大肠癌细胞中磷酸化和微管结合与 Tau 蛋白定位的测定
Summary
本手稿描述了测量 tau tau, 测量 tau 结合到微管的标准协议, 以及药物治疗后细胞内 tau 的定位。这些协议可以重复使用, 以筛选药物或其他化合物的目标 tau 或微管结合。
Abstract
微管相关蛋白 tau 是一种神经元蛋白, 本地化主要在轴突。一般来说, tau 是正常的神经功能的必要条件, 因为它涉及微管组装和稳定。除了神经元, tau 是在人的乳房, 前列腺, 胃, 结直肠癌和胰腺癌, 它显示几乎类似的结构和发挥类似功能的神经元 tau。tau 及其磷酸化的数量可以改变其作为微管的稳定剂的功能, 并导致在不同的神经退行性疾病, 如阿尔茨海默氏病的配对螺旋纤维的发展。确定 tau 的磷酸化状态及其微管结合特性是很重要的。此外, 检查 tau 的细胞内定位是重要的不同疾病。这份手稿详细的标准协议, 测量 tau 磷酸化和 tau 结合到微管的大肠癌细胞与或没有姜黄素和氯化治疗。这些治疗可以用来阻止癌细胞的增殖和发育。在使用低剂量的抗体时, 使用免疫组化和共聚焦显微镜检查 tau 的细胞内定位。这些化验可以反复用于筛选影响 tau tau 或微管结合的化合物。新的治疗方法用于不同的 tauopathies 或相关的抗癌药物, 可以潜在的特点使用这些协议。
Introduction
头最初被确定为热稳定的微管相关蛋白, co-purified 与蛋白1。头是完全表达在更高的真核生物2,3,4。tau 的主要功能是控制微管组件1,5,6。它还有助于聚合的微管的7, 轴突传输的8, 轴突直径9, 形成的神经瘤极性, 和神经10。Tau 也充当蛋白质支架来控制一些信号通路。鼠脑研究表明, tau 是神经元特异的, 它主要本地化在轴突11。由于 tau 是微管聚合和神经元发育的关键, 因此 tau 被假设在中枢神经系统的轴突发育中起主要作用;这一假说后来通过体外和活体实验来验证。除了神经元, tau 是表达在不同的神经细胞细胞, 包括肝脏, 肾脏和肌肉细胞12,13。Tau 也被表达在人的乳房, 前列腺, 大肠, 胃, 和胰腺癌细胞系和组织14,15,16,17,18,19. 头也被发现在包涵体肌炎作为扭 tubulofilaments 在包含身体20。
头可进行几修饰修改。在所有修饰的修饰中, 磷酸化是最常见的。增加的 tau 磷酸化降低了它对微管的亲和性, 最终破坏了细胞骨架。八十五磷酸化部位已被描述在 tau 蛋白从人类阿尔茨海默病的脑组织分离。这些网站中, 53% 构成丝氨酸, 41% 苏氨酸, 只有6% 酪氨酸残留物21,22,23。Tau 磷酸化影响其定位, 功能, 约束力, 溶解度, 其易感性的其他修饰的修改。并且 tau 磷酸化到超过正常程度 (或完全饱和与磷酸盐小组) 被称为 tau, 复制结构和功能特征的阿尔茨海默氏病24。Tau 保持轴突微管的正常功能, 并确保在生理条件下的神经功能。然而, hyperphosphorylated tau 没有保持一个组织良好的微管结合, 导致神经元的损失, 因为微管的拆卸。tau 磷酸化的正常水平是需要正确的 tau 功能, 但 tau 无法正常工作, 如果其特征磷酸化水平被改变, 如果它是 hyperphosphorylated25。在阿尔茨海默氏病和其他一些与年龄相关的神经退行性疾病中, tau 成为 hyperphosphorylated, 形成配对的螺旋纤维和纤维缠结26,27。因此, 确定 tau 磷酸化和微管结合的方法很重要。
结直肠癌, 一个年龄相关的癌症, 是第三个最常见的诊断癌症和第三个突出的死亡导致的男性和女性的癌症28。大肠癌是西方世界主要的致死性癌症之一,29。因为结肠直肠癌和阿尔茨海默病都与衰老有关, 这两种疾病主要发生在发达国家, 那里的人们享有类似的饮食习惯, 这两个疾病可能会有某种程度的关联。此外, tau 阳性和 tau 阴性癌细胞对化疗药物的反应不同,例如, 紫杉醇16。
姜黄素是姜黄姜黄的主要衍生物之一, 印度香料姜黄30。几个世纪以来, 南亚人口每天都在饮食中消耗姜黄。姜黄素用于治疗不同的疾病, 包括结直肠癌, 阿尔茨海默氏病, 糖尿病, 囊性纤维化, 炎症肠病, 关节炎, 高血脂, 动脉粥样硬化和缺血性心脏病31,32,33,34,35,36,37,38. 锂也能杀死大肠癌细胞或防止其增殖39。锂也可用于治疗阿尔茨海默氏病40 , 因为它减少 tau 聚集, 并防止其 tau 在转基因鼠标模型41,42,43中观察到, 44。
本手稿旨在: 1) 测量治疗细胞的总 tau 和磷头表达水平;2) 描述磷酸酶的测定, 以测量整体 tau 磷酸化;3) 检查 tau 的微管结合;4) 用姜黄素或氯化在大肠癌细胞系中用共焦显微镜定位 tau。结果表明, 细胞治疗的姜黄素, 这是一个所谓的良好的化疗药物的结肠癌, 和治疗氯化可以减少在大肠癌细胞系的总 tau 和磷酸化 tau 的表达。这些处理也可能导致头的核易位。然而, 出乎意料的是, 姜黄素未能改善 tau 与微管的结合。
Protocol
1. 试剂的制备 添加10和 #181; (1 毫米) 磺氟化物溶液, 10 和 #181; l (1x 从100x 库存) 蛋白酶抑制剂鸡尾酒溶液, 10 和 #181; l (1x 从100x 存货) 磷酸酶抑制剂鸡尾酒解决方案, 以1毫升的 1x radioimmunoprecipitation 检测 (帕) 缓冲区准备完整的帕裂解缓冲液. 准备10x 通用蛋白缓冲区 (PEM) 缓冲区. 添加800毫米 (6.0474 克) 哌嗪-n-氮和 #39;-bis-2-ethanesulfonic 酸, 10 毫米 (95.1 毫克) 乙二?…
Representative Results
用不同浓度的姜黄素或氯化 (图 1) 治疗后, 对全头和磷的表达进行了研究。三种不同浓度的姜黄素对细胞的治疗降低了 tau 表达水平;但是, 磷在低浓度姜黄素的治疗下增加, 但在处理姜黄素浓度较高的细胞时却减少。抗磷头 (Ser396) 用于检测磷头。在三种不同浓度的氯化 (图 1) 中, 总 tau 和磷的水平都降低了。先前的研究表明, tau ?…
Discussion
该手稿建立了不同的程序条件, 检测总 tau 和磷酸化 tau 在大肠癌细胞治疗姜黄素和氯化。为了评估蛋白样品中 tau 的总磷酸化状态, 本文描述了磷酸酶的测定方法。这种检测方法有可能用于检测任何蛋白质的磷酸化状态。
