陶子细胞定位作为研究疾病相关基因表达的工具的调制
Summary
Tau 是一种神经元蛋白，存在于细胞质中，它结合微管，在细胞核中，它发挥非常规功能，包括与阿尔茨海默氏病相关基因的调制。在这里，我们描述了一种研究核Tau功能的方法，同时排除了来自细胞质陶的任何干扰。
Abstract
Tau是一种在神经元中表达的微管结合蛋白，其主要已知功能与维持细胞骨骼稳定性有关。然而，最近的证据表明，Tau也存在于其他亚细胞室中，包括其与DNA保护、rRNA转录、逆转录泊剂流动性和结构组织有关的细胞核。核仁。我们最近已经证明，核Tau参与VGluT1基因的表达，建议一种分子机制，可以解释谷氨酸释放在阿尔茨海默氏病的早期阶段的病理增加。直到最近，由于技术限制，防止了细胞质Tau的贡献或其它影响，核陶参与调节目标基因的表达一直相对不确定和模糊。下游因素与核陶无关。为了克服这种不确定性，我们开发了一种方法，研究由核Tau蛋白专门调节的目标基因的表达。我们采用了一种协议，将定位信号和亚细胞分馏的使用耦合在一起，从而排除了细胞质Tau分子的干扰。最值得注意的是，该协议是容易的，由经典和可靠的方法组成，广泛适用于研究Tau在其他细胞类型和细胞条件下的核功能。
Introduction
Tau蛋白在核中的功能近年来引起了极大的兴趣，因为它已被证明与核酸1，2，3，4，5，6密切相关。事实上，最近的一项全基因组研究表明，Tau在体内结合基因和基因DNA序列。在核组织中的作用已被建议8，9，10，11。此外，Tau被建议参与DNA保护，免受氧化和高热应激5、10、12、13的影响，而突变的陶已与染色体不稳定和抗倍体14、15、16有关。
直至目前为止，研究陶在核舱的功能方面的挑战，由于难以剖析核陶在核质Tau的贡献中的具体贡献，因此，研究陶在核舱中所面临的挑战，几乎仍未解决。此外，迄今为止，核隔间中的Tau分子的功能只是相关的，因为它们缺乏核Tau蛋白直接参与的明确证明。事实上，陶参与逆子的流动或rRNA转录或DNA保护11、12、17、18、19，也可以由细胞质陶的贡献或与核陶无关的其他下游因素的影响来解释。
在这里，我们提供了一个方法，可以通过利用一个经典程序来隔离核隔间，结合使用带有核定位（NLS）或核出口信号（NES）标记的Tau构造0N4R来解决这个问题。这种方法消除了由于Tau分子从细胞质室溢出而可能产生的文物的复杂问题。此外，Tau-NLS和Tau-NES结构分别诱导Tau分子从核隔间中浓缩或排除，为核Tau分子参与特定功能提供正负控制。该协议在技术上是容易的，它由经典和可靠的方法组成，广泛适用于研究Tau在其他细胞类型中的核功能，有区别与否，如重新激活Tau表达20，21的癌细胞。此外，它也可以应用于细胞质和细胞核中存在的其他蛋白质，以解剖与不同隔间相关的生物功能。
Protocol
1. 细胞培养 培养SH-SY5Y细胞（人类神经母细胞瘤细胞系，CRL-2266）在完整的介质（Dulbeco的改性鹰培养基：营养混合物F12[DMEM/F-12]补充10%胎儿牛血清[FBS]，2mM L-谷氨酰胺，100 U/mL青霉素和100微克/mL链霉素）。将孵化器中的细胞保持在37°C和5%CO2。在10厘米的板中生长细胞，并在汇入时分裂。 2. 细胞分化 为了区分SH-SY5Y细胞，在电镀后的第二天，加入10μM视?…
Representative Results
图1描述了用于剖析核陶在基因表达中避免细胞质Tau蛋白贡献的策略。简单地说，标有NLS或NES标记的Tau蛋白分别在核隔间中积累或排除。这种不平衡的功能效应是作为VGluT1基因产物测量的基因表达的改变。 按照协议描述，SH-SY5Y细胞用RA处理5天，然后用BDNF治疗3天，以获得后线神经样细胞（?…
Discussion
我们描述了一种测量核Tau蛋白对基因表达影响的方法。有了这个协议，细胞质陶的贡献是强烈的限制。该协议的关键步骤如下：人类神经母细胞瘤SH-SY5Y细胞的分化、亚细胞分馏和Tau蛋白在核腔中的定位。
首先，如代表性结果部分所示，通过添加RA和BDNF来分化SH-SY5Y细胞对于在培养中获得神经元样细胞的良好制备至关重要。播种的细胞密度特别重要，因为较低的密度可能会影响…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作得到了斯库拉师范高级院（SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIO;SNS14_B_DIPRIMIOSNS16_B_DIPRIMIO）。
Materials
| Alexa Fluor 633 goat anti-mouse IgG | Life Technologies | A21050 | IF 1:500 |
| anti Actin Antibody | BETHYL LABORATORIE | A300-485A | anti-rabbit WB 1:10000 |
| anti GAPDH Antibody | Fitzgerald Industries International | 10R-G109a | anti-mouse WB 1:10000 |
| anti H2B Antibody | Abcam | ab1790 | anti-rabbit WB 1:15000 |
| anti Tau-13 Antibody | Santa Cruz Biotechnology | sc-21796 | anti-mouse WB 1:1000; IF 1:500 |
| anti Tubulin alpha Antibody | Thermo Fisher Scientific | PA5-16891 | anti-mouse WB 1:5000 |
| anti VGluT1 Antibody | Sigma-Aldrich | AMAb91041 | anti-mouse WB 1:500 |
