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 style="border-collapse:collapse;width:635pt" width="847">
	<colgroup>
		<col style="width:161pt" width="215" />
		<col style="width:92pt" width="122" />
		<col style="width:258pt" width="344" />
		<col style="width:125pt" width="166" />
	</colgroup>
	<tbody>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="width:161pt;height:30.0pt" width="215">Fernbach culture flask&#160;</td>
			<td class="xl18" style="width:92pt" width="122">Corning</td>
			<td class="xl19" style="width:258pt" width="344">4425-2XL</td>
			<td class="xl18" style="width:125pt" width="166">Pyrex, Capacity 2,800 ml, with 3 baffle indents</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Membrane Screw Cap&#160;</td>
			<td class="xl18" style="width:92pt" width="122">Schott</td>
			<td class="xl19">1088655</td>
			<td class="xl17">GL45</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Nutating Mixer</td>
			<td class="xl18" style="width:92pt" width="122">VWR</td>
			<td class="xl19">444-0148</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Separatory funnel</td>
			<td class="xl18" style="width:92pt" width="122">Nalgene</td>
			<td class="xl19">4300-1000</td>
			<td class="xl17">Capacity 1,000 ml</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">1 ml syringe&#160;</td>
			<td class="xl18" style="width:92pt" width="122">BD Plastipak</td>
			<td class="xl19">300013</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Gray needle, 27 G x &#189; &#34;, 0.4 mm x 13 mm</td>
			<td class="xl18" style="width:92pt" width="122">BD Microlance 3</td>
			<td class="xl19">300635</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Membrane filters 0.025 &#181;M</td>
			<td class="xl18" style="width:92pt" width="122">Millipore</td>
			<td class="xl19">VSWP04700</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">pH strip</td>
			<td class="xl18" style="width:92pt" width="122">Machery-Nagel</td>
			<td class="xl19">92110</td>
			<td class="xl17">pH-Fix 0-14</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Protease Inhibitor Cocktail</td>
			<td class="xl18" style="width:92pt" width="122">Roche</td>
			<td class="xl19">4693132001</td>
			<td class="xl16" style="width:125pt" width="166">Complete Mini EDTA-free tablets&#160;</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Octoxynol-9&#160;</td>
			<td class="xl18" style="width:92pt" width="122">Applichem</td>
			<td class="xl19">A1388</td>
			<td class="xl18" style="width:125pt" width="166">Triton X-100</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">4-Morpholineethanesulfonic acid (MES)</td>
			<td class="xl18" style="width:92pt" width="122">Sigma-Aldrich</td>
			<td class="xl19">M1317</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Nonylphenylpolyethylenglycol</td>
			<td class="xl18" style="width:92pt" width="122">Applichem</td>
			<td class="xl19">A1694</td>
			<td class="xl18" style="width:125pt" width="166">Nonidet P40 (NP40)</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">DNaseI</td>
			<td class="xl18" style="width:92pt" width="122">Roche</td>
			<td class="xl19">04716728001</td>
			<td class="xl18" style="width:125pt" width="166">recombinant, RNase free</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">RNaseA</td>
			<td class="xl18" style="width:92pt" width="122">Promega</td>
			<td class="xl19">A7973</td>
			<td class="xl18" style="width:125pt" width="166">solution</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Total protein blot staining</td>
			<td class="xl18" style="width:92pt" width="122">Thermofisher</td>
			<td class="xl19">S11791</td>
			<td class="xl18" style="width:125pt" width="166">Sypro Ruby protein blot stain</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Total protein gel staining</td>
			<td class="xl18" style="width:92pt" width="122">Thermofisher</td>
			<td class="xl19">S12001</td>
			<td class="xl18" style="width:125pt" width="166">Sypro Ruby protein gel stain</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">TCEP (tris (2-carboxyethyl) phosphine hydrochloride)</td>
			<td class="xl18" style="width:92pt" width="122">Serva</td>
			<td class="xl19">36970</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Iodoacetamide</td>
			<td class="xl18" style="width:92pt" width="122">Serva</td>
			<td class="xl19">26710</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Ammoniumbicarbonate</td>
			<td class="xl18" style="width:92pt" width="122">Sigma-Aldrich</td>
			<td class="xl19">09830</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Sequencing Grade Modified Trypsin</td>
			<td class="xl18" style="width:92pt" width="122">Promega</td>
			<td class="xl25" style="width:258pt" width="344">V5111</td>
			<td class="xl18" style="width:125pt" width="166"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Isobaric tags for relative and absolute quantitation</td>
			<td class="xl18" style="width:92pt" width="122">Sciex</td>
			<td class="xl19">4352135</td>
			<td class="xl18" style="width:125pt" width="166">iTRAQ Reagents Multiplex Kit</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Centrifuge Avanti J-26XP</td>
			<td class="xl18" style="width:92pt" width="122">Beckmann Coulter</td>
			<td class="xl19">393126</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Ultracentrifuge Optima Max-XP</td>
			<td class="xl18" style="width:92pt" width="122">Beckmann Coulter</td>
			<td class="xl19">393315</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Centrifuge 5424R</td>
			<td class="xl18" style="width:92pt" width="122">Eppendorf</td>
			<td class="xl19">5404000413</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Centrifuge 5702</td>
			<td class="xl18" style="width:92pt" width="122">Eppendorf</td>
			<td class="xl19">5702000329</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Centrifuge Megafuge 40R</td>
