得到瑞士国家科学基金会的支持;瑞士 Sinergia 计划;波兰-瑞士研究方案 (PSPB-094/2010);化学生物学中的 NCCR;LipidX 来自瑞士 SystemsX.ch 倡议, 由瑞士国家科学基金会 (j.g) 评估。o.m. 得到了 EMBO 长期研究金 (ALTF-516-2012) 的支持。

1. 解决方案和准备工作 注意: 所有的缓冲区和解决方案都应在双 (dd) H 2 O 中准备。由于蔗糖的水合状态不同, 所有蔗糖溶液的最终浓度必须用折射仪来确定. 
<ol> <li> 准备不含价阳离子的磷酸盐缓冲盐水 (PBS): 137 毫米盐、2.7 毫米氯化钾、1.5 mm 2 PO 4 和 6.5 mm Na 2 HPO 4 。调整 pH 值为7.4。灭菌 (1 循环在121和 #176; C 15 分钟) 和室温下贮存. </li>
	<li> 准备 300 mM 咪唑的库存解决方案: 0.204 克咪唑在10毫升的 ddH 2 o. 使用 HCl 将 pH 值调整为7.4 和 #176; c. 使用4和 #956 进行过滤灭菌; m 孔径过滤和存储在0.22 和 #176; c. </li>
	<li> 准备均匀化缓冲液 (HB; 准备大约100毫升): 250 毫米蔗糖在3毫米咪唑。调整 ph 值为 7.4, 在4和 #176; C 使用 ph 计。使用0.22 和 #956 的过滤器灭菌; m 孔径过滤器和存储在4和 #176; c. 补充 HB 前与鸡尾酒的蛋白酶抑制剂在最后浓度对应于10毫米 leupeptin, 1 毫米 pepstatin a, 和 10 ng/毫升抑肽酶. </li>
	<li> 为步骤浮选渐变, 准备 62%, 39%, 和35% 蔗糖溶液在3毫米咪唑, pH 7.4, 如下所述。使用折射仪精确确定所有蔗糖溶液的最终浓度. 
	<ol> <li> 在 3 mM 咪唑中制备100毫升62% 蔗糖溶液, pH 值7.4。通过搅拌80.4 克的蔗糖在 ddH 2 O 5ml 到35和 #176; c. 添加1毫升的 300 mM 咪唑库存溶液, 并填充到100毫升与 ddH 2 o. 将 pH 值调整为7.4 和 #176; c. 使用4和 #956 进行过滤灭菌; m 孔径过滤和贮存在4和 #176; C. </li>
		<li> 准备100毫升35% 蔗糖溶液在3毫米咪唑, pH 7.4。通过搅拌40.6 克的蔗糖在 ddH 2 o. 添加1毫升的 300 mM 咪唑库存溶液和填充到100毫升与 ddH 2 o. 将 pH 值调整为7.4 和 #176; c. 使用0.22 和 #956 的过滤器消毒; m 孔径过滤器, 存储在4和 #176; c. </li>
		<li> 准备50毫升39% 蔗糖在3毫米咪唑, pH 7.4。用35% 蔗糖溶液稀释62% 蔗糖溶液。存储在4和 #176; C. </li>
	</ol> </li> <li> 将3克甲醛 (粉煤灰) 通过100毫升的 PBS-5ml 搅拌到60和 #176; C 同时加入 1 M 氢氧化钠滴; 使用氢氧化钠将最终 pH 值调整到7.4。采用 0.22 #956; m 孔径过滤器和存储在-20 和 #176 上的过滤器灭菌; C. 
	警告: 3% 粉煤灰是一种有毒试剂. </li>
	<li> 准备1米氯化钾库存解决方案。在50毫升的 ddH 中搅拌3.73 克氯化钾, 2 o. 使用0.22 和 #956 的过滤器灭菌; m 孔径过滤器和室温下贮存. </li>
	<li> 准备50X 集中库存解决方案, 包含 0.625 M Hepes 在 pH 7.0 和 75 mM MgOAc 2 在 ddH 2 o 分和存储在-20 和 #176; C. </li>
	<li> 准备安装介质。混合搅拌2.4 克的聚 (乙烯醇) M w 〜 3.1万 (PVA) 与6克甘油。添加6毫升的 ddH 2 O, 并在室温下至少留2小时。添加12毫升0.2 米的三氯 (pH 8.5) 和热到约53和 #176; C 偶尔搅拌, 直到 PVA 溶解. </li>
	<li> 在室温下用离心法在 5000 x g 中澄清溶液的20分钟。收集上清液, 并加入 2.5% (含/v) 14-环-[2.2. 2] 辛烷 (DABCO), 以防止荧光检测过程中的漂白。漩涡为大约三十年代溶化。分到1.5 毫升塑料管和存储在-20 和 #176; C. </li>
</ol> 2。单元格区域性 <ol> <li> Dulbecco 和 #39 中的培养 HeLa 细胞, 补充了10% 胎小牛血清、10% 非必需氨基酸、10% l-谷氨酰胺和1% 青霉素-链霉素。保持在37和 #176; C 在 5% CO 2 孵化器中. </li>
	<li> 染在实验前24小时 GFP-RAB5 10 , 根据制造商和 #39 的说明使用商用转染试剂. </li>
	<li> 当需要时, 准备内涵的分数和胞从一个感兴趣的蛋白质 (, 即, 使用 rna 干扰或 CRISPR/Cas9) 和/或表达野生或突变形式的兴趣蛋白的细胞. </li>
</ol> 3。内涵分数准备 注意: 本协议描述了包含体和其他光膜的亚细胞分数的直接制备。如果需要, 纯化的 endosome 分数也可以使用 11 。准备2个培养皿 (10 厘米外径; 57 厘米 2 ), 将 GFP-RAB5 表达为起始物质的汇合细胞。2种培养皿中的汇合 HeLa 细胞总数大致相当于 2.5 x 10 7 细胞。当需要时, 体也可以从细胞中消耗的蛋白质的利益 ( 即, 使用 rna 干扰或 CRISPR/Cas9) 和/或表达的野生或突变形式的利益的蛋白质, 总是表达 GFP-RAB5. 
