作者要感谢Emilie Josse对手稿的批判性评论。这项工作得到了"癌症联盟"基金会、"癌症研究基金会"和"国家科学研究中心"（CNRS）的支持。

1. 大型单层囊泡（LUV）的制备
<ol><li>准备LUV，用作荧光泄漏测定的细胞膜模拟物。</li>
	<li>与汉密尔顿玻璃注射器磷脂酰胆碱（DOPC，786.11克/摩尔），鞘磷脂（SM，760.22克/摩尔）和胆固醇（Chol，386.65克/摩尔）以4:4:2的摩尔比混合。脂质溶液从在甲醇/氯仿（3/1;体积/体积）溶剂中以25mg/mL在25mL玻璃圆底烧瓶中溶解的每种脂质的储备溶液中获得。基于4μmol的DOPC，4μmol的SM和2μmol的Chol，通过分别混合126μL，117μL和31μL从储备溶液中获得脂质溶液。 注意:甲醇是一种有毒易燃溶剂，氯仿有毒且致癌。两者都应在引擎盖下进行适当的保护。</li>
	<li>在60°C下真空下使用旋转蒸发器蒸发甲醇/氯仿45-60分钟，直到形成干燥的脂膜。</li>
	<li>准备两种高效浓缩液缓冲液。通过混合20 mM HEPES（238.3 g / mol）和75 mM NaCl（58.44 g / mol）来制备HEPES缓冲液1，并将pH值调节至7.4。通过混合20 mM HEPES和145 mM NaCl来制备HEPES缓冲液2，并将pH调节至7.4。HEPES缓冲液可以在4°C下储存1个月。 注意:建议使用渗透压计检查缓冲液的渗透压。</li>
	<li>通过将膜不透性的荧光染料淬灭剂对，8-氨基萘-1，3，6-三磺酸，12.5mM（ANTS，427.33 g / mol）的二钠盐和对二甲苯 - 双吡啶溴化物以45mM（DPX，422.16 g / mol）溶解在HEPES缓冲溶液中来制备脂质水合溶液。将蚂蚁与DPX混合可得到黄色溶液。为了达到12.5mM的ANTS和45mM的DPX的浓度，分别溶解在4mLHEPES缓冲液1中21.4mg和76mg。 注意:脂质水合溶液可以通过用铝箔包裹管在4°C下储存2周。</li>
	<li>通过用1mL脂质水合溶液重悬干燥的脂质膜并通过涡旋直到干燥的脂质膜溶解来重建多层囊泡（MLV）。确保溶液完全溶解，因为小脂质聚集体会对上述步骤产生负面影响。另外，检查玻璃圆底烧瓶的壁，以确保没有残留的脂质膜。 注意:溶解后溶液会出现乳白色和淡黄色。</li>
	<li>将囊泡置于五个冷冻/解冻循环中，以获得单层囊泡。通过将玻璃圆底烧瓶在液氮中放置30秒以进行冷冻步骤，然后将其留在水浴中2分钟进行解冻步骤来执行每个循环。 注意:洗澡水的温度应比脂质的熔化温度高5-10°C。</li>
	<li>通过将两个过滤载体插入预先加湿的HEPES缓冲液在每个聚四氟乙烯（PTFE）挤出机片中置于金属挤出机罐中的固体挤出机来制备脂质挤出机。</li>
	<li>将HEPES加湿聚碳酸酯膜（孔径为0.1μm，直径25 mm）放在一个过滤器支架的顶部。</li>
	<li>组装两个金属挤出机罐并拧紧它们。</li>
	<li>将组装好的挤出机放入支架中，并在每个聚四氟乙烯挤出机片末端的适当孔中引入1 mL注射器。挤出对应于被测液体从一个注射器通过聚碳酸酯膜到另一个注射器的通道。</li>
	<li>使用装入 1 mL 注射器之一的 1 mL HEPES 缓冲液测试挤出机，以确保没有泄漏或问题。</li>
	<li>用MLV样品代替1 mL HEPES缓冲液。</li>
	<li>通过将MLV样品从一个注射器通过聚碳酸酯膜传递到另一个注射器至少21次来进行挤出，以获得相同尺寸的均匀LUV。 注意:挤出应在高于脂质混合物熔化温度的温度下进行。</li>
