この作品は、NIH R01 EB014869 によって支えられました。

1. イメージング用細胞を準備
注: 任意の付着性のセルのタイプの直接力プローブ (DFP) を使用できます。ここでは、NIH 3T3 マウス線維芽細胞は、このプロトコルのモデル細胞ラインとして使用されます。
<ol><li>文化 NIH 3T3 線維芽細胞でダルベッコ変更されたワシの媒体 (DMEM) 1% と 10% ドナー牛血清ペニシリン-ストレプトマイシン 35 ミリメートルのガラスの下に目的の confluency まで皿します。37 ° C と 5% の CO2の細胞を維持します。<ol>
<li>必ずイメージングのための NIH 3T3 細胞を播種する前に 5 μ g/ml のフィブロネクチン (または類似の ECM 蛋白質) のすべての 35 mm ガラス底培養皿をコートしてください。 注: セルする必要が完全に普及して実験用皿に付着。DFP メソッドが動作するための密度の面で制約があります。</li>
	</ol>
</li>
	<li>実験直前にセルを洗浄して完全な培地で一回の洗浄が続く PBS で 2 回。</li>
	<li>ガラス底皿に完全な成長培地 3 mL を追加します。</li>
</ol>2. 顕微鏡と画像の取得
注: 倒立蛍光顕微鏡 (または同等) 側の腕は、メーカーの推奨事項に従ってインストール マイクロマニピュレーターと。顕微鏡は、37 ° c、温度および 5% の CO2レベルを維持するために環境チャンバーを装備する必要があります。マイクロマニピュレーターとマイクロインジェクター顕微鏡に接続も必要です。油浸漬 40 x/1.3 NA または 60 x/1.49 NA (または同等の目標) は実験のためにお勧めします。顕微鏡は、除振台にマウントする必要があります。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58038/58038fig1.jpg"> 図 1.核変形と顕微鏡の焦点 A. 最大の核変形と核変形の緩和。最大の核の変形を計算する前に核の形態の背面のエッジが変形核の翻訳を修正する最初一致しました。ピペット先端部が抜け出る瞬間に核の形は引く前に初期の原子核の形にかぶせてありました。2 つの図形間の領域の違いを測定した Δ1。最大の核の変形は ΔA1 元の核の面積で割った値として定義されました。同様に、2 番目のパラメーター、Δ2、元の原子核の形でマイクロ ピペット剥離後最終的な定常状態の原子核の形を重ねることによって定義されるかもしれません。B. 焦点平面にセル、平面 B マイクロ ピペット チップを見つけるまで焦点の平面を移動します。画像の中に、マイクロ ピペットは右 (オレンジ色の矢印の方向) に翻訳しました。この図は、Neelamらから変更されています。15.<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58038/58038fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<ol><li>製造元のプロトコルごとマイクロインジェクターを入れます。</li>
	<li>対物レンズの上に液浸オイルの一滴を適用、浸漬オイル ドロッパーを使用します。</li>
	<li>料理を皿ホルダーにしっかりとクランプし、ステージ上に皿ホルダーをロードします。 注: セルは、実験を通じて CO2を 37 ° C、5% に保たれなければなりません。</li>
	<li>焦点 (平面 A図 1 b) にセルを持って目的の高さを調整します。</li>
	<li>目的のセルを検索する顕微鏡ステージに移動します。</li>
	<li>ピペット ホルダーをトップの位置に移動するマイクロマニピュレーターにジョイスティックを回転させます。0.5 μ m 径のヒント マイクロ ピペット ピペット ホルダーに読み込みます。<ol>
