Name: ligand nano-cluster arrays in a supported lipid bilayer
Uploaded: 2017-04-23T14:00:00.000+00:00
Duration: 10 min 34 s
Description: Watch this Scientific Journal Video about Ligand Nano-cluster Arrays in a Supported Lipid Bilayer at JoVE.com

我们感谢洛朗Limozin，皮尔·迪拉德和阿斯特丽德·瓦赫勒继续有关手机应用富有成效的讨论。我们也从PLANETE洁净室设备感谢弗雷德里克·贝杜他与SEM观察的帮助。这项工作是部分由欧洲研究委员会通过批准号307104 FP / 2007-2013 / ERC资助。

1.清洗玻璃盖，幻灯片和观察室<ol><li>安排在诸如聚四氟乙烯（PTFE）由惰性材料制成的多滑动托盘玻璃盖滑动。 </li><li>浸入表面活性剂溶液与滑动托盘和观察室（推荐用于清洁石英比色杯中是合适的任何产物）。 </li><li>使用在室温下30分钟的表面活性剂溶液的超声波浴，超声处理（20和30℃之间）。 </li><li>漂洗用超纯水5倍（18.2MΩ.cm，0.059μS/ cm）的。 </li><li>超声处理中，在室温下30分钟表面活性剂溶液（20和30℃之间）。 </li><li>冲洗用超纯水10倍。 </li><li>重复步骤1.5和1.6的两倍。 </li><li>储存在室温下（20和30℃之间）的水长达一周。 </li></ol> 2.蛋白质纳米点的制备<ol><li>珠沉积<ol><li> Deposi吨悬浮液的以15度的倾斜保持二氧化硅珠（2％V / V，70μL）通过在盖滑动滴（24×24毫米，厚170微米）。 </li><li>让该悬浮液散布约1分钟，同时以90°每隔15秒翻转玻璃。 </li><li>允许液体在环境条件下蒸发。 </li><li>储存在清洁的玻璃或PTFE室中为2天环境条件。 </li></ol></li><li>的铝沉积<ol><li>放置一个射频内的载玻片（2.1节中制备）（RF）磁控溅射设备8，在旋转台在约105 mm处的铝（99％）的距离:硅（1％）的目标。 </li><li>使用涡轮分子泵将淀积室抽空到2.6×10 -4 Pa以下。这一步对于去除可能的气体杂质重要。 </li><li>与0.8帕（6.6毫托）的压力下的10sccm的助熔剂引入纯氩气气氛（5N，99.999％）。 <br/&gt;注:传入的氩气流量由质量流控制器控制，并使用压力表进行监测的过程中的压力。氩气流量是连续的，并且通过入口和出口打开阀之间的平衡控制。 </li><li>交换机上的RF功率发生器。 注意:在此，使用400-600W¯¯射频功率的在13.56MHz频率的典型范围。 RF发生器一起使用以电容耦合等离子体模式中的匹配网络。反射功率被监控，并且使用网络适配最小化。 </li><li>等离子体稳定后，溅射2分钟保持关闭，以从靶的表面除去可能的杂质快门。 </li><li>打开快门并允许溅射继续60分钟，在玻璃上沉积滑动铝至厚度为200纳米。 </li><li>切断氩气的流动，关闭闸阀来隔离从沉积腔室中的涡轮分子泵和排气用干净n中的腔室itrogen获得了房间的压力。回收的铝 - 包被的载玻片。存储长达一个月的环境条件下，清洁和密封的玻璃或PTFE框。 </li></ol></li><li>有机硅烷的气相沉积<ol><li>沉浸在步骤2.2.6在超纯水中，在室温下和超声处理在超声浴中制备的30秒铝包覆滑动件（50 W，50/60赫兹，20和30℃之间）。 </li><li>存款0.5 mL，在干燥器的底部（3-氨基丙基） - 三乙氧基硅烷（APTES）的。 注意:APTES为有机硅烷，其蒸发容易且是有毒的。 APTES只应在流罩和手套进行处理。 </li><li>把玻璃载片（在2.3.1的方法制备）在干燥器内的陶瓷网格和地点。 </li><li>干燥器连接到隔膜泵和在最大功率下运行30分钟，以产生低真空。 </li><li>合上干燥器的阀并关闭泵。 </li><li>加热干燥器至约50℃，持续1个小时。 </li><li>打开干燥器和收集的幻灯片。 </li><li>转移到另一个清洁干燥器中储存在室温下（20至30℃）达48小时。 </li></ol></li><li>蛋白的第一层的沉积 - 用生物素标记的牛血清白蛋白（BSA - 生物素） <ol><li>放置在PTFE支撑在第2.3节中制备的载玻片中的一个。 </li><li>存款1毫升溶解在磷酸盐缓冲盐水（PBS）25微克/毫升BSA - 生物素的。留在室温下（20至30℃）30分钟。 </li><li>用PBS冲洗10次。在4℃下存储最多24个小时将样品从光源远。 </li></ol></li><li>铝掩模的去除<ol><li>孵育载玻片在NaOH在PBS过夜，在室温（20至30℃）溶液（通过滴加1M NaOH中的制备约100毫升PBS中，以得到pH≈12）。 警告。氢氧化钠具有腐蚀性，应使用手套来处理。 </li><li>冲洗在超纯水中的10倍。 </li></ol></li></ol> 3.