Name: in vitro polymerization of f-actin on early endosomes
Uploaded: 2017-08-28T14:00:00.000+00:00
Duration: 12 min 15 s
Description: Watch this Scientific Journal Video about In Vitro Polymerization of F-actin on Early Endosomes at JoVE.com

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>

<ol>
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C., 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=Moesin+and+cortactin+control+actin-dependent+multivesicular+endosome+biogenesis.">Moesin and cortactin control actin-dependent multivesicular endosome biogenesis.</a> Mol Biol Cell. 27 (21), 3305-3316 (2016).</li><li>Sonnichsen, B., De Renzis, S., Nielsen, E., Rietdorf, J., Zerial, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+membrane+domains+on+endosomes+in+the+recycling+pathway+visualized+by+multicolor+imaging+of+Rab4,+Rab5,+and+Rab11.">Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11.</a> J Cell Biol. 149 (4), 901-914 (2000).</li><li>Morel, E., Parton, R. 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+A2-dependent+polymerization+of+actin+mediates+endosome+biogenesis.">Annexin A2-dependent polymerization of actin mediates endosome biogenesis.</a> Dev Cell. 16 (3), 445-457 (2009).</li><li>Kamentsky, L., 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=Improved+structure,+function+and+compatibility+for+CellProfiler:+modular+high-throughput+image+analysis+software.">Improved structure, function and compatibility for CellProfiler: modular high-throughput image analysis software.</a> Bioinformatics. 27 (8), 1179-1180 (2011).</li><li>Meijering, E., 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=Design+and+validation+of+a+tool+for+neurite+tracing+and+analysis+in+fluorescence+microscopy+images.">Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images.</a> Cytometry A. 58 (2), 167-176 (2004).</li><li>Granger, E., McNee, G., Allan, V., Woodman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+cytoskeleton+and+molecular+motors+in+endosomal+dynamics.">The role of the cytoskeleton and molecular motors in endosomal dynamics.</a> Semin Cell Dev Biol. 31, 20-29 (2014).</li><li>Mayran, N., Parton, R. 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. P., Chavrier, P., Zerial, M., 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=rab5+controls+early+endosome+fusion+in+vitro.">rab5 controls early endosome fusion in vitro.