 警告: 分馏协议的所有步骤都应在冰上执行. 
<ol> <li> 将培养皿放置在一个扁平冰桶中的湿金属板上, 并立即用5毫升的 ice-cold PBS 清洗两次电池. </li>
	<li> 删除所有 pbs-从最后一次洗涤和添加3毫升的 ice-cold pbs 每道菜。细胞在处理过程中不应干燥: 如有必要, 将冰桶放在摇摆平台上, 以充分地覆盖有液体的细胞. </li>
	<li> 使用灵活的橡皮警察 ( 即, 自制的细胞刮板机) 从培养皿中机械地去除 PBS 中的细胞。在盘子的外面快速地、圆形的运动中先将这些细胞刮掉, 然后在盘子中间向下移动。轻轻地刮去获得和 #34; 床单和 #34; 附着的细胞。在冰上用一个15毫升的锥形聚丙烯管, 用一个塑料的巴斯德吸管轻轻地转移细胞. </li>
	<li> 离心5分钟, 在 175 x g 和 4 #176; C. </li>
	<li> 轻轻移除上清 (SN), 并在颗粒上加入 1-3 毫升的 HB。使用一个广泛的开放的塑料巴斯德吸管, 轻轻吸管向上和向下一次重的细胞. </li>
	<li> 离心 7-10 分钟在 1355 x g 和4和 #176; c. 轻轻移除 SN. </li>
	<li> 执行单元格均化. 
	注意: 条件应温和, 使细胞膜的细胞被打破, 但体保持完整。
	<ol> <li> 将已知的血红蛋白体积与蛋白酶抑制剂一起添加到每个细胞颗粒上 (大约100和 #956; 每细胞颗粒)。使用1000和 #181; 我微, 轻轻吸管向上和向下, 直到细胞悬浮。不要引入气泡. </li>
		<li> 准备一个1毫升结核菌素注射器与22G 针 pre-rinsed 与 HB 和没有空气或气泡. </li>
		<li> 将注射器与电池悬浮液相对缓慢地填充 (避免产生气泡), 将针的斜面尖端放在管壁上, 并迅速排出细胞悬浮液, 以剪切附着细胞的细胞膜. </li>
		<li> 取一小分匀浆 (大约3和 #181; l) 并将其稀释为50和 #181; 在玻璃滑梯上的 HB 的 l。用玻璃片。在装有20X 或40X 物镜的相衬显微镜下检查匀浆. 
		注意: 在最佳条件下, 大多数单元格都是断开的, 但核 (显示为深灰色和圆形结构) 不是 ( 图 1 ). </li>
		<li> 重复步骤3.7.3 和 3.7.4, 直到大多数细胞被破坏; 通常, 3-6 up-and-down 中风通过针是必要的。将匀浆的小分 (10-30 和 #181; L) 用于蛋白质测定. </li>
	</ol> </li> <li> 离心匀浆7分钟在 1355 x g 和4和 #176; C. 
	注: 离心后的颗粒 (定义为核颗粒;NP) 包含核和上清 (定义为 postnuclear 上清液;三七皂苷) 包含胞和细胞器释放后均匀化和自由悬浮. </li>
	<li> 仔细收集期票。保持小等分 (10-30 和 #181; L) 的总皂苷和 np 的蛋白质测定和丢弃的 np. </li>
	<li> 通过用 1:1. 1 的 PNS:62%sucrose 的比例来稀释62% 的蔗糖溶液, 将三七皂苷调节到40.6% 蔗糖。轻轻而彻底地混合, 不产生气泡。使用折射仪检查蔗糖浓度. </li>
	<li> 将三七总皂苷放在离心管底部的40.6% 蔗糖中。仔细覆盖2.5 毫升的35% 蔗糖溶液和填充离心管与 HB. 
	注: 蔗糖溶液应分层, 以便界面清晰可见. </li>
	<li> Ultracentrifuge 1 小时 #160 的渐变; #820; 16.5万 x g 和4和 #176; C. </li>
	<li> 小心地移除渐变顶部的白色层。接下来, 收集的接口含有体, 在 HB 和35% 蔗糖之间, 使用200和 #956; 我微的小费. 
	注: 内涵分数可直接用于在 体外 肌动蛋白聚合法或可在冰上 aliquoted, 在液氮中闪光冷冻, 储存在-80 和 #176; C. </li>
	<li> 确定三七总皂苷和内涵馏分的蛋白质浓度. 
	注: 此浓度应至少100和 #181;在2培养皿中生长的大约 2.5 x 10 7 细胞需要获得至少100和 #181 的三七皂苷; g/毫升 (见上面的说明). </li>
</ol> 4。胞准备 注意: 准备2个培养皿 (10 厘米直径; 57 厘米 2 ) 以汇合的 HeLa 细胞为起始材料。当需要时, 胞也可以从细胞中消耗的蛋白质的利益 ( 即, 使用 rna 干扰或 CRISPR/Cas9) 和/或表达的野生或突变形式的利益蛋白。至于 endosome 分馏协议, 所有的步骤应该在冰上执行. 
<ol> <li> 重复步骤 3.1-3.8. </li>
	<li> 将三七皂苷放在离心管中, ultracentrifuge 45 分钟, #820; 25万 x g 和4和 #176; C. </li>
	<li> 小心移除顶部的白色层, 并收集 SN (胞分数) 而不干扰微粒颗粒。保持分的胞分数的蛋白质测定. 
	注: 胞可直接用于 体外 肌动蛋白聚合测定, 或可在冰上 aliquoted, 在液氮中闪光冷冻, 贮存在-80 和 #176; C. </li>
	<li> 确定胞的蛋白质浓度. 
	注: 应至少3毫克/毫升. </li>
