这项工作得到了 DZNE 和居里夫人国际重返社会补助金 (322120 至 D.C.D.) 的资助。

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作者没有什么可透露的。

在这里, 我们报告了一种方法, 以隔离高不溶性蛋白聚合体的老化线虫受 rna 干扰分析的质谱法和西方印迹。我们表明通过减少 IIS 来改善 proteostasis 可以大大防止年龄依赖性的蛋白质聚集。通过在线虫中选择特定的聚集易过度蛋白, 可以进一步解剖调节固有蛋白质聚集的机制。
固有年龄依赖性蛋白质聚集与疾病相关蛋白聚集 目前的工作是根据先前的发现, 蛋白质聚集不限于特定的蛋白质聚合在疾病的背景。通过聚焦于内源性聚集易感蛋白而非 ectopically 表达的疾病相关蛋白, 期望能为聚集过程的生理调节提供新的洞察力。由于在不同的细胞位置5中发现了与年龄相关的蛋白质聚合体, 因此它们的研究也应该提供对隔室特定质量控制机制的洞察。总的来说, 控制固有蛋白质聚集的机制也可能与预防疾病蛋白聚集有关。值得注意的是, 虽然年龄依赖性蛋白质聚集类似于几个方面的疾病蛋白聚集, 但仍然不清楚这些聚合体是否含有淀粉样纤维, 这是许多疾病聚集的特征。
选择c. 线虫模型: 优点和限制 与哺乳动物模型相比, 使用C. 线虫来研究衰老相关特性的最大优点之一是它的寿命很短。在衰老过程中, 几百个蛋白质一直是高度聚集性的, 不的大量增加很容易被检测到, 即使是经过简化的生物化学萃取。然而, 使用C. 线虫研究年龄依赖性蛋白质聚集的一个主要限制是必须隔离大量的老龄动物。由于C. 线虫在其生殖阶段19中雌雄同体并产生大约220或更多的子代, 因此必须防止繁殖或消除后代以使种群老化。有几种方法可诱导不孕: 一种选择是使用化学 fluorodeoxyuridine (FUdR), 一种 DNA 合成抑制剂。然而, FUdR 导致许多副作用, 包括 proteostasis 和减少蛋白质聚合的变化20,21,22。重要的是, FUdR 诱导基因型特定的反应23。另一个选择是使用温度敏感的无菌突变体。例如, spe-9 (hc88), 15 (b26),有限元-1 (hc17), glp-1 (e2141),和尼泊尔-2 (q388)已被用于质谱分析的年龄依赖性聚合蛋白质组5, 8。
然而, 有几个限制与使用这些变种人有关。首先, 由于不育是温度依赖性, 有必要在25° c 下生长动物, 这是一个温和的压力, 加速衰老以及蛋白质聚集。相反, 我们观察到, gon-2、 glp-1和fem-1变种在25° c (未显示数据) 时与野生类型相比是 longer-lived 的。其次, 每种类型的生殖缺陷都有特定的局限性。使用精子缺陷突变体的一个缺点是未受精的卵母细胞的生产 (也产生于老年野生型雌雄同体)。这些卵母细胞的质量随着年龄的增长而下降, 在性腺24,25中积累。定量质谱分析表明, 精子缺陷突变体有几倍高的不溶性蛋白质积累与年龄相比, 无性腺或系缺陷突变。这表明蛋白质折叠和聚集位于性腺5, 与先前研究的协议12,13和在体内实验揭示了聚集蛋白位于异常卵母细胞群集5。因此, 性腺中过量的蛋白质聚集会掩盖体细胞组织中蛋白质聚集的细微变化。在比较不同程度的干预对生殖和躯体组织 proteostasis 的作用时也应考虑这一点。因此, 为了评估体细胞蛋白的聚集, 我们倾向于使用无性腺的gon-2突变体。另一种方法是使用系缺陷的glp-1变种。然而, 我们注意到, 这些动物的固有的蛋白质聚集与年龄相比, gon-2突变体5。这可能是由于信号从系缺陷性腺26,27,28的 proteostasis 在躯体组织中的改善。另一种选择是使用野生型动物, 并通过沉淀去除后代。然而, 这是一个问题, 由于难以完全消除所有的后代。
使用C. 线虫的另一个一般限制是它的小尺寸, 这使得特定的组织提取具有挑战性。由于这种全动物提取物不提供有关不同的条件如何调节特定组织中蛋白质聚集的相关信息。值得注意的是, 此问题可能会被利用最近开发的基于荧光活化的细胞排序的方法来分离, 以隔离C. 线虫29中的特定单元格。然而, 这一方法是否适合于获得不溶蛋白提取的足够材料, 是否适用于老龄动物还有待确定。另外, 组织特异性蛋白聚集及其调控可以通过另外的体内实验来研究。由于转基因动物表达了荧光标记的选择性聚集倾向蛋白, 可以快速生成, 由于动物是透明的, 因此检测蛋白质聚集原位相对容易。
总的来说, 在野生型背景下, 用活体实验来跟踪蛋白质组学结果是非常必要的。生物化学和 microscopy-based 特性的结合使对蛋白质聚集的综合研究和对感兴趣基因的作用的评估。在C. 线虫中, 通过通过喂养和整个基因组 rna 干扰库的可用性进行 rna 干扰来促进后者。此外, 大量具有特征性的突变体存在, 以证实通过 rna 干扰或使用而非 rna 干扰所获得的结果。
液体培养与生化萃取: 优势与局限 由于高度聚合的蛋白质只占总蛋白的一小部分, 因此有必要在大量的动物中开始提取。由于大量的不溶性蛋白, 因此, 进行广泛的多肽分馏, 以减少样品的复杂性, 质谱, 从而提高对低丰度蛋白质的鉴定是可行的。如果只需要少量的不溶性蛋白质, 另一种方法是在盘子上生长蠕虫, 用陶瓷珠子质它们。液体文化的缺点之一是, 在污染的情况下, 整个文化受到影响, 必须放弃。通常, C. 线虫的老化受到温度的强烈影响, 必须在液体培养中严格控制此参数, 以避免年龄依赖性蛋白质聚集的变化。在使用gon-2 (-)突变体时, 温度控制对于获得无性腺动物的同种种群也是必不可少的。
根据研究的目的, 重要的是要考虑不同的提取方法。这里提出的生物化学提取最初是根据协议来隔离疾病相关的聚合体11,30,31。我们选择了相对高浓度的洗涤剂, 如 0.5% SDS, 以隔离更不溶性的聚合体。此外, 通过将 0.5% SDS 不溶性集料以低离心速度制粒, 本议定书只将较大的骨料隔离。或者, 最近发布了一个不太严格的协议10。在这项研究中, 还提取了更多的可溶性和较小的骨料, 通过省略 SDS 和使用非常高的离心速度在 50万 x g。对不同协议的比较表明, 使用不太严格的协议7, 聚合蛋白质组中蛋白质的数量普遍增加。我们注意到, 长寿命的动物与减少的daf-2信号有效地阻止了 sds 可溶性和 sds 不溶性聚集物在性腺较少的动物中的积累7。
在体内年龄依赖性蛋白质聚集性分析 在构建适合于年龄依赖性蛋白质聚集的转基因模型时, 考虑在哪个组织中过度的聚集易感蛋白和表达水平是相关的。我们倾向于在头区的组织表达, 以避免干扰的自发荧光, 这是在老化肠道突出。为了使荧光标记的聚集体与 low-magnification, 聚集易发蛋白应表达在一个较高的水平。另一方面, 太高的表达会导致聚集易产生的蛋白质形成聚合体已经在幼小动物。
总之, 这些方法将帮助我们理解为什么部分蛋白质组聚集在一起的年龄, 并最终导致发展的战略, 促进健康的老龄化。

