Name: an integrated system to remotely trigger intracellular signal transduction by upconversion nanoparticle-mediated kinase photoactivation
Uploaded: 2017-08-30T14:00:00.000+00:00
Duration: 11 min 20 s
Description: Watch this Scientific Journal Video about An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation at JoVE.com

我们感谢中央研究院纳米科技项目和台湾科技部的资助 (101-2113 米-001-001-MY2; 103-2113 米-001-028-MY2)。

注意: 该协议描述了对转换辅助活化的一个详细的仪表修改, 这是一种生成笼式 PKA-UCNP 的合成过程, 它是由二氧化硅涂层的 UCNP 的透射电子显微镜 (TEM)和笼 PKA-UCNP 样品, 紫外线和近红外光解设置, 细胞制备, PKA UCNP 显微注射, 活化研究, 和 REF52 细胞的应力纤维染色. 
 1. 转换频谱测量的 Fluorimeter 设置 <ol> <li> 在荧光光谱仪的试管持有者旁边安装4X 物镜, 以便激光聚焦在试管的中间. </li>
	<li> 将可调谐的 0.5-至 8 W 980 nm 半导体激光二极管90和 #176; 与物镜匹配. 
	注意: 操作人员需要具备激光安全知识, 并佩戴近红外激光护目镜. </li>
	<li> 在物镜和激光器的光路相交处安装全反射镜。放置一个黑色阳极氧化金属板, 以阻止光谱的励磁窗口和保护内部部分. </li>
	<li> 将试管的二氧化硅涂层 UCNP 溶液 (0.2-0.6 毫克/毫升) 放在试管支架上, 使明亮的转换光束可以可视化, 并可用于调整镜像安装的最佳光对齐方式. </li>
	<li> 将光谱学转换为最小 PMT 电压设置的发光模式 (如果信号太弱则调整到更高的设置)。将镜像调整到最佳对准, 其中光束位于试管的中间, PMT 拾取最强的信号. </li>
	<li> 对齐后测量转换频谱, 并使用所需的电源设置。使用热堆功率计, 在激光电源上的电流读数上建立激光功率的校准曲线。不断检查激光性能, 确保性能在校准范围内. </li>
</ol> 2。显微镜设置 注意: 以下设置适用于配备双层光路的显微镜。如果用户打算在后端安装一个励磁光源开关, 使近红外激光器和卤化物灯共享同一端口, 则在后端端口的准直器将显著阻挡近红外光谱, 因为它是为减少样品加热而设计的。用户必须作出困难的选择: 保持准直器在和遭受非常低的转换效率, 或删除的准直和牺牲强度的励磁光的荧光. 
 注意: 显示修改后的光的光路方案的剖面图、转换发光和近红外解离模式的显微镜、荧光和 UV 光解模式, 以及实验设置用于此实验的说明如 图 2 所示. 
<ol> <li> 将荧光显微镜与金属卤化物灯导轨一起配置到后端. 
	注意: 在这个上层的光路中, 有一个立方体炮塔, 一个立方体的位置必须是空的, 允许从较低层光路径来的近红外光谱. </li>
	<li> 在右侧端口上安装可调谐的0.5 到 8.0 W 980 nm 激光二极管, 并打开侧面端口准直器. 
	注: 这将激光光斑放大到大约500和 #181; 当使用10X 物镜时, 直径为 m。除去这个准直器会产生微米范围的光点, 使近红外细胞照射变得不切实际。这种准直器是近红外透明的. </li>
	<li> 在下层光路中安装一个分色镜 (850 nm 短路) 和一个励磁滤镜 (950 nm long-pass). </li>
	<li> 使用 UCNP 的解决方案来可视化近红外光点。调整激光的位置, 使近红外光束在视野中居中, 以完成对准. </li>
</ol> 3。笼型 PKA UCNP 的制备与表征 <ol> <li> 孵化重组 PKA (9 和 #181; M) 与0.5 毫米 2-nitrobenzylbromide (NBB) 在锁定缓冲 (20 毫米三盐酸, 100 毫米氯化钠和10毫米氯化镁 2 ; 8.5-1 h 在2和 #176; c. 淬火过剩的 NBB 与10毫米 dthiothreitol (数码), 然后透析反对 4-(2 羟乙基) 哌嗪-1-磺酸 (HEPES) 缓冲 (10 毫米, pH 7.5) 去除多余的试剂和盐. 
	注: 在透析过程中, 锁定溶液的 ph 值从8.5 降低到 7.5, HEPES ph 值7.5 用于随后的固定化反应. </li>
	<li> 混合稀土盐-Y (CH 3 CO 2 ) 3 水合物 (99.9%, 0.4527 克, 1.38 摩尔), Yb (ch 3 CO 2 ) 3 水合物 (99.9%, 0.2533 g, 0.60 摩尔) 和 Tm (ch 3 co 2 ) 3 水合物 (99.9%, 0.0168 g, 0.02 摩尔)-在十八 (30 毫升) 和油酸 (12 毫升), 加热混合物到115和 #176; c 为30分钟, 冷却到50和 #176; c。然后, 将反应混合物溶解在甲醇溶液 (20 毫升) 中, 用0.2 克 (5.0 摩尔) 的氢氧化钠颗粒和0.2964 克 (8 摩尔) 的氟化铵超声为15分钟. 将反应温度提高到300和 #176; C 获取 UCNP 核心 (#946;-NaYF 4 : 1.0% Tm 3 + /30% Yb 3 + ) 纳米颗粒。
	<li> 溶解在环己烷 (10 毫升) CO-520 (0.5 毫升) 的核心纳米颗粒 (25 毫克)。添加氨水 (重量30% 伏/v, 0.08 毫升) 和 tetraethylorthosilicate (乙, 0.04 毫升), 并在室温下应用恒定搅拌12小时, 以获得二氧化硅涂层和 #946;-NaYF 4 : 1.0%Tm 3 + /30%Yb 3 + 核心纳米粒子. </li>
	<li> 孵育 PKA (6 nmol) 或笼 PKA (6 nmol) 与 UCNP (1 毫克) 15 在 HEPES 缓冲区 (10 毫米, pH 7.5) 在4和 #176; C 为 3 h 固定 PKA 在硅涂层的 UCNP 通过 静电相互作用. </li>
	<li> 使用 PVDF 过滤器过滤混合物 (0.45 和 #181; m), 离心机在 1.7万 x g 为10分钟在4和 #176; C, 和 redisperse HEPES (50 和 #181; L, 10 毫米, pH 7.5) 含有0.1% 蛋白稳定剂, 以获得纯化的 PKA-UCNP 复合物的颗粒。离心机在 1.7万 x g 为10分钟在4和 #176; C 和收集上清为布拉德福德化验在1.5 毫升管. </li>
	<li> 分析从步骤3.5 中保存的上清中的无界 PKA, 用布拉德福德法对 UCNP 粒子的 PKA 置换水平进行反计算, 后者通常具有 4.5 nmol 每毫克粒子的 PKA 的替代水平. 