</ol>2. 润滑油的净化
<ol><li>准备色谱柱纯化以除去未封装的ANTS和DPX过量。</li>
	<li>将交联葡聚糖凝胶（G-50）重悬于液相色谱柱（鲁尔锁，非夹套，1.0厘米×20 厘米，床体积16毫升）中，重悬于0.01%NaN 3（65克/摩尔）的水性培养基中，直至柱无色部分顶部下方1厘米处。</li>
	<li>打开水龙头，让液体流动以沉降交联葡聚糖凝胶。</li>
	<li>通过用20 mL HEPES缓冲液2洗脱来洗涤色谱柱，并丢弃色谱柱的输出流量。</li>
	<li>一旦柱上方溶剂的死体积最小化（<100μL），但足以避免交联葡聚糖凝胶的任何干燥，请关闭水龙头。</li>
	<li>将新鲜挤出的LUV（黄色）放在色谱柱上，让它们进入交联的葡聚糖凝胶中。</li>
	<li>连续向色谱柱中加入HEPES缓冲液2以执行LUV纯化。</li>
	<li>洗脱约2 mL HEPES缓冲液2（不要忘记定期填充色谱柱顶部以避免干燥交联葡聚糖凝胶）:游离的黄色ANTS和DPX溶液的迁移速度比脂质体慢。</li>
	<li>开始在试管（1.5 mL）中收集纯化的LUV。</li>
	<li>观察柱中的淋洗液滴，当它们变成乳白色时，它们含有脂质体。更换试管以回收含LUV的馏分。</li>
	<li>洗脱，直到滴剂不再乳白色（约1毫升）。之后，在单独的馏分中洗脱另外0.5 mL，然后停止洗脱。 注意:标准品现在提供多种分子量，如试剂盒或单个分子量，用于校准LUV的洗脱体积。</li>
	<li>用LUV包裹在铝箔中的管子，以避免荧光染料漂白。</li>
	<li>用20 mL HEPES缓冲液洗涤色谱柱 2.</li>
	<li>然后，LUV可以在4°C下储存一周。 注意:由于LUV稳定性可能取决于LUV浓度和组成以及离子强度，因此应使用动态光散射（DLS）仪器控制LUV的大小（见第4节）。在每次测试之前表征LUV尺寸和均匀性）。</li>
</ol>3. 量化LUV的浓度
<ol><li>通过磷脂定量试剂盒估计LUV浓度，从而可以评估胆碱浓度19。当含有胆碱的磷脂含量很大（>50%的LUV）时，可以应用该测定。</li>
	<li>通过将18mg染色原底物溶解在提供的3mL缓冲液中来制备颜色试剂。</li>
	<li>装入 10 x 10 x 45 mm 的聚苯乙烯比色皿，加入 3 mL 彩色试剂。</li>
	<li>使用纯色试剂作为空白条件（空白）。加入20μL LUV样品（测试）或20μL已知胆碱浓度的标准溶液（标准）。</li>
	<li>充分混合并在所有条件（空白，测试和标准）下在37°C下孵育5分钟。</li>
	<li>用分光光度计在600nm处以空白溶液作为对照，测量测试样品和标准溶液的吸光度（光密度，OD）。</li>
	<li>检查OD值，以便能够估计与已知浓度的标准相比，胆碱当量的LUV，C[LUV]的脂质浓度。</li>
	<li>使用以下等式执行计算: C[LUV] （摩尔 / 升） = （OD 样品 / OD 标准） x C[标准] （摩尔 / 升） 注意:磷脂定量试剂盒提供了氯化胆碱（139.6 mg / l）标准溶液，浓度为54 mg / dL，对应于C[标准]的摩尔浓度=3.87 mmol/ L. OD样品和OD标准品分别是在600nm处测量的LUV和胆碱溶液的吸光度。</li>
</ol>4. LUV尺寸和均匀性的表征
<ol><li>使用DLS仪器进行测量，以确定LUV尺寸（以nm为单位）和多分散指数（PdI）。</li>
	<li>通过指示溶剂/缓冲液和所用比色皿的粘度来对适当的"标准操作程序"（SOP）进行编程。</li>
