Name: preparation of mica supported lipid bilayers for high resolution optical microscopy imaging
Uploaded: 2014-06-07T15:00:00.000+00:00
Duration: 7 min 48 s
Description: Watch this Scientific Journal Video about Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging at JoVE.com

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1，云母和准备幻灯片<ol><li>号广场1½（0.17毫米）到盖玻片染色架。 </li><li>超声处理在60℃下在2％洗涤剂30分钟</li><li>用去离子水冲洗20次。 </li><li>使用镊子去除幻灯片及吹干用压缩空气或氮气。 </li><li>切云母片成10×10mm的正方形片用剪刀或刀片。 </li><li>剪下每个云母片成用刀片2-3更薄传单。 注:此步骤需要使用锋利的刀片。 </li></ol> 2，云母大会和会议厅安装<ol><li>用乙醇清洁显微镜载玻片上。 </li><li>云母胶切成单张步骤1.6到玻片上用光学胶。低粘度粘合剂应更好地传播下降和胶水的云母。 </li><li>在紫外灯固化，10分钟。 注:粘合剂是由UV光具有最大吸收在350nm的范围内，以380nm的和推荐的能量固化的热曲IRED完全固化是4.5焦耳/厘米2。然而，不同的光源可用于本步骤（参见由粘合剂供应商提供的资料）。 </li><li>通过消除与透明胶带最初几层露出干净的云母表面。 </li><li>将光学胶小降（〜20微米）到云母表面。 注意:在这一步中，高粘度胶粘剂，低自发荧光水平建议。我们的经验表明，使用低（步骤2.2）和高粘度的粘合剂组合增加了显著成效云母分裂（步骤2.7）。 </li><li>轻轻地放在刚清洗干净的盖玻片上的粘合剂的下降，避免产生气泡，让解决1分钟。 </li><li>在紫外灯固化，10分钟。 注意:在此步骤之后，载玻片，云母和盖玻片的夹心可存放相当长的时间周期（长达数周）。继续下一个步骤只是实际SLB前的准备，以确保云母表面是新鲜的。 注:将粘合剂固化，UV光具有最大吸收在350的范围内，以380nm的和完全固化的推荐能量为4.5J/cm 2。然而，不同的光源可用于本步骤（参见由粘合剂供应商提供的资料）。 </li><li>使用exacto刀，轻轻地从一边载玻片下马盖玻片所示的视频。在大多数情况下，薄且平坦层云母将保持附着在盖玻片。云母附着于载玻片可重复使用（从步骤2.4重复）。 </li><li>检查表面质量用肉眼或在解剖显微镜下，以确保云母层仍粘在盖玻片和未​​分裂在步骤2.8中完全除去。做一个小的擦伤用钳子或解剖针将有助于其具有由云母可检测的不同浓度的粘合剂之间进行区分。 </li><li>从1.5 ml的小瓶瓶盖取下橡胶密封和采用光学倒挂胶帽表面胶或指甲油，并用固化UV灯，或让空气干燥10分钟，分别。 注意:如果样品准备同步光（TIRFM或共焦）与原子力显微镜成像，1.5 ml的小瓶帽可能太小，无法安装原子力显微镜头显微镜舞台上。在这种情况下，帽可以由任何塑料O形环具有更大的直径，适于AFM头安装被取代。在实验时，需要不损伤云母表面上除去帽的情况下，可以用来代替胶或指甲油硅脂。 </li></ol> 3，支持的脂质双分子层（SLB）的形成<ol><li>将新鲜制备的脂质体溶液倒入腔与表面。需要SLB形成的最小体积为〜30微升。 注:有关脂质体和SLB形成更多详细信息，请参阅发布协议，如如袋等 ，2014 15。 </li><li>请继续使用所需的协议负载均衡的形成。在孵化和成像，该腔室可以被放置在一个热块或加热的显微镜载物台上，以维持保持高于其熔融温度所用的脂质所需的温度。 </li></ol>

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该协议描述了一种制备光滑而薄的云母表面的脂质双分子层沉积和高分辨率成像的方法。该技术只需要很少的手工技艺，大多限于仔细拆卸玻璃云母玻璃夹层（步骤2.8），这是获得高质量的云母表面至关重要的。总是需要新鲜剥离云母检查在这一点上，因为它有可能为云母，从光学粘合剂分离而没有解理，而使光学粘合剂的暴露区域。这可能导致在粘合剂而不是在云母上的双层的不必要的沉积。?...

