这项工作得到NIH R01 DK110355，DK093776 [NTS]，DK102450 [NTS]和P30 DK034987 [向UNC-Chapel Hill]的支持。作者感谢Deekshita Ramanarayanan协助qPCR和免疫印迹实验。

该协议是根据北卡罗来纳大学的机构动物保护和使用委员会（IACUC）批准和执行的。 准备工作<ol><li>制备Triton-X缓冲液（1％Triton X-100,5mM乙二胺四乙酸（EDTA）），在磷酸盐缓冲盐水（PBS），pH 7.4中调高体积）。制成500mL:将Triton X-100和500mM EDTA各5mL搅拌至490mL PBS（pH7.4）中。在4℃下储存Triton-X缓冲溶液。 </li><li>制备高盐缓冲液（10mM Tris-HCl，pH 7.6,140mM NaCl，1.5M KCl，5mM EDTA，0.5％Triton X-100，在双蒸馏（dd）H 2 O中调出体积）。制成500毫升:搅拌10mL 0.5M Tris-HCl（pH7.6），14mL 5M NaCl，55.9克KCl，5毫升0.5M EDTA和2.5毫升Triton X-100，将体积调至500 mL，使用双蒸水）。在4°C储存高盐缓冲溶液。 </li><li>在使用前，补充适量的Triton-X和高盐缓冲液<em&gt;例如，每25mg组织样品1mL）与蛋白酶或蛋白酶/磷酸酶抑制剂混合，并丢弃任何不含有抑制剂的缓冲液。 </li><li>在1×PBS，pH 7.4中制备5mM EDTA。制成500mL:将5mL的0.5M EDTA搅拌到495mL的1×PBS，pH7.4中。 </li><li>使用符合兽医指南和机构标准的认可方案，分离不同的小鼠器官（脑，心脏，肺，肝，胰腺，结肠，肠，肾，脾） 23 。组织采集程序应不超过5分钟，以保持富含蛋白酶的组织的RNA和蛋白质完整性（ 例如胰腺应先处理）。 </li><li>切割少量组织（〜5-20mg）并置于RNA储存溶液中，用于随后的RNA提取，cDNA合成和IF基因表达的定量实时PCR分析。将RNA储存溶液管与组织放置在4℃过夜，并遵循制造商协议进一步的折叠和隔离步骤。 </li><li>将其余的组织切成更小的碎片（ 例如 0.5厘米），并置于低温条件下。快速冷冻并将样品瓶储存在-80°C或液氮下长期储存。 </li></ol> IF基因表达分析<ol><li>从保存在RNA储存溶液试剂中的组织中提取RNA。根据制造商的方案使用任何合适/优选的RNA提取方法。 </li><li>定量RNA浓度，并使用2μgRNA根据制造商的方案使用合适的逆转录试剂盒产生cDNA。 </li><li>使用生成的cDNA和小鼠IF基因特异性引物设置qPCR反应，包括每个样品的三个技术重复以及根据制造商的方案的空白对照。 </li><li>将IF基因表达定量为比较不同条件（ 例如年轻组织和老组织）的折叠变化。 </li></ol> 3. P用于免疫印迹的总组织裂解物的修复<ol><li>在1mL的2x还原SDS样品缓冲液中均化25mg的组织。如果要进行比色蛋白测定以量化蛋白质浓度，则从样品缓冲液中除去溴酚蓝染料。 </li><li>加入5％（v / v）的2-巯基乙醇（2-ME）以制备还原样品。向200μL非还原样品中加入10μL2-ME。 </li><li>使用洗涤剂和还原剂相容的蛋白质测定法确定蛋白质浓度。如果染料已经包含在样品缓冲液中，蛋白质量可以通过许多技术运行凝胶之后估算，包括考马斯染色剂24 。 </li><li>涡旋所有样品并在95℃加热5分钟。 </li><li>在还原和非还原条件下进行Western印迹。短暂暴露膜（&lt;1分钟）以显示单体物质，并且更长（&gt; 1分钟）以显示高分子量复合物包含ng IF蛋白。如果监测聚集，检查整个凝胶/膜（包括凝胶孔的底部）。 </li><li>运行平行凝胶，并用蛋白质染色剂作为载体对照染色24 。在疾病模型或损伤实验中，总蛋白质染色应用作加载对照，而不是用于"内部"蛋白质（ 例如肌动蛋白，GAPDH）的免疫印迹，因为后者在不同应力条件下发生变化。 </li></ol> 4.组织特异性IF的洗涤剂可溶性和高盐提取物的制备<ol><li>将1mL冰冷的Triton X-100缓冲液加入玻璃管匀浆器中并置于冰上。 </li><li>从液氮储存中取出一小块薄片（约25 mg），并直接放入玻璃均化器中。使用聚四氟乙烯杵匀浆（50冲程），避免产生气泡。始终保持匀浆器和裂解液冷。 </li><li>将裂解液转移至1.5 mL micr离心管在冰上并在预冷却的离心机（4℃）中以20,000×g离心10分钟。 </li><li>将上清液部分收集到单独的管中。这是Triton X-可溶性级分，可用于免疫沉淀（ip）和可洗涤剂溶解的IF蛋白库的分析。注意，步骤4.1-4.4可以重复以实现来自脑组织的更清洁的IF提取物。 </li><li>将1 mL高盐缓冲液加入组织沉淀中，转移至干净的匀浆器中，并行100次。将匀浆转移回微量离心管，并将管置于冷藏室中的旋转振荡器上1小时。 </li><li>在4℃下以20,000×g离心匀浆20分钟。丢弃上清液。 </li><li>将1mL冰冷的PBS / EDTA缓冲液加入到沉淀中，并将其在干净的均化器中匀浆（20次），作为最终的清理步骤*。转移到新管中，在4℃下以20,000×g离心10分钟，得到IF蛋白 -丰富的高盐提取物（HSE）。 *任选地，在该步骤中涡旋而不是均质化。 </li><li>弃去上清液，并将丸粒溶解在300μL已预热的非还原性SDS样品缓冲液中。首先通过移液和涡旋破碎颗粒，然后在95℃下加热样品5分钟。 </li><li>涡流和移液管，以确保颗粒溶解。完全溶解颗粒可能需要几分钟的时间。 </li><li>将所有样品储存在-20°C直到分析。 </li></ol> 5.大容量实验中IF蛋白提取的自动组织裂解<ol><li>对于RNA提取，将裂解缓冲液（每25mg组织600μL缓冲液）置于含有裂解基质D（使用小陶瓷球）的管中，并在组织溶液中脉冲两次25秒。通过以20,000×g离心分离裂解物与基质，并分离进行下一步骤。 </li><li>对于蛋白质提取，放置Trit在X-100（或SDS样品缓冲液，如果准备总裂解物）在裂解管与裂解基质SS（使用单个不锈钢珠）。在测试多个矩阵之后，选择它是因为它产生的IF蛋白质提取物质量与传统的去抖动方法相同。请注意，自动化方法对胰腺和脾脏不是最佳的，标准均质方案应用于这些组织。 </li><li>要进行高盐提取物的制备，使用磁铁从管中取出不锈钢珠，并在4℃下以20,000 xg离心管10分钟。 </li><li>继续执行手册中的步骤4.4（以上）。 </li><li>将所有样品储存在-20°C直到分析。 </li></ol> 6.翻译后修饰的IF蛋白的免疫富集<ol><li>准备PBST缓冲液（0.02％Tween-20在PBS中）。加入10 mL Tween-20至50 mL PBS（pH 7.4）。 </li><li>制备PTM抗体溶液（200μLPBST中的1-10μg抗体）。一般来说，3μg抗体/反应是一个很好的起始条件，如果需要可进一步优化。 </li><li>对于每个反应，将50μL磁珠等分成微量离心管，置于磁体上并吸出珠子储存溶液。 </li><li>通过重新悬浮在抗体溶液中并在室温下孵育20分钟，将珠子与免疫沉淀抗体相结合，并在旋转器（端到端，确保小体积的混合物）上孵育20分钟。 </li><li>将管置于磁铁上并吸出抗体溶液。 </li><li>在200μLPBST中冲洗抗体缀合的珠粒一次，并清洗洗涤缓冲液。 </li><li>将0.6-1mL的组织裂解物加入珠中，通过温和移液混合并在冷室中在旋转器上孵育3小时。 </li><li>将管置于磁体上，除去溶胞产物，并用200μLPBST洗珠5次。最后洗涤步骤后，收集珠子在100 _6; L的PBS（不含Tween-20）并转移到干净的新管中。将管放在磁铁上。 </li><li>吸出PBS并加入100μL非还原样品缓冲液。取出50μL，加入2-ME（5％）以制备还原样品。将样品加热至95°C 5分钟。 </li><li>将ip分数与磁铁上的珠子分开，并将其收集到新的管中。 </li><li>将样品储存在-20°C直到分析。 </li></ol> 7.制备用于质谱分析的IF蛋白质样品<ol><li>在开始研究之前安排与蛋白质组学专家的磋商，因为质谱分析涉及到大量时间和成本。 </li><li>采取特殊预防措施，避免污染。用干净的手套处理所有凝胶，并在干净的容器中孵育，仅使用ddH 2 O（避免使用肥皂）洗涤。 </li><li>根据标准条件，在SDS-PAGE凝胶上运行20-50μL的HSE样品（来自第4和第5部分）。 </l我&gt; <li>用蛋白质污渍染色1小时。漂洗多次，并在ddH 2 O中脱色过夜。去蛋白后，IF蛋白带应易于看到。 </li><li>将凝胶放置在塑料片保护膜之间，扫描并标记将被切除并发送用于分析的条带。 </li><li>使用新的干净剃须刀消除IF蛋白质条带。 </li><li>将凝胶带置于干净的微量离心管中并转移到质谱仪中。 </li></ol>

