Name: quantification of endosome and lysosome motilities in cultured neurons using fluorescent probes
Uploaded: 2017-05-22T13:00:00
Description: Watch this Scientific Journal Video about Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes at JoVE.com

感谢Ricardo Dolmetsch博士（斯坦福大学医学院;现任隶属机构：诺华生物医学研究所），协助开展此项分析，马修·伍德博士，宝马爱原，以及董秀金博士对手稿的批判性阅读。

所有动物手术均经筑波大学动物护理与使用委员会（IACUC）批准进行。 解剖<ol><li>为了准备胚胎13-14日ICR或C57BL / 6胎儿，通过颈椎脱位安乐死怀孕的小鼠。取出子宫，并用汉克平衡盐溶液（HBSS）将其放入培养皿中。冲洗干净，以清除血液。 </li><li>使用镊子，将子宫转移到具有70％乙醇的新培养皿中以消毒。 </li><li>将子宫转移到带有HBSS的新培养皿中。使用消毒的解剖镊子和手...

<ol>
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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+of+FIG4+causes+neurodegeneration+in+the+pale+tremor+mouse+and+patients+with+CMT4J.">Mutation of FIG4 causes neurodegeneration in the pale tremor mouse and patients with CMT4J.</a> Nature. 448 (7149), 68-72 (2007).</li><li>Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+Vac14,+a+regulator+of+the+signaling+lipid+phosphatidylinositol+3,5-bisphosphate,+results+in+neurodegeneration+in+mice.">Loss of Vac14, a regulator of the signaling lipid phosphatidylinositol 3,5-bisphosphate, results in neurodegeneration in mice.</a> Proc Natl Acad Sci U S A. 104 (44), 17518-17523 (2007).</li><li>Mizushima, N., Yamamoto, A., Matsui, M., Yoshimori, T., Ohsumi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+analysis+of+autophagy+in+response+to+nutrient+starvation+using+transgenic+mice+expressing+a+fluorescent+autophagosome+marker.">In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker.</a> Mol Biol Cell. 15 (3), 1101-1111 (2004).</li><li>. Manual Tracking, a plug-in for ImageJ software [Internet] Available from: <a target="_blank" href="https://imagej.nih.gov/ij/plugins/track/track.html">https://imagej.nih.gov/ij/plugins/track/track.html</a> (2017)</li><li>Hu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exopolysaccharide-independent+social+motility+of+Myxococcus+xanthus.">Exopolysaccharide-independent social motility of Myxococcus xanthus.</a> PLoS One. 6 (1), e16102 (2011).</li><li>Tan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+four+type+IV+pilin+homologues+in+Stigmatella+aurantiaca+DSM17044+by+heterologous+expression+in+Myxococcus+xanthus.">Characterization of four type IV pilin homologues in Stigmatella aurantiaca DSM17044 by heterologous expression in Myxococcus xanthus.</a> PLoS One. 8 (9), e75105 (2013).</li><li>Choi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+variant+of+cortactin+linked+to+acute+lung+injury+impairs+lamellipodia+dynamics+and+endothelial+wound+healing.">A genetic variant of cortactin linked to acute lung injury impairs lamellipodia dynamics and endothelial wound healing.</a> Am J Physiol Lung Cell Mol Physiol. 309 (9), L983-L994 (2015).</li><li>Dahirel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Movement+propensity+and+ability+correlate+with+ecological+specialization+in+European+land+snails:+comparative+analysis+of+a+dispersal+syndrome.">Movement propensity and ability correlate with ecological specialization in European land snails: comparative analysis of a dispersal syndrome.</a> J Anim Ecol. 84 (1), 228-238 (2015).</li><li>Hu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+type+IV+pili+activity+and+exopolysaccharides+secretion+controls+motility+patterns+in+single+cells+of+Myxococcus+xanthus.">Interplay between type IV pili activity and exopolysaccharides secretion controls motility patterns in single cells of Myxococcus xanthus.</a> Sci Rep. 6, 17790 (2016).</li><li>. 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该协议介绍了囊泡运动量的定量程序。在原代神经元中，内含体和溶酶体往往在年轻神经元（4-6 DIV）中表现出较高的运动能力。鉴于神经元必须将一些成分递送到前端以延长神经过程，因此膜运输应在此阶段动态发生。因此，使用年轻神经元观察神经元树突中的动态运动是重要的。此外，即使在年轻的神经元中，较大的囊泡也不会表现出较高的运动性。囊泡大小的调节可能涉及融合裂变步骤的频...