</ol> 5。测定 Endosome 依赖性肌动蛋白聚合的实验研究 在体外 注意: Endosome 依赖性肌动蛋白聚合可以使用两种替代方法进行。在第一种方法中, 将材料混合在试管中, 固定, 并转移到片并进行分析。在这里描述的第二种方法中, 该分析可以直接在成像室进行, 并且可以不受机械扰动 ( 图 2C ) 进行固定和解析。在第二种方法中, 化验混合物不从试管转移到片, 因此保持不动, 减少了在转移过程中身体扰动的 f-肌动蛋白网络的危险。另外, 这种第二种方法与对 F 肌动蛋白聚合的延时分析相一致。值得注意的是, 这两种方法均未观察到 F 型肌动蛋白聚合的分析差异. 
注意: 在整个协议中使用剪切提示. 
<ol> <li> 在试管中 <ol> <li> 将 ice-cold 纯化的 GFP-RAB5 体 (步骤 3) 与胞 (步骤 4) 在 ice-cold 1.5 毫升锥形试管中的比例为 1:10 (蛋白质浓度). 
		注: 一个典型的实验是进行约 40-50 和 #181; L. </li>
		<li> 将 ice-cold 反应混合物调整为125毫米氯化钾的最终浓度 (使用1米氯化钾溶液)、12.5 mm Hepes 和 1.5 mm MgOAc 2 (使用50x 库存解决方案)。然后, 调整与蛋白酶抑制剂的混合物, 最终浓度为10毫米 leupeptin, 1 毫米 pepstatin A, 10 ng/毫升抑肽酶。轻轻地混合所有组件. </li>
		<li> 将含有反应混合物的试管放在37和 #176; C, 不摇晃, 并在所需时间内孵育. 
		注: 一般情况下, 在体和30分钟内检测肌动蛋白聚合需要大约3分钟, 以形成一个广泛的网络. </li>
		<li> 在所需的时间 ( 即, 3 分钟或30分钟), 通过将测试管放置在冰上并添加 1/10 (卷: 卷) 的冷3% 粉煤灰溶液来停止反应. </li>
		<li> 添加 0.3:10 (卷: 卷) 200 单位/毫升 (〜6.6 和 #956; M) 笔共轭的红橙色染料染色的聚合肌动蛋白. </li>
		<li> 将12和 #956; 将混合物的 l 放到12和 #956 上; 在微玻片上的 PVA 安装介质的 l。将 18 x 18 mm 2 玻璃 coverslipon 顶部. </li>
		<li> 用共焦显微镜分析样品. </li>
	</ol> </li> <li> 在成像室 <ol> <li> 中制作显微成像室, 使用等离子清洗器1分钟清洁18毫米直径的圆形片和一个直径为 20 mm 玻璃底部 (0.16-0. 19 mm) 的圆形 35 mm 的培养皿. </li>
		<li> 用1% 和 #946 孵育清洗过的表面;-酪蛋白为20分钟, 以尽量减少蛋白质与玻璃的结合。用3毫米咪唑洗两次. </li>
		<li> 添加 endosome 和胞, 在与步骤5.1.1 和5.1.2 相同的测定混合物中, 在冷 1.5 mL 试管中加入0.1 和 #956 的检测混合物; g/#956;
		<li> 使用39% 蔗糖溶液将混合物的最终折射率调整为 1.375 (26.5% 蔗糖). 
		注意: 通常, 添加相同的卷;然而, 这必须是经验主义地调整取决于被收集的分数的蔗糖集中, 确定使用 refractomer, 因为蔗糖集中可能轻微地改变从实验到实验. </li>
		<li> 将混合物放在经过预处理的 35 mm 盘上 (步骤 5.2.1), 并将其与经过预处理的 18 mm 片 (步骤 5.2.1) 覆盖. </li>
		<li> 将会议厅放置在37和 #176; C, 不摇晃. </li>
		<li> 使用荧光共聚焦显微镜分析样品. </li>
	</ol> </li> </ol> 6。图像交流肌动蛋白网络的 quisition 与分析 <ol> <li> 图像获取 <ol> 在反向扫描共聚焦显微镜上获取图像. <li>
		注: 图像采集设置优化的倒置扫描共聚焦显微镜与 63X 1.25 NA 油目标。调整激光功率 (通常为50% 左右), 以达到良好的信噪比. </li>
	对肌动蛋白网络的 </ol> </li> <li> 量化 <ol> <li> 分析荧光显微。使用开源软件 CellProfiler (v2.1.1) 12 来测量每个 endosome 的肌动蛋白量 (可使用替代软件)。
		<ol> <li> 首先, 使用 GFP-RAB5 信号将体标识为主要对象。然后, 用肌动蛋白信号 (红-橙染料共轭笔) 的传播作为辅助对象, 量化与单个体相关的 f-肌动蛋白. </li>
			<li> 将每个对象的关联材料的数量平均和规范化到控件条件. </li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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G., Gruenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annexin+II+regulates+multivesicular+endosome+biogenesis+in+the+degradation+pathway+of+animal+cells.">Annexin II regulates multivesicular endosome biogenesis in the degradation pathway of animal cells.</a> EMBO J. 22 (13), 3242-3253 (2003).</li><li>Gorvel, J. 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作者声明他们没有竞争的金融利益。

