（poro-）弹性收缩肌动球蛋白网络作为细胞骨架模型系统的机制
Summary
本工作采用 体外 重构方法研究动粒肌肽凝胶在受控条件下的多孔弹性。量化了肌动球蛋白凝胶和包埋溶剂的动力学，通过这些动力学证明了网络多孔弹性。我们还讨论了实验挑战，常见陷阱以及与细胞骨架力学的相关性。
Abstract
细胞可以主动改变它们的形状并变得运动，这一特性取决于它们主动重组其内部结构的能力。这一特征归因于细胞骨架的机械和动态特性，特别是肌动肌蛋白细胞骨架，它是极性肌动蛋白丝、肌球蛋白马达和辅助蛋白的活性凝胶，具有内在收缩特性。通常接受的观点是细胞骨架表现为粘弹性材料。然而，该模型不能总是解释实验结果，这些结果更符合将细胞骨架描述为多孔弹性活性材料 – 嵌入细胞质的弹性网络的图片。肌球蛋白马达产生的收缩梯度驱动细胞质在凝胶孔中的流动，这推断细胞骨架和细胞质的力学是紧密耦合的。多孔弹性的一个主要特征是网络中应力的扩散松弛，其特征在于有效扩散常数取决于凝胶弹性模量、孔隙率和细胞质（溶剂）粘度。由于细胞有许多方法来调节其结构和材料特性，我们目前对细胞骨架力学和细胞质流动动力学如何耦合的理解仍然知之甚少。在这里，采用 体外 重建方法来表征多孔弹性肌动球蛋白凝胶的材料特性，作为细胞骨架的模型系统。凝胶收缩由肌球蛋白运动收缩性驱动，这导致渗透溶剂流动的出现。本文描述了如何制备这些凝胶并进行实验。我们还讨论了如何在局部和全球范围内测量和分析溶剂流动和凝胶收缩。给出了用于数据量化的各种缩放关系。最后，讨论了实验挑战和常见陷阱，包括它们与细胞骨架力学的相关性。
Introduction
活细胞具有独特的机械性能。除了被动地对施加的力做出反应的能力外，它们还能够主动产生响应外部刺激的力1。这些特征对于各种细胞过程（特别是在细胞运动期间）至关重要，主要归因于细胞骨架的机械和动态特性，尤其是肌动肌蛋白细胞骨架，它是极性肌动蛋白丝、肌球蛋白分子马达和辅助蛋白的活性凝胶。这些肌动蛋白网络表现出由肌球蛋白驱动的内在自组织和收缩特性，肌球蛋白运动蛋白交联肌动蛋白丝并在ATP水解2的推动下在网络中积极产生机械应力。
已经进行了大量的实验和理论研究来研究细胞骨架的材料性质3。普遍接受的观点是细胞骨架表现为粘弹性材料4。这意味着在短时间尺度上，细胞骨架表现为弹性材料，而在长时间尺度上，由于交联蛋白和肌球蛋白运动脱离（和重新附着），它表现为粘性流体，这允许网络动态周转。然而，在许多情况下，粘弹性模型无法描述实验结果，这更符合描述细胞骨架的图片，更一般地说，细胞质被描述为多孔弹性活性材料5,6。这些类型的材料有两个主要特征。（i）第一个主要特征是通过肌球蛋白马达驱动的收缩梯度产生穿透性细胞质（“溶剂”）流过凝胶孔，这是细胞起泡7，运动性8和细胞形状振荡9等过程的基础。这种胞质流的出现可以是局部的，用于起泡，也可以是全球性的，如细胞运动。在后一种情况下，细胞后部施加的收缩应力驱动细胞溶质液流向细胞前部，从而补充板状伪足组装所需的蛋白质库8。（ii）第二个主要特征是应力的松弛是扩散的，其特征在于有效扩散常数，这取决于凝胶弹性模量、凝胶孔隙率和溶剂粘度5。多孔弹性扩散常数决定了系统对施加应力的响应速度。扩散常数越高，应力再分布越快。反过来，这决定了细胞内胞质液在施加机械应力（无论是外部还是内部）后在细胞内重新分布所需的时间，例如肌球蛋白马达产生的主动收缩应力。因此，这些例子表明，细胞骨架和细胞质的力学是紧密耦合的，不能分开处理3。
由于细胞可以通过多种方式调节其机械性能，因此网络力学和流体流动动力学之间的相互作用仍然知之甚少。一种强大的替代方法是使用体外重组系统，该系统允许完全控制各种微观成分和系统参数，这使得这些模型系统最适合物理分析10,11。该方法已成功用于研究蛋白质组成和系统几何形状对基于肌动蛋白的运动的影响12,13,14,15,16,17,18，肌动肌蛋白网络的2D图案化19,20,21,22，以及多孔弹性肌动球蛋白凝胶的网络收缩性和流体流动动力学之间的相互作用，这是本文的重点23。
在本手稿中，基于Ideses等人的工作，讨论了可控尺寸和材料性质的收缩弹性肌动球蛋白网络的制备23。分析和量化了收缩凝胶和排出溶剂的动力学，通过这些动力学肌肉蛋白凝胶可以描述为多孔弹性活性材料。研究溶剂粘度对应力扩散率的影响进一步证实了这些网络的多孔弹性性质。提供了用于数据量化的各种缩放关系。最后，讨论了实验挑战、常见陷阱以及实验结果与细胞骨架的相关性。
Protocol
1.玻璃表面处理及钝化： 注意：本节包括三个主要步骤（见 图1）：（i）清洁和亲水，（ii）硅烷化和（iii）表面钝化。 清洗和亲水使用食人鱼溶液清洁玻璃表面。注意：食人鱼溶液是30%H2O 2和70%H2 SO4（材料表）的混合物。其主要目标是去除盖玻片表面的有机污染物，并暴露玻璃表面上的OH基…
Representative Results
每个实验使用两个玻璃盖玻片。玻璃盖玻片经过清洁并用PEG聚合物钝化。钝化对于防止溶解的蛋白质在早期实验阶段粘附在玻璃表面上以及最大限度地减少收缩网络与玻璃壁的相互作用至关重要。未能实现良好的钝化会导致低效收缩，在极端情况下，甚至会抑制肌动蛋白网络的形成。 图1描述了表面处理程序的三个主要步骤。这些步骤包括以下内容：（i…
Discussion
在这里，采用体外方法来表征多孔弹性肌动肌肽凝胶的力学，作为细胞骨架的模型系统，更一般地说，细胞质的模型系统，其已被证明表现为多孔弹性材料3,5。细胞骨架（细胞质）的流变学特征在于多孔弹性扩散常数，该常数决定了细胞内胞质液在施加机械应力后在细胞内重新分布所需的时间。已经提出细胞使用这种机制来调节它们的形状<sup cla…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们要感谢Dina Aranovich的蛋白质纯化和标记。G.L.感谢以色列科学，技术和空间部提供的Jabotinsky博士奖学金。A.B.G.感谢以色列科学基金会（赠款2101/20）和以色列国科学技术部（赠款3-17491）的财政支持。
Materials
| (3-Mercaptopropyl)trimethoxysilane | Sigma-Aldrich Company | 175617 | Stored under Argon atmosphere at 4 °C |
| Acetic acid | Bio-Lab ltd | 1070521 | |
| Alexa-Fluor 488 | Invitrogene | A10254 | Diluted with DMSO, stored under Argon atmosphere at -20 °C |
| Alexa-Fluor 647 | Invitrogene | A20347 | Diluted with DMSO, stored under Argon atmosphere at -20 °C |
| BSA | Sigma -Aldrich Company | A3059 | Stored at 4 °C |