蛋白质折叠和聚集被认为是一些神经退行性疾病的标志, 如 AD, PD, 肌萎缩侧索硬化症 (ALS), 颞痴呆 (FTD), 和许多其他。例如, α-核组装成淀粉样纤维, 在帕金森病患者的黑质中积累为路易体体, 而 ALS 患者 TDP-43 或 FUS 错误在退化的运动神经元中形成细胞质聚集。在这些神经退行性疾病中, 维持蛋白质稳态或 proteostasis 的机制无法阻止错误蛋白的积累, 从而导致疾病。
Proteostasis 是至关重要的, 以确保细胞功能和正常情况下, 这些调控机制严格控制蛋白质的合成, 折叠和降解率。几项研究表明, 随着衰老, 许多细胞和器官保存蛋白质稳态的能力逐渐受到损害, proteostasis 网络的生理退化是一个重要的加重因素神经退行性疾病 (参考1,2,3)。事实上, 蛋白质的质量控制和细胞对展开的蛋白质应激的反应受到年龄的影响, 表明蛋白质折叠和聚集可能是衰老的一般后果。事实上, 我们和其他人已经证明, 蛋白质聚集不限于疾病, 而不是部分的蛋白质组成为高洗涤剂-不溶于老年动物4,5,6,7 ,8,9,10。计算和体内分析显示, 这些生理年龄相关的团聚体在几个方面类似于疾病聚集,5。发现内源性, 年龄依赖性的蛋白质聚集使我们有机会解剖的分子和细胞机制, 调节蛋白质聚集, 不使用 ectopically 表达的人类疾病相关的蛋白质。目前, 关于广泛的蛋白质不的调控, 以及这种失调对机体健康的影响, 只存在有限的信息。
线虫是在衰老研究中最广泛研究的模型生物之一, 因为这些动物的寿命相对较短, 并且在较高的生物体中表现出许多特征老化特征. 通过基于微分溶解度的序贯生化分馏技术, 研究了衰老对蛋白质不的影响, 广泛应用于神经研究领域中的疾病聚集物的提取 11.通过定量质谱法, 在没有疾病5的情况下, 显示了数百个蛋白质在C. 线虫中变得容易聚集。在这里, 我们详细地描述了在液体培养中生长大量蠕虫的协议, 以及用质谱法分离聚合蛋白质并通过印迹分析的顺序提取。由于错误和聚集倾向的蛋白质积累在老年的线虫性腺和其他躯体组织的面具变化5,12,13, 我们使用无性腺突变体来集中分析蛋白质不在非组织中。所提出的方法可以分析不溶于 0.5% SDS 的高不溶大团聚体, 并通过相对较低的离心速度进行颗粒化。另外, 在其他地方也发布了一个不太严格的提取协议来收集更小、更可溶解的聚合体,10。此外, 我们还描述了用于评估C. 线虫中的聚合在体内的方法。
总的来说, 这些方法与 rna 干扰结合, 可以评估一个基因在调节年龄依赖性蛋白质聚集中的作用。为此, 我们描述的分析, 从年轻和年老的蠕虫的提取物, 并没有被击倒的特定蛋白质的兴趣使用 rna 干扰。这些方法应该是一个强大的工具, 以确定哪些组成部分的 proteostasis 网络调节蛋白质不。一些干预措施, 如减少胰岛素/胰岛素样生长因子 (igf-i) 1 信号 (IIS) 已被证明显着延迟C. 线虫老化14。长寿途径往往诱导蛋白质质量控制机制, 因此这些途径可以积极影响蛋白质聚集率。例如, 我们在抑制 IIS 路径7时演示了在长寿命的动物中减少固有的蛋白质聚集。