本议定书的总体目标是展示了一种制备云母表面的云母支持的脂质双层（SLBs中）使用光学全内反射荧光显微镜（TIRFM）或共聚焦显微镜，它也可以用原子力结合高分辨率成像显微镜（AFM）。 SLBs中是一种广泛使用的模型为脂质聚类，相分离，双层的元件或多肽，蛋白质或其他化合物1-5及其相互作用的动力学的大量研究。不同底物可能用于SLB形成（ 即玻璃，云母，二氧化硅，聚合物）取决于研究4,6-8的性质。典型的膜研究依赖于显微镜为基础的成像技术，如TIRFM和原子力显微镜。因此，对于TIRFM成像，在玻璃表面是一个典型的选择，因为玻璃是透明的。是玻璃的制备比较容易，其结果的质量，主要是通过彻底的清洗表面前脂质囊泡的沉积来确定。原子力显微镜由于其较高的轴向分辨率要求云母表面。云母是一种硅酸盐矿物，具有接近完美的底面解理。因此，新鲜剥离的云母是原子级平整，允许甚至在亚纳米尺度9观察膜的高度差。 使用方法如荧光相关光谱（FCS），单分子跟踪（SMT），和光漂白（FRAP）后荧光恢复扩散的研究表明然而，该脂质膜动力学很大程度上依赖于表面在其上它们被沉积的类型，由此玻璃和云母能给广泛变化的结果10,11。这些不同之处包括该膜的探针不仅扩散系数，也是粒子具有不同的速率扩散的分离群体的检测，以及可能不同的状态之间进行切换。 因此，使用TIRFM和AFM技术获得的结果的直接比较是经常有问题，除非在同一表面（本例中的云母）被使用。虽然有一些研究，其中TIRFM和AFM双层成像于同云母表面12,13进行，云母很少使用，主要是因为处理问题的光学显微镜。云母编制要求切割手工成薄传单，然后用光学胶12粘在盖玻片。然而，这种方法需要一些练习，以达到满意的效果。此外，所得到的表面常常是波浪形而厚，使得它们难以与低的工作距离，高数值孔径的透镜使用。 如在此协议中所述制备云母表面非常薄（〜220微米，其中包括170微米盖玻片厚度）和极其平坦，从而避免"起伏"，这对于成功的高分辨率成像是至关重要的。它们可以被用来为TIRFM或共聚焦设置。此外，同样的样品可以被转移到的原子力显微镜，甚至同时成像的TIRFM /共聚焦和原子力显微镜。结合这两种技术可以与双层膜结构14扩散行为直接相关。由于云母表面的新鲜原木，他们是干净的，不需要费时，重复性差，并且有潜在危险的清洗程序（玻璃清洗协议通常包括化学物质，如Piranha溶液，硫酸，氢氧化钠/氢氧化钾）。安装一个小室中，在协议中还描述的，可减少所需的有效双层形成为小于50μl的小泡的体积。最后，表面组装的全过程是不费时（准备时间不超过30分钟），并且不需要手工技能的高度一样，传统的云母解理和胶合。

<table><tbody><tr><td>Bath Sonicator</td><td>Fisher Scientific</td><td>FB15051</td><td></td></tr><tr><td>Coverslips 24 x 50 mm - No H1.5</td><td>Marienfeld</td><td>102222</td><td></td></tr><tr><td>DOPC</td><td>Avanti Polar Lipids</td><td>850357</td><td></td></tr><tr><td>Hellmanex III (detergent)</td><td>Hellma Analytics</td><td>320.003</td><td></td></tr><tr><td>Mica V-1 Grade</td><td>SPI Suppliers</td><td>1872-CA</td><td></td></tr><tr><td>Optical Adhesive (high viscosity)</td><td>Norland Products</td><td>NOA63</td><td></td></tr><tr><td>Optical Adhesive (low viscosity)</td><td>Norland Products</td><td>NOA60</td><td></td></tr><tr><td>Sphingomyelin-ATTO647N</td><td>AttoTec</td><td>AD 647N-171</td><td></td></tr><tr><td>UV lamp</td><td>Synoptics Ltd.</td><td>GelVue GVM20</td><td>The lamp was set to 100% power</td></tr></tbody></table>

preparation of mica supported lipid bilayers for high resolution optical microscopy imaging

支持的脂质双层（SLBs中）被广泛用作模型用于研究膜性能（相分离，聚类，动力学）及其与其它化合物，例如药物或肽的相互作用。然而SLB特性不同，取决于所使用的支持。 常用的技术为SLB成像和测量的单分子荧光显微镜，FCS和原子力显微镜（AFM）。因为大多数光学成像研究，进行了在玻璃支撑体，而AFM需要一个非常平坦的表面（通常是云母），从这些技术的结果不能直接相比，由于这些材料的电荷和平滑特性强烈影响扩散。遗憾的是，所需的切割和粘合薄片云母至载玻片手巧的高电平代表一个障碍，以常规使用的云母为SLB制剂。虽然这将是选择的方法，例如制备云母表面往往最终会被不均匀（波浪），不易形象，尤其是小的工作距离，高数值孔径透镜。在这里，我们提出了制备薄，平的云母表面的脂质囊泡沉积和SLB准备一个简单的和可重复的方法。此外，我们的定制室只需要非常小的体积小泡的SLB形成。整个过程结果的高效，简单和廉价的生产高品质的脂质双分子层表面可直接媲美AFM研究使用。

在SLBs中的荧光脂质探针的扩散行为是根据不同的衬底上。 TIRFM结合的SMT技术是可视化的粒子运动并提取它们的扩散系数的一种有价值的方法。一个鞘磷脂-ATTO647N探针扩散在DOPC（1,2 -二油酰-sn-甘油基-3 -磷酸胆碱）支承于玻璃和云母双层的单分子的信号显示在附加的动画人物。在云母表面被根据这里提出的方案制备。为了估计光学像差，半高（FWHM）全宽进行测量，并利用马赛克二维PSF尺寸Ima...