</a> Cell. 64 (5), 915-925 (1991).</li><li>Wandinger-Ness, A., Zerial, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab+proteins+and+the+compartmentalization+of+the+endosomal+system.">Rab proteins and the compartmentalization of the endosomal system.</a> Cold Spring Harb Perspect Biol. 6 (11), a022616 (2014).</li><li>Balch, W. E., Glick, B. S., Rothman, 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。特别是, 我们用上述方法分析了 F-肌动蛋白聚合在早期体的顺序步骤, 并表征了分子水平的过程。
在这些实验中, 早期体被标记为 GFP-RAB5在体内, 通过亚细胞分离纯化, 并在胞存在的试管中孵育, 以允许 F 肌动蛋白聚合体外。在所需的时间, 反应停止, 反应混合物转移到微观幻灯片, 以分析荧光显微镜, 使用笔标签的 f-肌动蛋白。利用这一实验策略, 我们发现, 蛋白 A2 是必要的, 在早期体, 与 SPIRE1, 而 ARP2/3 介导的 f-肌动蛋白分支, 如预期。我们还表明, moesin 和 cortactin 是必要的形成的 f-肌动蛋白网络, 调解 ECV/MVB 生物和成熟沿哺乳动物细胞降解途径9,11。
重要的是要记住, 虽然低级别的 ectopically 表示 RAB5 不改变体任何重大程度的10, 这个小酶是一个关键的调节器早期 endosome 膜动力学16,17.在一些实验中, 它可能因此是适当的标签体在测试中使用替代协议。例如, 早期体可以方便地使用 endocytosed 荧光示踪剂标记, 如表皮生长因子或转铁蛋白4,14。
还应该注意到, 在技术上很难用传统的共焦显微镜来成像单个肌动蛋白丝。因此, 我们指的是 F-肌动蛋白结构在整个文本描述肌动蛋白网络的体。因此, 我们认为, 通过测量网络的荧光表面积而不是其强度, 可以更准确地量化 f-肌动蛋白网络的形成。
这是不容易完全排除的可能性, 肌动蛋白成核活动观察到在体外是介导的一些其他结构或细胞, 仍然绑定到 RAB5 体在纯化过程中。然而, 这种污染是极不可能的。虽然一些细胞器 co-purify 的梯度, 因为类似的物理性质, 他们不倾向于保持定量的相互结合后, 纯化。此外, 肌动蛋白成核活动严格依赖于蛋白 A24,14, 它存在于早期体中, 包含 RAB514。最后, 我们发现 F-肌动蛋白结构是有选择性的关联 RAB5 体在体内, 并没有发现在其他体或其他细胞膜4,14。最后, 肌动蛋白成核活动最有可能与 RAB5 体, 因为此活动 co-purifies 和 co-localizes 与 RAB5 体。
在这种分析中, 很像在其他的化验中, 重建复杂的生化反应, 控制体的动力学16或其他薄膜18 , 并在试管中执行, 这是很重要的, 以确保准备的细胞提取物是最佳的。细胞应在温和的条件下, 以减少对体和其他器官, 特别是细胞核和溶酶体的损害。此外, endosome 分数应保持在冰的条件下, 尽量减少渗透和离子应力, 以及自发肌动蛋白聚合。另外, 如步骤3.13 和4.4 分别所示, 在体和检测中需要相对较高浓度的 endosome 分数 (≥0.1 毫克/毫升) 和胞 (3 毫克/毫升), 以获得足够的 F-肌动蛋白聚合。同样, 这是很重要的进行的分析与1:10 的比例 endosome 胞 (蛋白质: 蛋白质), 正如在步骤5.1.1。最后, 移后的肌动蛋白聚合 (即,添加粉煤灰和笔) 应非常温和地做, 以避免崩溃或打破肌动蛋白网络。
如有必要,体外肌动蛋白聚合法也可直接在显微镜成像室中进行, 在加入纯化的罗丹明-肌动蛋白到测定混合物后, 限制肌动蛋白网络的机械扰动聚合到体 (请参见步骤5.2.3 和图 2)。在这种情况下, 最好将提取物调整到 26.5% (0.85 米) 蔗糖 (折射率为 1.375), 限制扩散和布朗样的运动 (步骤 5.2.3), 并删除可能的聚合纯化罗丹明-肌动蛋白的离心使用前。我们的观察, moesin 控制的形成 F 肌动蛋白网络的体是完全扼要使用此替代版本的我们的化验4,14。
无论是我们的协议版本, 在试管或成像室, 是最佳的全球分析 F 肌动蛋白成核和聚合的体。然而, 用这种方法来跟踪肌动蛋白聚合过程并不容易, 部分原因是体不是静止的。因此, 不容易监测单个纤维的生长, 从而确定肌动蛋白网络的历史。例如, 它可能很难区分 f-肌动蛋白分支从交联。然而, 我们有信心的条件, 实时监测 F-肌动蛋白聚合可改善在成像室, 通过优化条件固定体。同样, 用纯化成分从人工膜中监测 f-肌动蛋白聚合也会很有意义。事实上, 我们以前表明, F-肌动蛋白核和聚合可以发生在脂质体后结合 t成核因子蛋白 A2 到脂质体双层11。这一策略将有助于进一步剖析特定的脂质和蛋白质在 f-肌动蛋白成核和聚合过程中的作用, 并描述在内涵膜上形成 f-肌动蛋白网络所必需的最小分量。最后, 我们认为, 这一战略将是非常有用的研究其他 f-肌动蛋白依赖的过程中发生的 endosome 膜, 特别是招募和动员的 retromer/洗涤机械。