支持的脂质双层的沉积（SLB） <ol><li>清洁朗缪尔槽<ol><li>在使用泵的朗缪尔槽的PTFE外壳除去水。 </li><li>清洁用氯仿中浸过的无绒无纺一次性毛巾。 警告。氯仿是有毒的，并应在通风良好的区域进行操作，用掩模（或下一个流罩）中，用合适的手套。 </li><li>清洁4次用微温的超纯水（40〜50℃）。 </li><li>清洁至少6次用冷超纯水。 </li></ol></li><li>第一脂质层的沉积<ol><li>放置PTFE托盘在朗缪尔槽的PTFE外壳，然后用超纯水填充它。 </li><li>使用朗缪尔装置的控制软件来设置所测量的压力为0 mN / m的。 </li><li>使用气密的玻璃/金属注射器以沉积1mg / mL的脂质溶液的30微升（1，2- dioleoyl- SN中的水的表面上-glycero -3-磷酸胆碱（DOPC）的氯仿溶液）。将氯仿蒸发和脂质分子自发形成一个单层。 </li><li>使用控制软件通过关闭PTFE屏障直到所希望的压力（DOPC 27 mN / m的）达到以压缩脂质单层。 </li><li>使用控制软件浸在节2.5.2制备成使用机动夹具的PTFE外壳中的载玻片上。 </li><li>保持滑动，在夹具，垂直于空气 - 水界面。使用控制软件通过接口，以提高它慢慢（15毫米/分钟），同时保持恒定的压力在27 mN / m的。 </li><li>在干燥的环境的地方或者立即使用或储存于室温下（20和30℃之间）至24个小时。 </li></ol></li><li>第二脂质层的沉积<ol><li>维持朗缪尔槽的表面压力在27 mN / m的所需的值。压缩使用朗缪尔设备和/或控制软件添加脂质少量以实现期望的压力。 </li><li>放置携带脂质单层，在节3.2.6制备的，水平的水的表面上的玻璃玻片。确保每个滑动是浮动的PTFE托盘的上方。 </li><li>推玻璃向下滑动，一次一个滑动，到使用PTFE或金属镊子，使得它们被浸没在水中其相应的PTFE托盘。避免倾斜的幻灯片，同时推动。 </li><li>使用PTFE或金属镊子包含幻灯片的PTFE托盘转移到填充有超纯水确保幻灯片不暴露于空气中的结晶器中。 </li><li>使用PTFE或金属镊子来传输，而工作水下，双层涂覆的玻璃载片进观察室中。 注:观察腔室放置在水中结晶内部。观察室被定制，并且由PTFE环的用橡胶垫圈的ð钢外壳，该可组装的水下，使水密室，其底部表面从预先涂覆有脂质双层载玻片制成。 </li><li>合室中，同时继续在水下工作，并确保约1mL的水被截留在室的内部。 </li><li>就拿组装室出来的水。请确保它是防水的和无泄漏。 </li><li>通过添加和删除的PBS中的10倍500μL取代1mL的存在于与PBS的观察室的超纯水。确保该室是从未缺乏液体。 </li></ol></li><li> SLB封闭步骤<ol><li>在包含在3.3.6节中制备的载玻片上的观察室引入100微克/毫升的牛血清白蛋白（BSA）和孵育在室温下（20和30℃之间）30分钟。 </li><li>通过删除和添加的PBS中的10倍500μL冲洗双层。该双层可在4℃下保持24个小时。 </li></ol></li></ol> 4.功能化与配体<ol><li>在2μg/ mL的添加荧光或非荧光中性（NAV，抗生物素蛋白的去糖基化版本不在pH 7充电）到含有在室温下在部分3.4.2制备30分钟的滑动件（20和30°之间的腔室C）。 </li><li>通过添加和删除的PBS中的10倍500μL冲洗。 </li><li>在2μg/ mL的生物素化的添加抗CD3到含有在3.4.2节中制备的载玻片室中。对于SLB所述点的双官能化，以5μg/ mL的（这里，双层由DOPC的次氮基三乙酸（NTA）的脂质的+ 1％）添加的Fc-ICAM1 His标签。留在室温下（20和30℃之间）30分钟。 </li><li>通过添加和删除的PBS500μL的10倍冲洗</li><li>通过添加和移除细胞培养基的500微升10倍替换细胞培养基的PBS（PBS + 0.1％BSA）。 </li><li>离开细胞培养基的200微升的chambeR 2与滑动。 </li><li>添加细胞前，放置室，用于在37℃下10分钟。 </li></ol> 5.细胞沉积（详见参考文献7） <ol><li>仔细沉积400μL细胞悬浮液的腔室中。温育30分钟将细胞在37℃。 </li><li>固定用多聚甲醛的2％（PFA）的细胞在37℃下15分钟。通过反复删除和添加的PBS的500微升替换用PBS PFA。 </li></ol> 6.观察<ol><li> PROTEO脂质的纳米图案和SLB流动性<ol><li>使用适当的照明波长（639纳米）和滤波器立方体使用荧光显微镜图像在落射荧光模式的蛋白质图案（ 例如这里，EX TBP 483 + 564 + 642; BS TFT 506 + 582 + 659; EM TBP 526 + 601 688）。 注:该信号的强度可以被量化以估计内部和外部点的蛋白质的量。使用适当的照明波长（330纳米）和fILTER立方体图像双层（BP12分之365，FT 395，LP 397），这似乎与亮暗孔。 </li><li>使用连续光漂白（CPB）技术13,15以测量示踪剂的脂质双层中的扩散常数。