	<li>将500μLLUV溶液置于聚苯乙烯半微量比色皿中。</li>
	<li>将聚苯乙烯半微量比色皿插入DLS仪器中。</li>
	<li>在室温下，根据颗粒分布的平均尺寸（Z平均值）和均匀性（多分散指数，PdI）测量尺寸分布。</li>
	<li>所有结果都是通过三个重复周期进行的两次独立测量获得的。 注意:LUV 的标准值的平均大小为 137 ± 7 nm，PdI 为 0.149 ± 0.041。</li>
</ol>5. 制备肽溶液
<ol><li>准备肽的储备溶液，应对其进行泄漏测定分析。</li>
	<li>将肽粉（纯度>95%）溶解在纯水（例如，1mg肽在500μL纯水中）。 注意:建议在纯水中稀释肽并避免二甲基亚砜（DMSO）增溶，这可能会诱发伪影（例如，膜透化20）。</li>
	<li>涡旋肽溶液5 s。</li>
	<li>在水超声处理浴中超声处理肽溶液5分钟，然后在12，225×g下离心5分钟。 收集上清液进行浓度测定。</li>
	<li>测量三种独立肽稀释液在280nm处的吸光度，然后使用其摩尔消光系数ε（取决于肽序列中的色氨酸和酪氨酸含量）和Beer-Lambert规则计算肽浓度。 注意:如果肽含有色氨酸和酪氨酸，则根据色氨酸ε = 5，690 M -1 cm-1和酪氨酸ε = 1，280 M-1cm-1计算摩尔消光系数ε。如果肽序列不含色氨酸或酪氨酸，则可以进行其他比色测定来测量浓度（例如，BCA或Bradford）。</li>
	<li>将肽溶液在纯水中稀释至100μM的最终溶液并储存在4°C。 注意:在纯水中，在4°C储存期间不会发生肽降解。然而，肽浓度应每2周测量一次，以确保不会发生水分蒸发。</li>
</ol>6. 荧光泄漏测定
<ol><li>荧光泄漏测定在室温下在光谱仪上测量。激发和发射波长分别固定在Ex = 360 nm±3 nm和Em = 530 nm±5 nm。</li>
	<li>在石英荧光比色皿中将1 mL HEPES缓冲液2中的LUV稀释至终浓度为100μM。在实验过程中加入磁力搅拌器使溶液均质化。</li>
	<li>在前100秒内，在t = 0 s和t = 99 s之间单独测量LUV，以便访问背景荧光。 注意:在整个实验（15分钟）期间，也可以单独测量LUV，以便访问背景荧光和潜在的泄漏。</li>
	<li>此后，在接下来的900秒（15分钟）中，在加入肽溶液等分试样时，测量泄漏作为荧光强度的增加。该方案对测试的肽浓度从0.1μM到2.5μM进行。</li>
	<li>最后，通过加入1μLTriton X-100（0.1%，v / v）来溶解LUV，从而实现100%荧光，从而在t = 1，000 s和t = 1，100 s之间完全未淬火的探针。</li>
</ol>7. 泄漏量化
<ol><li>抑制 t = 1，090 s 后获得的值，以便为每个测试条件保持相同的点数。</li>
	<li>通过使t = 0 s和t = 49 s（仅LUV）之间的50个点的平均值来计算最小荧光Fmin。</li>
	<li>计算最大荧光Fmax，方法是在t = 1，041 s和t = 1，090 s之间取50个点的平均值（使用Triton X-100的LUV）。</li>
	<li>根据以下等式计算每个时间点（t = x）的泄漏百分比（%泄漏）: 泄漏百分比（t=x） = （F（t=x） - F最小值 ） / （F最大 值 - F最小值） x 100</li>
	<li>计算相同条件下使用不同 LUV 制备（n ≥ 2）获得的值的平均值和标准偏差。</li>
	<li>绘制泄漏百分比 %Leak（t=x），以时间 （s） 的函数表示。</li>
</ol>