<table><tbody><tr><td>Fernbach culture flask&amp;nbsp;</td><td>Corning</td><td>4425-2XL</td><td>Pyrex, Capacity 2,800 ml, with 3 baffle indents</td></tr><tr><td>Membrane Screw Cap&amp;nbsp;</td><td>Schott</td><td>1088655</td><td>GL45</td></tr><tr><td>Nutating Mixer</td><td>VWR</td><td>444-0148</td><td></td></tr><tr><td>Separatory funnel</td><td>Nalgene</td><td>4300-1000</td><td>Capacity 1,000 ml</td></tr><tr><td>1 ml syringe&amp;nbsp;</td><td>BD Plastipak</td><td>300013</td><td></td></tr><tr><td>Gray needle, 27 G x &amp;frac12; &quot;, 0.4 mm x 13 mm</td><td>BD Microlance 3</td><td>300635</td><td></td></tr><tr><td>Membrane filters 0.025 &amp;micro;M</td><td>Millipore</td><td>VSWP04700</td><td></td></tr><tr><td>pH strip</td><td>Machery-Nagel</td><td>92110</td><td>pH-Fix 0-14</td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Roche</td><td>4693132001</td><td>Complete Mini EDTA-free tablets&amp;nbsp;</td></tr><tr><td>Octoxynol-9&amp;nbsp;</td><td>Applichem</td><td>A1388</td><td>Triton X-100</td></tr><tr><td>4-Morpholineethanesulfonic acid (MES)</td><td>Sigma-Aldrich</td><td>M1317</td><td></td></tr><tr><td>Nonylphenylpolyethylenglycol</td><td>Applichem</td><td>A1694</td><td>Nonidet P40 (NP40)</td></tr><tr><td>DNaseI</td><td>Roche</td><td>04716728001</td><td>recombinant, RNase free</td></tr><tr><td>RNaseA</td><td>Promega</td><td>A7973</td><td>solution</td></tr><tr><td>Total protein blot staining</td><td>Thermofisher</td><td>S11791</td><td>Sypro Ruby protein blot stain</td></tr><tr><td>Total protein gel staining</td><td>Thermofisher</td><td>S12001</td><td>Sypro Ruby protein gel stain</td></tr><tr><td>TCEP (tris (2-carboxyethyl) phosphine hydrochloride)</td><td>Serva</td><td>36970</td><td></td></tr><tr><td>Iodoacetamide</td><td>Serva</td><td>26710</td><td></td></tr><tr><td>Ammoniumbicarbonate</td><td>Sigma-Aldrich</td><td>09830</td><td></td></tr><tr><td>Sequencing Grade Modified Trypsin</td><td>Promega</td><td>V5111</td><td></td></tr><tr><td>Isobaric tags for relative and absolute quantitation</td><td>Sciex</td><td>4352135</td><td>iTRAQ Reagents Multiplex Kit</td></tr><tr><td>Centrifuge Avanti J-26XP</td><td>Beckmann Coulter</td><td>393126</td><td></td></tr><tr><td>Ultracentrifuge Optima Max-XP</td><td>Beckmann Coulter</td><td>393315</td><td></td></tr><tr><td>Centrifuge 5424R</td><td>Eppendorf</td><td>5404000413</td><td></td></tr><tr><td>Centrifuge 5702</td><td>Eppendorf</td><td>5702000329</td><td></td></tr><tr><td>Centrifuge Megafuge 40R</td><td>Thermo Scientific</td><td>75004518</td><td></td></tr><tr><td>Concentrator Plus</td><td>Eppendorf</td><td>5305000304</td><td>Centrifugal evaporator</td></tr><tr><td>Fluorescent stereo-microscope M165 FC&amp;nbsp;</td><td>Leica</td><td></td><td>With Planapo 2.0x objective</td></tr><tr><td>Dissection microscope</td><td>Leica&amp;nbsp;</td><td></td><td>Leica S6E</td></tr><tr><td>High magnification microscope Zeiss Axio Observer Z1</td><td>Zeiss</td><td></td><td>With PlanAPOCHROMAT 20x objective and Zeiss Axio Cam MRm</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Image analysis software</td><td>ImageJ</td><td></td><td></td></tr><tr><td>Analysis of mass spectrometry data</td><td>Protein Prospector</td><td></td><td>http://prospector.ucsf.edu/prospector/mshome.htm</td></tr><tr><td>&lt;strong&gt;E.coli strain&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>OP50</td><td>CGC</td><td></td><td></td></tr><tr><td>&lt;strong&gt;RNAi bacteria&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>L4440</td><td>Julie Ahringer RNAi library</td><td></td><td></td></tr><tr><td>&lt;strong&gt;&lt;em&gt;C. elegans&lt;/em&gt; mutants&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CF2253</td><td>CGC, strain name: EJ1158&amp;nbsp;</td><td></td><td>Genotype: &lt;em&gt;gon-2(q388)&lt;/em&gt;</td></tr><tr><td>&lt;strong&gt;&lt;em&gt;C. elegans&lt;/em&gt; transgenics&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DCD214</td><td>Della David&#039;s lab at DZNE T&amp;uuml;bingen</td><td></td><td>Genotype: &lt;em&gt;N2; uqIs24[Pmyo-2::tagrfp::pab-1]&lt;/em&gt;</td></tr><tr><td>DCD215</td><td>Della David&#039;s lab at DZNE T&amp;uuml;bingen</td><td></td><td>Genotype: &lt;em&gt;daf-2(e1370) III; uqIs24[Pmyo-2::tagrfp::pab-1]&lt;/em&gt;</td></tr></tbody></table>