作者宣称他们没有竞争的经济利益。

使IF蛋白的生物化学表征的方法可用于了解哺乳动物系统中的许多病理生理现象，因为IF蛋白既是细胞和组织应激的标志物和调节剂。目前的方法背后的原理是基于20世纪70年代和80年代开发的初步程序，从细胞和组织中分离，分离和重建IF蛋白，一般采用低盐和高盐溶液和Triton-X100洗涤剂25,30,31,32 ， 33,34,35 。对于支持IF蛋白生化分离的研究的历史见解，请参见最近的评论<su...

<html> IF是一系列蛋白质，在人类中由73个基因编码并分为六种主要类型:I-IV型是细胞质（ 例如上皮和毛发角蛋白（K），肌细胞结蛋白，神经丝，胶质纤维酸性蛋白（GFAP）和别的）; V型是核脂肪;并且VI型是眼镜1中的IF。在其分子组织方面，IF蛋白质具有三个共同的结构域:高度保守的螺旋线圈"棒"结构域，以及球状"头"和"尾"结构域。 IF蛋白质四聚体组装形成短丝前体，其最终并入成熟细丝中，其形成涉及机械保护的动态细胞骨架和核结构结构2 ，应激感测3,4 ，调节转录5和生长以及其他关键细胞功能"&gt; 1,6,7 。 由IF基因错义突变引起的许多人类疾病的存在突出了IF系统的功能重要性，包括神经病，肌病，皮肤脆性障碍，代谢功能障碍和早衰症候群8 。一些IF基因突变不会引起疾病进展的倾向，如肝脏疾病中的简单上皮角蛋白9 。后者是由于上皮中的IF的临界应力保护功能。一般来说，IF在基础条件下是最丰富的细胞蛋白，但在各种类型的应激10中进一步强烈诱导。例如，最近对线虫线虫的蛋白质组范围变化进行的研究表明，多个IF被高度上调并且易于聚集在有机体老化11,12 。由于维持适当的IF结构对于细胞对各种形式的应力10的抵抗是至关重要的，所以IF聚集也可能导致衰老过程中的功能下降。然而，缺乏在不同组织中进行压力检测多种哺乳动物IF蛋白的有机体水平的研究。 IF是高度动态的结构，可以适应蜂窝需求。例如，角蛋白经历可溶性（非丝状）和不溶性（丝状）蛋白质池13之间的生物合成非依赖性循环。在正常的生理条件下，约5％的K8 / K18总库可以在无洗涤剂的缓冲液中提取，相比之下可以溶解在非离子型洗涤剂Nonidet P-40中的约20％，这是与Triton- X100 14 ， </s&gt; 15 。在有丝分裂期间，简单型上皮K8和K1814的溶解度显着增加，这在表皮角蛋白中不太明显，但在波形蛋白和其他III型IF蛋白15,16 中更为明显。 IF蛋白的溶解性质通过磷酸化来进行严格调控，磷酸化是针对灯丝重排和溶解度的关键的翻译后修饰（PTM）17,18,19,20。大多数IF受到保护地点的多个PTM的广泛监管，导致功能变化17 。 这种方法的目的是引入新的IF领域的研究人员进行生物化学提取和多个小鼠组织中IF蛋白研究的分析方法。具体盟军，我们专注于使用高盐提取方法分离IF蛋白，并通过质谱法和PTM靶向抗体评估PTMs的变化。这些方法基于以前发布的程序21，但包括用于提取不同IF蛋白类型的修饰，以揭示通过IF家族调节的常见机制。例如，特定赖氨酸残基处的K8乙酰化调节细丝组织，而超乙酰化促进K8不溶性和聚集体形成22 。最近的全球蛋白质组学分析研究还发现，大多数组织特异性IF蛋白质也是乙酰化的靶标，大多数IF乙酰化位点被限制在高度保守的杆结构域。这突出表明需要适合IF系统的全局分析的方法。我们还介绍了使用Opti自动均质化从多组织分离IF蛋白的快速方法大小溶解基质。所得制剂适用于通过质谱法和其他方法进行下游PTM分析。

<table><tbody><tr><td>Dynabeads Protein G</td><td>ThermoFisher Scientific</td><td>10009</td><td>immunoprecipitation beads</td></tr><tr><td>PBS</td><td>ThermoFisher Scientific</td><td>10010049</td><td>for buffers</td></tr><tr><td>Purelink RNA mini kit</td><td>ThermoFisher Scientific</td><td>12183018</td><td>RNA extraction from tissue</td></tr><tr><td>Purelink DNAse set</td><td>ThermoFisher Scientific</td><td>12185010A</td><td>on column DNA digestion</td></tr><tr><td>Dynamag-2</td><td>ThermoFisher Scientific</td><td>12321D</td><td>magnet for use with dynabeads</td></tr><tr><td>GelCode Blue Stain Reagent</td><td>ThermoFisher Scientific</td><td>24592</td><td>mass spectrometry-compatible gel