	注意: 布拉德福德化验显示了一个强烈的蓝颜色的颗粒, 这意味着高水平和稳定的蛋白质固定不脱附, 而笼 PKA-UCNP 溶液的上清分数显示一种颜色类似 HEPES 缓冲区 ( 图 4 c-4e ). </li>
</ol> 4。特性 注意: 激酶测定用于量化丙酮酸激酶的特定活性。简言之, 与丙酮酸激酶和乳酸脱氢酶结合的肽的磷酸化, 导致 NADH 氧化。后者的形成是通过测量 NADH 在 340 nm 的吸光度下降来监测的. 
<ol> <li> 确定60和 #181 中的 PKA 和 PKA UCNP 的活动; 含 4-morpholinepropanesulfonic 酸 (拖把, 100 毫米, pH 7.5), 氯化钾 (100 毫米), 丙酮 (PEP, 1 毫米), 三磷酸腺苷 (ATP, 1 毫米), 和 #946 的测定混合物的总容积。烟酰胺腺嘌呤核苷酸, 减少的形式 (NADH, 0.8 毫米), 氯化镁 (氯化镁 2 , 1 毫米), kemptide (0.4 毫米) 和丙酮酸激酶/乳酸脱氢酶混合物 (PK/LDH 的30/40 单位). </li>
	<li> 测量 PKA 溶液 (2-#181; L) 加入测定混合物后的 NADH 吸收量的减少。计算直线的斜率, 以直接反映被测激酶的活动. </li>
	<li> 测量 PKA-UCNP 15 和整个活动的活动, 该行为应与本机 PKA 特定活动的乘法乘积以及计算的 PKA 表面涂层量匹配. </li>
</ol> 5。光解设置 <ol> <li> 多边环境协定使用热堆传感器和计算功率密度 (PD = 功率 (W)/面积 (cm 2 )) 的所有光源功率, 基于光点区域的 18 . </li>
	<li> 执行 体外 紫外线光解实验在一个商业系统与 365 nm LED (200 兆瓦/cm 2 ) 15 。
	<li> 对于显微镜阶段的 UV 活化, 用商业立方体过滤的激发光照亮 PKA UCNP 溶液 15 . 
	注: 功率密度为 ~ 15 兆瓦/cm 2 , 无物镜聚焦. </li>
	<li> For 体外 近红外活化在显微镜阶段, 用 980 nm 激光照射 PKA-UCNP 溶液, 在下层光路上安装有定制的转换滤镜/镜像设置, 分色镜 (850 nm 短路),励磁过滤器 (950 nm long-pass). 
	注: 在4X 物镜聚焦前, 输出功率密度为5瓦特/厘米 2 。在4X 物镜聚焦后, 功率密度估计为68瓦特/cm 2 . </li>
</ol> 6。PKA-UCNP 配合物的细胞样品制备及显微注射 <ol> <li> 通过0.45 和 #181 对样本进行预筛选; m PKA 固定后的过滤器 (步骤 3.5). </li>
	<li> 在注射期内孵育含有 L-15 培养基的10% 胎牛血清 (FBS) 中的细胞, 以便在恒温箱外进行稳定的 pH 控制. 
	注: 要确认微注射完成, co-inject 原 PKA-UCNP, 笼 PKA UCNP, 或乙二醇 UCNP (7.5 毫克/毫升) 15 具 5 (6)-四 (TAMRA) (10 和 #181; M) 到单元格中. </li>
	<li> 调整 PKA UCNP 颗粒的浓度, 使其在微注射后, 胞浆浓度为 1-3 和 #956; M PKA-一个细胞内 PKA 的生理浓度范围, 假设注射量是1/10 的细胞体积.</li>
	<li> 使用带有单轴液压微 ( 图 3 ) 的微量设备对单元 (步骤 6.2) 执行微注射. </li>
	<li> 使用数字压力表监测微量所施加的压力在操作范围内 (50-200 hPa), 并在定制加压室中密封分配器阀, 以提供微注射的补偿压力。使用拉拔器将针向后拉出. </li>
	<li> 保持50和 75 hpa 之间的注射压力, 注入时间为200-300 毫秒, 15 hpa 的补偿压力, 以防止培养基通过毛细管作用回流到针头中. </li>
	<li> 检查针头的尖端开口, 以确保它们的外径介于1.3 和1.5 和 #181 之间; m, 可以通过使用40x 物镜拍摄图像来确认. </li>
	<li> 用2毫升的 L-15 培养基在注射后用 10% FBS 冲洗细胞。观察 5 (6)-carboxytetramethyl-罗丹明 (TAMRA) 的细胞荧光成像, 以确认微注射完成. </li>
</ol> 7。在活细胞中使用紫外或近红外光的活化笼 PKA UCNP <ol> <li> 在 PKA-UCNP 微注射后, 在3.5 和 #181 上生长 REF52 细胞; m 盘, 将它们放在显微镜舞台上, 然后用 uv 光照射它们立方体为3分钟, 没有物镜聚焦. 
	注: REF52 细胞保持在 DMEM 10% FBS 37 和 #176; C 在 5% CO 2 孵化器中, 当70-100 每90% 天达到2-3 时, 细胞被继. </li>
	<li> 对于 PKA-UCNP 微注射后的近红外活化, 请按照步骤5.4 中的说明, 从较低层的滤镜/镜像设置中照射 980 nm 的激光器上的单元格. 
	注: 功率密度输出为5瓦特/厘米 2 前10X 物镜对焦和估计是300瓦特/厘米 2 后10x 物镜聚焦。转换是一个需要高光子密度的过程, 特别是当需要更大的反斯托克位移时。因此, 在活化中需要集中. </li>
	<li> 当需要更大的光点 (650 和 #181; m x 520 和 #181 m) 时, 用10x 物镜聚焦的近红外光谱照射这些细胞, 使准直器为 15 min. 允许在每5分钟照射后5分钟的暗间隔, 以避免发热. 
	注意: 当需要高空间分辨率 (亚细胞光点) 时, 使用40X 物镜, 并在导光指南的出口端安装针孔, 以生成亚细胞尺寸的光点 (5 和 #181; m x 4 和 #181; m)。在这种情况下, 1 s 的辐照是足够的近红外活化由于高通量的光子. </li>
	<li> 在 UV 或近红外光解之后, 允许单元格恢复在 5% CO 2 孵化器在37和 #176; C 为 1 h 在完全培养基产生细胞反应。随后, 将它们固定在1毫升的4% 甲醛/磷酸盐缓冲盐水 (PBS) 20 分钟前进一步染色. 
	警告: 甲醛是剧毒的;避免接触皮肤、眼睛或粘膜。在测量和准备过程中避免吸入粉末. </li>
</ol> 8。近红外活化引起的应力纤维崩解的可视化 <ol> <li> 在固定后, 使用 Alexa 594-笔 (添加5和 #181; l 6.6 和 #181; M 库存解决方案, 以200和 #181; 我的 PBS 染色; 在室温下孵育25分钟) 染色可视化应力纤维。使用滤镜集可视化 Alexa 594-笔 (前 581 nm/Em 609 nm) 的图像. </li>
	<li> 在室温下孵育4和 #39 中的细胞5分钟;, 6-diamidino-2-吲 (DAPI)。染色后, 用 PBS 清洗样品, 分别取应力纤维和细胞核的荧光图像. </li>
</ol>