methods to study changes in inherent protein aggregation with age in caenorhabditis elegans

在过去的几十年中, 神经退行性疾病的流行, 如阿尔茨海默氏病 (AD) 和帕金森氏症 (PD), 已经增长。这些年龄相关的疾病的特点是在这些患者的大脑中出现了纤维结构的蛋白质聚集。确切地说, 为什么通常可溶性蛋白质会经历一个聚集过程仍然是不清楚的。发现蛋白质聚集不限于疾病过程, 而是正常衰老过程的一部分, 使研究的分子和细胞机制, 调节蛋白质聚集, 不使用 ectopically 表达人类疾病相关的蛋白质。在这里, 我们描述了通过互补的方法来检查线虫线虫中固有的蛋白质聚集的方法。首先, 我们将研究如何培养大量的年龄同步的线虫来获取衰老的动物, 我们提出了分离高不溶性大团聚体的生物化学程序。结合有针对性的基因击倒, 它可以解剖的作用, 一个基因的兴趣, 以促进或防止年龄依赖性的蛋白质聚集通过使用的综合分析与定量质谱或candidate-based 分析与抗体。然后通过体内分析, 用转基因动物表达荧光标记的聚集易发蛋白来证实这些结果。这些方法应该有助于澄清为什么某些蛋白质容易聚集与年龄和最终如何保持这些蛋白质完全功能。

