这项工作得到了 NIH R01 EB014869 的支持。

1. 准备用于成像的细胞
注: 直接力探头 (DFP) 可用于任何粘附单元类型。在这里, NIH 3T3 小鼠成纤维细胞被用作该协议的模型细胞线。
<ol><li>培养 NIH 3T3 成纤维细胞在 Dulbecco 的改良鹰的培养基 (DMEM) 补充10% 捐赠牛血清和1% 青霉素-链霉素在35毫米玻璃底部菜, 直到理想的融合。保持细胞在37°c 和 5% CO2。<ol>
<li>一定要涂上所有35毫米玻璃底菜与5µg/毫升纤维连接蛋白 (或类似的 ECM 蛋白质), 然后播种 NIH 3T3 细胞进行成像。 注: 这些细胞必须在盘子上充分传播和附着, 以进行实验。对于 DFP 方法的工作融合, 没有任何限制。</li>
	</ol>
</li>
	<li>在实验之前, 先用 PBS 冲洗细胞两次, 然后用完全生长培养基进行一次冲洗。</li>
	<li>添加3毫升完全生长培养基的玻璃底部菜。</li>
</ol>2. 显微和图像采集
注: 根据制造商的建议, 用机器人安装在侧臂上的倒置荧光显微镜 (或等效)。该显微镜还应配备一个环境室, 以保持37摄氏度的温度, 并2水平在5%。还需要机器人和 microinjector 显微镜。试验中建议采用油浸泡 40 x/1.3 na 或 60 x/1.49 na (或等效目标)。该显微镜应安装在隔振台上。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58038/58038fig1.jpg"> 图 1.核形变与显微镜聚焦 a. 最大的核形变和放松核形变。在计算最大核形变之前, 核形状的后缘首先与变形核的平移相吻合。在微尖端脱离时, 细胞核的形状被叠加在最初的核形状上, 然后拉起。两个形状之间的面积的差异被测量为Δ1。最大核形变被定义为Δ1 除以原核区。同样, 第二个参数, Δ2, 可以定义的叠加最终稳态核形状后, 微支队的原始核形状。b. 将单元格集中在平面 A 上, 然后将焦点平面移至 B 平面, 以找到微尖。在成像过程中, 微被翻译成右侧 (橙色箭头方向)。这个数字已经从处处长 neelam. 15.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58038/58038fig1large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
<ol><li>根据制造商的协议打开 microinjector。</li>
	<li>使用浸入式油吸管, 在物镜的顶端涂一滴浸入油。</li>
	<li>把盘子紧紧夹在盘子里, 把盘子放在台上。 注: 在整个实验中, 细胞必须保持在37摄氏度和 5% CO2 。</li>
	<li>调整目标的高度以使单元格成为焦点 (平面 A,图 1B)。</li>
	<li>移动显微镜阶段找到感兴趣的细胞。</li>