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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=A+UV-Blocking+Polymer+Shell+Prevents+One-Photon+Photoreactions+while+Allowing+Multi-Photon+Processes+in+Encapsulated+Upconverting+Nanoparticles.">A UV-Blocking Polymer Shell Prevents One-Photon Photoreactions while Allowing Multi-Photon Processes in Encapsulated Upconverting Nanoparticles.</a> Angew Chem Int Ed. 52 (42), 11106-11109 (2013).</li><li>Zhou, L., Chen, Z. W., Dong, K., Yin, M. L., Ren, J. S., Qu, X. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-mediated+Construction+of+Hollow+Upconversion+Nanoparticles+for+Protein+Harvesting+and+Near-Infrared+Light+Triggered+Release.">DNA-mediated Construction of Hollow Upconversion Nanoparticles for Protein Harvesting and Near-Infrared Light Triggered Release.</a> Adv Mater. 26 (15), 2424-2430 (2014).</li><li>Gao, H. -. D., 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=Construction+of+a+Near-Infrared-Activatable+Enzyme+Platform+To+Remotely+Trigger+Intracellular+Signal+Transduction+Using+an+Upconversion+Nanoparticle.">Construction of a Near-Infrared-Activatable Enzyme Platform To Remotely Trigger Intracellular Signal Transduction Using an Upconversion Nanoparticle.</a> ACS Nano. 9 (7), 7041-7051 (2015).</li><li>Wehbi, V. L., Task&#233;n, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Mechanisms+for+cAMP-Mediated+Immunoregulation+in+T+cells+-+Role+of+Anchored+Protein+Kinase+A+Signaling+Units.">Molecular Mechanisms for cAMP-Mediated Immunoregulation in T cells - Role of Anchored Protein Kinase A Signaling Units.</a> Front Immunol. 7, (2016).</li><li>Pitchiaya, S., Heinicke, L. A., Custer, T. C., Walter, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Molecule+Fluorescence+Approaches+Shed+Light+on+Intracellular+RNAs.">Single Molecule Fluorescence Approaches Shed Light on Intracellular RNAs.</a> Chem Rev. 114 (6), 3224-3265 (2014).</li><li>Gao, D., Tian, D., Zhang, X., Gao, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Quasi-one+dimensional+Propagationand+Tuning+of+Upconversion+Luminescence+Through+Waveguide+Effect.">Simultaneous Quasi-one dimensional Propagationand Tuning of Upconversion Luminescence Through Waveguide Effect.</a> Scientific Rep. 6, 22433-22442 (2016).</li><li>Roger, P. P., Rickaert, F., Huez, G., Authelet, M., Hofmann, F., Dumont, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+catalytic+subunit+of+cyclic+AMP-dependent+protein+kinases+triggers+acute+morphological+changes+in+thyroid+epithelial+cells.">Microinjection of catalytic subunit of cyclic AMP-dependent protein kinases triggers acute morphological changes in thyroid epithelial cells.</a> FEBS Lett. 232 (2), 409-413 (1988).</li><li>Curley, K., Lawrence, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivation+of+a+Signal+Transduction+Pathway+in+Living+Cells.">Photoactivation of a Signal Transduction Pathway in Living Cells.</a> J Am Chem Soc. 120 (33), 8573-8574 (1998).</li><li>Carling, C. J., Nourmohammadian, F., Boyer, J. C., Branda, N. 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作者声明他们没有竞争的金融利益。