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Binding mechanism and thermodynamic analysis.</a> Biochemistry. 42 (30), 9185-9194 (2003).</li><li>Thor&#233;n, P. E. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorus+assay+in+column+chromatography.">Phosphorus assay in column chromatography.</a> Journal of Biological Chemistry. 234 (3), 466-468 (1959).</li><li>Stewart, J. C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+determination+of+phospholipids+with+ammonium+ferrothiocyanate.">Colorimetric determination of phospholipids with ammonium ferrothiocyanate.</a> Analytical Biochemistry. 104 (1), 10-14 (1980).</li><li>Spinella, S. A., Nelson, R. B., Elmore, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+peptide+translocation+into+large+unilamellar+vesicles.">Measuring peptide translocation into large unilamellar vesicles.</a> Journal of Visualized Experiments: JoVE. (59), e3571 (2012).</li><li>Wimley, W. 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作者没有利益冲突。

所提出的荧光泄漏测定是一种简单快速的方法，用于解决细胞穿透肽的膜不稳定。它很容易做到，还可以间接比较不同的膜相互作用肽或其他膜相互作用分子。关于方案的关键步骤，由于该测定提供了基线（仅LUV）和最大荧光释放（Triton条件）之间的相对值，我们通常使用磷脂定量试剂盒评估LUV的浓度，该试剂盒仅估计LUV的胆碱贡献。然而，也可以包括更准确地测量LUV浓度，方法是通过Rouser及其?...