	<li>旋转机器人上的操纵杆, 将吸管保持架移动到顶部位置。将0.5 µm 直径尖端微加载到吸管支架上。<ol>
<li>为避免细胞黏附于微, 在室温下用0.3 毫克/毫升锁相环-g PEG 溶液对微尖端进行预处理。通过接触微到原子核而没有任何吸入压力来测试附着力, 然后将微从原子核中平移。缺乏附着力可以从完全缺乏核形变和翻译中看出。 注: 请按照生产建议打开包装。</li>
	</ol>
</li>
	<li>调整精细控制 (图 1B, 见步骤 2.4), 将平面 A 和单元格顶部的目标焦平面提升为平面 B。</li>
	<li>将机器人设置为粗控。通过观察微的剪影, 慢慢地把微下到飞机 B, 直到微完全进入焦点。</li>
	<li>一旦微提示集中, 将机器人设置为 "精细控制"。</li>
	<li>将目标降低到单元格的赤道平面 (平面 a、图 1B) 并将微降低到平面 a (图 1B、虚线微) 的大约15µm。</li>
	<li>将 microinjector 的补偿压力 (Pc) 设置为所需的压力;等待几秒钟的压力, 以稳定。 注: 最佳压力设定点取决于细胞类型和实验的具体目标。在大多数情况下, 300 的 hPa 将是一个很好的起点。</li>
	<li>确保微不堵塞, 使用机器人面板上的清洁设置, 并检查以确保气泡出现从微尖端。</li>
	<li>通过逐渐降低微直到尖端轻轻接触核表面, 将尖端插入到细胞中。 注意: 当降低微时, 微尖的轮廓将变得清晰, 因为它进入焦点。在微接触核之前, 提高目标焦点, 并将微尖端与核 (相同的 x y 坐标, 更高的 z 平面) 对齐。将焦点返回到原子核的赤道平面 (平面 A,图 1B), 并逐渐降低微尖端。</li>
	<li>通过从微注射系统中拔下压力供应管, 在微尖端和核膜之间建立一个密封, 从而打开微管的末端到大气层。这一步产生的负压等于在核表面上的Pc 。</li>
	<li>利用显微镜图像采集软件获取图像。在图像收集软件中设置 avi 捕获 (视频) 或 nd 捕获 (图像)。 注: 对于任何图像获取软件, 设置实时视频成像或时间推移图像采集短时间间隔。</li>
	<li>切换到相应的荧光成像通道 (如GFP、RFP等) 并开始成像。</li>
	<li>将微尖从细胞体 (右图 1B) 平移, 直到原子核与微分离。 注: 沿正向 x 方向 (在视野中的右侧) 拉出笔尖。拉速可通过计算机编程控制, 或者操纵杆可以手动移动。我们还没有发现拉速和核形变15之间的任何关联, 这暗示着对力的主要弹性反应。</li>
</ol>3. 数据分析
<ol><li>使用任何基本的图像处理软件进行图像分析。核形变的程度可以通过长度应变 (Ɛ) 或区域应变来量化 (图 1A)。用方程1来量化长度应变, 其中 l 和 l0分别表示最大变形和初始位置的原子核长度。 <img alt="Equation 1" src="/files/ftp_upload/58038/58038eq1.jpg">(等式 1)</li>
</ol>

