自下而上的 体外 方法测定隔膜的超微结构组织、膜重塑和曲率敏感性行为
1Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University,Sorbonne Université, 2Institut Fresnel, CNRS UMR7249,Aix Marseille Univ, Centrale Marseille, 3Department of Bionanoscience, Kavli Institute of Nanoscience Delft,Delft University of Technology, 4Department of Chemical Engineering,Imperial College London, 5Sorbonne Université, CNRS, Institut de Biologie Paris-Seine (IBPS),Service de microscopie électronique (IBPS-SME), 6Laboratoire Matière et Systèmes Complexes (MSC),Université Paris Cité, 7Institute of Biotechnology,Czech Academy of Sciences, BIOCEV
Summary
败类是细胞骨架蛋白。它们与脂质膜相互作用，可以感测但也会产生微米级的膜曲率。我们在该协议中描述了用于分析膜变形，曲率敏感的隔膜结合和隔膜长丝超微结构的自下而上的 体外 方法。
Abstract
膜重塑不断发生在质膜和细胞器内。为了充分剖析环境的作用（离子条件，蛋白质和脂质组成，膜曲率）以及与特定膜重塑过程相关的不同伙伴，我们采用 体外 自下而上的方法。近年来，人们一直对揭示与主要疾病相关的隔膜蛋白的作用有着浓厚的兴趣。隔膜是与质膜相互作用的必需且无处不在的细胞骨架蛋白。它们与细胞分裂，细胞运动，神经形态发生和精子发生以及其他功能有关。因此，重要的是要了解隔膜上如何相互作用和组织以随后诱导膜变形，以及它们如何对特定的膜曲率敏感。本文旨在破译分子水平上隔膜的超结构与微米级膜重塑之间的相互作用。为此，对萌芽酵母和哺乳动物隔膜复合物进行重组表达和纯化。然后使用 体外 测定的组合来分析隔膜处的自组装。使用支撑的脂质双分子层（SLBs），巨型单层囊泡（GUV），大型单层囊泡（LUV）和波浪基质来研究隔膜自组装，膜重塑和膜曲率之间的相互作用。
Introduction
败类是与脂质膜相互作用的细胞骨架细丝形成蛋白。败类在真核生物中无处不在，对许多细胞功能至关重要。它们已被确定为萌芽酵母和哺乳动物细胞分裂的主要调节因子1，2。它们参与膜重塑事件，纤毛发生3和精子发生4。在哺乳动物细胞中，隔膜蛋白还可以以Rho GTPas酶（BORG）依赖性方式的结合剂8与肌动蛋白和微管5，6，7相互作用。在各种组织（神经元9，纤毛3，精子10）中，隔膜蛋白已被鉴定为膜结合组分11的扩散屏障的调节因子。隔膜也被证明可以调节膜起泡和突出形成12。败类是多任务蛋白质，与各种流行疾病的出现有关13.他们的失调与癌症14和神经退行性疾病15的出现有关。
根据生物体的不同，几个隔膜亚基（秀丽隐杆线虫中的两个到人类的13个）聚集在一起形成复合物，其组织以组织依赖性方式变化16。基本的隔膜构建块聚集了两到四个亚基，以两个副本存在并以棒状回文方式自我组装。在萌芽酵母中，七分素是八聚体17，18。在原位，隔膜通常局限于具有微米曲率的部位;它们被发现在分裂收缩部位，纤毛和树突的底部，以及精子19，20的环空。在膜上，隔膜的作用似乎是双重的：它们与重塑脂质双层和维持膜完整性有关21。因此，研究隔膜上形成隔膜的长丝蛋白和/或亚基的生物物理性质对于理解它们的作用至关重要。为了在控制良好的环境中解剖隔膜蛋白的特定性质，自下而上的体外方法是合适的。到目前为止，只有少数小组在体外描述了隔膜蛋白20，22，23的生物物理性质。因此，与其他细胞骨架丝相比，目前关于隔膜蛋白在体外行为的知识仍然有限。
该协议描述了如何分析隔膜长丝的组织，膜重塑和曲率敏感性19。为此，使用了光学和电子显微镜方法（荧光显微镜，冷冻电子显微镜[冷冻电镜]和扫描电子显微镜[SEM]）的组合。使用荧光光学显微镜可视化微米级巨型单层囊泡（GUV）的膜重塑。使用冷冻EM对与脂质囊泡结合的隔膜丝的排列和超微结构进行分析。使用SEM对沉积在可变曲率的波浪基板上的固体负载脂质双分子层结合的隔膜蛋白丝的行为，对隔膜曲率敏感性进行分析，从而可以分析正曲率和负曲率的曲率敏感性。与之前的分析20，24相比，本文建议结合使用多种方法，彻底分析隔膜自组装、协同变形膜和曲率敏感。该方案被认为是有用的，并且适用于任何对膜具有亲和力的丝状蛋白质。
Protocol
1. 使用巨型单层囊泡 （GUV） 测定膜重塑 注意：在本节中，生成GUV是为了模拟隔膜在细胞环境中可能由隔膜引起的膜变形。事实上，在细胞中，隔膜蛋白经常在具有微米曲率的位点发现。GUV的尺寸从几微米到几十微米不等，可能会变形。因此，它们适用于测定任何微米级隔膜蛋白引起的变形。荧光脂质以及荧光标记的隔膜蛋白（使用绿色荧光蛋白[GFP]）用于 通?…
Representative Results
无人机变形在隔膜蛋白聚合的条件下， 图3显示了与隔膜蛋白一起孵育后重塑的GUV的典型共聚焦荧光图像。裸露的GUV（图3A）是完全球形的。在与超过50 nM的萌芽酵母隔膜丝孵育时，囊泡出现变形。浓度高达100nM的芽酵母隔膜八聚体，囊泡出现小平面，变形保持静态，因此不波动（图3B）。高于200nM的发芽酵母隔板，?…
Discussion