此前, 霍夫曼和同事发现, 在自由 PKA19的微注射后, 在 REF52 细胞中观察到戏剧性的形态学改变。在另一项研究中, 劳伦斯小组证明, 笼 PKA 可以激活在体内, 导致形态学变化和应力纤维的解体时, 受到紫外线光解20。早先关于开发 upconverted 紫外光的报告活化显示了激活的几个 UCNP 协助, 笼, 小分子 bioeffectors21,22,</...

化学修饰的蛋白质, 可以光 (例如, PKA 笼的蛋白质) 已经发展成为一个新兴的领域, 在化工生物学性操作细胞间生化过程1,2 ,3。当激活这些笼中的蛋白质时, 利用光作为刺激提供了绝佳的时空分辨率。然而, 紫外线会导致不需要的形态学改变, 细胞凋亡和 DNA 损伤4,5。因此, photocaging 组的设计最近的发展重点是使 photocleavage 在较长波长或双光子激励下减少光, 以及增加深层穿透6,7。响应较长波长的锁定组允许我们选择合适的 uncaging 波长 (即、通道), 以便在两个或多个锁定组存在7时有选择性地激活 bioeffectors。鉴于这些有用的特征, 开发新的红光 photocaging 小组是非常重要的上游工作, 在光化学方法的生物研究范围内, 从探索的反应机制, 以控制细胞活动8。然而, 一个双光子锁定群通常过于疏水性由于融合芳香环结构, 和可见光锁定组通常是有机金属, 与芳香配体。当 bioeffector 是蛋白质或酵素时, 这种疏水/芳香物产不适合, 因为它使酵素或蛋白质的激活站点和导致作用损失, 即使共轭和光解仍然工作在化工水平上2 ,9。
UCNPs 是将近红外激发光转换为紫外线的有效传感器。这一独特而迷人的 UCNPs 的财产提供了现实的解决方案, 以应对与活化和触发控制释放小分子, 包括叶酸10, 顺铂衍生物11, DNA/siRNA12, 共聚物泡13, 空心粒子14。然而, 据我们所知, 目前为止, UCNP 辅助的酶或蛋白质活化还没有经过测试。因为没有成功的案例使用红光或近红外光谱来光酶, 我们被提示执行的近红外触发活化的蛋白质/酶结构组成的化学改性笼酶复合物与二氧化硅涂层,掺杂稀土的 UCNP15。在这项研究中, UCNP 被共轭的快速反应信号转导激酶的形式笼 PKA。PKA 控制糖原合成和骨架调节, 响应外部刺激通过循环腺苷磷酸 (阵营) 调节在胞16。本文研究了近红外辐照下细胞实验中酶激活的时间和空间方式的可行性。这种 UCNP 辅助的活化平台是一种新的方法, photoactivate 的酶使用近红外光谱和避免不受欢迎的信号转导反应由传统的紫外线照射2,4。
在细胞膜上转移大的 bioeffectors (例如,蛋白质) 很难控制细胞活动。虽然粒子固定化的蛋白质可能更容易通过吞转移到胞, 吞可能被破坏或退化通过内涵诱捕和随后的溶酶体退化2,4。即使笼状蛋白在膜移位后仍然功能正常, 移位量也不足以触发细胞响应2,17。与之形成鲜明对比的是, 微注射是一种直接和定量的方法, 可以将大的 bioeffectors 到细胞的细胞质中。此外, UCNP 固定化的 bioeffector 需要 upconverted 光来激活。因此, 光学仪器需要进一步的修改来测量、可视化和利用转换光。在这项工作中, 提供一个笼 PKA-UCNP 复合物的细胞使用显微注射和以下基本光谱学和显微镜修改的近红外活化将详细介绍。