<ol>
	<li>Chow, K. H., Factor, R. E., &#38; Ullman, K. S. The nuclear envelope environment and its cancer connections. Nature Reviews Cancer. 12 (3), 196-209 (2012).</li>
	<li>Zink, D., Fischer, A. H., &#38; Nickerson, J. A. Nuclear structure in cancer cells. Nature Reviews Cancer. 4 (9), 677-687 (2004).</li>
	<li>Bank, E. M., &#38; Gruenbaum, Y. The nuclear lamina and heterochromatin: a complex relationship. Biochemical Society Transactions. 39 (6), 1705-1709 (2011).</li>
	<li>Lammerding, J. et al. Lamins A and C but not lamin B1 regulate nuclear mechanics. Journal of Biological Chemistry. 281 (35), 25768-25780 (2006).</li>
	<li>Dahl, K. N., Engler, A. J., Pajerowski, J. D., &#38; Discher, D. E. Power-law rheology of isolated nuclei with deformation mapping of nuclear substructures. Biophysical Journal. 89 (4), 2855-2864 (2005).</li>
	<li>Crisp, M. et al. Coupling of the nucleus and cytoplasm: role of the LINC complex. Journal of Cell Biology. 172 (1), 41-53 (2006).</li>
	<li>Sosa, B. A., Rothballer, A., Kutay, U., &#38; Schwartz, T. U. LINC complexes form by binding of three KASH peptides to domain interfaces of trimeric SUN proteins. Cell. 149 (5), 1035-1047 (2012).</li>
	<li>Tapley, E. C., &#38; Starr, D. A. Connecting the nucleus to the cytoskeleton by SUN-KASH bridges across the nuclear envelope. Current Opinion in Cell Biology. 25 (1), 57-62 (2013).</li>
	<li>Arsenovic, P. T. et al. Nesprin-2G, a Component of the Nuclear LINC Complex, Is Subject to Myosin-Dependent Tension. Biophysical Journal. 110 (1), 34-43 (2016).</li>
	<li>Rowat, A. C., Lammerding, J., &#38; Ipsen, J. H. Mechanical properties of the cell nucleus and the effect of emerin deficiency. Biophysical Journal. 91 (12), 4649-4664 (2006).</li>
	<li>Rowat, A. C., Foster, L. J., Nielsen, M. M., Weiss, M., &#38; Ipsen, J. H. Characterization of the elastic properties of the nuclear envelope. Journal of the Royal Society Interface. 2 (2), 63-69 (2005).</li>
	<li>Pagliara, S. et al. Auxetic nuclei in embryonic stem cells exiting pluripotency. Nature Materials. 13 (6), 638-644 (2014).</li>
	<li>Liu, H. et al. In situ mechanical characterization of the cell nucleus by atomic force microscopy. ACS Nanotechnology. 8 (4), 3821-3828 (2014).</li>
	<li>Krause, M., Te Riet, J., &#38; Wolf, K. Probing the compressibility of tumor cell nuclei by combined atomic force-confocal microscopy. Physical Biology. 10 (6), 065002 (2013).</li>
	<li>Neelam, S. et al. Direct force probe reveals the mechanics of nuclear homeostasis in the mammalian cell. Proceedings of the National Academy of Sciences of the United States of America. 112 (18), 5720-5725 (2015).</li>
	<li>Pajerowski, J. D., Dahl, K. N., Zhong, F. L., Sammak, P. J., &#38; Discher, D. E. Physical plasticity of the nucleus in stem cell differentiation. Proceedings of the National Academy of Sciences of the United States of America. 104 (40), 15619-15624 (2007).</li>
	<li>Lammerding, J. et al. Lamin A/C deficiency causes defective nuclear mechanics and mechanotransduction. Journal of Clinical Investigation. 113 (3), 370-378 (2004).</li>
	<li>Chancellor, T. J., Lee, J., Thodeti, C. K., &#38; Lele, T. Actomyosin tension exerted on the nucleus through nesprin-1 connections influences endothelial cell adhesion, migration, and cyclic strain-induced reorientation. Biophysical Journal. 99 (1), 115-123 (2010).</li>
	<li>Neelam, S., Dickinson, R. B., &#38; Lele, T. P. New approaches for understanding the nuclear force balance in living, adherent cells. Methods. 94 27-32 (2016).</li>
</ol>