			<td class="xl18" style="width:92pt" width="122">Thermo Scientific</td>
			<td class="xl19">75004518</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Concentrator Plus</td>
			<td class="xl18" style="width:92pt" width="122">Eppendorf</td>
			<td class="xl19">5305000304</td>
			<td class="xl17">Centrifugal evaporator</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Fluorescent stereo-microscope M165 FC&#160;</td>
			<td class="xl18" style="width:92pt" width="122">Leica</td>
			<td class="xl19"></td>
			<td class="xl18" style="width:125pt" width="166">With Planapo 2.0x objective</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Dissection microscope</td>
			<td class="xl18" style="width:92pt" width="122">Leica&#160;</td>
			<td class="xl19"></td>
			<td class="xl17">Leica S6E</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="80" rowspan="2" style="width:161pt;height:60.0pt" width="215">High magnification microscope Zeiss Axio Observer Z1</td>
			<td class="xl18" height="80" rowspan="2" style="width:92pt;height:60.0pt" width="122">Zeiss</td>
			<td class="xl19" height="80" rowspan="2" style="width:258pt;height:60.0pt" width="344"></td>
			<td class="xl16" height="80" rowspan="2" style="width:125pt;height:60.0pt" width="166">With PlanAPOCHROMAT 20x objective and Zeiss Axio Cam MRm</td>
		</tr>
		<tr height="40" style="height:30.0pt">
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl32" height="40" style="height:30.0pt">Software</td>
			<td class="xl33" style="width:92pt" width="122"></td>
			<td class="xl32"></td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Image analysis software</td>
			<td class="xl18" style="width:92pt" width="122">ImageJ</td>
			<td class="xl19"></td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Analysis of mass spectrometry data</td>
			<td class="xl18" style="width:92pt" width="122">Protein Prospector</td>
			<td class="xl136" style="width:258pt" width="344"></td>
			<td class="xl137">http://prospector.ucsf.edu/prospector/mshome.htm</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl32" height="40" style="height:30.0pt">E.coli strain</td>
			<td class="xl33" style="width:92pt" width="122"></td>
			<td class="xl19"></td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">OP50</td>
			<td class="xl18" style="width:92pt" width="122">CGC</td>
			<td class="xl19"></td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl32" height="40" style="height:30.0pt">RNAi bacteria</td>
			<td class="xl33" style="width:92pt" width="122"></td>
			<td class="xl19"></td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">L4440</td>
			<td class="xl18" style="width:92pt" width="122">Julie Ahringer RNAi library</td>
			<td class="xl19"></td>
			<td class="xl17"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl32" height="42" rowspan="2" style="width:161pt;height:31.5pt" width="215">C. elegans mutants</td>
			<td class="xl33" height="42" rowspan="2" style="width:92pt;height:31.5pt" width="122"></td>
			<td class="xl32" height="42" rowspan="2" style="width:258pt;height:31.5pt" width="344"></td>
			<td class="xl19" height="42" rowspan="2" style="width:125pt;height:31.5pt" width="166"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl17" height="42" style="height:31.5pt">CF2253</td>
			<td class="xl18" style="width:92pt" width="122">CGC, strain name: EJ1158&#160;</td>
			<td class="xl40" style="width:258pt" width="344"></td>
			<td class="xl17">Genotype: gon-2(q388)</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl33" height="42" rowspan="2" style="width:161pt;height:31.5pt" width="215">C. elegans transgenics</td>
			<td class="xl33" height="42" rowspan="2" style="width:92pt;height:31.5pt" width="122"></td>
			<td class="xl32" height="42" rowspan="2" style="width:258pt;height:31.5pt" width="344"></td>
			<td class="xl19" height="42" rowspan="2" style="width:125pt;height:31.5pt" width="166"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl18" height="42" rowspan="2" style="width:161pt;height:31.5pt" width="215">DCD214</td>
			<td class="xl25" height="42" rowspan="2" style="width:92pt;height:31.5pt" width="122">Della David&#39;s lab at DZNE T&#252;bingen</td>
			<td class="xl40" height="42" rowspan="2" style="width:258pt;height:31.5pt" width="344"></td>
			<td class="xl68" height="42" rowspan="2" style="width:125pt;height:31.5pt" width="166">Genotype: N2; uqIs24[Pmyo-2::tagrfp::pab-1]</td>
		</tr>
		<tr height="21" style="height:15.75pt">
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="42" rowspan="2" style="width:161pt;height:31.5pt" width="215">DCD215</td>
			<td class="xl87" height="42" rowspan="2" style="width:92pt;height:31.5pt" width="122">Della David&#39;s lab at DZNE T&#252;bingen</td>
			<td class="xl40" height="42" rowspan="2" style="width:258pt;height:31.5pt" width="344"></td>
			<td class="xl68" height="42" rowspan="2" style="width:125pt;height:31.5pt" width="166">Genotype: daf-2(e1370) III; uqIs24[Pmyo-2::tagrfp::pab-1]</td>
		</tr>
		<tr height="21" style="height:15.75pt">
		</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

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

实心板为基础的饮食限制在线虫</em

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

科研

生物学

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

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 ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

在活细胞中蛋白质聚集的4D成像

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

前列腺素的提取与分析<em&gt;线虫</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

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 ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

体外聚集测定使用tau蛋白的蛋白质

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

栽培<em&gt;秀丽隐杆线虫</em&gt;在三个维度实验室

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

线虫的核苷酸和蛋白质提取联合

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

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 ...