stain</td></tr><tr><td>Pierce ECL Western Blotting Substrate</td><td>ThermoFisher Scientific</td><td>32106</td><td>for use in western blot</td></tr><tr><td>High Capacity cDNA reverse transcription kit</td><td>ThermoFisher Scientific</td><td>4368813</td><td>for use in gene expression analysis</td></tr><tr><td>Proflex 3 x 32-well PCR System</td><td>ThermoFisher Scientific</td><td>4484073</td><td>PCR system</td></tr><tr><td>PVDF transfer membrane&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>88520</td><td>for western blot</td></tr><tr><td>Power Up SYBR master mix</td><td>ThermoFisher Scientific</td><td>A25778</td><td>for qPCR analysis</td></tr><tr><td>RNAlater</td><td>ThermoFisher Scientific</td><td>AM7020</td><td>solution for tissue storage prior to RNA isolation</td></tr><tr><td>Novex 4-20% Tris Glycine Gel</td><td>ThermoFisher Scientific</td><td>XP04205BOX</td><td>Precast protein gel</td></tr><tr><td>Anti-Keratin 8 antibody (TS1)</td><td>ThermoFisher Scientific</td><td>MA514428</td><td>for western blot detection of K8</td></tr><tr><td>Anti-Vimentin</td><td>ThermoFisher Scientific</td><td>MA511883</td><td>for western blot detection of vimentin</td></tr><tr><td>Tris Glycine Transfer Buffer (25x)</td><td>ThermoFisher Scientific</td><td>LC3675</td><td>for wet transfer of protein gels</td></tr><tr><td>2x SDS Sample Buffer</td><td>ThermoFisher Scientific</td><td>LC2676</td><td>for preparing protein gel samples</td></tr><tr><td>Tris Glycine SDS Running Buffer (10x)</td><td>ThermoFisher Scientific</td><td>LC26755</td><td>for running protein gels</td></tr><tr><td>Lysing beads - Matrix D</td><td>MP Biomedicals</td><td>116913100</td><td>Lysis beads and matrix tubes for tissue disruption and RNA extraction</td></tr><tr><td>Lysing beads - Matrix SS</td><td>MP Biomedicals</td><td>116921100</td><td>Lysis beads and matrix tubes for tissue disruption and protein extraction</td></tr><tr><td>NanoDrop Lite Spectrophotometer</td><td>ThermoFisher Scientific</td><td>ND-LITE</td><td>measurement of protein and nucleic acid</td></tr><tr><td>Precellys 24 homogenizer</td><td>Bertin Instruments</td><td>EQ03119</td><td>Automated tissue homogenizer</td></tr></tbody></table>