作者没有什么可透露的。

测量细胞核与细胞骨架的机械结合是目前大多数方法的挑战, 如微吸入16, 因为它们需要两个孤立的原子核 (在细胞核与骨架分离的地方) 或悬浮细胞中的细胞核 (如牵引力, 无细胞外力)。通过将双轴应变应用于附着在膜17、18的细胞中, 将力应用于细胞核;然而, 这种技术是有限的事实, 在原子核的力量是未知的。原子力显微镜 (AFM) 探针被用来在完整的细胞中缩进细胞核;这些提供了优势, 他们揭示了核的力学性质, 而它与细胞骨架结合。为了增加12、13、14核的现有体内技术, 我们开发了一种简单而健壮的方法来机械地探测活体黏附细胞中的细胞核, 而它仍然是整合到周围的细胞骨架。这项技术的一个关键特点是, 原子核上的力是精确已知和控制的。
通过应用 DFP, 我们估计纳米牛顿尺度力足以在活体、粘附细胞中变形和转化细胞核。此外, 通过系统研究各种骨架元素的贡献, 我们发现, 波形的中间丝是细胞细胞骨架的主要组成部分, 负责加强细胞核在细胞15。
一些注意事项适用于这些实验。重要的是定期检查微提示堵塞或破损在实验中, 因为两个堵塞的微或骨折的提示将导致未知的变化, 对应用于细胞的吸入压力。要检查微是否堵塞, 请使用机械手上的清洁设置来施加最大压力, 并检查从微尖端出现的气泡。如果微提示断开或断开, 请在开始下一个实验之前替换微。此外, 重要的是要确认, 核应变变化的力量, 并零的力量 (见处处长 neelam等。15). 如果在实验中没有观察到这一点, 那么微尖端与核表面之间的非特定粘附可能会导致核形变。这项技术的另一个局限性是, 将微插入细胞本身可能会导致局部破坏骨架结构。
测试核形变/平移程度与加载速率之间的相关性是有益的。缺乏相关性将意味着主要的弹性阻力。事实上, 这是我们发现的情况下, 成纤维细胞核15。当原子核的力已知时, 该方法不允许计算像杨氏模量这样的参数。我们主要用它来解剖细胞结构对核形变和细胞中的转化的抗性贡献。虽然我们已经报告了二维核形状15,19, 它可能是有用的, 以量化的完整的三维的形状, 原子核的力量。
微的吸入压力是已知的, 但它比实际压力大, 因为水流通过毛孔10的核表面。然而, 我们已经表明, 通过微, 通过核孔隙流动的阻力要大得多 (参见处处长 neelam等的计算支持信息。15) 合理的膜渗透和微尺寸。因此, 尽管存在流动, 外膜表面的压力应该等于吸力压力。
最后, DFP 允许使用者将粘附细胞中的核细胞骨架的机械反应量化为已知的和受控制的力。该方法可用于解剖细胞核和细胞质中分子成分对细胞内细胞核力学行为的贡献。

如癌症等病症涉及对核形态和结构1,2的改变, 通常伴随着 "软化" 的核心3,4。对机械变形的核阻力一般是将力应用于孤立原子核5。
细胞核在细胞是分子连接到细胞骨架由 Nucleoskeleton 和细胞骨架 (林肯) 复杂6,7,8,9的链接器。因此, 细胞核是机械地与细胞骨架结合, 并通过细胞基质粘连, 细胞外基质。机械地探测黏附细胞内的细胞核可以提供对这种机械整合的洞察力。在活细胞中操纵细胞核的方法包括微吸入10、11和原子力显微镜12、13、14。我们最近描述了一个直接的力量探针 (DFP), 直接地应用机械力量在原子核在活黏附力细胞15。
在这里, 我们概述了使用显微注射系统的程序, 通常是在显微镜的设施, 以应用已知的, 纳米级的机械力直接到核在黏附细胞。femtotip (0.5 µm 直径微尖端) 安装并连接到微注射系统由一个管。尖端, 定位在45°角度相对于养殖皿的表面, 被降低, 直到相邻的核表面。管子然后被断开并且被打开对大气, 在核表面创造一个负吸力压力并且封印微尖端反对核表面。通过微尖端的翻译, 原子核变形, 最终 (取决于施加的力的大小), 脱离微。当由原子核和细胞施加的恢复力 (抵抗) 力等于微所施加的吸力时, 这种分离就会发生。分析可以通过测量原子核的位移、长度应变 (方程 1) 或区域应变 (图 1A) 来进行。

<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

原子核的力学性质决定了它对细胞中产生的机械力的反应。由于细胞核是细胞骨架的连续分子, 因此需要方法来探讨其在黏附细胞中的力学行为。在这里, 我们讨论直接力探针 (DFP) 作为一个工具, 以力量直接向细胞核在一个活生生的黏附细胞。我们用吸力将一个狭窄的微附着在核表面。微从原子核中被翻译出来, 使原子核变形和转化。当恢复力等于吸力时, 原子核分离, 弹性松弛。因为吸入压力是已知的, 核表面上的力是已知的。该方法表明, 纳米尺度力足以使粘附细胞中的细胞核变形和转化, 并确定骨架元素, 使得原子核能够抵抗力。DFP 可用于解剖细胞和核成分对活细胞核力学性能的贡献。