肌动蛋白在 endosome 膜动力学中起关键作用4,14。我们以前报道, 肌动蛋白成核和聚合发生在早期体, 形成小的 f-肌动蛋白补丁或网络。这些 f-肌动蛋白网络是绝对需要的膜运输超越早期体沿降解途径。干扰此成核和聚合过程的任何步骤, 防止 endosome 成熟, 从而对后期的体 andlysosomes9,11,15...

在更高的真核细胞中, 蛋白质和脂质被内化为早期的体, 在那里进行分类。一些蛋白质和脂质, 这是注定要利用, 被纳入到管状区域的早期体, 然后运到等离子膜或转移到高尔基体网络 (TGN)1,2。相比之下, 其他的蛋白质和脂质被选择性地包装到早期体的区域, multivesicular 出现。这些地区扩展, 并在脱离早期内涵膜, 最终成熟成自由内涵载体囊泡或 multivesicular 体 (ECV/MVBs), 负责货物运输对晚体1, 2。
肌动蛋白在与内涵分选能力和 endosome 生物相关的膜重塑过程中起着至关重要的作用。蛋白质的分类沿着回收途径的等离子膜或 TGN 取决于 retromer 复合物和相关的蛋白质。这种分拣机械似乎是耦合到回收小管通过相互作用的 retromer 复合体, 与黄蜂和疤痕同系物 (洗涤) 复杂和分支肌动蛋白3,4,5.相反, 注定要降解的分子, 特别是活化的信号受体, 被分类成腔内囊泡 (ILVs) 的内涵分拣复合物所需的运输 (ESCRT)2,6, 7。尽管肌动蛋白在 ESCRT 依赖性分选过程中可能扮演的角色尚不清楚, 但 F-肌动蛋白在 ECV/MVBs 的生物和早期体的运输中起着重要作用。特别是, 我们发现蛋白 A2 结合胆固醇丰富地区早期 endosome, 并与 spire1, 核 F-肌动蛋白聚合。在体上观察到的分支肌动蛋白网络的形成, 需要蛋白 (ARP) 2/3 复合体的分支活性, 以及蛋白质 moesin 和肌动蛋白结合蛋白 cortactin8,9。
在这里, 我们描述了一个 microscopy-based 的体外测定, 每天的核化和聚合的 f-肌动蛋白在早期内涵膜的试管。该方法已被用于研究蛋白 A2 在 f-肌动蛋白成核和 moesin 和 cortactin 形成内涵肌动蛋白网络中的作用8,9。通过这种体外协议, 在肌动蛋白聚合过程中体发生的一系列复杂反应, 可以对过程的顺序步骤进行生物化学和分子分析, 包括肌动蛋白成核、线性聚合、分枝和交联。