<table><tbody><tr><td>&lt;strong&gt;Reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>Sigma</td><td>154563</td><td></td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Sigma</td><td>M9272</td><td></td></tr><tr><td>MOPS</td><td>Sigma</td><td>M1254</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H4034</td><td></td></tr><tr><td>Sodium chloride&amp;nbsp;</td><td>Sigma</td><td>31434</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>12636</td><td></td></tr><tr><td>Yttrium acetate&amp;nbsp;hydrate</td><td>Sigma</td><td>326046</td><td>Y(C&lt;sub&gt;2&lt;/sub&gt;H&lt;sub&gt;3&lt;/sub&gt;CO&lt;sub&gt;2&lt;/sub&gt;)&lt;sub&gt;3&lt;/sub&gt; &amp;middot; xH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Thulium(III) acetate hydrate</td><td>Alfa Aesar</td><td>14582</td><td>Tm(CH&lt;sub&gt;3&lt;/sub&gt;CO&lt;sub&gt;2&lt;/sub&gt;)&lt;sub&gt;3&lt;/sub&gt; &amp;middot; xH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Ytterbium(III) acetate tetrahydrate</td><td>Sigma</td><td>326011</td><td>Yb(C&lt;sub&gt;2&lt;/sub&gt;H&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;)&lt;sub&gt;3&lt;/sub&gt; &amp;middot; 4H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>1-Octadecene</td><td>Sigma</td><td>O806</td><td></td></tr><tr><td>Oleic acid</td><td>Sigma</td><td>364525</td><td></td></tr><tr><td>Methanol&amp;nbsp;</td><td>macron</td><td>304168</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma</td><td>30620</td><td></td></tr><tr><td>Ammonium fluoride</td><td>J.T.Baker</td><td>69804</td><td></td></tr><tr><td>IGEPAL&amp;nbsp;CO-520</td><td>Sigma</td><td>238643</td><td></td></tr><tr><td>Cyclohexane</td><td>J.T.Baker</td><td>920601</td><td></td></tr><tr><td>Ammonium hydroxide (28% - 30%)</td><td>J.T.Baker</td><td>972101</td><td>Ammonia</td></tr><tr><td>Tetraethyl orthosilicate (TEOS)</td><td>Sigma</td><td>8658</td><td></td></tr><tr><td>DL-Dithiothreitol (DTT)</td><td>Sigma</td><td>D0632</td><td></td></tr><tr><td>N-hydroxymaleimide (NHM)</td><td>Sigma</td><td>226351</td><td>PKA activity blocking reagent</td></tr><tr><td>Prionex protein stabilizer solution from hog collagen</td><td>Sigma</td><td>81662</td><td>Protein stabilizer solution</td></tr><tr><td>2-nitrobenzyl bromide (NBB)</td><td>Sigma</td><td>107794</td><td>PKA caging reagent</td></tr><tr><td>8-(4-Chlorophenylthio)adenosine 3&amp;prime;,5&amp;prime;-cyclic monophosphate sodium salt</td><td>Sigma</td><td>C3912</td><td>8-CPT-cAMP</td></tr><tr><td>Pyruvate Kinase/Lactic Dehydrogenase enzymes from rabbit muscle</td><td>Sigma</td><td>P0294</td><td>PK/LDH</td></tr><tr><td>Adenosine 5&#039;-triphosphate disodium</td><td>Sigma</td><td>A2387</td><td>ATP</td></tr><tr><td>&amp;beta;-NADH reduced from dipotassium</td><td>Sigma</td><td>N4505</td><td></td></tr><tr><td>Phosphoenolpyruvate</td><td>Sigma</td><td>P7127</td><td>PEP</td></tr><tr><td>Coomassie Protein Assay Reagent, 950 mL</td><td>Thermo Scientific</td><td>23200</td><td>Bradford assay reagent</td></tr><tr><td>cAMP-dependent protein kinase</td><td>Promega</td><td>V5161</td><td>PKA activity control</td></tr><tr><td>pET15b-PKACAT plasmid</td><td>Addgene</td><td>#14921</td><td></td></tr><tr><td>pKaede-MC1 plasmid</td><td>CoralHue</td><td>AM-V0012</td><td></td></tr><tr><td>Phosphate buffered saline (PBS), pH 7.4</td><td>Thermo Scientific</td><td>10010023</td><td></td></tr><tr><td>DMEM, high glucose, pyruvate</td><td>Gibco</td><td>12800-017</td><td>Cell culture medium</td></tr><tr><td>Leibovitz L-15 Medium</td><td>Biological Industries</td><td>01-115-1A</td><td>Cell culture medium</td></tr><tr><td>Fetal Bovine Serum</td><td>Biological Industries</td><td>04-001-1A</td><td></td></tr><tr><td>Paraformaldehyde</td><td>ACROS</td><td>416785000</td><td></td></tr><tr><td>DAPI</td><td>Invitrogen</td><td>D1306</td><td>Nucleus staining dye</td></tr><tr><td>Alexa 594-phalloidin</td><td>Invitrogen</td><td>A12381</td><td>F-actin staining dye</td></tr><tr><td>5(6)-Carboxytetramethylrhodamine&amp;nbsp;</td><td>Novabiochem</td><td>8.51030.9999</td><td></td></tr><tr><td>Pierce Coomassie (Bradford) Protein Assay Kit</td><td>Thermo Scientific</td><td>23200</td><td></td></tr><tr><td>CelluSep T4 Tubings/Nominal filter rating MWCO 12,000 - 14,000 Da</td><td>Membrane Filtration Products, Inc.</td><td>1430-33</td><td>Dialysis membrane</td></tr><tr><td>Millex-HV Syringe Filter Unit, 0.45 &amp;micro;m, PVDF, 13 mm, gamma sterilized</td><td>EMD Milipore</td><td>SLHVX13NL</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dynamic Light Scattering/Zetapotential Zetasizer nano-ZS</td><td>Malvern</td><td>M104</td><td></td></tr><tr><td>Transmission Electron Microscope</td><td>JEOL</td><td>JEM-1400</td><td></td></tr><tr><td>Fluorescence Spectrophotometer</td><td>Agilent Technologies</td><td>10075200</td><td>Cary Eclipse&amp;nbsp;</td></tr><tr><td>UV-Vis Spectrophotometer</td><td>Agilent Technologies</td><td>10068900</td><td>Cary 50&amp;nbsp;</td></tr><tr><td>Fluorescence Microscope</td><td>Olympus</td><td>IX-71</td><td></td></tr><tr><td>950 nm longpass filter&amp;nbsp;</td><td>Thorlabs</td><td>FEL0950</td><td></td></tr><tr><td>850 nm dichroic mirror shortpass</td><td>Chroma</td><td>NC265609</td><td></td></tr><tr><td>RT3 color CCD system</td><td>SPOT</td><td>RT2520</td><td></td></tr><tr><td>Fluorescence Illumination</td><td>PRIOR</td><td>Lumen 200</td><td></td></tr><tr><td>980 nm Infra-red diode laser</td><td>CNI</td><td>MDL-N-980-8W</td><td></td></tr><tr><td>UV LED Spot Light Source</td><td>UVATA</td><td>UVATA-UPS412</td><td>With a UPH-056-365 nm LED at 200 mW/cm&lt;sup&gt;2&lt;/sup&gt;</td></tr><tr><td>Thermal pile sensor</td><td>OPHIR</td><td>12A-V1-ROHS</td><td></td></tr><tr><td>Picospritzer III</td><td>Parker Hannifin</td><td>052-0500-900</td><td>Intracellular Microinjection Dispense Systems</td></tr><tr><td>PC-10 Needle puller</td><td>Narishige</td><td>PC-10</td><td></td></tr><tr><td>MANOMETER Digital pressure gauge</td><td>Lutron</td><td>PM-9100</td><td></td></tr><tr><td>One-axis Oil Hydraulic Micromanipulator</td><td>Narishige</td><td>MMO-220A</td><td></td></tr><tr><td>Heraeus Fresco 17 Centrifuge, Refrigerated</td><td>Thermo Scientific</td><td>75002421</td><td></td></tr></tbody></table>