图 2A显示了 NIH 3T3 小鼠成纤维细胞核的强迫。当微尖端被翻译成右侧时, 原子核会变形, 最终与微尖端分离。随着抽吸力的增加, 原子核的长度应变增加 (图 2B)。原子核的前缘 (微拉边) 形成核突起, 尾部边缘从原位置偏移。突起的长度远远大于尾部边缘位移 (图 2C), 表明细胞核与周围细胞质紧密结合。时间尺度是短期的放松的核前沿 (< 1 s) 和核后缘 (< 2 s) (图 2D)。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58038/58038fig2.jpg"> 图 2.核形变的表征 DFP 的活体、黏附细胞中细胞核的变形和位移。NIH 3T3 成纤维细胞核被拉扯了与 6 nN 力量。图像显示核形状在指定的时间。刻度条: 5 µm b. 核形变按长度应变进行量化。核形变随作用力的增加而增大。误差条表示平均值的标准误差 (SEM);n > 6。在前缘的位移大于尾部的位移。长度应变松弛比后边缘松弛快得多。*P < 0.05;误差条表示 SEM;n=10。这个数字已经从处处长 neelam. 15.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58038/58038fig2large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
我们使用了 DFP 方法来确定骨架力是如何导致核电阻在细胞中变形的。虽然在 F 肌动蛋白 (由细胞松弛素) 或微管中断 (nocodazole) 后发现核形变或翻译没有显著差异, 但通过 siRNA 的击倒减少波形的表达, 大大提高了核翻译和变形 (图 3)。这表明, 成纤维细胞中的波形中间丝是帮助细胞核抵抗局部力的主要骨架元素。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58038/58038fig3.jpg"> 图 3.核形变的荧光图像 DFP 被用来确定骨架力对核形变的贡献。细胞核被 SYTO 59 染料染色。荧光图像在所述条件下显示核的叠加 (红色、伪色) 和后 (绿色、伪色)。CTRL, 控制单元格;细胞, 细胞细胞松弛素治疗;用 nocodazole 治疗的细胞;滚, 用炒 siRNA 转染的细胞;vim siRNA, 细胞转染 siRNA 靶向波形。这个数字已经从处处长 neelam. 15.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58038/58038fig3large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>

Watch this Scientific Journal Video about A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton at JoVE.com

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

测量细胞核与骨架间机械一体化的直接力探针

在本协议中, 我们描述了一种微方法, 直接将受控力应用于活细胞中的细胞核。这项化验允许审讯活体, 黏附细胞的核机械性质。

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

Nanyang Technological University

Research

JoVE Journal

Biochemistry

15.2K 次观看.  University of Florida. 在本协议中, 我们描述了一种微方法, 直接将受控力应用于活细胞中的细胞核。这项化验允许审讯活体, 黏附细胞的核机械性质。

视频: 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.

生物膜荧光探针力:受体 - 配体动力学并行和定量结合诱导细胞内信号作用于单细胞

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

科研

生物工程

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.

一种利用共振能量转移（FRET）的生物传感器-force议定书衡量所有核LINC复杂的机械力

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.

使用光学镊子测量禁闭中的亚细胞力学的直接力

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

发育生物学

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.

荧光技术结合测定活细胞中的力敏感蛋白动力学

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.

生物物理检测探头相间细胞核的力学性能：基材应变的应用和微针操纵

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.

DNA张力探针用于绘制免疫细胞的瞬时皮牛顿受体力

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.

凸出力显微镜: 一种量化细胞突起力的方法

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.

使用纳米-牛顿分辨率力转换器从骨骼肌肉活检中隔离肌异物并确定收缩函数

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.

直接测量重组活性微管束内的力

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.

结合3D磁力致动器和多功能荧光成像来研究细胞核力学生物学

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