<li>ピペットへの細胞接着を避けるためには、あらかじめ室温で 0.3 mg/ml の 1 h の PLL g ペグ ソリューション マイクロ ピペット チップを扱います。任意の吸引圧力なし核にマイクロ ピペットを触れるし、核からピペットを翻訳密着性をテストします。密着性の不在は、核変形と翻訳の完全な欠如から識別できます。 注: パッケージを開く製造の提案に従ってください。</li>
	</ol>
</li>
	<li>細かい制御を調整することによって面 A と面 B のセルの上部の客観的焦点面を上げる (図 1 b、ステップ 2.4 を参照)。</li>
	<li>粗制御に変えればエンブリオバイオプシーに対応を設定します。ゆっくりピペットが焦点に完全にくるまをマイクロ ピペットのシルエット見て平面 B にマイクロ ピペットをもたらします。</li>
	<li>マイクロ ピペットのチップは、フォーカス、マニピュレーターを微調整に設定します。</li>
	<li>(平面 A、図 1 b) 細胞の赤道面に目標を下げるし、(図 1 b、破線ピペット) 平面の上約 15 μ m のマイクロ ピペットを下げます。</li>
	<li>圧が得られる; マイクロインジェクター on 補正圧力 (Pc)圧力を安定させるために数秒を待ちます。 注: 最適な圧セット ポイントはセルの種類および実験の特定の目的に依存します。ほとんどの場合、300 hPa は良い出発点になります。</li>
	<li>マイクロマニピュレーター パネルのクリーン設定を使用して、マイクロ ピペット チップから出てくる空気の泡を確認するためにチェックして、ピペットが詰まっていないことを確認します。</li>
	<li>先端は核の表面を軽く触れるまでマイクロ ピペットを徐々 に下げることによって、先端をセルに挿入します。 注: マイクロ ピペットを下げるとマイクロ ピペット チップのシルエットが焦点になるよう明確ななります。マイクロ ピペットに触れる核、前に上げる目的のフォーカス、核 (同じ x と y 座標、z 面の高い) とマイクロ ピペット チップを合わせます。フォーカスを核 (平面 A、図 1 b) の赤道面に戻り、マイクロ ピペット チップを徐々 に下げます。</li>
	<li>それにより大気中にマイクロ ピペット チューブの端を開くマイクロインジェクション システムから圧力供給管を切断してマイクロ ピペット チップと核膜のシールを作成します。この手順は、核の表面にPcに等しい負圧を作成します。</li>
	<li>顕微鏡画像コレクション ソフトウェアが画像を取得します。Avi ファイルの取得 (ビデオの) または nd 取得 (イメージ) をコレクションの画像ソフトで設定します。 注: 任意のイメージング ソフトウェアを取得、リアルタイムのビデオ画像や短い時間間隔でタイムラプス映像買収を設定します。</li>
	<li>対応するイメージング チャンネルに切り替え (すなわちGFP、RFP、等)、イメージングを開始。</li>
	<li>(右側、図 1 b) にセルの体からマイクロ ピペット チップを翻訳核は、マイクロ ピペットから外れるまで引っ張ります。 注: は、(ビューのフィールドの右側) に肯定的な x 方向のヒントを引き出します。引き率のプログラムやコンピューターによって制御されるジョイスティックを手動で移動することがでくことができます。引っ張って相関が見つかっていない率と核変形15を強制的に主に弾性応答を示唆しています。</li>
</ol>3. データ分析
<ol><li>利用できる基本的な画像処理ソフトウェアで画像解析を実行します。長さひずみ (Ɛ) または領域のひずみ (図 1 a) のいずれかによって核変形の程度を定量化することができます。式 1 を使用して長さひずみを定量化、L と L0はそれぞれ最大変形と初期位置で核の長さを表します。 <img alt="Equation 1" src="/files/ftp_upload/58038/58038eq1.jpg">(関係式 1)</li>
</ol>

