Name: assay to measure nucleocytoplasmic transport in real time within motor neuron-like nsc-34 cells
Uploaded: 2017-05-16T15:00:00
Description: Watch this Scientific Journal Video about Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells at JoVE.com

我们感谢AI实验室的所有成员的有用的意见和建议。我们要感谢Mark Hipp教授（马克斯·普朗克生物化学研究所）为我们提供了GFP-GFP载体。这项工作得到了以色列科学基金会（ISF＃124/14），滨海科技基金会（BSF＃2013325），FP7玛丽·居里职业融合赠款（CIG＃333794）和以色列国家心理生物学研究所NIPI＃b133-14 / 15）。

细胞系制备<ol><li>解冻储存在液氮容器中的约1,000,000只小鼠神经母细胞瘤脊髓细胞（NSC-34细胞）。使用37°C的水培养箱，直到细胞完全解冻。 </li><li>加入新鲜，温和的培养基（含有10％胎牛血清（FBS），1％L-谷氨酰胺和1％pen-strep抗生素的DMEM培养基）。 </li><li>以500×g离心解冻细胞（SL16R）5分钟，形成细胞沉淀。弃去培养基，加入1 mL新培养基用于细胞再悬浮。 </li><li>将再悬浮的细胞置于15mL烧瓶中?...

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该方案证明了一种高度敏感和定量的新的检测方法来评估核细胞质运输的微小变化。使用该系统，可以实时观察和测量核细胞质运输及其功能障碍，不仅会导致蛋白质分布发生显着变化的大缺陷。该测定不仅具有灵敏度的优点，而且还需要很少的制备，便宜，并且是非常容易且低参与度的测定。 为了测试该系统的效率，使用了先前显示引起核细胞质运输缺陷的C9orf72蛋白，聚?...

核孔复合体（NPC）控制大分子进出细胞核的进出口。与不受调节1进入细胞核的小分子相反，较大分子的转运受到NPC的强烈控制。这些大分子，如蛋白质和RNA，与运输因子（包括进口和出口）相关，从核心进口和出口2 。为了进口，蛋白质必须含有一个小的肽基序，通常被称为核定位信号（NLS），其由进口蛋白2约束。这些氨基酸序列用作标签，并且在组合物3,4 中是不同的。由于它们与出口有关，蛋白质可以从核出口到细胞质s，其结合信号序列，通常称为核导出信号（NES） 2 。由于Ras相关核蛋白GTPase（Ran） 2的调节，进口和出口都能运输货物。 Ran已被证明存在于不同的构象状态，这取决于它是否符合GTP或GDP。当绑定到GTP时，Ran可以绑定到importins或exportins。在绑定到RanGTP后，进口商发放货物，而出口商必须绑定RanGTP，以形成与其出口货物的综合体。 Ran的主要核苷酸结合状态取决于其在细胞核（RanGTP）或细胞质（RanGDP）中的位置。 最近，一些研究显示了核细胞质通路的损伤与肌萎缩性侧索硬化（ALS）和额颞叶痴呆（FTD） 5,6 </sup&gt; ， 7，8，9，10，11，12 。 ALS是进行性和致命的神经变性疾病，影响上下运动神经元（MN） 13 ，并导致瘫痪，最终死亡，平均在2-5年内。大多数ALS分类为散发性（SALS），缺乏任何明显的遗传连锁，但10％以主要方式（家族性ALS; FALS）遗传。最近，染色体9开放阅读框72（C9orf72）基因中的六核苷酸重复扩增（HREs）被鉴定为14,15作为ALS和FTD的遗传原因。这些突变体占FALS病例的30-40％ 16 ，不同的研究有争议它们通过影响核细胞质运输5,6,7,8,9,10,11,12造成毒性。 这些研究主要表现出HREs对核细胞质运输的最终结果和表现，但并不能实时显示核胞质中断。结果，只有严重的核细胞质运输缺陷已被评估11,17,18。 这个协议描述了一个新的和very敏感测定法实时评估和定量核细胞质运输功能障碍。使用荧光显微镜可以实时定量NLS-NES-GFP蛋白（穿梭GFP）的导入速率。如前所述，这是使用exportin抑制剂完成的，因此允许穿梭GFP仅进入细胞核。使用荧光显微镜和能够实时定量荧光变化的软件，可以量化位于细胞核中的穿梭GFP的荧光强度的逐渐变化。结果，可以制造出在任何给定时间表示核穿梭GFP的量的荧光时间轴的Michaelis-Menten样饱和曲线。通过使用曲线的初始线性速率，可以产生斜率，其表示在荧光饱和之前进入细胞核的速率。 验证测定，翻译已经报道已经报道破坏核细胞质运输的d C9orf72二肽重复序列，聚（GR）和聚（PR）被用于5,7,8,10,12。使用该测定，与对照相比，观察到核进口率降低了50％。使用该系统，可以检查核细胞质运输的微小变化。此外，可以确定不同因素拯救核细胞质运输缺陷的能力。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mouse Motor Neuron-Like hybrid Cell Line (NSC-34)</td>
			<td>tebu-bio</td>
			<td>CLU140-A</td>
		</tr>
		<tr>
			<td>DMEM 4.5g/L L-glucose</td>
			<td>Biological Industries</td>
			<td>01-055-1A</td>
		</tr>
		<tr>
			<td>FBS European Grade</td>
			<td>Biological Industries</td>
			<td>04-007-1A</td>
		</tr>
		<tr>
			<td>SL 16R Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004030</td>
		</tr>
		<tr>
			<td>Thermo Forma Series ii Water Jacketed CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>3111</td>
		</tr>
		<tr>
			<td>Cover glass 12mm dia. thick 0.13-0.17mm</td>
			<td>Bar Naor LTD</td>
			<td></td>
		</tr>
		<tr>
			<td><a href="https://www.thermofisher.com/order/catalog/product/R0531">TurboFect Transfection Reagent</a></td>
			<td>Thermo Scientific</td>
			<td><a href="https://www.thermofisher.com/order/catalog/product/R0531">R0531</a></td>
		</tr>
		<tr>
			<td>Axiovert 100 inverted microscope</td>
			<td>Zeiss</td>
			<td></td>
		</tr>
		<tr>
			<td>IMAGING WORKBENCH 2&#160;</td>
			<td>Axon Instruments</td>
			<td></td>
		</tr>
		<tr>
			<td>Leptomycin B</td>
			<td>Sigma</td>
			<td>L2913</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Biological Industries</td>
			<td>02-023-1A</td>
		</tr>
		<tr>
			<td>Homogenizer motor/control/chuck/90-degree clamp</td>
			<td>Glas-Col</td>
			<td>099C K5424CE</td>
		</tr>
		<tr>
			<td>MicroCL 17R Centrifuge, Refrigerated</td>
			<td>Thermo Scientific</td>
			<td>75002455</td>
		</tr>
	</tbody>
</table>