这一观察刚沉积SLB后优选由。 注意:为了量化扩散常数，两个参数在CPB方法测定:将染料（拉紧）和周围的漂白区（TAUD）形成晕圈的荧光分布的衰减长度的特定漂白时间。首先是通过在照明场的期间CPB过程中心绘制的荧光强度随时间的变化而获得。第二个是通过在照射区（在达到稳态后平均超过12行和第5个图像）的边缘绘制的强度分布而获得。曲线被装配以获得绷紧和TAUD。扩散常数由TA给出UD 2 /拉紧（D = <img alt="方程" src="/files/ftp_upload/55060/55060eq1.jpg" /> ）。 </li></ol></li></ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+spreading+and+focal+adhesion+dynamics+are+regulated+by+spacing+of+integrin+ligands.">Cell spreading and focal adhesion dynamics are regulated by spacing of integrin ligands.</a> Biophys J. 92 (8), 2964-2974 (2007).</li><li>Schvartzman, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanolithographic+Control+of+the+Spatial+Organization+of+Cellular+Adhesion+Receptors+at+the+Single-Molecule+Level.">Nanolithographic Control of the Spatial Organization of Cellular Adhesion Receptors at the Single-Molecule Level.</a> Nano Lett. 11 (3), 1306-1312 (2011).</li><li>Mossman, K., Groves, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterned+supported+membranes+as+tools+for+quantitative+studies+of+the+immunological+synapse.">Micropatterned supported membranes as tools for quantitative studies of the immunological synapse.</a> Chem.Soc.Rev. 36 (1), 46-54 (2007).</li><li>Furlan, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatase+CD45+both+positively+and+negatively+regulates+T+cell+receptor+phosphorylation+in+reconstituted+membrane+protein+clusters.">Phosphatase CD45 both positively and negatively regulates T cell receptor phosphorylation in reconstituted membrane protein clusters.</a> J Biol Chem. 289 (41), 28514-28525 (2014).</li><li>Hsu, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand+mobility+modulates+immunological+synapse+formation+and+T+cell+activation.">Ligand mobility modulates immunological synapse formation and T cell activation.</a> PloS One. 7 (2), e32398 (2012).</li><li>Yu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+integrin+binding+to+Arg-Gly-Asp+peptide+activates+actin+polymerization+and+contractile+movement+that+stimulates+outward+translocation.">Early integrin binding to Arg-Gly-Asp peptide activates actin polymerization and contractile movement that stimulates outward translocation.</a> Proc Natl Acad Sci U S A. 108 (51), 20585-20590 (2011).</li></ol>