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著者が明らかに何もありません。

彼らはいずれかの隔離された核 (核は細胞骨格から分離) を必要とするためピペット吸引16など、最新の方法のための挑戦は、細胞骨格に核の機械的統合を測定または核浮遊細胞 (細胞外の力、牽引力などは欠席)。力は、軸ひずみを膜17,18; に付着性のセルに適用することで、核に適用されています。ただし、この手法は、核の力が?...

癌などの疾患は、核の形状と構造1,2, '' 核3,4の軟化に伴う一般的に変化を含みます。原子力機械変形抵抗は、隔離された核5に力を適用することで一般的に特徴づけられています。
細胞核は細胞骨格に Nucleoskeleton リンカーと細胞骨格 (バイオス) 複雑な6,7,8,9分子接続されます。その結果、核は、細胞骨格と、細胞-基質間の癒着、細胞外マトリックスを機械的に統合されています。機械的に付着性細胞の中の核を探るこの機械的統合への洞察力を提供できます。マイクロ ピペット吸引10、11、および原子間力顕微鏡12,13,14生きた細胞中の核を操作するメソッドが含まれます。最近生活付着性のセル15に核に直接機械的な力を適用する直接力プローブ (DFP) について述べる。
ここでは、我々 は付着性細胞の核に直接知られているナノ機械的な力を適用する顕微鏡施設で利用可能なインジェクション システムを使用する手順を概説します。Femtotip (0.5 μ m 径マイクロ ピペット チップ) がマウントされ、チューブでのインジェクション システムに接続されています。まで核の表面に隣接して、培養皿の表面に対して 45 ° の角度で配置されている、先端が低下します。チューブは切断し、核の表面に負の吸引圧を作成し、核の表面に対してマイクロ ピペット先端をシール大気開放します。マイクロ ピペット チップの翻訳を通じて核は変形し、最終的に (力の大きさ) によって、マイクロ ピペットを切り離します。この剥離は、核および細胞、によって出される、復元 (耐熱) 力、ピペットによって適用される吸引力の等しい場合に発生します。核の変位を測定することによって分析を行うことが長さひずみ (式 1)、または領域のひずみ (図 1 a)。

<table><tbody><tr><td>FluoroDish</td><td>WPI</td><td>FD35</td><td></td></tr><tr><td>SYTO 59</td><td>ThermoFisher Scientific</td><td>S11341</td><td></td></tr><tr><td>Femtotips&amp;nbsp;</td><td>Eppendorf</td><td>930000043</td><td></td></tr><tr><td>InjectMan NI2</td><td>Eppendorf</td><td>NA</td><td>discontinued, current equivalent model: InjectMan 4</td></tr><tr><td>FemtoJet</td><td>Eppendorf</td><td>NA</td><td>Current model FemtoJet 4i</td></tr><tr><td>Plan Fluor oil immersion 40x</td><td>Nikon</td><td>NA</td><td></td></tr><tr><td>Apo TIRF oil immersion 60x</td><td>Nikon</td><td>NA</td><td></td></tr><tr><td>Donor Bovine Serum (DBS)</td><td>ThermoFisher Scientific</td><td>16030074</td><td>NIH 3T3 serum</td></tr><tr><td>Dulbecco&#039;s Modification of Eagle&#039;s (DMEM)</td><td>Mediatech cellgro</td><td>MT10013CVRF</td><td>NIH 3T3 medium</td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>Mediatech</td><td>MT30004CIRF</td><td>NIH 3T3 medium supplement</td></tr><tr><td>Immersion Oil Type LDF Non-Fluorescing</td><td>Nikon</td><td>77007</td><td>Immersion oil for objective lens&amp;nbsp;</td></tr></tbody></table>

a direct force probe for measuring mechanical integration between the nucleus and the cytoskeleton