isolation of intermediate filament proteins from multiple mouse tissues to study aging-associated post-translational modifications

中间丝（IFs）与肌动蛋白丝和微管一起形成细胞骨架 - 每个细胞的关键结构元件。正常功能的IF为细胞提供机械和应激弹性，而功能失调的IF细胞骨架损害细胞健康，并与许多人类疾病相关。翻译后修饰（PTM）对生理变化和应激条件下的IF动力学进行了严格的调节。因此，监测IFM的PTM签名变化的能力可以有助于在细胞损伤期间作为应激反应者的IF系统的更好的功能理解和最终调节。然而，由超过70个个体基因编码并以组织依赖性方式表达的大量IF蛋白质是排除不同PTM相对重要性的主要挑战。为此，可以监测IF蛋白上的PTMs的方法在生物体层面上，而不是孤立的家庭成员，可以加速这一领域的研究进展。在这里，我们提出生物化学方法，从9种不同的小鼠组织（脑，心脏，肺，肝，小肠，大肠，胰腺，肾脏和）分离IF蛋白的总，洗涤剂可溶性和去污剂抗性分数脾）。我们进一步展示了通过使用裂解基质和不同小鼠组织的自动均质化来快速分离IF蛋白的优化方案。自动化方案对于在具有高样品体积的实验中（例如涉及多个动物和实验组的疾病模型）中的IF进行分析是有用的。所得样品可用于各种下游分析，包括基于质谱的PTM分析。利用这些方法，我们提供新的数据，显示不同小鼠组织（脑和肝）中的IF蛋白在其表达方面发生平行变化老龄化期间的PTMs。

一种使用裂解基质从多个小鼠组织中高度盐提取IF蛋白的新的快速方法。 在这里修饰了分离大部分中间丝蛋白部分与上皮组织的传统方法25,26以包括9种不同的器官和更快速的组织溶解过程。虽然传统方法需要3个手动均质步骤，但修改过程只有1个手动均质步骤，可以?...

