Name: assay to measure nucleocytoplasmic transport in real time within motor neuron-like nsc-34 cells
Uploaded: 2017-05-16T15:00:00.000+00:00
Duration: 8 min 53 s
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烧瓶中并加入额外的5mL培养基。在37℃的5％CO 2的无菌培养箱中孵育细胞过夜。 </li><li>平行地，在60mm培养皿中放置8个未涂覆的盖玻片（盖玻片:12mm，0.13-0.17mm厚），并使用70％乙醇灭菌并暴露于30分钟的UV光直至干燥。向每个菜中加入培养基，处理残留的乙醇。</li><li>将胰蛋白酶缓冲液（2.5g胰蛋白酶和0.2g EDTA在1升Hanks平衡盐溶液中）在达到约90％汇合（每个烧瓶约600万个细胞）后，将细胞分离。 注意:NSC-34细胞是敏感的，并且仅使用培养基解离之前需要15秒的胰蛋白酶缓冲液温育和1分钟的无胰蛋白酶孵育。 </li><li>将细胞放置在已经含有盖玻片的60mm培养皿上，其中培养基为3mL，每碟约35万。将它们放在培养箱中24小时，以允许细胞附着在盖玻片上。 </li></ol>细胞转染注意:大约24小时后，NSC-34细胞将达到40％汇合（约80万个细胞）并准备转染。 <ol><li>在无菌微量离心桶中制备纯DMEM培养基（每个60 mm培养皿300μLDMEM培养基）ES。 </li><li>向每个含有DMEM的微量离心管中加入2μg转染所需的每种质粒。 注意:穿梭GFP质粒在所有细胞转染中都是必需的。 </li><li>向所有含有所需质粒的微量离心管中加入转染试剂。在每个2μgDNA中加入4μL商业转染试剂。 </li><li>旋转含有DMEM，质粒和转染试剂的微量离心管，并在室温下孵育25分钟。 </li><li>将每个微量离心管加入培养皿中，轻轻搅拌，形成均匀的溶液。将盘放回培养箱中48小时。 注意:由于穿梭GFP应在所有细胞中表达，穿梭GFP转染效率可以部分检查，并在过夜转染后使用荧光显微镜进行预先测试。可以使用免疫印迹分析进行非荧光表达蛋白质的全面检查是。 </li></ol>显微镜<ol><li>将含有转染的NSC-34细胞的盖玻片连接到盖玻片支架上，并用磷酸盐缓冲盐水（PBS）轻轻洗涤以除去分离的死细胞。 </li><li>使用具有倒置显微镜的绿色荧光过滤器识别合适的细胞贴片。 注意:合适的细胞贴片被定义为显示大量细胞（场中为20-30个细胞）的显微镜视野，并且其中核在细胞中被清楚地定义。细胞膜片内的细胞核首先由眼睛识别，并由成像软件手动标记。 </li><li>通过在显微镜下滴加盖玻片上的缓冲液，将200μL含有10ng / mL LMB的PBS的出口蛋白-1抑制剂Leptomycin B（LMB）缓冲液加入到盖玻片中。 注意:此时间点设置为间隔为零。 </li><li>将标记的核的荧光强度以3秒的间隔量化大约400-500秒。 </li><li&gt;在每个单独的实验中为每个转染组测试3至5个盖板。 </li></ol>分析注意:由于不同的细胞表达不同水平的荧光穿梭GFP，重要的是将每个细胞的核荧光强度归一化为其自身的初始荧光强度。 <ol><li>通过将每个核的3s荧光间隔与该核的初始六个间隔的平均值相除来标准化标记的核的荧光强度。 </li><li>在每个细胞核正常化后，产生一个荧光强度曲线，代表每个细胞贴片作为时间的函数的核荧光变化。为此，计算每个细胞贴片中所有细胞的平均值（由每个贴片中约20-30个细胞组成）。 注意:核荧光强度曲线类似于Michaelis-Menten饱和曲线，其在增加的ti内更好地观察到我的时期这是由于由于核运输而导致的随着时间的飞梭GFP的胞质量的减少。这种减少统计上降低了穿梭GFP转运到细胞核中的可能性，从而在核荧光中达到饱和。从曲线的线性初始速率得到的斜率产生进入核的V max进入速率。 </li><li>使用图形软件分析从三到五个独立实验得到的每个细胞贴片的V max斜率，以产生一个表示每个实验组的斜率。使用单因素方差分析统计分析进行定量。 </li></ol>蛋白质表达验证<ol><li>将含有残留细胞（未在上述实验中使用）的60mm培养皿从冰上每个实验组放置。 </li><li>处理培养基并用PBS洗涤细胞两次以稀释并洗涤剩余的培养基。 </li><li>用200-400处理细胞μL含有PBS，0.5％Triton和蛋白酶抑制剂鸡尾酒片（PI）的裂解缓冲液。刮擦使用细胞刮刀。 </li><li>在微量离心管中收集裂解缓冲液中的细胞，并在匀浆器中以4000rpm在冰下均质化1分钟。 </li><li>将匀浆细胞以2500×g离心10分钟。收集上清液，使用Bradford测定法定量蛋白质浓度，并保存至-20°C使用。 </li><li>收集30-70μg总蛋白（取决于蛋白质），并将其加载到SDS-PAGE凝胶中，以使用免疫印迹法检测蛋白质表达。 </li></ol>