核の機械的性質は、細胞で生成された機械的な力への応答を決定します。核が細胞骨格の分子に連続しているため付着細胞での力学的挙動を調べるための方法が必要です。ここでは、付着性細胞の核に直接力を適用するためのツールとして直接力プローブ (DFP) について述べる。我々 は、吸込みを伴う核の表面に細いピペットを添付します。マイクロ ピペットは、翻訳を変形させ、核を引き起こす核から変換されます。復元力が吸引力に等しい、核はデタッチし、弾性を緩和します。吸引圧が正確に知られているために、核の表面に働く力が知られています。このメソッドは、ナノの力が変形し、付着細胞で核を翻訳するのに十分なされ、力に抵抗する核を可能にする骨格の要素を明らかにしました。DFP を使用して、細胞内の核の力学特性に細胞や原子力部品の貢献を分析できます。

図 2 aは、NIH 3T3 マウス線維芽細胞核の強制を示しています。マイクロ ピペット チップの右側に翻訳核変形、最終的にマイクロ ピペット チップから切り離します。吸引力 (図 2 b) とともに増加する核の長さのひずみが見られます。核 (端を引っ張ってマイクロ ピペット) の前面の端を形成する核の突起とトレーリング エッ?...

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

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

このプロトコルでは生きている細胞の核に直接制御力を適用するマイクロ ピペット法について述べる。このアッセイでは、生活、付着性の細胞で核の力学特性の尋問をことができます。

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

a-direct-force-probe-for-measuring-mechanical-integration-between

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

15.1K ビュー.  University of Florida. このプロトコルでは生きている細胞の核に直接制御力を適用するマイクロ ピペット法について述べる。このアッセイでは、生活、付着性の細胞で核の力学特性の尋問をことができます。

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

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

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

原子力LINCコンプレックス全体に機械的な力を測定するために、蛍光共鳴エネルギー移動（FRET）-forceのバイオセンサーを使用するためのプロトコル

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

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 ...

発生生物学

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

蛍光技術の組み合わせを用いた生細胞内力感受性タンパク質ダイナミクスの測定

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

相間細胞核の力学特性を探査する生物物理学的なアッセイ：基板の歪みのアプリケーションおよびマイクロニードル操作

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

生物学

DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical for regulating many processes ranging from development to immunology. Therefore, developing the tools to study these forces at the molecular scale is critical. Our group developed a suite of molecular tension sensors to quantify and visualize the forces generated by cells and transmitted to specific ligands. The most sensitive class of molecular tension sensors are comprised of nucleic acid stem-loop hairpins. These sensors use fluorophore-quencher pairs to report on the mechanical extension and unfolding of DNA hairpins under force. One challenge with DNA hairpin tension sensors is that they are reversible with rapid hairpin refolding upon termination of the tension and thus transient forces are difficult to record. In this article, we describe the protocols for preparing DNA tension sensors that can be "locked" and prevented from refolding to enable "storing" of mechanical information. This allows for the recording of highly transient piconewton forces, which can be subsequently "erased" by the addition of complementary nucleic acids that remove the lock. This ability to toggle between real-time tension mapping and mechanical information storing reveals weak, short-lived, and less abundant forces, that are commonly employed by T cells as part of their immune functions.

This paper describes a detailed protocol for using DNA-based tension probes to image the receptor forces applied by immune cells. This approach can map receptor forces &gt;4.7pN in real-time and can integrate forces over time.

免疫細胞による一過性ピコニュートン受容体力をマッピングするためのDNA張力プローブ

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical ...

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

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction forces have been well-characterized, but there is a lack of techniques allowing the measurement of the protrusion forces exerted by cells orthogonally to their substrate. We designed an experimental setup to measure the protrusion forces exerted by adherent cells on their substrate. Cells plated on a compliant Formvar sheet deform this substrate and the resulting topography is mapped by atomic force microscopy (AFM) at the nanometer scale. Force values are then extracted from an analysis of the deformation profile based on the geometry of the protrusive cellular structures. Hence, the forces exerted by the individual protruding units of a living cell can be measured over time. This technique will enable the study of force generation and its regulation in the many cellular processes involving protrusion. Here, we describe its application to measure the protrusive forces generated by podosomes formed by human macrophages.