| BCA Protein Assay Kit | Euroclone | EMPO14500 | |
| BDNF | Alomone Labs | B-250 | |
| Blotting-Grade Blocker | Biorad | 1706404 | Non-fat dry milk |
| BOVIN SERUM ALBUMIN | Sigma-Aldrich | A4503-50g | |
| cOmplete Mini | Roche | 11836170001 | protease inhibitor |
| Criterion TGX 4-20% Stain Free, 10 well | Biorad | 5678093 | |
| DAPI | Thermo Fisher Scientific | 62247 | |
| DMEM/F-12 | GIBCO | 21331-020 | |
| Dulbecco's Modified Eagle's Medium Low Glucose | Euroclone | ECM0060L | |
| EDTA | Sigma-Aldrich | 0390-100ml | pH=8 0.5M |
| Foetal Bovine Serum | Euroclone | EC50182L | |
| Glycerol | Sigma-Aldrich | G5516-500ml | |
| Goat anti-mouse IgG-HPR | Santa Cruz Biotechnology | sc-2005 | WB 1:1000 |
| Goat anti-rabbit IgG-HPR | Santa Cruz Biotechnology | sc-2004 | WB 1:1000 |
| IGEPAL CA-630 | Sigma-Aldrich | I8896-50ml | Octylphenoxy poly(ethyleneoxy)ethanol |
| Immobilon Western | MERCK | WBKLS0500 | |
| Lab-Tech Chamber slide 8 well glass slide | nunc | 177402 | |
| L-glutamine | Euroclone | ECB3000D | 100X |
| Lipofectamine 2000 transfection reagent | Thermo Fisher Scientific | 12566014 | cationic lipid |
| Methanol | Sigma-Aldrich | 322415-6X1L | |
| MgCl2 | Sigma-Aldrich | M8266-100G | |
| NaCl | Sigma-Aldrich | S3014-1kg | |
| Opti-MEM reduced serum medium | Gibco | 31985070 | |
| PEI | Sigma-Aldrich | 40,872-7 | |
| Penicillin/Streptomycin | Thermo Fisher Scientific | 15140122 | 10,000 U/ml, 100ml |
| Phosphate Buffered Saline (Dulbecco A) | OXOID | BR0014G | |
| PhosStop | Roche | 4906837001 | phosphatase inhibitor |
| QIAGEN Plasmid Maxi Kit | Qiagen | 12163 | Step 3.10 |
| Retinoic acid | Sigma-Aldrich | R2625-100mg | |
| Subcellular Protein Fractionation Kit for cultured cells | Thermo Fisher Scientific | 78840 | |
| Supported Nitrocellulose membrane | Biorad | 1620097 | |
| TC-Plate 6well | SARSTEDT | 833,920 | |
| TCS SP2 laser scanning confocal microscope | Leica | N/A | |
| Triton x-100 | Sigma-Aldrich | X100-500ml | Non-ionic surfactant |
| Trypsin-EDTA | Thermo Fisher Scientific | 15400054 | 0.50% |
| Tween-20 | Sigma-Aldrich | P9416-100ml | |
| VECTASHIELD antifade mounting medium | Vector Laboratories | H-1000 | |
| Wizard Plus SV Minipreps DNA Purification Systems | Promega | A1330 | Step 3.5 |
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Citar este artículo
Siano, G., Caiazza, M. C., Varisco, M., Calvello, M., Quercioli, V., Cattaneo, A., Di Primio, C. Modulation of Tau Subcellular Localization as a Tool to Investigate the Expression of Disease-related Genes. J. Vis. Exp. (154), e59988, doi:10.3791/59988 (2019).