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作者没有什么可以披露的。

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

assay-to-measure-nucleocytoplasmic-transport-real-time-within-motor

Research

JoVE Journal

Neuroscience

8.6K 次观看.  Ben-Gurion University of the Negev. 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。 

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

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted area of the brain, neural stem cells proliferate and constantly generate neuroblasts that migrate tangentially in chains along the rostral migratory stream (RMS) to reach the olfactory bulb (OB). Once in the OB, neuroblasts switch to radial migration and then differentiate into mature neurons able to incorporate into the preexisting neuronal network. Proper neuroblast migration is a fundamental step in neurogenesis, ensuring the correct functional maturation of newborn neurons. Given the ability of SVZ-derived neuroblasts to target injured areas in the brain, investigating the intracellular mechanisms underlying their motility will not only enhance the understanding of neurogenesis but may also promote the development of neuroregenerative strategies.
This manuscript describes a detailed protocol for the transfection of primary rodent RMS postnatal neuroblasts and the analysis of their motility using a 3D in vitro migration assay recapitulating their mode of migration observed in vivo. Both rat and mouse neuroblasts can be quickly and efficiently transfected via nucleofection with either plasmid DNA, small hairpin (sh)RNA or short interfering (si)RNA oligos targeting genes of interest. To analyze migration, nucleofected cells are reaggregated in &#39;hanging drops&#39; and subsequently embedded in a three-dimensional matrix. Nucleofection per se does not significantly impair the migration of neuroblasts. Pharmacological treatment of nucleofected and reaggregated neuroblasts can also be performed to study the role of signaling pathways involved in neuroblast migration.

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Neuroblast migration is a crucial step in postnatal neurogenesis. The protocol described here can be used to investigate the role of candidate regulators of neuroblast migration by employing DNA/small hairpin RNA (shRNA) nucleofection and a 3D migration assay with neuroblasts isolated from the rodent postnatal rostral migratory stream.

鼠神经母细胞核转染到研究神经细胞迁移<em&gt;在体外</em

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted ...

科研

神经科学

A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system.

Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.

高含量的成像检测方法的肉毒毒素抑制剂的鉴定

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic ...