作者什么都没有透露。

上述方案中的关键步骤是通过一个支撑脂双层相关的蛋白质纳米点或这些点周围的空间的回填的形成。相对于蛋白质纳米点的第一个关键步骤是胎圈掩模的制备方法。盖滑动的清洁是至关重要的。将载玻片需要与被推荐用于清洗石英比色杯中，或用氧等离子体的洗涤剂溶液中或者清洗。其它清洁技术，如浸渍在乙醇或异丙醇处理不会使玻璃充分亲水，并因此不支持大覆盖珠单层的形成。与此同时?...

<html>细胞粘附通过专门的细胞粘附分子（凸轮），蛋白质存在于细胞膜，其能够在细胞外基质或在另一细胞结合到其对应的发生。上粘附的细胞，最粘附分子包括整联无处不和钙粘蛋白，在簇1中的形式存在。 T淋巴细胞（T细胞）与抗原呈递细胞（的APCs）的相互作用提供的在两个小区之间的界面形成受体簇的重要性特别引人注目插图 - 通常被称为免疫突触。在形成与充当信令的平台2，3，4，并最终集中的T细胞形式微米尺度簇的表面上的APC，T细胞受体（TCR）的第一接触，以形成一个较大的中心超分子簇（CSMAC ）小姑娘= "外部参照"&gt; 5，6，7。最近，已表明在APC上侧，所述TCR的配体也聚集8。 在T细胞的APC相互作用，混合动力系统的部署，其中APC通过与相关蛋白官能化的人造表面模仿的上下文中，已大大提高了我们突触接口2，3，4，5，6，7的理解。在这种情况下，它是设计APC模拟表面可捕捉到目标小区的一个或多个方面密切相关。例如，如果配体是在支持的脂质双层接枝，它们可以以双层的平面扩散，模仿APC表面上的情况，并在同一时间允许的形成CSMAC 6,7。类似地，APC上的簇已经通过在聚合物9，10，11，12，13，14的海创建配体的岛屿模仿。然而，这两个功能迄今尚未合并。 在这里，我们描述了一种新技术，以创建由脂质双层与扩散脂质包围抗CD3（靶向TCR复合物的抗体）的纳米点。双层使用朗缪尔-布罗杰特/朗缪尔-谢弗技术7，15，16沉积并如果需要的话，可以用一个特定的蛋白质被官能化-例如，T细胞整联配体（称为ICAM1）。此外，抗CD3蛋白点腠LD与另一种抗体或CAM来代替。虽然我们选择的蛋白质如T细胞黏附的研究平台将来使用，这里详细的策略可以适用于任何的蛋白质和DNA，甚至。