内源 - 溶酶体运输的精确控制对于调节神经元功能是必不可少的。值得注意的是，这些囊泡的动态运动是神经元形态，发育和生存调节的关键因素。该系统的缺陷引起严重的神经元疾病1,2 。将囊泡运输与神经元疾病相关联的分子机制被认为是复杂的，并且有几个组织试图检查这种相关性。例如，已经报道，扰动的晚期内体运动性与尼曼 - 皮克C病3 （由溶酶体缺陷引起的遗传性神经变性疾病）显着相关。另一个例子是溶酶体Ca 2+通道的突变， trpml1 ，其损害溶酶体运动，导致溶酶体贮积病4,5 ， </sup&gt; 6 。我们的研究小组报道，PtdIns（3,5）P 2转换的失调抑制了神经元中的内体和溶酶体运动，导致应激反应脆弱性增加7,8 。 PtdIns（3,5）P 2的代谢调节，其主要定位于晚期内体和溶酶体上，在多种细胞功能中起重要作用，包括囊泡运输和融合裂变过程9,10 。由于受损的PtdIns（3,5）P 2周转导致严重的神经变性11,12 ，内体 - 溶酶体运动的异常调节可能是了解神经变性发病机理的关键因素。对囊泡运动基础的分子机制的调查可能因此提供有希望的线索，可以加深我们的不便了解几种神经元疾病。 在本文中，我们引入了一种有价值的方法来量化神经元中的囊泡运动，使用自由软件包称为手动跟踪。目的是开发一种快速量化方法来分析囊泡运动。该定量是通过点击延时电影的每个帧中的参考点的标准方法进行的。与其他应用程序不同，使用手动跟踪软件使此方法非常简单，具有广泛的实用性。此外，该方法也适用于其他细胞，例如胶质细胞。虽然这种方法是原始的，但它可以应用于各种分析，包括细胞运动和形态变化。例如，在跨越图像序列定义参考点之后，可以使用数据分析和图像处理软件从连续图像中提取关于参考点的位置和每个位置处的时间的信息洁具。综合起来，这种方法简单而有力，有助于提高基于膜运输的研究的效率的发展，例如检查内体 - 溶酶体功能的研究。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Neurobasal medium</td>
			<td>Thermo Fisher</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle medium&#160;</td>
			<td>Wako</td>
			<td>044-29765</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Thermo Fisher</td>
			<td>31985070</td>
			<td>Serum free medium</td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution</td>
			<td>Thermo Fisher</td>
			<td>14170112</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin Streptmycin</td>
			<td>Thermo Fisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Thermo Fisher</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplements</td>
			<td>Thermo Fisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5% Trypsine</td>
			<td>Thermo Fisher</td>
			<td>15090046</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-D-lysine hydrobromide</td>
			<td>Sigma</td>
			<td>P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-L-ornithine&#160;</td>
			<td>Sigma</td>
			<td>P4957</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon cell strainer</td>
			<td>Corning</td>
			<td>431750</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Thermo Fisher</td>
			<td>11668027</td>
			<td>Transfection reagent</td>
		</tr>
		<tr>
			<td>Polyethyleneimine &#34;Max&#34;</td>
			<td>polysciences</td>
			<td>24765</td>
			<td>Transfection reagent. As an alternative to Lipofectamine2000, 0.1mg/ml Polyethyleneimine dissolved in sterilized water is available. But it is low efficiency and high toxicity.</td>
		</tr>
		<tr>
			<td>BIOREVO BZ-9000</td>
			<td>Keyence</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubation system INUG2-K13</td>
			<td>Tokay Hit</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism version 6.0</td>
			<td>GraphPad Software</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel version 15</td>
			<td>Microsoft</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ verion 1.47</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of endosome and lysosome motilities in cultured neurons using fluorescent probes

在大脑中，膜运输系统在调节神经元功能，如神经元形态，突触可塑性，存活和胶质细胞通讯中发挥重要作用。迄今为止，许多研究报道，这些系统中的缺陷引起各种神经元疾病。因此，了解囊泡动力学的机制可能会提供有助于治疗几种神经元疾病的有影响力的线索。在这里，我们使用ImageJ平台的软件插件描述了一种量化囊泡运动的方法，例如运动距离和运动速度。为了获得定量图像，我们用EGFP标记的囊泡标记蛋白标记神经元内体 - 溶酶体结构，并使用延时显微镜观察囊泡的运动。该方法非常有用，简化了神经突，如轴突和树突，以及神经元和神经胶质细胞的神经元测量囊泡运动。 Furthermo这种方法可以应用于其他细胞系，如成纤维细胞和内皮细胞。这种方法可以为我们对膜运输的理解提供有价值的推动。

该测定设计用于测量体外培养中的囊泡动力学。该测定用于确定与神经元形态和存活相关的囊泡运动性。 图1A和B显示了显示神经元溶酶体运动性的代表性数据。皮质神经元用溶酶体标记LAMP-EGFP转染，并使用标准荧光显微镜观察。以前曾报道，即使在静息神经元中，特异性内体溶酶体囊泡也是高度移动的。实际上，这些结果表明在相同的枝?...

Watch this Scientific Journal Video about Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes at JoVE.com

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

膜运输的调查对于了解神经元功能至关重要。在这里，我们介绍一种量化神经元囊泡运动性的方法。这是一种方便的方法，可以适应于神经系统膜运输的量化。

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, ...

Research

JoVE Journal

Neuroscience

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

漂白后荧光标记蛋白的荧光恢复（FRAP）在培养海马神经元的树突棘

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

在培养的中枢神经系统的神经元树突棘形态分析

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

使用调频染料培养的小脑颗粒神经突触小泡池补充的定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

诱导培养的交感神经元的树突状增长

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

移动硅探针的神经行为鼠害的大型记录

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

横向扩散和培养的神经元膜蛋白的胞吐评估使用荧光恢复和荧光损失漂白

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

培养成年的背根神经节神经元轴突再生的遗传研究

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

检测蛋白质棕榈酰化培养海马神经元通过免疫沉淀和酰基-生物素交易所（ABE）

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...