在九十年代发现它们之后，细胞穿透肽（CPPs）被开发用于促进货物通过质膜的高效细胞递送1，2。CPP通常是短肽，通常为8至30个氨基酸，具有多种来源。它们首先被定义为"直接易位"携带者，这意味着它们能够穿过质膜并将货物转移到细胞中，而不受任何内吞途径的影响，既不要求能量也不参与受体。然而，进一步的研究表明，这些最初的观察结果主要来自由于实验伪影和/或使用甲醇3的固定方案而导致的荧光高估。如今，人们普遍认为CPP摄取是通过内吞作用和能量无关的易位4，5，6，7发生的，这取决于不同的参数，例如货物的性质，CPP和货物之间的使用链接，研究的细胞系等。
CPP可以根据两种策略用作转染剂，要么涉及CPP与其货物之间的化学链接（共价策略）或静电/疏水相互作用（非共价策略）8，9，10，11。尽管这两种策略都显示出它们在几种货物的细胞转移中的效率，但对CPP内化机制的理解仍然存在争议，并且内吞作用途径或直接渗透之间的平衡仍然难以测量12，13。虽然有一套实验工具和策略可以清楚地解决内吞过程的参与问题，但直接易位似乎更难表征，因为它意味着与质膜组分的更离散的相互作用。生物膜通常由许多组分组成，从磷脂到膜蛋白，这些组分可能根据细胞类型和/或环境（应激条件，细胞分裂等）而变化。这种组成的多样性，以及因此缺乏通用细胞膜模型，无法以单一方式进行研究。然而，为了规避这些限制，用人造膜或膜提取物开发了分步方法。从小型单层囊泡到单层方法，每个模型显然都与回答特定问题14，15相关。其中，大型单层囊泡（LUV）构成适当的膜模拟模型，以研究肽/膜相互作用作为内化过程的关键点。
在这种情况下，以下方案描述了通过监测阴离子荧光染料及其封装在脂质体中的相应多阳离子淬灭剂来研究肽和肽/膜相互作用对LUV完整性的影响。该工具用于研究CPP /膜相互作用，以了解它们是否能够进行直接膜易位。虽然通常用于比较不同的膜相互作用肽，但这种LUV荧光泄漏测定也可用于研究CPPs-cargo偶联物（共价策略）和CPP:cargo复合物（非共价策略）。
因此，本方案将首先以最近开发的色氨酸（W）-和精氨酸（R）富两性肽（WRAP）16为例。WRAP能够形成基于肽的纳米颗粒，以快速有效地在几个细胞系中递送小干扰RNA（siRNA）16。监测单独使用WRAP肽或siRNA负载的WRAP基纳米颗粒的荧光泄漏特性，以表征其细胞内化机制。结果表明，它们的内化机制主要涉及直接易位7。在第二个例子中，WRAP肽与蛋白质/蛋白质干扰肽iCAL36（WRAP-iCAL36）17共价偶联，并且在荧光泄漏测定中将其破坏膜稳定性的能力与iCAL36偶联到另一种CPP的Interrin 18（Penetratin-iCAL36）进行比较。
最后，将从技术角度和生物学相关性的角度讨论该方法的优点和局限性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >25 mL glass round-bottom flask</td>
			<td >Pyrex</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >8-aminonaphthalene-1, 3, 6-trisulfonic acid, disodium salt (ANTS)</td>
			<td >Invitrogen</td>
			<td >A350</td>
			<td>Protect from light&#160;</td>
		</tr>
		<tr >
			<td >Chloroform&#160;</td>
			<td >Sigma-Aldrich</td>
			<td >288306</td>
			<td></td>
		</tr>
		<tr >
			<td >Cholesterol</td>
			<td >Sigma-Aldrich</td>
			<td >C8667</td>
			<td></td>
		</tr>
		<tr >
			<td >DOPC (dioleoylphosphatidylcholine)</td>
			<td >Avanti Polar</td>
			<td>850375P</td>
			<td>Protect from air</td>
		</tr>
		<tr >
			<td >Extruder</td>
			<td >Avanti Polar</td>
			<td >610000</td>
			<td></td>
		</tr>
		<tr >
			<td >Fluorimeter</td>
			<td >PTI Serlabo</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >50 &#181;L glass syringe</td>
			<td >Hamilton</td>
			<td >705N</td>
			<td></td>
		</tr>
		<tr >
			<td >HEPES</td>
			<td >Sigma-Aldrich</td>
			<td >H3375</td>
			<td></td>
		</tr>
		<tr >
			<td >LabAssay Phospholipid&#160;</td>
			<td >WAKO&#160;</td>
			<td >296-63801</td>
			<td></td>
		</tr>
		<tr >
			<td >liquid chromatography column</td>
			<td >Sigma-Aldrich</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Methanol</td>
			<td >Carlo Erba</td>
			<td >414902</td>
			<td></td>
		</tr>
		<tr >
			<td >Nuclepore polycarbonate membrane (0.1 &#181;m pore size, 25 mm diameter)</td>
			<td >Whatman</td>
			<td >800309</td>
			<td></td>
		</tr>
		<tr >
			<td >polystyrene cuvette, 10 x 10 x 45 mm</td>
			<td >Grener Bio-One</td>
			<td >614101</td>
			<td></td>
		</tr>
		<tr >
			<td >polystyrene semi-micro cuvette, DLS</td>
			<td >Fisher Scientific</td>
			<td >FB55924</td>
			<td></td>
		</tr>
		<tr >
			<td >p-xylene-bispyridinium bromide (DPX)</td>
			<td >Invitrogen</td>
			<td >X1525</td>
			<td>Protect from light&#160;</td>
		</tr>
		<tr >
			<td >quartz fluorescence cuvette</td>
			<td >Hellma</td>
			<td >109.004F-QS</td>
			<td></td>
		</tr>
		<tr >
			<td >rotavapor system&#160;</td>
			<td >Heidolph</td>
			<td >Z334898</td>
			<td></td>
		</tr>
		<tr >
			<td >Sephadex G-50 resin</td>
			<td >Amersham</td>
			<td >17-0042-01</td>
			<td></td>
		</tr>
		<tr >
			<td >Sodium azide (NaN3)</td>
			<td >Sigma-Aldrich</td>
			<td >S2002</td>
			<td></td>
		</tr>
		<tr >
			<td >Sodium chlorid (NaCl)</td>
			<td >Sigma-Aldrich</td>
			<td >S5886</td>
			<td></td>
		</tr>
		<tr >
			<td >Sonicator bath USC300T</td>
			<td >VWR</td>
			<td >142-6001</td>
			<td></td>
		</tr>
		<tr >
			<td >Sphingomyelin</td>
			<td >Avanti Polar</td>
			<td>860062P</td>
			<td>Protect from air</td>
		</tr>
		<tr >
			<td >Triton X-100&#160;</td>
			<td >Eromedex</td>
			<td >2000-B</td>
			<td></td>
		</tr>
		<tr >
			<td >Zetaziser NanoZS&#160;</td>
			<td >Malvern</td>
			<td >ZEN3500</td>
			<td></td>
		</tr>
	</tbody>
</table>

fluorescent leakage assay to investigate membrane destabilization by cell-penetrating peptide