<table><tbody><tr><td>Glass coverslips</td><td>Assistent, Karl Hecht KG&amp;nbsp;</td><td></td><td></td></tr><tr><td>Observation chamber</td><td>Home made</td><td></td><td></td></tr><tr><td>Alkaline surfactant concentrate (Hellmanex)</td><td>Hella Analytics</td><td>9-307-011-4-507</td><td></td></tr><tr><td>Ultra-sonicator</td><td>ThermoFisher</td><td></td><td></td></tr><tr><td>Desiccator</td><td>Labbox</td><td></td><td></td></tr><tr><td>Crystallizer&amp;nbsp;</td><td>Shott</td><td></td><td></td></tr><tr><td>Neutravidine</td><td>Thermo Fischer Scientifique</td><td>84607</td><td></td></tr><tr><td>PBS&amp;nbsp;</td><td>Sigma-aldrich</td><td>P3813</td><td></td></tr><tr><td>Water MQ&amp;nbsp;</td><td>ELGA, Veolia France</td><td></td><td></td></tr><tr><td>Silica beads</td><td>Corpuscular Inc</td><td>147114-10</td><td></td></tr><tr><td>APTES</td><td>Sigma-aldrich</td><td>A3648</td><td></td></tr><tr><td>BSA-Biotin</td><td>Sigma-aldrich</td><td>A8549</td><td></td></tr><tr><td>DOPC</td><td>Avanti Polar Lipids</td><td>850375C</td><td></td></tr><tr><td>Dansyl-PE</td><td>Avanti Polar Lipids</td><td>810330C</td><td></td></tr><tr><td>Chloroform</td><td>Sigma-aldrich</td><td>650471</td><td></td></tr><tr><td>Gastight syringe&amp;nbsp;</td><td>Dominique Dustcher , France</td><td>74453</td><td></td></tr><tr><td>Film balance</td><td>NIMA</td><td>Medium</td><td></td></tr><tr><td>Microscope</td><td>Zeiss, Germany</td><td>TIRF-III system</td><td></td></tr><tr><td>Aluminium Target&amp;nbsp;</td><td>Kurt J. Lesker Compagny, USA</td><td></td><td></td></tr><tr><td>Radio Frequency Magnetron sputtering Syst&amp;egrave;me&amp;nbsp;</td><td>modified SMC 600 tool by ALCATEL , France</td><td></td><td></td></tr></tbody></table>

ligand nano-cluster arrays in a supported lipid bilayer

目前，处于钝化表面的细胞生物学研究的海洋产生黏附蛋白岛的有序阵列相当大的兴趣。在过去几年里，它已成为越来越清楚地看到活细胞反应，不仅能呈现给他们的分子的生化性质，而且这些分子的显示方式。创建蛋白微图案因此现在在许多生物学实验室的标准;纳米图案也更方便。然而，在细胞间相互作用的背景下，有必要图案不仅蛋白质，而且脂质双层。这种双PROTEO脂质的图案至今没有得到方便。我们提供了一个浅显的技术来创建支持玻璃蛋白质纳米点，提出了一种方法，用支撑脂双层（SLB）回填点间的空间。从示踪剂的光漂白包括在SLB荧光脂质，我们证明了双层的-PL表现出相当大的ANE流动性。用荧光基团功能化的蛋白点，使我们能够像他们，并表明他们在规则六边形网格排列。典型的点尺寸是大约800nm和这里展示的间距为2微米。这些基板有望成为用于细胞粘附，迁移和机械 - 感测研究，作为有用的平台。