Watch this Scientific Journal Video about Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging at JoVE.com

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

我们提出了制备云母支持的脂质双层膜为高分辨率显微镜的方法。云母是在原子尺度透明且平坦，但很少采用，因为处理困难成像;我们的制剂导致的云母薄片甚至沉积，并且降低了在双分子层制备中使用的材料。

支持的脂双层云母制备的高分辨率光学显微成像

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

preparation-mica-supported-lipid-bilayers-for-high-resolution-optical

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Bioengineering

13.3K 次观看.  Nanyang Technological University. 我们提出了制备云母支持的脂质双层膜为高分辨率显微镜的方法。云母是在原子尺度透明且平坦，但很少采用，因为处理困难成像;我们的制剂导致的云母薄片甚至沉积，并且降低了在双分子层制备中使用的材料。 

视频: Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions between lymphocytes and antigen presenting cells, we use Ni2+-chelating lipids to anchor recombinant cell adhesion and MHC proteins to the upper leaflet of a bilayer with poly-histidine tags. Planar bilayers are generated by preparing lipid, treating the glass surfaces where the bilayer will form, and then forming the bilayer in a specialized chamber containing a flow-cell where the lymphocytes will be added.  Then, bilayers are charged with Ni and his-tagged recombinant proteins are added.  Finally,  lymphocytes are injected into the flow cell and TIRF microscopy can be used to image synapse formation and the mechanisms that control T cell locomotion, sites of receptor sorting,  and sites of receptor degradation.

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse. Here, we show methods for anchoring cell adhesion proteins known to modulate synapse formation to the upper leaflet of the lipid bilyer and visualize synapse formation using TIRF microscopy.

形成免疫突触和Kinapse研究的支持平面双层膜

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions ...

科研

生物学

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

生物膜的微阵列用刮墨刀型组合方式形成

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

生物工程

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

原子力显微镜成像和支持的脂质双层力谱

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

生物膜制作的溶剂辅助脂质双分子层（SALB）方法

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

单分子荧光显微镜在平面支持双层膜

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

的自然杀伤细胞免疫突触上的玻璃支承平面脂双层超分辨率成像

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

免疫与感染

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

聚合物拥挤脂质膜上的单分子扩散和组装

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...

生物化学

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.

This protocol describes the formation of cell mimicking uni-lipid and multi-lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. These in vitro models can be adapted to incorporate a variety of lipid types and can be used to investigate various molecule and macromolecule interactions.

细胞模拟支持和悬浮脂质双分子层模型的组装用于分子相互作用研究

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial ...

Reconstitution of Membrane-Tethered Minimal Actin Cortices on Supported Lipid Bilayers

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane with the underlying actin cortex. In the past decades, reconstituted, minimal systems based on supported lipid bilayers in combination with actin filament networks have proven to be very instrumental in unraveling basic mechanisms and consequences of membrane-tethered actin networks, as well as in studying the functions of individual membrane-associated proteins. Here, we describe how to reconstitute such active composite systems in vitro that consist of fluid supported lipid bilayers coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors that can be readily observed via total internal reflection fluorescence microscopy. An open-chamber design allows one to assemble the system in a step-by-step manner and to systematically control many parameters such as linker protein concentration, actin concentration, actin filament length, actin/myosin ratio, as well as ATP levels. Finally, we discuss how to control the quality of the system, how to detect and troubleshoot commonly occurring problems, and some limitations of this system in comparison with the living cell surface.

This protocol describes the formation of supported lipid bilayers and the addition of cytoskeletal filaments and motor proteins to study the dynamics of reconstituted, membrane-tethered cytoskeletal networks using fluorescence microscopy.

在支持的脂质双层上重建膜系的最小肌动蛋白皮质

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane ...

Planar Supported Lipid Bilayer Assay: A Microscopy-Based In Vitro Technique to Investigate Septin Assembly on Biological Membrane Mimics

Source: Curtis, B. N. et al. <a href="https://www.jove.com/v/64090">Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions.</a> J. Vis. Exp. (2022)
In this video, we demonstrate a procedure to investigate septin organization on a glass-supported lipid bilayer in vitro using total internal reflection fluorescence microscopy.

平面支持的脂质双层测定:一种基于显微镜的体外技术，用于研究生物膜模拟物上的 septin 组装

Source: Curtis, B. N. et al. Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions. J. Vis. Exp. (2022)
In this ...