我们使用这里提出的方法来评估如何长寿命的动物与减少的 IIS 调节年龄依赖性蛋白质聚集。由西部污点 (参见步骤 2.2, 快速不溶蛋白提取为西部污点分析), 我们分析了总和不溶蛋白内容的年轻人 (3 天成人) 和年龄 (18 天成人) 蠕虫在控制 rna 干扰和以 rna 干扰为目标胰岛素IGF-1-like 受体daf-2。我们观察到, 在总蛋白水平上, 年龄或控制与daf-2 rna 干扰条件 (图 2A) 之间没有大的变化。正如预期的那样, 我们检测到控制动物中高度不溶性蛋白水平的强年龄相关增加 (图 2B)。在比较长寿的动物在daf-2 rna 干扰, 聚集倾向大大减少。不溶性提取物的全蛋白染色显示, 与年龄匹配的控件相比, 在daf-2 rna 干扰中, 18 天老蠕虫提取物中的蛋白质带模式较不复杂。定量质谱分析表明, 长寿命的动物与减少的 IIS 有全面较少年龄相关的蛋白质聚集和重要的是, daf-2抑制完全废除了年龄依赖性的不蛋白质的特定子集7。为了对这些发现进行后续研究, 我们将重点放在这些易于聚集的蛋白质之一 PAB-1。PAB-1 是一种 RNA 结合蛋白, 具有预测的 "朊病毒样" 域。抗体染色证实长寿动物维持 PAB-1 可溶于年龄 (图 2C)。
对于体内PAB-1 聚集的分析, 我们生产了表达 tagRFP 的转基因动物: 咽肌:P AB-1。在幼小动物, tagRFP::P AB-1 主要广泛位于整个咽 (图 3A, "低" 聚合水平), 但随着年龄的增长, 我们观察到在明亮的点开始在后咽球的渐进积累 (图 3一个"中间" 聚合级别)。在衰老过程中, 我们还观察到在越来越多的动物中, 咽前球的聚集 (图 3A, "高" 聚合水平)。通过将动物分成不同的类别, 我们在人口中 PAB-1 聚集的比率。在成年的2天, 大多数的蠕虫 (88%) 没有或极少数的点, 没有检测到高聚集水平的动物。已经在5天, 19% 的人口显示中间和16% 高聚集水平, 而在天7超过70% 的蠕虫呈现中间和高聚合水平。为了分析长寿对 tagRFP:PAB-1 年龄依赖性聚集的影响, 我们生成了转基因 tagRFP: 用daf-2突变:P AB-1 蠕虫。这些动物显着减少 tagRFP:PAB-1 点形成与老化, 少于15% 的人口表现中间和高聚集水平甚至在7天 (图 3B)。
通过使用这里描述的不同的方法, 我们发现, 减少的 IIS 防止年龄依赖性的蛋白质聚集到不同的程度和长寿的动物是特别有效的恢复动态的 PAB-17。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56464/56464fig1.jpg"> 图 1: 工作流示意图, 用于研究与年龄有关的固有蛋白质聚集的变化C. 线虫 主要步骤是粗体。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56464/56464fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56464/56464fig2.jpg"> 图 2: 蛋白质不随着控制动物年龄的增长而增加, 但在动物中却没有减少daf-2信号.(A) 总分数的总蛋白染色从天3和天 18 gon-2 (-)突变体受控制和daf-2 rna 干扰 (生长在25° c)。(B) 从3天到 18 gon-2 (-)突变体对控制和daf-2 rna 干扰 (在25° c 下生长) 的 SDS 不溶性分数的总蛋白染色。面板从引用7重新发布。(C) 免疫检测 SDS-不溶性分数中的 PAB-1, 由红色箭头指示的相关蛋白带。面板从引用7重新发布。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56464/56464fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56464/56464fig3.jpg"> 图 3:减少daf-2信号阻止 tagRFP 的累积::P AB-1 点.(A) 有代表性的蠕虫的图片与 tagRFP::P AB-1 过度表达在咽肌 (高倍放大显微镜, tagRFP 检测设置: 激发 558 nm, 发射 583 nm)。动物低 (0-10 tagRFP::P AB-1 点在咽后球), 中间 (超过10点在咽后球), 或高聚集水平 (超过10点在前咽球) 显示。缩放条 = 20 µm. (B) 不同 tagRFP 的量化: 在野生和daf-2突变背景下的:P AB-1 聚合水平显示强烈延迟 tagRFP::P AB-1 聚集与年龄在长寿的动物。2天、5天和7天daf-2 (-)与野生背景相比, 低于中级和高聚合级别: 费舍尔的精确测试, **p < 0.0001。面板从引用7重新发布。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56464/56464fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>库存解决方案</td>
			<td>量</td>
			<td>最终浓度</td>
			<td>评论/说明</td>
		</tr>
<tr>
<td colspan="4">液体培养, 总容量300毫升</td>
		</tr>
<tr>
<td>S 基部 (500 毫升: 5.9 克氯化钠, 50 毫升1米磷酸钾 pH 6)</td>
			<td>200毫升</td>
			<td></td>
			<td>1 M 磷酸钾 pH 值 6: 1 升: 129 g2PO4二, 52 g K2HPO4二元无水</td>
		</tr>
<tr>
<td>1 M 枸橼酸钾 6 (400 毫升: 13.1 克柠檬酸一水合物, 134.4 g 三钾柠檬酸一水合物)</td>
			<td>3毫升</td>
			<td>10毫米</td>
			<td></td>
		</tr>
<tr>
<td>微量金属溶液 (1 升: 1.86 克乙酸乙酸 (EDTA), 0.69 g FeSO4 · 7 h2O, 0.2 g MnCl2 · 4 H2o, 0.29 g ZnSO4 · 7 H2o, 0.025 g 丘索4· 5 h2o)</td>
			<td>3毫升</td>
			<td></td>
			<td>库存解决方案在黑暗中在4° c</td>
		</tr>
<tr>
<td>1 M MgSO4 (500 毫升: 123.3 g)</td>
			<td>900µL</td>
			<td>3毫米</td>
			<td></td>
		</tr>
<tr>
<td>1 M CaCl2 (500 毫升: 73.5 g)</td>
			<td>900µL</td>
			<td>3毫米</td>
			<td></td>
		</tr>
<tr>
<td>200µg/毫升多菌灵 (用于50毫升:10 毫克甲醇)</td>
			<td>150µL</td>
			<td>100 ng/毫升</td>
			<td></td>
		</tr>
<tr>
<td>5毫克/毫升胆固醇 (100 毫升: 0.5 克在乙醇中)</td>
			<td>300µL</td>
			<td>5µg/毫升</td>
			<td></td>
		</tr>
<tr>
<td colspan="4">重组 (盐) 萃取缓冲器, 30 毫升</td>
		</tr>
<tr>
<td>1米 4-Morpholineethanesulfonic 酸 (MES)</td>
			<td>3毫升</td>
			<td>100毫米</td>
			<td>库存缓冲组件</td>
		</tr>
<tr>
<td>0.5 米 Ethyleneglycoltetraacetic 酸 (EGTA)</td>
			<td>60µL</td>
			<td>1毫米</td>
			<td>库存缓冲组件</td>
		</tr>
<tr>
<td>0.5 米乙酸乙酸 (EDTA)</td>
			<td>6µL</td>
			<td>0.1 毫米</td>
			<td>库存缓冲组件</td>
		</tr>
<tr>
<td>1 M MgSO4
</td>
			<td>15µL</td>
			<td>0.5 毫米</td>
			<td>库存缓冲组件</td>
		</tr>
<tr>
<td>5 M 氯化钠</td>
			<td>4.5 毫升</td>
			<td>750毫米</td>
			<td>库存缓冲组件</td>
		</tr>
<tr>
<td>1米氟化钠 (氟)</td>
			<td>600µL</td>
			<td>20毫米</td>
			<td>库存缓冲组件</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td></td>
			<td>完成到30毫升共计与 ddH2O</td>
		</tr>
<tr>
<td>50x 蛋白酶抑制剂鸡尾酒</td>
			<td>*</td>
			<td>2x</td>
			<td rowspan="4">* 添加试剂后, 采取的数量, 是需要的布的缓冲 (为提取的质谱6毫升)。</td>
		</tr>
<tr>
<td>100毫米磺氟化物 (PMSF) 在异丙醇,-20 ° c</td>
			<td>*</td>
			<td>1毫米</td>
		</tr>
<tr>
<td>10 U/µL DNaseI</td>
			<td>*</td>
			<td>200 U/毫升</td>
		</tr>
<tr>
<td>4毫克/毫升 RNaseA</td>
			<td>*</td>
			<td>100µg/毫升</td>
		</tr>
<tr>
<td colspan="4">1米蔗糖, 30 毫升</td>
		</tr>
<tr>
<td>2米蔗糖 (50 毫升: 34.2 克)</td>
			<td>15毫升</td>
			<td>1 M</td>
			<td>添加除 DNaseI 和 RNaseA 外的缓冲试剂</td>
		</tr>
<tr>
<td colspan="4">Radioimmunoprecipitation 测定 (帕) 缓冲液, 30 毫升</td>
		</tr>
<tr>
<td>1 M 三羟甲基氨基 (三) pH 值8</td>
			<td>1.5 毫升</td>
			<td>50毫米</td>
			<td>帕库存缓冲组件</td>
		</tr>
<tr>
<td>5 M 氯化钠</td>
			<td>0.9 毫升</td>
			<td>150毫米</td>
			<td>帕库存缓冲组件</td>
		</tr>
<tr>
<td>0.5 M EDTA</td>
			<td>0.3 毫升</td>
			<td>5毫米</td>
			<td>帕库存缓冲组件</td>
		</tr>
<tr>
<td>10% 十二烷基硫酸钠 (SDS)</td>
			<td>1.5 毫升</td>
			<td>0.50%</td>
			<td>帕库存缓冲组件</td>
		</tr>
<tr>
<td>10% 胆酸钠 (SDO)</td>
			<td>1.5 毫升</td>
			<td>0.50%</td>
			<td>帕库存缓冲组件</td>
		</tr>
<tr>
<td>Nonylphenylpolyethylenglycol (NP40)</td>
			<td>0.3 毫升</td>
			<td>1%</td>
			<td>帕库存缓冲组件</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td></td>
			<td>完成到30毫升共计与 ddH2O</td>
		</tr>
<tr>
<td>100毫米 PMSF</td>
			<td>*</td>
			<td>1毫米</td>
			<td rowspan="2">* 在服用所需的帕存量缓冲剂后添加试剂 (用于质谱10毫升的萃取)。</td>
		</tr>
<tr>
<td>50x 蛋白酶抑制剂鸡尾酒</td>
			<td>*</td>
			<td>1x</td>
		</tr>
<tr>
<td colspan="4">尿素/SDS 缓冲液, 10 毫升</td>
		</tr>
<tr>
<td>尿素</td>
			<td>4.8 克</td>
			<td>8 M</td>
			<td></td>
		</tr>
<tr>
<td>10% SDS</td>
			<td>2毫升</td>
			<td>2%</td>
			<td></td>
		</tr>
<tr>
<td>糖 (德勤)</td>
			<td>77毫克</td>
			<td>50毫米</td>
			<td></td>
		</tr>
<tr>
<td>1 M 三 pH 8</td>
			<td>0.5 毫升</td>
			<td>50毫米</td>
			<td></td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td></td>
			<td>完成到10毫升共计与 ddH2O</td>
		</tr>
<tr>
<td colspan="4">透析缓冲, 1 L</td>
		</tr>
<tr>
<td>三</td>
			<td>6克</td>
			<td>50毫米</td>
			<td></td>
		</tr>
<tr>
<td>dtt</td>
			<td>154毫克</td>
			<td>1毫米</td>
			<td></td>
		</tr>
<tr>
<td>100毫米 PMSF</td>
			<td>1毫升</td>
			<td>0.1 毫米</td>
			<td></td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td></td>
			<td>调整到 pH 值7.5 和完成到 1 L 共计与 ddH2O</td>
		</tr>
</tbody></table>表 1: 液体培养、不溶蛋白提取和透析的缓冲。