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><tbody><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>71380</td><td></td></tr><tr><td>KCl</td><td>Acros Organics</td><td>196770010</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>AppliChem</td><td>A1042</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Acros Organics</td><td>424370025</td><td></td></tr><tr><td>Hepes</td><td>AppliChem</td><td>A3724</td><td></td></tr><tr><td>Magnesiun acetate tetrahydrate</td><td>Fluka</td><td>63047</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>AppliChem</td><td>A2948</td><td></td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>10125</td><td></td></tr><tr><td>NaOH</td><td>Fluka</td><td>71690</td><td></td></tr><tr><td>Sucrose</td><td>Merck Millipore</td><td>107687</td><td></td></tr><tr><td>Leupeptin</td><td>Roche</td><td>11017101001</td><td></td></tr><tr><td>Pepstatin</td><td>Roche</td><td>10253286001</td><td></td></tr><tr><td>Aprotinin</td><td>Roche</td><td>10236624001</td><td></td></tr><tr><td>Paraformaldehide</td><td>Polysciences. Inc</td><td>380</td><td></td></tr><tr><td>Alexa Fluor 555 phalloidin</td><td>Molecular Probes</td><td>A34055</td><td></td></tr><tr><td>Actin rhodamine</td><td>Cytoskeleton. Inc</td><td>APHR-A</td><td></td></tr><tr><td>Mowiol 4-88</td><td>Sigma-Aldrich</td><td>81381</td><td>poly(vinyl alcohol), Mw ~31 000</td></tr><tr><td>DABCO</td><td>Sigma-Aldrich</td><td>D-2522</td><td></td></tr><tr><td>Tris-HCl</td><td>AppliChem</td><td>A1086</td><td></td></tr><tr><td>&amp;beta;-casein</td><td>Sigma-Aldrich</td><td>C6905</td><td></td></tr><tr><td>Filter 0.22 &amp;mu;m</td><td>Millex</td><td>SL6V033RS</td><td></td></tr><tr><td>Round 10 cm dishes for cell culture</td><td>Thermo Fisher Scientific</td><td>150350</td><td></td></tr><tr><td>Plastic Pasteur pipette</td><td>Assistent</td><td>569/3 40569003</td><td></td></tr><tr><td>15-mL polypropylen tube</td><td>TPP</td><td>91015</td><td></td></tr><tr><td>Hypodermic Needle 22G Black 30mm</td><td>BD Microlance</td><td>300900</td><td></td></tr><tr><td>Sterile Luer-slip 1mL Syringes without needle</td><td>BD Plastipak</td><td>300013</td><td></td></tr><tr><td>Micro glass slides</td><td>Assistent</td><td>2406</td><td></td></tr><tr><td>18 x 18-mm glass coverslip</td><td>Assistent</td><td>1000/1818</td><td></td></tr><tr><td>SW60 centrifuge tube</td><td>Beckman coulter</td><td>344062</td><td></td></tr><tr><td>TLS-55 centrifuge tube</td><td>Beckman coulter</td><td>343778</td><td></td></tr><tr><td>200-&amp;mu;L yellow tip</td><td>Starlab</td><td>S1111-0706</td><td></td></tr><tr><td>1,000-&amp;mu;L Blue Graduated Tip</td><td>Starlab</td><td>S1111-6801</td><td></td></tr><tr><td>1.5-mL test tube</td><td>Axygen</td><td>MCT-175-C 311-04-051</td><td></td></tr><tr><td>18-mm diameter round coverslip</td><td>Assistent</td><td>1001/18</td><td></td></tr><tr><td>35-mm diameter round dish with a 20-mm glass bottom (0.16 - 0.19 mm)</td><td>In vitro Scientific</td><td>D35-20-1.5-N</td><td></td></tr><tr><td>Refractometer</td><td>Carl Zeiss</td><td>79729</td><td></td></tr><tr><td>Plasma cleaner</td><td>Harrick Plasma</td><td>PDC-32G</td><td></td></tr><tr><td>Sorvall WX80 Ultracentrifuge</td><td>Thermo Fisher Scientific</td><td>46900</td><td></td></tr><tr><td>Tabletop ultracentrifuge</td><td>Beckman coulter</td><td>TL-100</td><td></td></tr><tr><td>SW60 rotor</td><td>Beckman coulter</td><td>335649</td><td></td></tr><tr><td>TLS-55 rotor</td><td>Beckman coulter</td><td>346936</td><td></td></tr><tr><td>Confocal microscopy</td><td>Carl Zeiss</td><td>LSM-780</td><td></td></tr><tr><td>Fugene HD transfection reagent</td><td>Promega</td><td>E2311</td><td></td></tr><tr><td>Protein