Watch this Scientific Journal Video about Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications at JoVE.com

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

在这种方法中，我们提出了从多个小鼠组织快速高效地分离中间丝（IF）蛋白质的生物化学方法。分离的IF可用于通过质谱法和其他生化测定法研究翻译后修饰的变化。

从多个小鼠组织分离中间丝状蛋白以研究与老化相关的翻译后修饰

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

isolation-intermediate-filament-proteins-from-multiple-mouse-tissues

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

8.4K 次观看.  University of North Carolina at Chapel Hill. 在这种方法中，我们提出了从多个小鼠组织快速高效地分离中间丝（IF）蛋白质的生物化学方法。分离的IF可用于通过质谱法和其他生化测定法研究翻译后修饰的变化。 

视频: Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances in both basic and clinical research. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. Specific housekeeping proteins have traditionally been used to normalize protein levels for quantification, however, these have a number of limitations and have therefore been increasingly criticized over the past few years. Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints. By using a fluorescent total protein stain and introducing the use of an internal loading standard, it is possible to overcome existing limitations in the number of samples that can be compared within experiments and systematically compare protein levels across a range of experimental conditions. This approach expands the use of traditional western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

利用标准化定量西方印迹技术, 对跨组织和整个开发过程中的蛋白质水平进行可靠比较

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

科研

生物化学

Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. Specifically, gait analysis allows recapturing disease relevant phenotypes that are observed in human patients, especially in neurodegenerative diseases that affect motor abilities such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease (AD), amyotrophic lateral sclerosis (ALS), and others. In early studies along this line, the measurement of gait parameters was laborious and depended on factors that were hard to control (e.g., running speed, continuous running). The development of ventral plane imaging (VPI) systems made it feasible to perform gait analysis at a large scale, making this method a useful tool for the assessment of motor behavior in rodents. Here, we present an in-depth protocol of how to use kinematic gait analysis to examine the age-dependent progression of motor deficits in mouse models of neurodegeneration; mouse lines with decreased levels of endophilin, in which neurodegenerative damage progressively increases with age, are used as an example.

In this study, we demonstrate the use of kinematic gait analysis based on ventral plane imaging to monitor the subtle changes in motor coordination as well as the progression of neurodegeneration with advancing age in mouse models (e.g., endophilin mutant mouse lines).

变性小鼠年龄依赖性运动障碍的步态分析

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. ...

发育生物学

Quantitative Microtubule Fractionation Technique to Separate Stable Microtubules, Labile Microtubules, and Free Tubulin in Mouse Tissues

Microtubules, composed of &#945;/&#946;-tubulin dimers, are a crucial component of the cytoskeleton in eukaryotic cells. These tube-like polymers exhibit dynamic instability as tubulin heterodimer subunits undergo repetitive polymerization and depolymerization. Precise control of microtubule stability and dynamics, achieved through tubulin post-translational modifications and microtubule-associated proteins, is essential for various cellular functions. Dysfunctions in microtubules are strongly implicated in pathogenesis, including neurodegenerative disorders. Ongoing research focuses on microtubule-targeting therapeutic agents that modulate stability, offering potential treatment options for these diseases and cancers. Consequently, understanding the dynamic state of microtubules is crucial for assessing disease progression and therapeutic effects.
Traditionally, microtubule dynamics have been assessed in vitro or in cultured cells through rough fractionation or immunoassay, using antibodies targeting post-translational modifications of tubulin. However, accurately analyzing tubulin status in tissues using such procedures poses challenges. In this study, we developed a simple and innovative microtubule fractionation method to separate stable microtubules, labile microtubules, and free tubulin in mouse tissues.
The procedure involved homogenizing dissected mouse tissues in a microtubule-stabilizing buffer at a 19:1 volume ratio. The homogenates were then fractionated through a two-step ultracentrifugation process following initial slow centrifugation (2,400 &#215; g) to remove debris. The first ultracentrifugation step (100,000 &#215; g) precipitated stable microtubules, while the resulting supernatant was subjected to a second ultracentrifugation step (500,000 &#215; g) to fractionate labile microtubules and soluble tubulin dimers. This method determined the proportions of tubulin constituting stable or labile microtubules in the mouse brain. Additionally, distinct tissue variations in microtubule stability were observed that correlated with the proliferative capacity of constituent cells. These findings highlight the significant potential of this novel method for analyzing microtubule stability in physiological and pathological conditions.