Watch this Scientific Journal Video about Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans at JoVE.com

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

本文所提出的方法的目的是在模型生物体的正常衰老过程中, 探索蛋白质的聚集性, 即线虫。该协议代表了一个强大的工具, 以研究高度不溶性的大骨料, 形成与年龄和确定如何改变 proteostasis 影响蛋白质聚集。

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

methods-to-study-changes-inherent-protein-aggregation-with-age

Nanyang Technological University

Research

JoVE Journal

Biology

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

8.4K 次观看.  German Center for Neurodegenerative Diseases (DZNE). 本文所提出的方法的目的是在模型生物体的正常衰老过程中, 探索蛋白质的聚集性, 即线虫。该协议代表了一个强大的工具, 以研究高度不溶性的大骨料, 形成与年龄和确定如何改变 proteostasis 影响蛋白质聚集。

视频: Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These amyloid fibrils can sequester endogenous metastable proteins as well as components of the proteostasis network (PN) and thereby exacerbate protein misfolding in the cell. There are a limited number of tools available to assess the aggregation process of amyloid proteins within an animal. We present a protocol for fluorescence lifetime microscopy (FLIM) that allows monitoring as well as quantification of the amyloid fibrilization in specific cells, such as neurons, in a noninvasive manner and with the progression of aging and upon perturbation of the PN. FLIM is independent of the expression levels of the fluorophore and enables an analysis of the aggregation process without any further staining or bleaching. Fluorophores are quenched when they are in close vicinity of amyloid structures, which results in a decrease of the fluorescence lifetime. The quenching directly correlates with the aggregation of the amyloid protein. FLIM is a versatile technique that can be applied to compare the fibrilization process of different amyloid proteins, environmental stimuli, or genetic backgrounds in vivo in a non-invasive manner.