| Catalase | Sigma -Aldrich Company | C9322 | The stock bottle is kept under dry atmosphere (silica gel) at -20 °C |
| Coverslips | Mezel-glaser | CG2222-1.5 | Kept in milliQ-water after the Piranha treatment and used within 3 weeks |
| Creatine kinase | Roche Life Science Products | 10736988001 | Prepared fresh in glycine buffer, kep on ice, and used within 3 days. The stock bottle is kept under dry atmosphere (silica gel) at 4 °C |
| Creatine phosphate | Roche Life Science Products | 10621714001 | When dissolved should be kept at -20 °C and used within 3 months. The stock bottle is kept under Argon atmosphere and stored at 4 °C |
| DTT | Roche Life Science Products | 10708984001 | When dissolved should be kept at -20 °C and used within 3 months |
| Dual view Simultaneous Imaging System | Photometrics | DV2-CUBE | |
| EGTA | MP Biomedicals | 195174 | |
| EM-CCD Camera | Andor Technology Ltd | DV 887 | |
| EM-CCD Camera | Photometrics | Evolve Delta | |
| Ethanol | Bio-Lab ltd | 525050300 | |
| Flourescence Lamp | Rapp Optoelectronic | ||
| Fluoresbrite YG Microspheres | Polysciences | 17151-10 | 200 nm diameter |
| Glucose | ICN Biomedicals Inc | 194024 | When dissolved should be kept at -20 °C and used within 3 months. |
| Glucose oxidase | Sigma-Aldrich Company | G7141 | Kept in -20 °C and used within 3 months. The stock powder is kept under Argon atmosphere and kept at -20 °C |
| Glycerol | ICN Biomedicals Inc | 800687 | |
| Glycine | MP Biomedicals | 808822 | |
| Hydrogen Peroxide | Sigma-Aldrich Company | 216763 | Stored at 4 °C |
| KCl | EMD Millipore Corp. | 529552 | |
| Methanol | Bio-Lab ltd | 1368052100 | |
| MgCl2 | EMD Millipore Corp. | 442615 | |
| Microscope | Leica Microsystems | DMI3000 | |
| mPEG-mal | Nanocs | PG1-ML-5k | Mw = 5000 Da. Divided to small batches by weight. Stored under Argon atmosphere at -20 °C |
| Nile red microspheres | Spherotech | FP-2056-2 | 2300 nm diameter |
| Objective (10x) | Leica Germany | HC PL AP0 | UPlanFL Numerical Aperture = 0.3 |
| Objective (2.5x) | Leica Germany | 506304 | Plan-NEOFLUAR Numerical Aperture = 0.075 |
| Oven | WTC Binder | ||
| Parafilm | Amcor | PM-996 | |
| PBS Buffer | Sigma-Aldrich Company | P4417 | |
| Shutter Driver | Vincet Associates | VMM D1 | |
| Silica gel | Merck | 1.01907.5000 | |
| Sonicator | Elma | Elmasonic P | |
| Sulfuric acid | Carlo Erba reagents | 410301 | |
| DV2 Dual-Channel Simultaneous-Imaging System | Photometrics | ||
| TRIS | MP Biomedicals | 819620 | |
| UV-VIS Spectrophotometer | Pharmacia | Ultraspec 2100 pro | |
| MICROMAN E | Gilson | FD10001 | 1–10 uL |
| MATLAB R2017b | MathWorks | Data quantification | |
| MetaMorph | Molecular devices | Control software of the optical imaging system; data quantification (particle tracking analysis, network mesh size) |
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Citer Cet Article
Choudhary, S., Livne, G., Gat, S., Bernheim-Groswasser, A. The Mechanics of (Poro-)Elastic Contractile Actomyosin Networks As a Model System of the Cell Cytoskeleton. J. Vis. Exp. (193), e64377, doi:10.3791/64377 (2023).