Microtubules, which are tubulin polymers, play a crucial role as a cytoskeleton component in eukaryotic cells and are known for their dynamic instability. This study developed a method for fractionating microtubules to separate them into stable microtubules, labile microtubules, and free tubulin to evaluate the stability of microtubules in various mouse tissues.

定量微管分离技术分离小鼠组织中稳定的微管、不稳定的微管和游离微管蛋白

Microtubules, composed of &#945;/&#946;-tubulin dimers, are a crucial component of the cytoskeleton in eukaryotic cells. These tube-like polymers exhibit ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

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 ...

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

单肌纤维隔离与文化从埃默里-德雷福斯肌肉萎缩的早期后纳塔尔发展穆林模型

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

医学

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

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

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

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

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

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 ...

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 ...

可变长度小鼠肌肉纤维的酶促分离和表征

Intact Short, Intermediate, and Long Skeletal Muscle Fibers Obtained by Enzymatic Dissociation of Six Hindlimb Muscles of Mice: Beyond Flexor Digitorum Brevis

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal with the short fibers of the flexor digitorum brevis (FDB), which restrains the scope of results dealing with fiber types, limits the amount of biological material available, and impedes a clear connection between cellular physiological phenomena and previous biochemical and dynamical knowledge obtained in other muscles.
This paper describes how to obtain intact fibers from six muscles with different fiber type profiles and lengths. Using C57BL/6 adult mice, we show the muscle dissection and fiber isolation protocol and demonstrate the suitability of the fibers for Ca2+ transient studies and their morphometric characterization. The fiber type composition of the muscles is also presented. When dissociated, all muscles rendered intact, living fibers that contract briskly for more than 24 h. FDB gave short (&#60;1 mm), peroneus digiti quarti (PDQA) and peroneus longus (PL) gave intermediate (1-3 mm), while extensor digitorum longus (EDL), extensor hallucis longus (EHL), and soleus muscles released long (3-6 mm) fibers.
When recorded with the fast dye Mag-Fluo-4, Ca2+ transients of PDQA, PL, and EHL fibers showed the fast, narrow kinetics reminiscent of the morphology type II (MT-II), known to correspond to type IIX and IIB fibers. This is consistent with the fact that these muscles have over 90% of type II fibers compared with FDB (~80%) and soleus (~65%). Moving beyond FDB, we demonstrate for the first time the dissociation of several muscles, which render fibers spanning a range of lengths between 1 and 6 mm. These fibers are viable and give fast Ca2+ transients, indicating that the MT-II can be generalized to IIX and IIB fast fibers, regardless of their muscle source. These results increase the availability of models for mature skeletal muscle studies.

作者聚焦：从小鼠后肢肌肉中分离长肌肉纤维，用于研究跨纤维类型的兴奋-收缩耦合

We describe a protocol to obtain enzymatically dissociated fibers of different lengths and types from six muscles of adult mice: three of them already described (flexor digitorum brevis, extensor digitorum longus, soleus) and three of them successfully dissociated for the first time (extensor hallucis longus, peroneus longus, peroneus digiti quarti).

通过酶解离小鼠六块后肢肌肉获得的完整短、中和长骨骼肌纤维：超越趾短屈肌

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal ...

从多个小鼠组织分离中间丝状蛋白以研究与老化相关的翻译后修饰

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此视频中的章节

利用标准化定量西方印迹技术, 对跨组织和整个开发过程中的蛋白质水平进行可靠比较

变性小鼠年龄依赖性运动障碍的步态分析

定量微管分离技术分离小鼠组织中稳定的微管、不稳定的微管和游离微管蛋白

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

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

单肌纤维隔离与文化从埃默里-德雷福斯肌肉萎缩的早期后纳塔尔发展穆林模型

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

量化秀丽隐杆线虫中组织特异性蛋白静剂下降

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

通过酶解离小鼠六块后肢肌肉获得的完整短、中和长骨骼肌纤维：超越趾短屈肌