Here, we detail the experimental techniques used to evaluate the protrusion forces that podosomes apply on a compliant film, from the preparation of the film to the automated analysis of topographical images.

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 ...

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

骨格筋生検からミオフィブリルを分離し、ナノニュートン分解力トランスデューサーによる収縮機能を決定する

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of ...

Directly Measuring Forces Within Reconstituted Active Microtubule Bundles

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as specialized arrays during mitosis to regulate chromosome segregation. Proteins that interact with microtubules include motors such as kinesins and dynein, which can generate active forces and directional motion, as well as non-motor proteins that crosslink filaments into higher-order networks or regulate filament dynamics. To date, biophysical studies of microtubule-associated proteins have overwhelmingly focused on the role of single motor proteins needed for vesicle transport, and significant progress has been made in elucidating the force-generating properties and mechanochemical regulation of kinesins and dyneins. However, for processes in which microtubules act both as cargo and track, such as during filament sliding within the mitotic spindle, much less is understood about the biophysical regulation of ensembles of the crosslinking proteins involved. Here, we detail our methodology for directly probing force generation and response within crosslinked microtubule minimal networks reconstituted from purified microtubules and mitotic proteins. Microtubule pairs are crosslinked by proteins of interest, one microtubule is immobilized to a microscope coverslip, and the second microtubule is manipulated by an optical trap. Simultaneous total internal reflection fluorescence microscopy allows for multichannel visualization of all the components of this microtubule network as the filaments slide apart to generate force. We also demonstrate how these techniques can be used to probe pushing forces exerted by kinesin-5 ensembles and how viscous braking forces arise between sliding microtubule pairs crosslinked by the mitotic MAP PRC1. These assays provide insights into the mechanisms of spindle assembly and function and can be more broadly adapted to study dense microtubule network mechanics in diverse contexts, such as the axon and dendrites of neurons and polar epithelial cells.

Here, we present a protocol for reconstituting microtubule bundles in vitro and directly quantifying the forces exerted within them using simultaneous optical trapping and total internal reflection fluorescence microscopy. This assay allows for nanoscale-level measurement of the forces and displacements generated by protein ensembles within active microtubule networks.

再構成された活性微小管束内の力を直接測定

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as ...

Combining 3D Magnetic Force Actuator and Multi-Functional Fluorescence Imaging to Study Nucleus Mechanobiology

A fundamental question in mechanobiology is how living cells sense extracellular mechanical stimuli in the context of cell physiology and pathology. The cellular mechano-sensation of extracellular mechanical stimuli is believed to be through the membrane receptors, the associated protein complex, and the cytoskeleton. Recent advances in mechanobiology demonstrate that the cell nucleus in cytoplasm itself can independently sense mechanical stimuli simultaneously. However, a mechanistic understanding of how the cell nucleus senses, transduces, and responds to mechanical stimuli is lacking, mainly because of the technical challenges in accessing and quantifying the nucleus mechanics by conventional tools. This paper describes the design, fabrication, and implementation of a new magnetic force actuator that applies precise and non-invasive 3D mechanical stimuli to directly deform the cell nucleus. Using CRISPR/Cas9-engineered cells, this study demonstrates that this tool, combined with high-resolution confocal fluorescent imaging, enables the revelation of the real-time dynamics of a mechano-sensitive yes-associated protein (YAP) in single cells as a function of nucleus deformation. This simple method has the potential to bridge the current technology gap in the mechanobiology community and provide answers to the knowledge gap that exists in the relation between nucleus mechanotransduction and cell function.

This study presents a new protocol to directly apply mechanical force on the cell nucleus through magnetic microbeads delivered into the cytoplasm and to conduct simultaneous live-cell fluorescent imaging.

3D磁力アクチュエータと多機能蛍光イメージングを組み合わせて核メカノバイオロジーを研究

A fundamental question in mechanobiology is how living cells sense extracellular mechanical stimuli in the context of cell physiology and pathology. The ...