an integrated system to remotely trigger intracellular signal transduction by upconversion nanoparticle-mediated kinase photoactivation

转换纳米粒子 (UCNP) 介导的活化是一种新的方法远程控制 bioeffectors 与更少的光和更深的组织渗透。然而, 现有的仪器在市场上是不容易兼容的转换应用。因此, 修改商业上可用的仪器是这项研究的关键。本文首先阐述了传统的 fluorimeter 和荧光显微镜的改进, 使其与光子转换实验相兼容。然后, 我们描述了一个近红外线 (近红外) 触发笼蛋白激酶催化亚基 (PKA) 固定在 UCNP 复合物的合成。还报告了显微注射和近红外活化程序的参数。在笼 PKA-UCNP 微量成 REF52 成纤维细胞后, 近红外辐射明显优于传统的紫外线照射, 有效地触发了活细胞中的 PKA 信号传导通路。同时, 正、负控制实验证实, PKA 诱导的应力纤维分解通路是由近红外辐射引起的。因此, 使用蛋白质修饰的 UCNP 提供了一种创新的方法, 远程控制 light-modulated 细胞实验, 其中直接暴露在紫外线必须避免。

在图 1中说明了笼式酶 UCNP 结构的设计。PKA 酶第一反应与 2-硝基苄溴生成一个非活性笼 PKA, 然后静电固定在 UCNP 表面。UCNPs 发出 upconverted 光, 因此 photolytically 在胱氨酸199和胱氨酸343上切割邻硝基苄基基团, 生成激活的 PKA。TEM 图像和布拉德福德化验证实, PKA 和笼 PKA 被固定在 UCNPs 表面, 并在紫外 UCNP 检测后的笼 PKA 活化溶液的激酶活性 (<strong class="xfig"...

Watch this Scientific Journal Video about An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation at JoVE.com

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

在这个协议中, 笼状蛋白激酶 a (PKA), 细胞信号转导 bioeffector, 被固定在纳米粒子表面, 微量进入胞, 并激活由 upconverted 紫外线光近红外线 (近红外) 照射, 诱导下游应力纤维解体的胞。

转换纳米粒子介导的激酶活化远程触发细胞内信号转导的集成系统

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

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Research

JoVE Journal

Bioengineering

7.4K 次观看.  Academia Sinica. 在这个协议中, 笼状蛋白激酶 a (PKA), 细胞信号转导 bioeffector, 被固定在纳米粒子表面, 微量进入胞, 并激活由 upconverted 紫外线光近红外线 (近红外) 照射, 诱导下游应力纤维解体的胞。

视频: An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

A 'Plug and Play' Method to Create Water-dispersible Nanoassemblies Containing an Amphiphilic Polymer, Organic Dyes and Upconverting Nanoparticles

In this protocol, we first describe a procedure to synthesize lanthanide doped upconverting nanoparticles (UCNPs). We then demonstrate how to generate amphiphilic polymers in situ, and describe a protocol to encapsulate the prepared UCNPs and different organic dye molecules (porphyrins and diarylethenes) using polymer shells to form stable water-dispersible nanoassemblies. The nanoassembly samples containing both the UCNPs and the diarylethene organic dyes have interesting photochemical and photophysical properties. Upon 365 nm UV irradiation, the diarylethene group undergoes a visual color change. When the samples are irradiated with visible light of another specific wavelength, the color fades and the samples return to the initial colorless state. The samples also emit visible light from the UCNPs upon irradiation with 980 nm near-infrared light. The emission intensity of the samples can be tuned through alternate irradiation with UV and visible light. Modulation of fluorescence can be performed for many cycles without observable degradation of the samples. This versatile encapsulation procedure allows for the transfer of hydrophobic molecules and nanoparticles from an organic solvent to an aqueous medium. The polymer helps to maintain a lipid-like microenvironment for the organic molecules to aid in preservation of their photochemical behavior in water. Thus this method is ideal to prepare water-dispersible photoresponsive systems. The use of near-infrared light to activate upconverting nanoparticles allows for lower energy light to be used to activate photoreactions instead of more harmful ultraviolet light.

Organic dye molecules and oleic acid coated upconverting nanoparticles are not water-soluble. This protocol describes a &#8216;plug and play&#8217; method that enables the transfer of organic dye molecules and upconverting particles from their initial hydrophobic solvent to water.

A&#39;即插即用&#39;的方法来创建水分散性Nanoassemblies包含两亲聚合物，有机染料和上变频纳米粒子

In this protocol, we first describe a procedure to synthesize lanthanide doped upconverting nanoparticles (UCNPs). We then demonstrate how to generate ...