的荧光图像进行分析，以测量点的间距和尺寸。典型间隔被发现是1900±80 nm和典型点大小为600±100纳米（ 图1G）。的间距用的用于掩模珠的大小设置。的点尺寸是由珠尺寸以及沉积条件设定。 SLB被唯一地围绕蛋白质点和不淀积在它们（ 图2），在SLB成像通道看到的孔，并在NAV成像通道看到的点之间的完美的互补性。连续光漂白数据的分析表明，在?...

Watch this Scientific Journal Video about Ligand Nano-cluster Arrays in a Supported Lipid Bilayer at JoVE.com

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

我们提出了一个协议官能玻璃与由流体脂质双层包围的纳米蛋白贴剂。这些基材与先进的光学显微镜兼容，并有望作为细胞黏附和迁移的研究平台。

在支撑脂双层配体纳米簇阵列

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

ligand-nano-cluster-arrays-in-a-supported-lipid-bilayer

Research

JoVE Journal

Bioengineering

6.9K 次观看.  CNRS, UMR 7325, CINaM. 我们提出了一个协议官能玻璃与由流体脂质双层包围的纳米蛋白贴剂。这些基材与先进的光学显微镜兼容，并有望作为细胞黏附和迁移的研究平台。 

视频: Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

方法可视化和用透射电子显微镜分析膜蛋白的相互作用

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

科研

生物化学

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

内部自组装脂质颗粒的简易制备碳纳米管稳定

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

化学

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

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

用于膜蛋白-蛋白质相互作用分析的原生细胞膜纳米粒子系统

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.

自动化脂双层膜的形成使用聚二甲基硅氧烷薄膜

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

Self-Assembly of Hybrid Lipid Membranes Doped with Hydrophobic Organic Molecules at the Water/Air Interface

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers can be readily functionalized and have been used in various applications such as bio-sensors and bio-devices. In this study, we introduced a planar organic molecule: copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) to dope lipid membranes. The CuPc/lipid hybrid membrane forms at the water/air interface by self-assembly. In this membrane, the hydrophobic CuPc molecules are located between the hydrophobic tails of lipid molecules, forming a lipid/CuPc/lipid sandwich structure. Interestingly, an air-stable hybrid lipid bilayer can be readily formed by transferring the hybrid membrane onto a Si substrate. We report a straightforward method for incorporating nanomaterials into a lipid bilayer system, which represents a new methodology for the fabrication of biosensors and biodevices.

We report a protocol for producing a hybrid lipid membrane at the water/air interface by doping the lipid bilayer with copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) molecules. The resulting hybrid lipid membrane has a lipid/CuPc/lipid sandwich structure. This protocol can also be applied to the formation of other functional nanomaterials.

在水/空气界面上掺杂与疏水有机分子混合脂膜的自组装

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers ...

Sample Preparation using a Lipid Monolayer Method for Electron Crystallographic Studies

Electron crystallography is a powerful tool for high-resolution structure determination. Macromolecules such as soluble or membrane proteins can be grown into highly ordered two-dimensional (2D) crystals under favorable conditions. The quality of the grown 2D crystals is crucial to the resolution of the final reconstruction via 2D image processing. Over the years, lipid monolayers have been used as a supporting layer to foster the 2D crystallization of peripheral membrane proteins as well as soluble proteins. This method can also be applied to 2D crystallization of integral membrane proteins but requires more extensive empirical investigation to determine detergent and dialysis conditions to promote partitioning to the monolayer. A lipid monolayer forms at the air-water interface such that the polar lipid head groups remain hydrated in the aqueous phase and the non-polar, acyl chains, tails partition into the air, breaking the surface tension and flattening the water surface. The charged nature or distinctive chemical moieties of the head groups provide affinity for proteins in solution, promoting binding for 2D array formation. A newly formed monolayer with the 2D array can be readily transfer into an electron microscope (EM) on a carbon-coated copper grid used to lift and support the crystalline array. In this work, we describe a lipid monolayer methodology for cryogenic electron microscopic (cryo-EM) imaging.