assay reagent A</td><td>Bio-Rad</td><td>500-0113</td><td></td></tr><tr><td>Protein assay reagent B</td><td>Bio-Rad</td><td>500-0114</td><td></td></tr><tr><td>Protein assay reagent S</td><td>Bio-Rad</td><td>500-0115</td><td></td></tr><tr><td>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><td>Refractometer</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Minimum Essential Medium Eagle (MEM)</td><td>Sigma-Aldrich</td><td>M0643</td><td></td></tr><tr><td>FCS</td><td>Thermo Fisher Scientific</td><td>10270-106</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids (NEAA)</td><td>Thermo Fisher Scientific</td><td>11140-035</td><td></td></tr><tr><td>L-Glutamine</td><td>Thermo Fisher Scientific</td><td>25030-024</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>pH Meter 691</td><td>Metrohm</td><td></td><td></td></tr><tr><td>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) 显示了 F-肌动蛋白。肌动蛋白在早期内涵膜上的成核以及随后的聚合过程迅速发生。实际上, F-肌动蛋白在反应开始 (图 3A) 后的1min 内已经可以在体上可视化。 这些肌动蛋白结构, 最初是短的, 迅速变得更长, 分枝和连接到另一个导致形成一个交织网络的肌动蛋白丝 (图 3A), 大概反映不平衡肌动蛋白动力学的体外。在用纯化的罗丹明标记的肌动蛋白和细胞胞进行测定时, 观察到了类似的核化和聚合率。这是在玻璃底的菜肴, 使结构可以直接成像, 在没有任何扰动 (图 3B)。在这里所描述的, 早期体核的肌动蛋白聚合选择性。事实上, 当体外反应在没有体或胞的情况下进行时, 肌动蛋白聚合没有发生。因此可以得出结论, 早期体具有支持肌动蛋白花丝成核和随后聚合的基本能力9,11。
为了分析体的肌动蛋白聚合量, 利用 CellProfiler (图 4A) 对不同时间获得的图像进行了分析。每 endosome 的肌动蛋白聚合量被测量为肌动蛋白周围的一个单一的早期 endosome。在该过程开始时, 肌动蛋白聚合率较高, 随时间减少 (图 4B)。 肌动蛋白网络可以更详细地分析在早期时点后的反应开始。在这些早期的时点, 个体肌动蛋白包含的结构可能仍然被区分从彼此。为了评估肌动蛋白网络的复杂组织, 从每个 endosome 中产生的肌动蛋白丝数 (即主丝) 计数。同样, 从原丝 (即, 次花丝) 中产生的肌动蛋白丝的数量, 以及所有其他可以在网络中识别的肌动蛋白丝的数量, 并没有起源于体 (即, 其他细丝) (图 4C-e。一旦肌动蛋白丝被跟踪, 其他参数, 如单个肌动蛋白丝的长度和体连接到相同肌动蛋白丝的数量, 可以很容易地计算 (图 4F)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55829/55829fig1.jpg"> 
图 1: HeLa 细胞匀浆的相衬显微图像.均匀化后, 将萃取物安装在显微镜滑动和片之间。为出版, 显微被夺取了以100X 目标在油浸泡以后。然而, 在常规检查中, 细胞提取物在20X 或40X 目标下是可视的, 没有油浸。给出了匀浆高倍视图的两个例子 (A-b)。核 (星) 出现作为圆的结构深灰色在颜色, 没有附上细胞残余。箭头指向不同大小、形状和半透明的特征颗粒, 它们在细胞同质化时释放, 对应于细胞内的物质 (即器)。缩放栏 = 10 µm.<a href="http://ecsource.jove.com/files/ftp_upload/55829/55829fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55829/55829fig2.jpg"> 
图 2: 协议的示意图表示形式.用于准备内涵提取物 (A) 和胞提取物 (B) 的协议的示意图说明, 并从体体外(C) 监视肌动蛋白聚合。上清 (SN)、均质缓冲 (HB)、postnuclear 上清 (三七)、核颗粒 (NP) 和甲醛 (粉煤灰)。<a href="http://ecsource.jove.com/files/ftp_upload/55829/55829fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55829/55829fig3.jpg"> 
图 3: 早期体中 F 肌动蛋白的成核和聚合体外。(一个) 体纯化的 hela 细胞表达 GFP-Rab5 (绿色) 和 hela 胞分别制备。在试验中, 早期体 (EEs) 与胞混合, 并在指定时间内孵育。然后固定, 用笔 (红色) 标记 F 肌动蛋白, 并通过荧光共焦显微镜分析。栏:10 µm (顶部面板) 和5µm (底部面板)。(B) EEs 纯化的 hela 细胞表达 GFP-Rab5 (绿色) 和 hela 胞分别制备。用纯化的罗丹明-肌动蛋白 (red) 在显微镜室中对这些细胞提取物进行培养, 并用共聚焦显微镜对其进行了分析。缩放条 = 10 µm. (C) 实验是在A中执行的。hela 胞 (无 EEs) 和 hela 体 (绿色) 标有 GFP-Rab5 (没有胞) 分别孵育 (左和中间板) 或一起 (右面板) 为30分钟, 固定, 标签与笔 (红色) 标记 F-肌动蛋白, 并分析荧光共焦显微镜。缩放栏 = 10 µm.<a href="http://ecsource.jove.com/files/ftp_upload/55829/55829fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55829/55829fig4.jpg"> 
图 4: 肌动蛋白网络分析(A) CellProfiler 分析的工作流。(B) 聚合肌动蛋白的数量相对于使用 CellProfiler 软件的体数量而言是定量的。数据是中值± s.e.m. (n = 3)。数据是指±法令 (n = 3)。5 min 相对于 15 min (ns p= 0.0736), 5 分钟对 30 min (* p = 0.0195), 和 15 min vs 30 min (ns p = 0.3129)。(C)分别制备 GFP-Rab5 和 hela 胞的 hela 细胞纯化的早期体 (EEs), 并在图 2A中进行检测。样品在7分钟后固定。中间的面板显示相同的显微图像, 与 F-肌动蛋白结构的痕迹重叠使用 NeuronJ 插件13的 ImageJ 软件绘制。右侧面板仅显示跟踪。缩放条 = 10 µm. (D) 表示的 F-肌动蛋白结构编号序列中使用的量化与 ImageJ 软件。(E) 定量化肌动蛋白丝的数量, 如C-d中所定义的那样。数据是指±法令 (n = 3)。主分支与辅助分支 (ns p = 0.5262)、辅助分支与其他分支 (ns p = 0.6815) 和主分支与其他分支 (ns p = 0.2254)。(f) F 肌动蛋白结构的长度和每个肌动蛋白结构的 EEs 数显示为其他参数的例子, 而不是用C-d中定义的量化指标来分析的。<a href="http://ecsource.jove.com/files/ftp_upload/55829/55829fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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 ...