科研

化学

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

在细胞分化过程中光介导的可逆调节丝裂原活化蛋白激酶途径<em&gt;爪蟾</em&gt;胚胎发育

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

发育生物学

Synthesis of Core-shell Lanthanide-doped Upconversion Nanocrystals for Cellular Applications

Lanthanide-doped upconversion nanocrystals (UCNs) have attracted much attention in recent years based on their promising and controllable optical properties, which allow for the absorption of near-infrared (NIR) light and can subsequently convert it into multiplexed emissions that span over a broad range of regions from the UV to the visible to the NIR. This article presents detailed experimental procedures for high-temperature co-precipitation synthesis of core-shell UCNs that incorporate different lanthanide ions into nanocrystals for efficiently converting deep-tissue penetrable NIR excitation (808 nm) into a strong blue emission at 480 nm. By controlling the surface modification with biocompatible polymer (polyacrylic acid, PAA), the as-prepared UCNs acquires great solubility in buffer solutions. The hydrophilic nanocrystals are further functionalized with specific ligands (dibenzyl cyclooctyne, DBCO) for localization on the cell membrane. Upon NIR light (808 nm) irradiation, the upconverted blue emission can effectively activate the light-gated channel protein on the cell membrane and specifically regulate the cation (e.g., Ca2+) influx in the cytoplasm. This protocol provides a feasible methodology for the synthesis of core-shell lanthanide-doped UCNs and subsequent biocompatible surface modification for further cellular applications.

A protocol is presented for the synthesis of core-shell lanthanide-doped upconversion nanocrystals (UCNs) and their cellular applications for channel protein regulation upon near-infrared (NIR) light illumination.

核壳镧系掺杂转换纳米晶的合成及其在蜂窝中的应用

Lanthanide-doped upconversion nanocrystals (UCNs) have attracted much attention in recent years based on their promising and controllable optical ...

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate light-gated ion channels and pumps in neurons. While this bioluminescent optogenetics (BL-OG) approach confers a chemogenetic component to optogenetic tools, it is not limited to use in neuroscience. Rather, bioluminescence can be harnessed to activate any photosensory protein, thus enabling the manipulation of a multitude of light-mediated functions in cells. A variety of luciferase-luciferin pairs can be matched with photosensory proteins requiring different wavelengths of light and light intensities.
Depending on the specific application, efficient light delivery can be achieved by using luciferase-photoreceptor fusion proteins or by simple co-transfection. Photosensory proteins based on light-dependent dimerization or conformational changes can be driven by bioluminescence to effect cellular processes from protein localization, regulation of intracellular signaling pathways to transcription. The protocol below details the experimental execution of bioluminescence activation in cells and organisms and describes the results using bioluminescence-driven recombinases and transcription factors. The protocol provides investigators with the basic procedures for carrying out bioluminescent optogenetics in vitro and in vivo. The described approaches can be further extended and individualized to a multitude of different experimental paradigms.

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence-light emitted by a luciferase enzyme oxidizing a small-molecule substrate, a luciferin-can be harnessed to activate photosensory proteins, thereby adding another dimension to light stimulation and enabling the manipulation of a multitude of light-mediated functions in cells across temporal and spatial scales.

生物发光光遗传学2.0:利用生物发光在体外和体内激活光敏蛋白

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate ...

生物化学

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

生物学

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological synapse, a stereotyped cell-cell junction that regulates T cell activation and directionally targets effector responses. To study these processes effectively, an imaging approach that is tailored to capturing fast, polarized responses is necessary. This protocol describes such a system, which is based on a photoactivatable peptide-major histocompatibility complex (pMHC) that is non-stimulatory until it is exposed to ultraviolet light. Targeted decaging of this reagent during videomicroscopy experiments enables precise spatiotemporal control of TCR activation and high-resolution monitoring of subsequent cellular responses by total internal reflection (TIRF) imaging. This approach is also compatible with genetic and pharmacological perturbation strategies. This allows for the assembly of well-defined molecular pathways that link TCR signaling to the formation of the polarized cytoskeletal structures that underlie the immunological synapse.

This protocol describes an imaging-based method to activate T lymphocytes using photoactivatable peptide-MHC, enabling precise spatiotemporal control of T cell activation.

Photoactivatable 激动剂对 T 细胞活化的时空控制

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological ...

免疫与感染

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. Despite their importance, technologies for tracking neuromodulatory events with cellular resolution are just beginning to emerge. Neuromodulators, such as dopamine, norepinephrine, acetylcholine, and serotonin, trigger intracellular signaling events via their respective G protein-coupled receptors to modulate neuronal excitability, synaptic communications, and other neuronal functions, thereby regulating information processing in the neuronal network. The above mentioned neuromodulators converge onto the cAMP/protein kinase A (PKA) pathway. Therefore, in vivo PKA imaging with single-cell resolution was developed as a readout for neuromodulatory events in a manner analogous to calcium imaging for neuronal electrical activities. Herein, a method is presented to visualize PKA activity at the level of individual neurons in the cortex of head-fixed behaving mice. To do so, an improved A-kinase activity reporter (AKAR), called tAKAR&#945;, is used, which is based on F&#246;rster resonance energy transfer (FRET). This genetically-encoded PKA sensor is introduced into the motor cortex via in utero electroporation (IUE) of DNA plasmids, or stereotaxic injection of adeno-associated virus (AAV). FRET changes are imaged using two-photon fluorescence lifetime imaging microscopy (2pFLIM), which offers advantages over ratiometric FRET measurements for quantifying FRET signal in light-scattering brain tissue. To study PKA activities during enforced locomotion, tAKAR&#945; is imaged through a chronic cranial window above the cortex of awake, head-fixed mice, which run or rest on a speed-controlled motorized treadmill. This imaging approach will be applicable to many other brain regions to study corresponding behavior-induced PKA activities and to other FLIM-based sensors for in vivo imaging.