Lipid monolayers have been used as a foundation for forming two-dimensional (2D) protein crystals for structural studies for decades. They are stable at the air-water interface and can serve as a thin supporting material for electron imaging. Here we present the proven steps on preparing lipid monolayers for biological studies.

使用脂质单层方法进行电子晶体学研究的样品制备

Electron crystallography is a powerful tool for high-resolution structure determination. Macromolecules such as soluble or membrane proteins can be grown ...

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

Membrane curvature plays important roles&#160;in various essential processes of cells, such as cell migration, cell division, and vesicle trafficking. It is not only passively generated by cellular activities, but also actively regulates protein interactions and is involved in many intracellular signaling. Thus, it is of great value to examine the role of membrane curvature in regulating the distribution and dynamics of proteins and lipids. Recently, many techniques have been developed to study the relationship between the curved membrane and protein in vitro. Compared to traditional techniques, the newly developed nanobar-supported lipid bilayer (SLB) offers both high-throughput and better accuracy in membrane curvature generation by forming a continuous lipid bilayer on patterned arrays of nanobars with a pre-defined membrane curvature and local flat control. Both the lipid fluidity and protein sensitivity to curved membranes can be quantitatively characterized using fluorescence microscopy imaging. Here, a detailed procedure on how to form a SLB on fabricated glass surfaces containing nanobar arrays&#160;and the characterization of curvature-sensitive proteins on such SLB are introduced. In addition, protocols for nanochip reusing and image processing are covered. Beyond the nanobar-SLB, this protocol is readily applicable to all types of nanostructured glass chips for curvature sensing studies.

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

Here, a nanobar-supported lipid bilayer system is developed to provide a synthetic membrane with a defined curvature that enables the characterization of proteins with curvature sensing ability in vitro.

纳米巴支持的脂质双层系统，用于体外研究膜曲率传感蛋白

Membrane curvature plays important roles&#160;in various essential processes of cells, such as cell migration, cell division, and vesicle trafficking. It ...

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 ...

脂质膜上的光控可逆蛋白质图案

Dynamic Light-Induced Protein Patterns at Model Membranes

The precise localization and activation of proteins at the cell membrane at a certain time gives rise to many cellular processes, including cell polarization, migration, and division. Thus, methods to recruit proteins to model membranes with subcellular resolution and high temporal control are essential when reproducing and controlling such processes in synthetic cells. Here, a method is described for fabricating light-regulated reversible protein patterns at lipid membranes with high spatiotemporal precision. For this purpose, we immobilize the photoswitchable protein iLID (improved light-inducible dimer) on supported lipid bilayers (SLBs) and on the outer membrane of giant unilamellar vesicles (GUVs). Upon local blue light illumination, iLID binds to its partner Nano (wild-type SspB) and allows the recruitment of any protein of interest (POI) fused to Nano from the solution to the illuminated area on the membrane. This binding is reversible in the dark, which provides dynamic binding and release of the POI. Overall, this is a flexible and versatile method for regulating the localization of proteins with high precision in space and time using blue light.

作者聚焦:用于人工细胞系统中可逆模式的光可切换蛋白质募集

Here, a protocol is described for generating light-regulated and reversible protein patterns with high spatiotemporal precision at artificial lipid membranes. The method consists of the localized photoactivation of the protein iLID (improved light-inducible dimer) immobilized on model membranes that, under blue light, binds to its partner protein Nano (wild-type SspB).