如上所述，已经使用了脂质混合物来增强脂质双层内的PI（4，5）P2 掺入，从而促进隔膜相互作用。事实上，我们已经在其他地方展示了25 ，萌芽的酵母隔膜以PI（4，5）P2特异性方式与囊泡相互作用。该脂质组成通过筛选多种组合物进行经验调整，现在被作者广泛使用。PI（4，5）P2 脂质必须小心处理。储备溶液必须小体积等分，以便特定小瓶不会打开两?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢帕特里夏·巴塞罗和丹尼尔·莱维的有益建议和讨论。这项工作得益于国家研究机构（ANR-13-JSV8-0002-01）、ANR SEPTIMORF ANR-17-CE13-0014和“SEPTSCORT”项目ANR-20-CE11-0014-01的支持。B. Chauvin由巴黎高等医学院“ED564：法兰西岛的体质”和医学研究基金会资助。K.中泽得到了索邦大学（AAP新兴）的支持。G.H.科恩德林克得到了荷兰组织联合会（NWO/OCW）通过“BaSyC-构建合成细胞”的支持。引力授予（024.003.019）。我们感谢拉博细胞（n）量表（ANR-11-LABX0038）和巴黎科学与文学（ANR-10-IDEX-0001-02）。我们感谢法国国家研究基础设施法国生物成像（ANR10-INBS-04）成员居里研究所的细胞和组织成像（PICT-IBiSA）。
Materials
| 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine | Avanti Polar Lipids | 850725 | |
| 1,2-dioleoyl-sn-glycero-3-phospho-L-serine | Avanti Polar Lipids | 840035 | |
| Bath sonicator | Elma | Elmasonic S10H | |
| Bodipy-TR-Ceramide | invitrogen, Thermo Fischer scientific | 11504726 | |
| Chemicals: NaCl, Tris-HCl, sucrose, KCl, MgCl2, B-casein, chloroform, sodium cacodylate, tannic acid, ethanol | Sigma Aldrich | ||
| Confocal microscope | nikon | spinning disk or confocal | |
| Critical point dryer | Leica microsystems | CPD300 | |
| Deionized water generator | MilliQ | F1CA38083B | MilliQ integral 3 |
| Egg L-α-phosphatidylcholine | Avanti Polar Lipids | 840051 | |
| Field Emission Gun SEM (FESEM) | Carl Zeiss | Gemini SEM500 | |
| Glutaraldehyde 25 %, aqueous solution | Thermo Fischer scientific | 50-262-19 | |
| High vacuum grease, Dow corning | VWR | ||
| IMOD software | https://bio3d.colorado.edu/imod/ | software suite for tilted series image alignment and 3D reconstruction | |
| Lacey Formvar/carbon electron microscopy grids | Eloise | 01883-F | |
| Lipids | Avanti Polar Lipids | ||
| L-α-phosphatidylinositol-4,5-bisphosphate | Avanti Polar Lipids | 840046 | |
| Metal evaporator | Leica microsystems | EM ACE600 | |
| NOA (Norland Optical Adhesives), NOA 71 and NOA 81 | Norland Products | NOA71, NOA81 | |
| Osmium tetraoxyde 4% | delta microscopies | 19170 | |
| Osmometer | Löser | 15 M | |
| Plasma cleaner | Alcatel | pascal 2005 SD | |
| Plasma generator | Electron Microscopy Science | ||
| Plunge freezing equipment | leica microsystems | EMGP | |
| Transmission electron microscope | Thermofischer | Tecnai G2 200 kV, LaB6 | |
| Uranyl acetate | Electron Microscopy Science | 22451 | this product is not available for purchase any longer |
| Wax plates, Vitrex | VWR |
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Citazione di questo articolo
Chauvin, B., Nakazawa, K., Beber, A., Di Cicco, A., Hajj, B., Iv, F., Mavrakis, M., Koenderink, G. H., Cabral, J. T., Trichet, M., Mangenot, S., Bertin, A. Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins. J. Vis. Exp. (186), e63889, doi:10.3791/63889 (2022).