assay to measure nucleocytoplasmic transport in real time within motor neuron-like nsc-34 cells

核细胞质运输是指从细胞核进入和输出大分子。最近，一些研究已经显示了肌萎缩性侧索硬化（ALS）与核细胞质通路中的损伤之间的联系。 ALS是一种神经退行性疾病，影响运动神经元，导致瘫痪，最终死亡，平均在2-5年内。大多数ALS病例是零星的，缺乏明显的遗传联系，但10％以遗传为主。最近，染色体9开放阅读框72（C9orf72）基因中的六核苷酸重复扩增（HREs）被鉴定为ALS和额颞叶痴呆（FTD）的遗传原因。重要的是，不同的群体最近提出这些突变体影响核细胞质运输。这些研究大多表明HREs对核细胞质运输的最终结果和表现，但并不表现出核转运功能障碍即时的。结果，只有严重的核细胞质运输缺陷可以被确定，主要是由于高过度表达或外源蛋白质插入。 该方案描述了一种新的非常灵敏的测定方法，用于实时评估和定量核细胞质运输功能障碍。使用荧光显微镜可以实时定量NLS-NES-GFP蛋白（穿梭GFP）的导入速率。这通过使用输出蛋白抑制剂进行，从而允许穿梭GFP仅进入细胞核。为了验证测定，使用了先前显示为破坏核胞质运输的C9orf72 HRE翻译的二肽重复序列，聚（GR）和聚（PR）。使用所述测定，与对照相比，观察到核进口率降低了50％。使用该系统，可以检查核细胞质运输的微小变化，以及不同因素拯救（甚至部分）核细胞质的能力ansport缺陷可以确定。

使用本文介绍的方法，用NLS-NES-GFP（穿梭GFP）蛋白转染运动神经元样NSC-34细胞。这种具有NLS和NES信号序列（ 图1 ）的蛋白质可以在细胞核和细胞质之间穿梭。通用GFP只能通过扩散而在没有任何定位信号标签的情况下进入或离开细胞核，因此分布在细胞核和细胞质之间（ 图2A ）。相比之下，由于NES和NLS信号影响其分散，穿梭GFP蛋白主要存在于细胞质...

Watch this Scientific Journal Video about Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells at JoVE.com

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

该方案描述了一种通过量化NLS-NES-GFP蛋白的核进口的实时变化来敏感地测量运动神经元样NSC-34细胞内的核胞质转运速率的方法。

测定在运动神经元样NSC-34细胞中实时测量核细胞质运输

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

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