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Fluorescence lifetime imaging monitors, quantifies and distinguishes the aggregation tendencies of proteins in living, aging, and stressed C. elegans disease models.

使用荧光寿命成像在老化C.Elegans中淀粉样蛋白结构的表征

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These ...

科研

生物化学

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis elegans is a well-established model organism that has been widely used in the field to study protein aggregation and toxicity. Its optical transparency enables the direct visualization of protein aggregation by fluorescence microscopy. Moreover, the fast reproductive cycle and short lifespan make the nematode a suitable model to screen for genes and molecules that modulate this process.
However, the quantification of aggregate load in living animals is poorly standardized, typically performed by manual inclusion counting under a fluorescence dissection microscope at a single time point. This approach can result in high variability between observers and limits the understanding of the aggregation process. In contrast, amyloid-like protein aggregation in vitro is routinely monitored by thioflavin T fluorescence in a highly quantitative and time-resolved fashion.
Here, an analogous method is presented for the unbiased analysis of aggregation kinetics in living C. elegans, using a high-throughput confocal microscope combined with custom-made image analysis and data fitting. The applicability of this method is demonstrated by monitoring inclusion formation of a fluorescently labeled polyglutamine (polyQ) protein in the body wall muscle cells. The image analysis workflow allows the determination of the numbers of inclusions at different timepoints, which are fitted to a mathematical model based on independent nucleation events in individual muscle cells. The method described here may prove useful to assess the effects of proteostasis factors and potential therapeutics for protein aggregation diseases in a living animal in a robust and quantitative manner.

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Here, a method is presented for the analysis of protein aggregation kinetics in the nematode Caenorhabditis elegans. Animals from an age-synchronized population are imaged at different time points, followed by semiautomated inclusion counting in CellProfiler and fitting to a mathematical model in AmyloFit.

使用秀丽隐杆线虫中的自动包涵体计数监测体内蛋白质聚集动力学

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

基本的<em&gt;线虫</em&gt;方法：同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

生物学

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s disease (HD) is principally driven by a CAG nucleotide repeat that encodes an expanded glutamine tract in huntingtin protein. Thus, the inhibition of polyglutamine (polyQ) aggregation and, in particular, aggregation-associated neurotoxicity is a useful strategy for the prevention of HD and other polyQ-associated conditions. This paper introduces generalized experimental protocols to assess the neuroprotective capacity of test compounds against HD using established polyQ transgenic Caenorhabditis elegans models. The AM141 strain is chosen for the polyQ aggregation assay as an age-associated phenotype of discrete fluorescent aggregates can be easily observed in its body wall at the adult stage due to muscle-specific expression of polyQ::YFP fusion proteins. In contrast, the HA759 model with strong expression of polyQ-expanded tracts in ASH neurons is used to examine neuronal death and chemoavoidance behavior. To comprehensively evaluate the neuroprotective capacity of target compounds, the above test results are ultimately presented as a radar chart with profiling of multiple phenotypes in a manner of direct comparison and direct viewing.

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Here, we present a protocol to assess the neuroprotective activities of test compounds in Caenorhabditis elegans, including polyglutamine aggregation, neuronal death, and chemoavoidance behavior, as well as an exemplary integration of multiple phenotypes.

秀丽隐杆线虫 作为发现生物活性化合物对抗聚谷氨酰胺介导的神经毒性的模型系统

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s ...

神经科学

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

量化秀丽隐杆线虫中组织特异性蛋白静剂下降

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their detrimental effects on the human life quality and the healthcare system, the majority of human neurodegenerative disorders still remain incurable and non-preventable. Therefore, the development of novel therapeutic interventions against such maladies is becoming a pressing urgency. Age-associated deterioration of neuronal circuits and function is evolutionarily conserved in organisms as diverse as the lowly worm Caenorhabditis elegans and humans, signifying similarities in the underlying cellular and molecular mechanisms. C. elegans is a highly malleable genetic model, which offers a well-characterized nervous system, body transparency and a diverse repertoire of genetic and imaging techniques to assess neuronal activity and quality control during ageing. Here, we introduce and describe methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis (e.g., deg-3(d) and mec-4(d)) and protein aggregate (e.g., &#945;-syunclein and poly-glutamate)-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-related neuronal breakdown. A combination of these animal neurodegeneration models, together with genetic and pharmacological screens for cell death modulators will lead to an unprecedented understanding of age-related breakdown of neuronal function and will provide critical insights with broad relevance to human health and quality of life.

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Here, we introduce and describe widely accessible methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis and protein aggregate-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-associated neurodegenerative diseases.