in-vitro-polymerization-of-f-actin-on-early-endosomes

Nanyang Technological University

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biochemistry

In Vitro Polymerization of F-actin on Early Endosomes

Unit 1

The Cytoskeleton I: Actin and Microfilaments

SCIENTISTS IN ACTION

9.0K 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

Monitoring Actin Disassembly with Time-lapse Microscopy

Okay, so what I'd like to do today is to show you how to construct a flow chamber, a fusion chamber for imaging on an upright microscope. What then I then do is I create two spaces using film. So what I do is I take the para film, I cut it into small strips.Next I'm going to, this will basically form the channels for the, the walls, and I dry them with a tilted air. I put the cover slip on top of the para, And here's the finished. Then I heat it on a heat block to melt the param and basically create a seal.So what this is, is the, the cover glass here, and then on top of the cover glass is two layers of phim as you see right here. And then on top of this, again, there's a, it's a 22 by two two mil meter cover slip. This one right here, which I pressed against the param to fall the seal.Okay, so the liquid goes in in one direction here and I can suck it out with a filter paper here in this direction. And I'll show you how to do that in a bit. So what I like to do is I like to put it in a humidified chamber, which is very simple.Take two kind of elevated strips and have a filter of paper underneath. And to keep it noisy as I, I put the cover, cover slip on the flow chamber on. Now the way you introduce a solution to the chamber is that you just put the pipet tip at the entrance of the chamber and you just split.Now what I do is I incubate the chamber with the lid closed for 10 minutes. What I usually do to suck the liquid out from the other side is to use it, get a filter paper and cut it into thin strips like this. And I take one of these thin strips and I simultaneously apply the solution into the entrance while putting the filter paper at the exit to suck the exchange, the solution.So you'll see the liquid flowing through the solution. And then I wait for five minutes for the mid close. Now what I'm ready to do is to actually put that the actin it and the actin, I actually have to polymerize, I polymerize inside the flow chamber and it's just the same procedures before.So because like this, and then now that the action is polymerized, I wash out all the un excess un polymerized action using a large volume of just buffer. So here I have about a hundred microliters and it's a few reaction chamber volumes worth of buffer. Just, you know, when the filter paper becomes saturated, I have to change the direction.Okay?And so I'm ready to do imaging with this. So perfusion chamber with actin attached to it. Now I take the slide, mount it under the microscope, and since I'm imaging with an oil objective, I'm gonna put some immersion oil on the top surface of the cover, grass touch, such so assess solution or protein or anything on acting inside.So what I do is I can actually exchange the solution side while I'm doing the time lapse imaging. As such.