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

单分子成像核运输

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

生物学

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

使用荧光探针定量培养的神经元中的内体和溶酶体运动

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

A High-content Assay for Monitoring AMPA Receptor Trafficking

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different stimuli. The &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are responsible for fast excitatory synaptic transmission in neurons, are trafficked to and from the postsynaptic surface to dynamically alter neuronal excitability. AMPA receptor trafficking is essential for synaptic plasticity and can be disrupted in neurological disease. However, prevalent approaches for quantifying receptor trafficking ignore entire receptor pools, are overly time- and labor-intensive, or potentially disrupt normal trafficking mechanisms and therefore complicate the interpretation of resulting data. We present a high-content assay for the quantification of both surface and internal AMPA receptor populations in cultured primary hippocampal neurons using dual fluorescent immunolabeling and a near-infrared fluorescent 96-well microplate scanner. This approach facilitates the rapid screening of bulk internalized and surface receptor densities while minimizing sample material. However, our method has limitations in obtaining single-cell resolution or conducting live cell imaging. Finally, this protocol may be amenable to other receptors and different cell types, provided proper adjustments and optimization.

Neuronal excitability can be modulated through a dynamic process of endo- and exocytosis of excitatory ionotropic glutamate receptors. Described here is an accessible, high-content assay for quantifying surface and internal receptor population pools.

一种用于监测 ampa 受体贩运的高含量检测方法

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different ...

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutes&#160;and microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

切除小鼠神经神经纤维神经丝的成像与分析

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...

Nuclear Transport Assays in Permeabilized Mouse Cortical Neurons

Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases. Moreover, there is a growing recognition of cell-specific differences in nuclear pore complex structure, prompting a need to adapt nuclear transport methods for use in neurons. Permeabilized cell assays, in which the plasma membrane is selectively perforated by digitonin, are widely used to study passive and active nuclear transport in immortalized cell lines but have not been applied to neuronal cultures. In our initial attempts, we observed the rapid loss of nuclear membrane integrity in primary mouse cortical neurons exposed to even low concentrations of digitonin. We hypothesized that neuronal nuclear membranes may be uniquely vulnerable to the loss of cytoplasmic support. After testing multiple approaches to improve nuclear stability, we observed optimal nuclear integrity following hypotonic lysis in the presence of a concentrated bovine serum albumin cushion. Neuronal nuclei prepared by this approach reliably import recombinant fluorescent cargo in an energy-dependent manner, facilitating analysis of nuclear import by high content microscopy with automated analysis. We anticipate that this method will be broadly applicable to studies of passive and active nuclear transport in primary neurons.

We have developed a reliable method of selective plasma membrane permeabilization of primary mouse cortical neurons for high content automated analysis of neuronal nucleocytoplasmic transport.

Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases. Moreover, there is a growing ...

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

肌萎缩性侧索硬化症模型中突触功能的实时荧光测量

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative.
This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

扩展轴突转运体内成像工具包

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal ...

Measuring Axonal Cargo Transport in Mouse Primary Cortical Cultured Neurons

Neuronal cells are highly polarized cells that stereotypically harbor several dendrites and an axon. The length of an axon necessitates efficient bidirectional transport by motor proteins. Various reports have suggested that defects in axonal transport are associated with neurodegenerative diseases. Also, the mechanism of the coordination of multiple motor proteins has been an attractive topic. Since the axon has uni-directional microtubules, it is easier to determine which motor proteins are involved in the movement. Therefore, understanding the mechanisms underlying the transport of axonal cargo is crucial for uncovering the molecular mechanism of neurodegenerative diseases and the regulation of motor proteins. Here, we introduce the entire process of axonal transport analysis, including the culturing of mouse primary cortical neurons, transfection of plasmids encoding cargo proteins, and directional and velocity analyses without the effect of pauses. Furthermore, the open-access software "KYMOMAKER" is introduced, which enables the generation of a kymograph to highlight transport traces according to their direction and allow easier visualization of axonal transport.

The present protocol describes the whole procedure of analysis for axonal transport. In particular, the calculation of transport velocity, not including the pausing, and the visualization method using open-access software "KYMOMAKER" are shown here.