细胞穿透肽（CPP）被定义为能够穿过质膜并将货物转移到细胞中的载体。这种活性所需的主要共同特征之一是CPP与质膜（脂质）的相互作用，特别是与膜本身的细胞外基质（硫酸肝素）的相互作用。事实上，独立于直接易位或内吞作用依赖性内化，脂质双层在质膜水平和细胞内转量水平（内体囊泡）参与内化过程。在本文中，我们提出了一个详细的方案，描述了大型单层囊泡（LUV）配方，纯化，表征和在荧光泄漏测定中的应用的不同步骤，以检测可能的CPP膜不稳定/相互作用，并解决它们在内化机制中的作用。产生具有反映质膜含量的脂质组合物的LUV，以便封装荧光染料和淬灭剂。因此，在阴囊外培养基中添加肽和诱导LUV上的肽 - 膜相互作用可能以剂量依赖性方式诱导荧光的显着增加，从而揭示泄漏。这里提供了最近开发的色氨酸（W）和精氨酸（R）丰富的两性肽（WRAP）的例子，它们在各种细胞系中显示出快速有效的siRNA递送。最后，讨论了这些相互作用的性质和对脂质的亲和力，以了解和改善膜易位和/或内体逃逸。

荧光泄漏测定的原理如图 1所示。详细地，用感兴趣的生物分子处理封装荧光染料和淬灭剂（无荧光信号）的大型单层囊泡（LUV）。由于肽与脂质膜的相互作用，这可能意味着膜通透性，重组甚至破裂，荧光染料和淬灭剂从LUV中释放出来。缓冲液中的后续稀释导致荧光信号增加。
尽管该方案显示了对游离...

Watch this Scientific Journal Video about Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide at JoVE.com

Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide

荧光泄漏测定是一种简单的方法，可以研究肽/膜相互作用，以了解它们在几个生物过程中的参与，特别是细胞穿透肽在直接细胞易位过程中扰乱磷脂双层的能力。

荧光泄漏测定研究细胞穿透肽对膜的破坏稳定性

Cell-penetrating peptides (CPPs) are defined as carriers that are able to cross the plasma membrane and to transfer a cargo into cells. One of the main ...

fluorescent-leakage-assay-to-investigate-membrane-destabilization

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Biochemistry

6.1K 次观看.  INSERM U1046 - CNRS UMR 9214 - University of Montpellier. 荧光泄漏测定是一种简单的方法，可以研究肽/膜相互作用，以了解它们在几个生物过程中的参与，特别是细胞穿透肽在直接细胞易位过程中扰乱磷脂双层的能力。

视频: Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a determinant phosphoinositide of the plasma membrane (PM), and it is required to modulate ion channels, actin dynamics, exocytosis, endocytosis, intracellular signaling, and many other cellular processes. However, the spatial organization of PI(4,5)P2 in the PM is controversial, and its nanoscale distribution is poorly understood due to the technical limitations of research approaches. Here by utilizing single molecule localization microscopy and the Pleckstrin Homology (PH) domain based dual color fluorescent probes, we describe a novel method to visualize the nanoscale distribution of PI(4,5)P2 in the PM in fixed membrane sheets as well as live cells.

PI(4,5)P2 regulates various cellular functions, but its nanoscale organization in the cell plasma membrane is poorly understood. By labeling PI(4,5)P2 with a dual-color fluorescent probe fused to the Pleckstrin Homology domain, we describe a novel approach to study the PI(4,5)P2 spatial distribution in the plasma membrane at the nanometer scale.

在质膜用新型荧光探针磷脂酰-4,5-二磷酸的单分子超分辨率成像

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a ...

科研

生物化学

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

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 ...