拆装时间推移显微镜监测肌动蛋白

科研

生物学

Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally expressed molecular guidance cues that bind growth cone receptors and regulate the dynamics and organization of the growth cone cytoskeleton3-6. The main target of these navigational signals is the actin filament meshwork that fills the growth cone periphery and that drives growth cone motility through continual actin polymerization and dynamic remodeling7. Positive or attractive guidance cues induce growth cone turning by stimulating actin filament (F-actin) polymerization in the region of the growth cone periphery that is nearer the source of the attractant cue. This actin polymerization drives local growth cone protrusion, adhesion of the leading margin and axonal elongation toward the attractant.
Actin filament polymerization depends on the availability of sufficient actin monomer and on polymerization nuclei or actin filament barbed ends for the addition of monomer. Actin monomer is abundantly available in chick retinal and dorsal root ganglion (DRG) growth cones. Consequently, polymerization increases rapidly when free F-actin barbed ends become available for monomer addition. This occurs in chick DRG and retinal growth cones via the local activation of the F-actin severing protein actin depolymerizing factor (ADF/cofilin) in the growth cone region closer to an attractant8-10. This heightened ADF/cofilin activity severs actin filaments to create new F-actin barbed ends for polymerization. The following method demonstrates this mechanism. Total content of F-actin is visualized by staining with fluorescent phalloidin. F-actin barbed ends are visualized by the incorporation of rhodamine-actin within growth cones that are permeabilized with the procedure described in the following, which is adapted from previous studies of other motile cells11, 12. When rhodamine-actin is added at a concentration above the critical concentration for actin monomer addition to barbed ends, rhodamine-actin assembles onto free barbed ends. If the attractive cue is presented in a gradient, such as being released from a micropipette positioned to one side of a growth cone, the incorporation of rhodamine-actin onto F-actin barbed ends will be greater in the growth cone side toward the micropipette10.
Growth cones are small and delicate cell structures. The procedures of permeabilization, rhodamine-actin incorporation, fixation and fluorescence visualization are all carefully done and can be conducted on the stage of an inverted microscope. These methods can be applied to studying local actin polymerization in migrating neurons, other primary tissue cells or cell lines.

A method to visualize and quantify F-actin barbed ends in neuronal growth cones is described. After culturing neurons on glass coverslips, cells are permeabilized with a saponin-containing solution. Then, a short incubation with the saponin buffer containing rhodamine-actin incorporates fluorescent actin onto free actin barbed ends.

罗丹明-肌动蛋白标记F -肌动蛋白在通透的神经生长锥刺完

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally ...

神经科学

Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor individual reactions live in well-controlled conditions. To do so, live single-filament experiments have emerged over the past 20 years, mostly using total internal reflection fluorescence (TIRF) microscopy, and have provided a trove of key results. In 2011, in order to further expand the possibilities of these experiments and to avoid recurring problematic artifacts, we introduced simple microfluidics in these assays. This study details our basic protocol, where individual actin filaments are anchored by one end to the passivated coverslip surface, align with the flow, and can be successively exposed to different protein solutions. We also present the protocols for specific applications and explain how controlled mechanical forces can be applied, thanks to the viscous drag of the flowing solution. We highlight the technical caveats of these experiments and briefly present possible developments based on this technique. These protocols and explanations, along with today&#39;s availability of easy-to-use microfluidics equipment, should allow non-specialists to implement this assay in their labs.

We present protocols for simple actin filament microfluidic assays, in combination with fluorescence microscopy, that allow one to accurately monitor individual actin filaments in real-time while sequentially exposing them to different protein solutions.

利用微流体和荧光显微镜研究单根肌动蛋白丝和束的组装动力学

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor ...

生物化学

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and regulated by different suites of binding proteins unique for each polymer. Many studies now demonstrate that the dynamics of both cytoskeletal polymers are intertwined and that this crosstalk is required for most cellular behaviors. A number of proteins involved in actin-microtubule interactions have already been identified (i.e., Tau, MACF, GAS, formins, and more) and are well characterized with regard to either actin or microtubules alone. However, relatively few studies showed assays of actin-microtubule coordination with dynamic versions of both polymers. This may occlude emergent linking mechanisms between actin and microtubules. Here, a total internal reflection fluorescence (TIRF) microscopy-based in vitro reconstitution technique permits the visualization of both actin and microtubule dynamics from the one biochemical reaction. This technique preserves the polymerization dynamics of either actin filament or microtubules individually or in the presence of the other polymer. Commercially available Tau protein is used to demonstrate how actin-microtubule behaviors change in the presence of a classic cytoskeletal crosslinking protein. This method can provide reliable functional and mechanistic insights into how individual regulatory proteins coordinate actin-microtubule dynamics at a resolution of single filaments or higher-order complexes.

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence TIRF Microscopy

This protocol is a guide for visualizing dynamic actin and microtubules using an in vitro total internal fluorescence (TIRF) microscopy assay.

通过全内反射荧光（TIRF）显微镜 在体外 可视化肌动蛋白和微管耦合动力学

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and ...

