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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 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?…
Representative Results
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"…
Discussion
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,</…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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)。
Materials
| Reagent | |||
| Tris(hydorxymethyl)aminomethane | Sigma | 154563 | |
| Magnesium chloride hexahydrate | Sigma | M9272 | |
| MOPS | Sigma | M1254 | |
| HEPES | Sigma | H4034 | |
| Sodium chloride | Sigma | 31434 | |
| Potassium chloride | Sigma | 12636 | |
| Yttrium acetate hydrate | Sigma | 326046 | Y(C2H3CO2)3 · xH2O |
| Thulium(III) acetate hydrate | Alfa Aesar | 14582 | Tm(CH3CO2)3 · xH2O |
| Ytterbium(III) acetate tetrahydrate | Sigma | 326011 | Yb(C2H3O2)3 · 4H2O |
| 1-Octadecene | Sigma | O806 | |
| Oleic acid | Sigma | 364525 | |
| Methanol | macron | 304168 | |
| Sodium hydroxide | Sigma | 30620 | |
| Ammonium fluoride | J.T.Baker | 69804 | |
| IGEPAL CO-520 | Sigma | 238643 | |
| Cyclohexane | J.T.Baker | 920601 | |
| Amomonium hydroxide (28%-30%) | J.T.Baker | 972101 | Ammonia |
| Tetraethyl orthosilicate (TEOS) | Sigma | 8658 | |
| DL-Dithiothreitol (DTT) | Sigma | D0632 | |
| N-hydroxymaleimide (NHM) | Sigma | 226351 | PKA activity blocking reagent |
| Prionex protein stabilizer solution from hog collagen | Sigma | 81662 | Protein stabilizer solution |
| 2-nitrobenzyl bromide (NBB) | Sigma | 107794 | PKA caging reagent |
| 8-(4-Chlorophenylthio)adenosine 3′,5′-cyclic monophosphate sodium salt | Sigma | C3912 | 8-CPT-cAMP |
| Pyruvate Kinase/Lactic Dehydrogenase enzymes from rabbit muscle | Sigma | P0294 | PK/LDH |
| Adenosine 5'-triphosphate disodium | Sigma | A2387 | ATP |
| β-NADH reduced from dipotassium | Sigma | N4505 | |
| Phosphoenolpyruvate | Sigma | P7127 | PEP |
| Coomassie Protein Assay Reagent, 950 ml | Thermo Scientific | 23200 | Bradford assay reagent |
| cAMP-dependent protein kinase | Promega | V5161 | PKA activity control |
| pET15b-PKACAT plasmid | Addgene | #14921 | |
| pKaede-MC1 plasmid | CoralHue | AM-V0012 | |
| Phosphate buffered saline (PBS), pH 7.4 | Thermo Scientific | 10010023 | |
| DMEM, high glucose, pyruvate | Gibco | 12800-017 | Cell culture medium |
| Leibovitz L-15 Medium | Biological Industries | 01-115-1A | Cell culture medium |
| Fetal Bovine Serum | Biological Industries | 04-001-1A | |
| Paraformaldehyde | ACROS | 416785000 | |
| DAPI | Invitrogen | D1306 | Nucleus staining dye |
| Alexa 594-phalloidin | Invitrogen | A12381 | F-actin staining dye |
| 5(6)-Carboxyfluorescein | Novabiochem | 8.51082.0005 | |
| 5(6)-Carboxytetramethylrhodamine | Novabiochem | 8.51030.9999 | |
| Pierce Coomassie (Bradford) Protein Assay Kit | Thermo Scientific | 23200 | |
| CelluSep T4 Tubings/Nominal filter rating MWCO 12000-14000 Da | Membrane Filtration Products, Inc. | 1430-33 | Dialysis membrane |
| Millex-HV Syringe Filter Unit, 0.45 µm, PVDF, 13 mm, gamma sterilized | EMD Milipore | SLHVX13NL | |
| Equipment | |||
| Dynamic Light Scattering/Zetapotential Zetasizer nano-ZS | Malvern | M104 | |
| Transmission Electron Microscope | JEOL | JEM-1400 | |
| Fluorescence Spectrophotometer | Agilent Technologies | 10075200 | Cary Eclipse |
| UV-Vis Spectrophotometer | Agilent Technologies | 10068900 | Cary 50 |
| Fluorescence Microscopy | Olympus | IX-71 | |
| 950 nm longpass filter | Thorlabs | FEL0950 | |
| 850 nm dichroic mirror shortpass | Chroma | NC265609 | |
| RT3 color CCD system | SPOT | RT2520 | |
| Fluorescence Illumination | PRIOR | Lumen 200 | |
| 980nm Infra-red diode laser | CNI | MDL-N-980-8W | |
| UV LED Spot Light Source | UVATA | UVATA-UPS412 | With a UPH-056-365 nm LED at 200 mW/cm2 |
| Thermal pile sensor | OPHIR | 12A-V1-ROHS | |
| Picospritzer III | Parker Hannifin | 052-0500-900 | Intracellular Microinjection Dispense Systems |
| PC-10 Needle puller | Narishige | PC-10 | |
| MANOMETER Digital pressure gauge | Lutron | PM-9100 | |
| One-axis Oil Hydraulic Micromanipulator | Narishige | MMO-220A | |
| Heraeus Fresco 17 Centrifuge, Refrigerated | Thermo Scientific | 75002421 |
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Citer Cet Article
Gao, H., Thanasekaran, P., Chen, T., Chang, Y., Chen, Y., Lee, H. An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation. J. Vis. Exp. (126), e55769, doi:10.3791/55769 (2017).