建模年龄相关的神经退行性疾病在 卡诺哈布迪蒂斯埃利根

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and degradation instigates a multitude of age-related diseases. Decline of proteostasis quality control mechanisms is a hallmark of ageing. Biochemical methods to detect de novo protein synthesis are still limited, have several disadvantages and cannot be performed in live cells or animals. Caenorhabditis elegans, being transparent and easily genetically modified, is an excellent model to monitor protein synthesis rates by using imaging techniques. Here, we introduce and describe a method to measure de novo protein synthesis in vivo utilizing fluorescence recovery after photobleaching (FRAP). Transgenic animals expressing fluorescent proteins in specific cells or tissues are irradiated by a powerful light source resulting in fluorescence photobleaching. In turn, assessment of fluorescence recovery signifies new protein synthesis in cells and/or tissues of interest. Hence, the combination of transgenic nematodes, genetic and/or pharmacological interventions together with live imaging of protein synthesis rates can shed light on mechanisms mediating age-dependent proteostasis collapse.

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Here, we introduce and describe a nonradioactive and noninvasive method to assess de novo protein synthesis in vivo, utilizing the nematode Caenorhabditis elegans and fluorescence recovery after photobleaching (FRAP). This method can be combined with genetic and/or pharmacological screens to identify novel modulators of protein synthesis.

卡诺哈布迪蒂斯 伊利根斯新蛋白合成率评估

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and ...

Quantification of Insoluble Protein Aggregation in Caenorhabditis elegans during Aging with a Novel Data-Independent Acquisition Workflow

We and others have shown that the aging process results in a proteome-wide accumulation of insoluble proteins. Knocking down genes encoding the insoluble proteins over 40% of the time results in an extension of the lifespan in C. elegans, suggesting that many of these proteins are key determinants of the aging process. Isolation and quantitative identification of these insoluble proteins are crucial to understand key biological processes that occur during aging. Here, we present a modified and improved protocol that details how to extract and isolate the SDS-insoluble proteins (insolublome) from C. elegans more efficiently to streamline mass spectrometric workflows via a novel label-free quantitative proteomics analysis. This improved protocol utilizes a highly efficient sonicator for worm lysis that greatly increases efficiency for protein extraction and allows us to use significantly less starting material (approximately 3,000 worms) than in previous protocols (typically using at least 40,000 worms). Subsequent quantitative proteomic analysis of the insolublome was performed using data-dependent acquisition (DDA) for protein discovery and identification and data-independent acquisition (DIA) for comprehensive and more accurate protein quantification. Bioinformatic analysis of quantified proteins provides potential candidates that can be easily followed up with other molecular methods in C. elegans. With this workflow, we routinely identify more than 1000 proteins and quantify more than 500 proteins. This new protocol enables efficient compound screening with C. elegans. Here, we validated and applied this improved protocol to wild-type C. elegans N2-Bristol strain and confirmed that aged day-10 N2 worms showed greater accumulation of the insolublome than day-2 young worms.

Quantification of Insoluble Protein Aggregation in Caenorhabditis elegans during Aging with a Novel Data-Independent Acquisition Workflow

This novel workflow efficiently extracts and isolates SDS-insoluble proteins (insolublome) from Caenorhabditis elegans with minimal starting material for quantitative differential proteomic analysis. The protocol uses a comprehensive data-independent acquisition mass spectrometry analysis to quantify the insolublome and bioinformatic analysis to gain biological insights into aging mechanisms and pathologies.

We and others have shown that the aging process results in a proteome-wide accumulation of insoluble proteins. Knocking down genes encoding the insoluble ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

自动化秀丽隐杆线虫的聚集定量

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

摘要

探索更多视频

此视频中的章节

使用荧光寿命成像在老化C.Elegans中淀粉样蛋白结构的表征

使用秀丽隐杆线虫中的自动包涵体计数监测体内蛋白质聚集动力学

基本的

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

秀丽隐杆线虫作为发现生物活性化合物对抗聚谷氨酰胺介导的神经毒性的模型系统

量化秀丽隐杆线虫中组织特异性蛋白静剂下降

建模年龄相关的神经退行性疾病在卡诺哈布迪蒂斯埃利根

卡诺哈布迪蒂斯伊利根斯新蛋白合成率评估

Quantification of Insoluble Protein Aggregation in Caenorhabditis elegans during Aging with a Novel Data-Independent Acquisition Workflow

自动化秀丽隐杆线虫的聚集定量

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

摘要

探索更多视频

此视频中的章节

使用荧光寿命成像在老化C.Elegans中淀粉样蛋白结构的表征

使用秀丽隐杆线虫中的自动包涵体计数监测体内蛋白质聚集动力学

基本的

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

秀丽隐杆线虫 作为发现生物活性化合物对抗聚谷氨酰胺介导的神经毒性的模型系统

量化秀丽隐杆线虫中组织特异性蛋白静剂下降

建模年龄相关的神经退行性疾病在 卡诺哈布迪蒂斯埃利根

卡诺哈布迪蒂斯 伊利根斯新蛋白合成率评估

Quantification of Insoluble Protein Aggregation in Caenorhabditis elegans during Aging with a Novel Data-Independent Acquisition Workflow

自动化秀丽隐杆线虫的聚集定量

秀丽隐杆线虫作为发现生物活性化合物对抗聚谷氨酰胺介导的神经毒性的模型系统

建模年龄相关的神经退行性疾病在卡诺哈布迪蒂斯埃利根

卡诺哈布迪蒂斯伊利根斯新蛋白合成率评估