<table border="0" cellpadding="0" cellspacing="0" width="1287">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:477px;">NaCl</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:145px;">71380</td>
			<td style="width:443px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td>Acros Organics</td>
			<td>196770010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KH2PO4</td>
			<td>AppliChem</td>
			<td>A1042</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Na2HPO4</td>
			<td>Acros Organics</td>
			<td>424370025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hepes</td>
			<td>AppliChem</td>
			<td>A3724</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesiun acetate tetrahydrate</td>
			<td>Fluka</td>
			<td>63047</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dithiothreitol (DTT)</td>
			<td>AppliChem</td>
			<td>A2948</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>10125</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaOH</td>
			<td>Fluka</td>
			<td>71690</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose</td>
			<td>Merck Millipore</td>
			<td>107687</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leupeptin</td>
			<td>Roche</td>
			<td>11017101001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pepstatin</td>
			<td>Roche</td>
			<td>10253286001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Aprotinin</td>
			<td>Roche</td>
			<td>10236624001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehide</td>
			<td>Polysciences. Inc</td>
			<td>380</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexa Fluor 555 phalloidin</td>
			<td>Molecular Probes</td>
			<td>A34055</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Actin rhodamine</td>
			<td>Cytoskeleton. Inc</td>
			<td>APHR-A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mowiol 4-88</td>
			<td>Sigma-Aldrich</td>
			<td>81381</td>
			<td>poly(vinyl alcohol), Mw ~31 000&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DABCO</td>
			<td>Sigma-Aldrich</td>
			<td>D-2522</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris-HCl</td>
			<td>AppliChem</td>
			<td>A1086</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#946;-casein</td>
			<td>Sigma-Aldrich</td>
			<td>C6905</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Filter 0.22um</td>
			<td>Millex&#160;</td>
			<td>SL6V033RS</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Round 10cm dishes for cell culture</td>
			<td>Thermo Fisher Scientific</td>
			<td>150350</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic Pasteur pipette</td>
			<td>Assistent</td>
			<td>569/3 40569003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15-ml polypropylen tube&#160;</td>
			<td>TPP</td>
			<td>91015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hypodermic Needle 22G Black 30mm&#160;</td>
			<td>BD Microlance</td>
			<td>300900</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile Luer-slip 1ml Syringes without needle</td>
			<td>BD Plastipak</td>
			<td>300013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro glass slides&#160;</td>
			<td>Assistent</td>
			<td>2406</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">18X18-mm glass coverslip</td>
			<td>Assistent</td>
			<td>1000/1818</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SW60 centrifuge tube</td>
			<td>Beckman coulter</td>
			<td>344062</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TLS-55 centrifuge tube</td>
			<td>Beckman coulter</td>
			<td>343778</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">200-&#956;l yellow tip</td>
			<td>Starlab</td>
			<td>S1111-0706</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1000-&#956;l Blue Graduated Tip</td>
			<td>Starlab</td>
			<td>S1111-6801</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5-ml test tube</td>
			<td>Axygen</td>
			<td>MCT-175-C 311-04-051</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">18-mm diameter round coverslip&#160;</td>
			<td>Assistent</td>
			<td>1001/18</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35-mm diameter round dish with a 20-mm glass bottom (0.16-0.19&#160;mm)&#160;</td>
			<td>In vitro Scientific</td>
			<td>D35-20-1.5-N</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Refractometer</td>
			<td>Carl Zeiss</td>
			<td>79729</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-32G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sorvall WX80 Ultracentrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>46900</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tabletop ultracentrifuge</td>
			<td>Beckman coulter</td>
			<td>TL-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SW60 rotor</td>
			<td>Beckman coulter</td>
			<td>335649</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TLS-55 rotor</td>
			<td>Beckman coulter</td>
			<td>346936</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal microscopy</td>
			<td>Carl Zeiss</td>
			<td>LSM-780</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fugene HD transfection reagent</td>
			<td>Promega</td>
			<td style="width:145px;">E2311</td>
			<td style="width:443px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Protein assay reagent A</td>
			<td>Bio-Rad</td>
			<td>500-0113</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Protein assay reagent B</td>
			<td>Bio-Rad</td>
			<td>500-0114</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Protein assay reagent S</td>
			<td>Bio-Rad</td>
			<td>500-0115</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell scraper</td>
			<td>Homemade</td>
			<td></td>
			<td>Silicone rubber piece of about 2 cm, cut at a very sharp angle and attached to a metal bar or held with forceps</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Refractometer</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Minimum Essential Medium Eagle (MEM)</td>
			<td>Sigma-Aldrich</td>
			<td>M0643</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FCS</td>
			<td>Thermo Fisher Scientific</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM Non-Essential Amino Acids (NEAA)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pH Meter 691</td>
			<td>Metrohm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ImageJ software</td>
			<td></td>
			<td></td>
			<td>NIH, Bethesda MD</td>
		</tr>
	</tbody>
</table>