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell deformation, division, migration, and adhesion. However, studying the dynamics and structure of the actin network in vivo is complicated by the biochemical and genetic regulation within live cells. To build a minimal model devoid of intracellular biochemical regulation, actin is encapsulated inside giant unilamellar vesicles (GUVs, also called liposomes). The biomimetic liposomes are cell-sized and facilitate a quantitative insight into the mechanical and dynamical properties of the cytoskeleton network, opening a viable route for bottom-up synthetic biology. To generate liposomes for encapsulation, the inverted emulsion method (also referred to as the emulsion transfer method) is utilized, which is one of the most successful techniques for encapsulating complex solutions into liposomes to prepare various cell-mimicking systems. With this method, a mixture of proteins of interest is added to the inner buffer, which is later emulsified in a phospholipid-containing mineral oil solution to form monolayer lipid droplets. The desired liposomes are generated from monolayer lipid droplets crossing a lipid/oil-water interface. This method enables the encapsulation of concentrated actin polymers into the liposomes with desired lipid components, paving the way for in vitro reconstitution of a biomimicking cytoskeleton network.

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

In this manuscript, we demonstrate the experimental techniques to encapsulate the F-actin cytoskeleton into giant unilamellar lipid vesicles (also called liposomes), and the method to form a cortex-biomimicking F-actin layer at the inner leaflet of the liposome membrane.

体外 重组巨型单层囊泡内的肌动蛋白细胞骨架

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell ...

生物工程

Reconstitution of Actin-Based Motility with Commercially Available Proteins

Many cell movements and shape changes and certain types of intracellular bacterial and organelle motility are driven by the biopolymer actin that forms a dynamic network at the surface of the cell, organelle, or bacterium. The biochemical and mechanical basis of force production during this process can be studied by reproducing actin-based movement in an acellular manner on inert surfaces such as beads that are functionalized and incubated with a controlled set of components. Under the appropriate conditions, an elastic actin network assembles at the bead surface and breaks open due to the stress generated by network growth, forming an "actin comet" that propels the bead forward. However, such experiments require the purification of a host of different actin-binding proteins, often putting them beyond the reach of non-specialists. This article details a protocol for reproducibly obtaining actin comets and motility of beads using commercially available reagents. Bead coating, bead size, and motility mixture can be altered to observe the effect on bead speed, trajectories, and other parameters. This assay can be used for testing the biochemical activities of different actin-binding proteins, and for performing quantitative physical measurements that shed light on active matter properties of actin networks. This will be a useful tool for the community, enabling the study of in vitro actin-based motility without expert knowledge in actin-binding protein purification.

This protocol describes how to produce actin comets on the surfaces of beads using commercially available protein ingredients. Such systems mimic the protrusive structures found in cells, and can be used to examine physiological mechanisms of force production in a simplified way.

用市售蛋白质重建基于肌动蛋白的运动

Many cell movements and shape changes and certain types of intracellular bacterial and organelle motility are driven by the biopolymer actin that forms a ...

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin occurs in equilibrium in its two forms: monomeric globular (G-actin) and polymerized filamentous (F- actin). At the synaptic terminals, actin cytoskeleton forms the basis for critical pre- and post-synaptic functions. Moreover, dynamic changes in the actin polymerization status (interconversion between globular and filamentous forms of actin) are closely linked to plasticity-related alterations in synaptic structure and function. We report here a modified fluorescence-based methodology to assess polymerization status of actin in ex vivo conditions. The assay employs fluorescently labelled phalloidin, a phallotoxin that specifically binds to actin filaments (F-actin), providing a direct measure of polymerized filamentous actin. As a proof of principle, we provide evidence for the suitability of the assay both in rodent and post-mortem human brain tissue homogenates. Using latrunculin A (a drug that depolymerizes actin filaments), we confirm the utility of the assay in monitoring alterations in F-actin levels. Further, we extend the assay to biochemical fractions of isolated synaptic terminals wherein we confirm increased actin polymerization upon stimulation by depolarization with high extracellular K+.

We report a simple, time-efficient and high-throughput fluorescence spectroscopy-based assay for the quantification of actin filaments in ex vivo biological samples from brain tissues of rodents and human subjects.

用于评估啮齿动物和人类脑组织中行为素聚合状态的基于时间的荧光光谱分析

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin ...

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

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

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

在支持的脂质双层上重建膜系的最小肌动蛋白皮质

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

Reconstituting and Characterizing Actin-Microtubule Composites with Tunable Motor-Driven Dynamics and Mechanics

The composite cytoskeleton, comprising interacting networks of semiflexible actin filaments and rigid microtubules, restructures and generates forces using motor proteins such as myosin II and kinesin to drive key processes such as migration, cytokinesis, adhesion, and mechanosensing. While actin-microtubule interactions are key to the cytoskeleton's versatility and adaptability, an understanding of their interplay with myosin and kinesin activity is still nascent. This work describes how to engineer tunable three-dimensional composite networks of co-entangled actin filaments and microtubules that undergo active restructuring and ballistic motion, driven by myosin II and kinesin motors, and are tuned by the relative concentrations of actin, microtubules, motor proteins, and passive crosslinkers. Protocols for fluorescence labeling of the microtubules and actin filaments to most effectively visualize composite restructuring and motion using multi-spectral confocal imaging are also detailed. Finally, the results of data analysis methods that can be used to quantitatively characterize non-equilibrium structure, dynamics, and mechanics are presented. Recreating and investigating this tunable biomimetic platform provides valuable insight into how coupled motor activity, composite mechanics, and filament dynamics can lead to myriad cellular processes from mitosis to polarization to mechano-sensation.