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

便于操作的体系结构基于蛋白质的凝胶中为单元格文化应用程序

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.

This protocol describes the characterization of DNA polymerase synthesis of modified DNA through observation of changes to near-infrared fluorescently labeled DNA using gel electrophoresis and gel imaging. Acrylamide gels are used for high resolution imaging of the separation of short nucleic acids, which migrate at different rates depending on size.

用丙烯酰胺凝胶电泳法可视化近红外荧光标记 dna 聚合酶活性

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

化细胞穿透肽研究胞内蛋白-蛋白质相互作用

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage by extracellular oxidants. This process of transporting electrons from intracellular reductants to extracellular oxidants is not well defined. Here we present spectrophotometric assays by C2C12 myotubes to monitor tPMET utilizing the extracellular electron acceptors: water-soluble tetrazolium salt-1 (WST-1) and 2,6-dichlorophenolindophenol (DPIP or DCIP). Through reduction of these electron acceptors, we are able to monitor this process in a real-time analysis. With the addition of enzymes such as ascorbate oxidase (AO) and superoxide dismutase (SOD) to the assays, we can determine which portion of tPMET is due to ascorbate export or superoxide production, respectively. While WST-1 was shown to produce stable results with low background, DPIP was able to be re-oxidized after the addition of AO and SOD, which was demonstrated with spectrophotometric analysis. This method demonstrates a real-time, multi-well, quick spectrophotometric assay with advantages over other methods used to monitor tPMET, such as ferricyanide (FeCN) and ferricytochrome c reduction.

The goal of this protocol is to spectrophotometrically monitor trans-plasma membrane electron transport utilizing extracellular electron acceptors and to analyze enzymatic interactions that may occur with these extracellular electron acceptors.

C2C12 Myotubes 测量反式等离子体膜电子传输

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often viruses exhibit cell and tissue tropism and are adapted to specific cell or tissue proteases. Moreover, these viruses can introduce mutations or insertions into their genome during replication that may affect the cleavage, and thus can contribute to adaptations to a new host. Here, we present a fluorogenic peptide cleavage assay that allows a rapid screening of peptides mimicking the cleavage site of viral fusion proteins. The technique is very flexible and can be used to investigate the proteolytic activity of a single protease on many different substrates, and in addition, it also allows exploration of the activity of multiple proteases on one or more peptide substrates. In this study, we used peptides mimicking the cleavage site motifs of the coronavirus spike protein. We tested human and camel derived Middle East Respiratory Syndrome coronaviruses (MERS-CoV) to demonstrate that single and double substitutions in the cleavage site can alter the activity of furin and dramatically change cleavage efficiency. We also used this method in combination with bioinformatics to test furin cleavage activity of feline coronavirus spike proteins from different serotypes and strains. This peptide-based method is less labor- and time intensive than conventional methods used for the analysis of proteolytic activity for viruses, and results can be obtained within a single day.

We present a fluorogenic peptide cleavage assay that allows a rapid screening of the proteolytic activity of proteases on peptides representing the cleavage site of viral fusion peptides. This method can also be used on any other amino acid motif within a protein sequence to test for the protease activity.

一种用于蛋白质溶解活性的荧光肽裂解检测: 冠状病毒穗蛋白活化的应用

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP production. The amphiphilic nature of membrane proteins requires particular attention to their handling, and reconstitution into the natural lipid environment is indispensable when studying membrane transport processes like proton translocation. Here, we detail a method that has been used for the investigation of the proton-pumping mechanism of membrane redox enzymes, taking cytochrome bo3 from Escherichia coli as an example. A combination of electrochemistry and fluorescence microscopy is used to control the redox state of the quinone pool and monitor pH changes in the lumen. Due to the spatial resolution of fluorescent microscopy, hundreds of liposomes can be measured simultaneously while the enzyme content can be scaled down to a single enzyme or transporter per liposome. The respective single enzyme analysis can reveal patterns in the enzyme functional dynamics that might be otherwise hidden by the behavior of the whole population. We include a description of a script for automated image analysis.

Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

电化学和荧光显微镜在质子泵膜酶研究中的单脂质体测定

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...