in vitro polymerization of f-actin on early endosomes

许多早期的 endosome 功能, 特别是货物蛋白分选和膜变形, 依赖于内涵膜上有核的短的 f-肌动蛋白纤维的补丁。我们已经建立了一个 microscopy-based 的体外检测, 每天的核化和聚合的 f-肌动蛋白在早期内涵膜的试管, 从而呈现这一系列复杂的反应, 可遗传和生物化学操作.从表达早期内涵蛋白 GFP-RAB5 的细胞中浮选蔗糖梯度制备内涵分数。胞浆组分是由分离的细胞分批制备的。如果需要的话, 内涵和胞浆组分都可以在液氮中冷冻保存。在试验中, 内涵和胞浆组分混合, 在适当的条件下 (例如,离子强度, 还原环境) 在37° c 下孵育混合物。在所需的时间, 反应混合物是固定的, 和 f-肌动蛋白是揭示与笔。然后用荧光显微镜对肌动蛋白成核和聚合进行分析。在这里, 我们报告, 这种方法可以用来研究的作用, 涉及的因素, 在膜上的肌动蛋白成核, 或在随后的伸长, 分支, 或交联的 f-肌动蛋白丝。

为了深入了解早期 endosome 膜上的 F 肌动蛋白补丁的形成, 我们遵循了图 2中概述的协议。简单地, 细胞被转染与 GFP-RAB5, 然后早期体是由亚单位分离制备的。这些纯化早期体的培养与胞, 以提供肌动蛋白本身以及其他因素可能涉及的反应。在潜伏期结束时, 通过固定的混合物停止反应。然后采集样品并在显微镜下沉积。最后, 笔 (图 3A</str...