This paper presents protocols for engineering and characterizing tunable three-dimensional composite networks of co-entangled actin filaments and microtubules. Composites undergo active restructuring and ballistic motion, driven by myosin II and kinesin motors, and are tuned by the relative concentrations of actin, microtubules, motor proteins, and passive crosslinkers.

重组和表征具有可调电机驱动动力学和力学的肌动蛋白-微管复合材料

The composite cytoskeleton, comprising interacting networks of semiflexible actin filaments and rigid microtubules, restructures and generates forces ...

Actin Polymerization and Cell Motility

Actin is a family of globular proteins that are highly abundant in eukaryotic cells. It makes up approximately 1-5% of total cell protein concentration. Actin monomers polymerize to form a complex network of polarized filaments, the actin cytoskeleton, that plays a crucial role in many cellular processes, including cell motility, division, endocytosis, and metastasis of cancer cells.
Actin cytoskeleton dynamics can produce pushing, pulling, and resistance forces that help the cell to migrate. This is achieved by forming different types of membrane protrusions, depending on the cell type and the extracellular signals. Primarily, cells move by recurrent cycles of protrusion and attachment of the cell front to the substratum, followed by detachment and retraction at their rear end.
The human genome encodes six highly conserved actin proteins&#8212;ACTC1, ACTA1, ACTA2, ACTG1, ACTG2, and ACTB; all expressed in different cell types. Mutations in any of these six actin genes or the genes encoding actin-binding proteins can lead to disease. For example, mutations in the ACTA1 gene can lead to muscle weakness, especially in the respiratory muscles, which can cause breathing difficulties &#8212; a condition called Nemaline Myopathy. Similarly, mutations in the WAS gene, a key regulator of actin filament nucleation, which is important for cell adhesion, chemotaxis, and phagocytosis of immune cells, causes Wiskott Aldrich syndrome in humans.

肌动蛋白聚合和细胞运动

Actin is a family of globular proteins that are highly abundant in eukaryotic cells. It makes up approximately 1-5% of total cell protein concentration. ...

体外F-肌动蛋白在早期体的聚合

摘要

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拆装时间推移显微镜监测肌动蛋白

罗丹明-肌动蛋白标记F -肌动蛋白在通透的神经生长锥刺完

利用微流体和荧光显微镜研究单根肌动蛋白丝和束的组装动力学

通过全内反射荧光（TIRF）显微镜在体外可视化肌动蛋白和微管耦合动力学

体外重组巨型单层囊泡内的肌动蛋白细胞骨架

用市售蛋白质重建基于肌动蛋白的运动

用于评估啮齿动物和人类脑组织中行为素聚合状态的基于时间的荧光光谱分析

在支持的脂质双层上重建膜系的最小肌动蛋白皮质

重组和表征具有可调电机驱动动力学和力学的肌动蛋白-微管复合材料

肌动蛋白聚合和细胞运动

体外F-肌动蛋白在早期体的聚合

摘要

探索更多视频

此视频中的章节

拆装时间推移显微镜监测肌动蛋白

罗丹明-肌动蛋白标记F -肌动蛋白在通透的神经生长锥刺完

利用微流体和荧光显微镜研究单根肌动蛋白丝和束的组装动力学

通过全内反射荧光（TIRF）显微镜 在体外 可视化肌动蛋白和微管耦合动力学

体外 重组巨型单层囊泡内的肌动蛋白细胞骨架

用市售蛋白质重建基于肌动蛋白的运动

用于评估啮齿动物和人类脑组织中行为素聚合状态的基于时间的荧光光谱分析

在支持的脂质双层上重建膜系的最小肌动蛋白皮质

重组和表征具有可调电机驱动动力学和力学的肌动蛋白-微管复合材料

肌动蛋白聚合和细胞运动

通过全内反射荧光（TIRF）显微镜在体外可视化肌动蛋白和微管耦合动力学

体外重组巨型单层囊泡内的肌动蛋白细胞骨架