这种方法是基于磷酸化蛋白比其 non-phosphorylated 状态慢的原理。本协议采用碱性磷酸酶和碱性磷酸酶缓冲剂。在将化验成分加入到细胞裂解后, 样品应在特定的…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究是作为项目的一部分, 题为 ‘ 开发和产业化的高价值的海洋微藻的化妆品原料 ‘, 由海洋和渔业部, 韩国, 并得到了内部资助 (2Z04930) 的支持来自 KIST 江陵天然产物研究所。
Materials
| HCT 116 cell | ATCC | CCL-247 | |
| MEM (EBSS) | Hyclone | SH30024.01 | |
| Fetal Bovine Serum (FBS) | ThermoFisher (Gibco) | 16000044 | Store at -20 °C |
| penicillin-streptomycin | Hyclone | SV30010 | |
| Trypsin-EDTA solution | WelGene | LS 015-01 | |
| 100 mm dish | Corning | 430161 | |
| 6 well plate | Corning | Coster 3516 | |
| Anti-Tau 13 antibody | abcam | ab19030 | |
| Dithiothreitol (DTT) | Roche | 10 708 984 001 | Storage Temperature 2–8 °C |
| Microlitre Centrifuges | Hettich Zentrifugen | MIKRO 200 R | |
| Paclitaxel | Sigma-Aldrich | T1912 | Storage Temperature 2–8 °C |
| Curcumin | Sigma-Aldrich (Fluka) | 78246 | Storage Temperature 2–8 °C |
| Microtubules (MT) | Cytoskeleton | MT001 | Store at 4 °C (desiccated) |
| Mounting Medium with DAPI | Vector Laboratories | H-1200 | Store at 4 °C in the dark |
| Sodium hydroxide | Sigma | 72068 | |
| Magnesium Chloride | Sigma-Aldrich | M2670 | |
| GTP | Sigma-Aldrich | G8877 | Store at -20 °C |
| DPBS | WelGene | LB 001-02 | |
| Sonic Dismembrator | Fisher Scientific | Model 500 | |
| Ultracentrifuge | Beckman Coulter | Optima L-100 XP | |
| PIPES | Sigma | P1851 | |
| Bovine serum Albumin (BSA) | Sigma | A7906 | |
| Molecular Imager | Bio-Rad | ChemiDoc XRS+ | Store at 4 °C |
| Protein assay dye reagent | Bio-Rad | 500-0006 | |
| α-tubulin (11H10) Rabbit mAb | Cell signalling | 2125 | |
| GAPDH (14C10) Rabbit mAb | Cell signalling | 2118 | |
| Anti-Tau (phospho S396) antibody | abcam | ab109390 | |
| EGTA | Sigma | E3889 | Store at room temperature |
| FastAP Thermosensitive Alkaline Phosphatase | Thermo Scientific | EF0651 | Store at -20 °C |
| PMSF | Sigma | P7626 | Store at room temperature |
| Phosphatase Inhibitor Cocktail | Cell Signalling | 5870 | Store at 4 °C |
| Protease Inhibitor Cocktail | Cell Signalling | 5871 | Store at 4 °C |
| RIPA Buffer | Sigma | R 0278 | Storage Temperature 2–8 °C |
| Tau-352 human | Sigma | T 9950 | Store at -20 °C |
| Triton X-100 | Sigma-Aldrich | X – 100 | Store at around 25 °C |
| PVDF membrane | Bio-Rad | 162-0177 | |
| Goat anti-mouse IgG Secondary Antibody | ThermoFisher | A-11005 | Store at 4 °C in the dark |
| Confocal Microscopy | Leica Microsystem | Leica TCS SP5 | |
| Sodium Dodecyl Sulfate (SDS) | Affymetrix | 75819 | |
| Protein Assay | Bio-Rad | 500-0006 | Store at 4 °C |
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Cite This Article
Huda, M. N., Erdene-Ochir, E., Pan, C. Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells. J. Vis. Exp. (128), e55932, doi:10.3791/55932 (2017).