Watch this Scientific Journal Video about In Vitro Polymerization of F-actin on Early Endosomes at JoVE.com

In Vitro Polymerization of F-actin on Early Endosomes

早期 endosome 功能依赖于 F-肌动蛋白聚合。在这里, 我们描述了一个 microscopy-based 的体外测定, 每天的核化和聚合的 f-肌动蛋白在早期内涵膜的试管, 从而呈现这一系列复杂的反应, 可生化和遗传操作.

体外F-肌动蛋白在早期体的聚合

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto ...

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In Vitro Polymerization of F-actin on Early Endosomes

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The Cytoskeleton I: Actin and Microfilaments

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8.9K 次观看.  University of Geneva. 早期 endosome 功能依赖于 F-肌动蛋白聚合。在这里, 我们描述了一个 microscopy-based 的体外测定, 每天的核化和聚合的 f-肌动蛋白在早期内涵膜的试管, 从而呈现这一系列复杂的反应, 可生化和遗传操作.

视频: In Vitro Polymerization of F-actin on Early Endosomes

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

的pH依赖性活性的检测<em&gt;大肠杆菌</em&gt;陪护HdeB<em&gt;体外</em&gt;和<em&gt;在体内</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

科研

生物化学

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Measuring Protein Binding to F-actin by Co-sedimentation

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, cross-linking and/or disassembly. This protocol describes a technique &#8211; the actin co-sedimentation, or pelleting, assay &#8211; to determine whether a protein or protein domain binds F-actin and to measure the affinity of the interaction (i.e., the dissociation equilibrium constant). In this technique, a protein of interest is first incubated with F-actin in solution. Then, differential centrifugation is used to sediment the actin filaments, and the pelleted material is analyzed by SDS-PAGE. If the protein of interest binds F-actin, it will co-sediment with the actin filaments. The products of the binding reaction (i.e., F-actin and the protein of interest) can be quantified to determine the affinity of the interaction. The actin pelleting assay is a straightforward technique for determining if a protein of interest binds F-actin and for assessing how changes to that protein, such as ligand binding, affect its interaction with F-actin.

This protocol describes a method to test the ability of a protein to co-sediment with filamentous actin (F-actin) and, if binding is observed, to measure the affinity of the interaction.

通过共沉淀测量蛋白与F-肌动蛋白的结合

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

电化学检测氘动力学同位素对希瓦氏菌 oneidensis MR-1 细胞外电子传输的影响

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

体外肠道宿主-微生物界面合成细菌联合的可行性评价

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such systems allows for detailed studies of their underlying mechanisms which would not be feasible in vivo. Here, we provide a protocol for the in vitro reconstitution of the MinCDE system of Escherichia coli, which positions the cell division septum in the cell middle. The assay is designed to supply only the components necessary for self-organization, namely a membrane, the two proteins MinD and MinE and energy in the form of ATP. We therefore fabricate an open reaction chamber on a coverslip, on which a supported lipid bilayer is formed. The open design of the chamber allows for optimal preparation of the lipid bilayer and controlled manipulation of the bulk content. The two proteins, MinD and MinE, as well as ATP, are then added into the bulk volume above the membrane. Imaging is possible by many optical microscopies, as the design supports confocal, wide-field and TIRF microscopy alike. In a variation of the protocol, the lipid bilayer is formed on a patterned support, on cell-shaped PDMS microstructures, instead of glass. Lowering the bulk solution to the rim of these compartments encloses the reaction in a smaller compartment and provides boundaries that allow mimicking of in vivo oscillatory behavior. Taken together, we describe protocols to reconstitute the MinCDE system both with and without spatial confinement, allowing researchers to precisely control all aspects influencing pattern formation, such as concentration ranges and addition of other factors or proteins, and to systematically increase system complexity in a relatively simple experimental setup.

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

We provide a protocol for in vitro self-organization assays of MinD and MinE on a supported lipid bilayer in an open chamber. Additionally, we describe how to enclose the assay in lipid-clad PDMS microcompartments to mimic in vivo conditions by reaction confinement.

体外支持性脂质双层自组织蛋白模式的重组

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such ...

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

低聚活性的体外定量

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

核糖体组蛋白的体外泛化和二次基质测定

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

体外F-肌动蛋白在早期体的聚合

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的pH依赖性活性的检测