical variables for generating forces for growth cone advance and navigation. In addition, the present study describes a detailed protocol of traction force microscopy, which can quantify 3) traction force generated by growth cones. Thus, by coupling the analyses of&#160;single speckle imaging and traction force microscopy, investigators can monitor the molecular mechanics underlying growth cone advance and navigation.

To advance, growth cones must exert traction forces against the external environment. The generation of traction forces is dependent on actin dynamics and clutch coupling. The present study describes methods for analyzing actin dynamics, clutch coupling and traction forces for growth cone advance.

アクチンダイナミクス、クラッチカップリング、成長コーンアドバンスの牽引力の解析

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes ...

突起力顕微鏡: 細胞突起が開発した力を定量化する方法

要約

さらに動画を探す

この動画の章

核と細胞骨格との間の機械的統合を測定するための直接力プローブ

コネクシン43構造破壊による内皮バイオメカニクスの摂動

蛍光生体膜フォースプローブ：単一細胞上の受容体 - リガンド速度論の同時定量との結合によって誘導される細胞内シグナル伝達

ショウジョウバエの胚のビーズ無料光ピンセットプローブ細胞力学

光ピンセットを用いた閉じ込めにおける細胞下力学の直接力測定

GFP -アクチンを発現するライブ内皮細胞におけるアクチン細胞骨格の研究

リファレンスフリートラクションフォース顕微鏡プラットフォームの製造と実装

Bリンパ球活性化を研究するための牽引力顕微鏡

マイクロパターントラクション顕微鏡のパターン生成

アクチンダイナミクス、クラッチカップリング、成長コーンアドバンスの牽引力の解析

突起力顕微鏡: 細胞突起が開発した力を定量化する方法

要約

さらに動画を探す

この動画の章

核と細胞骨格との間の機械的統合を測定するための直接力プローブ

コネクシン43構造破壊による内皮バイオメカニクスの摂動

蛍光生体膜フォースプローブ：単一細胞上の受容体 - リガンド速度論の同時定量との結合によって誘導される細胞内シグナル伝達

ショウジョウバエの胚のビーズ無料光ピンセット プローブ細胞力学

光ピンセットを用いた閉じ込めにおける細胞下力学の直接力測定

GFP -アクチンを発現するライブ内皮細胞におけるアクチン細胞骨格の研究

リファレンスフリートラクションフォース顕微鏡プラットフォームの製造と実装

Bリンパ球活性化を研究するための牽引力顕微鏡

マイクロパターントラクション顕微鏡のパターン生成

アクチンダイナミクス、クラッチカップリング、成長コーンアドバンスの牽引力の解析

ショウジョウバエの胚のビーズ無料光ピンセットプローブ細胞力学