A procedure is presented to visualize protein kinase A activities in head-fixed, behaving mice. An improved A-kinase activity reporter, tAKAR&#945;, is expressed in cortical neurons and made accessible for imaging through a cranial window. Two-photon fluorescence lifetime imaging microscopy is used to visualize PKA activities in vivo during enforced locomotion.

可视化蛋白质激酶 在头部固定行为小鼠中使用 Vivo 双光子荧光寿命成像显微镜的活性

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. ...

神经科学

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase (ERK) Signaling or Mitochondrial Events in Target Cells

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation can simultaneously activate multiple cellular molecular signaling pathways. In this protocol, we show that through coupling femtosecond laser into a confocal microscope, cells can be stimulated precisely by the tightly-focused laser. Some molecular processes that can be simultaneously observed are subsequently activated. We present detailed protocols of the photostimulation to activate extracellular signal regulated kinase (ERK) signaling pathway in Hela cells. Mitochondrial flashes of reactive oxygen species (ROS) and other mitochondrial events can be also stimulated if focusing the femtosecond laser pulse on a certain mitochondrial tubular structure. This protocol includes pretreating cells before photostimulation, delivering the photostimulation by a femtosecond laser flash onto the target, and observing/identifying molecular changes afterwards. This protocol represents an all-optical tool for related biological researches.

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase ERK Signaling or Mitochondrial Events in Target Cells

Tightly-focused femtosecond laser can deliver precise stimulation to cells by being coupled into a confocal microscopy enabling the real-time observation and photostimulation. The photostimulation can activate cell molecular events including ERK signaling pathway and mitochondrial flashes of reactive oxygen species.

飞秒激光的光刺激激活目标细胞中的细胞外信号调节激酶(ERK)信号或线粒体事件

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation ...

A Light-Stimulation Grid Approach to Study Cellular Optogenetic Protein

Mapping the Cellular Distribution of an Optogenetic Protein Using a Light-Stimulation Grid

Our goal was to accurately track the cellular distribution of an optogenetic protein and evaluate its functionality within a specific cytoplasmic location. To achieve this, we co-transfected cells with nuclear-targeted cAMP sensors and our laboratory-developed optogenetic protein, bacterial photoactivatable adenylyl cyclase-nanoluciferase (bPAC-nLuc). bPAC-nLuc, when stimulated with 445 nm light or luciferase substrates, generates adenosine 3&#39;,5&#39;-cyclic monophosphate (cAMP). We employed a solid-state laser illuminator connected to a point scanning system that allowed us to create a grid/matrix pattern of small illuminated spots (~1 &#181;m2) throughout the cytoplasm of HC-1 cells. By doing so, we were able to effectively track the distribution of nuclear-targeted bPAC-nLuc and generate a comprehensive cAMP response map. This map accurately represented the cellular distribution of bPAC-nLuc, and its response to light stimulation varied according to the amount of protein in the illuminated spot. This innovative approach contributes to the expanding toolkit of techniques available for investigating cellular optogenetic proteins. The ability to map its distribution and response with high precision has far-reaching potential and could advance various fields of research.

This protocol involves transfecting cAMP sensors and bPAC-nLuc, an optogenetic protein, to accurately track its cellular distribution and response to light stimulation. The innovative approach of creating a cAMP response map using a point scanning system holds the potential for advancing research with optogenetic proteins in different fields.

转换纳米粒子介导的激酶活化远程触发细胞内信号转导的集成系统

摘要

探索更多视频

此视频中的章节

A'即插即用'的方法来创建水分散性Nanoassemblies包含两亲聚合物，有机染料和上变频纳米粒子

在细胞分化过程中光介导的可逆调节丝裂原活化蛋白激酶途径

核壳镧系掺杂转换纳米晶的合成及其在蜂窝中的应用

生物发光光遗传学2.0:利用生物发光在体外和体内激活光敏蛋白

在现场的斑马鱼胚胎的基于双光子Photoactivation

Photoactivatable 激动剂对 T 细胞活化的时空控制

可视化蛋白质激酶在头部固定行为小鼠中使用 Vivo 双光子荧光寿命成像显微镜的活性

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

飞秒激光的光刺激激活目标细胞中的细胞外信号调节激酶(ERK)信号或线粒体事件

Mapping the Cellular Distribution of an Optogenetic Protein Using a Light-Stimulation Grid

转换纳米粒子介导的激酶活化远程触发细胞内信号转导的集成系统

摘要

探索更多视频

此视频中的章节

A'即插即用'的方法来创建水分散性Nanoassemblies包含两亲聚合物，有机染料和上变频纳米粒子

在细胞分化过程中光介导的可逆调节丝裂原活化蛋白激酶途径

核壳镧系掺杂转换纳米晶的合成及其在蜂窝中的应用

生物发光光遗传学2.0:利用生物发光在体外和体内激活光敏蛋白

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

Photoactivatable 激动剂对 T 细胞活化的时空控制

可视化蛋白质激酶 在头部固定行为小鼠中使用 Vivo 双光子荧光寿命成像显微镜的活性

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

飞秒激光的光刺激激活目标细胞中的细胞外信号调节激酶(ERK)信号或线粒体事件

Mapping the Cellular Distribution of an Optogenetic Protein Using a Light-Stimulation Grid

在现场的斑马鱼胚胎的基于双光子Photoactivation

可视化蛋白质激酶在头部固定行为小鼠中使用 Vivo 双光子荧光寿命成像显微镜的活性