调查的传播和毒性朊病毒样蛋白使用后生动物模式生物<em>温度。线虫</em
Summary
Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.
Abstract
Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.
Introduction
许多神经变性疾病，包括阿尔茨海默氏病（AD），帕金森氏病（PD），肌萎缩性侧索硬化症（ALS），和传染性海绵状脑病（海绵状脑病），都与聚集倾向的蛋白质相关联，并且因而是统称为蛋白质错误折叠疾病（PMDS ）。的TSE或朊病毒病构成一类独特的PMD的，因为它们可以是传染性在人类和动物1。在分子水平上，朊病毒复制招募和单体α螺旋丰富的主机编码细胞朊蛋白（PRP C）转换成病理β折叠丰富的朊蛋白构象钪2,3。自我繁殖的蛋白质聚集体已经还确定了真菌，它与哺乳动物朊病毒4,5共享的重要特征。此外，哺乳动物朊病毒能够从细胞至细胞移动及感染幼稚细胞6,7。
而超视距的PMD呃不是传染性海绵状脑病是不会传染的，他们与朊病毒疾病8,9都有一个共同的致病原理。虽然链接到每个所述的PMD的蛋白质的结构或功能是不相关的，它们经由一个结晶状过程中的所有形式的聚集体称为有核或接种聚合;而且蛋白质的种子长出通过招募他们的可溶性亚型2,10,11。效率，以自我传播体内而变化，这取决于蛋白的固有性质，连同另外的细胞因子，如分子伴侣最终确定聚合成核，播种，碎裂和扩频12-15的速率。因此，必须存在这些因素之间，允许蛋白质聚集的有效传播一个微妙的平衡。这可能也解释了为什么只有某些淀粉样蛋白聚集怀有朊病毒的特点，因而并不是所有的PMD都具有传染性。朊病毒似乎代表“顶级表演'O广发自我复制的蛋白质聚集的频谱，这使得他们的强大工具来研究的PMD 8,13。
有趣的是，与疾病相关的聚集相关的毒性经常具有非细胞自主组件16,17。这意味着，它们影响邻近的细胞不表达相应的基因，而相比之下，严格细胞自主的效果，这意味着只有该细胞表达该基因表现出特定的表型。这是令人信服由组织特异性表达证实或敲在神经变性疾病18-26的众多模型的各蛋白质的向下。各种机制被建议作为用于这种非细胞中的PMD自主毒性，包括减少营养供给，失衡神经信号，谷氨酸兴奋毒性，和神经炎症16,27,28的基础。此外，疾病联的聚集体的细胞之间的朊病毒样运动migh吨有助于这方面的29,30。越来越多的证据表明，其它蛋白质夹杂比朊病毒可以从细胞-细胞传递，这可以解释病理学在许多的PMD 30-36观察的特征传播。然而，它尚未确定是否有疾病的蛋白质的细胞间移动和对邻近细胞的毒性效果之间的明确的因果关系。因此，更好地理解所依据的细胞至细胞传播和非细胞自主毒性的细胞途径的是必要的和必不可少的新疗法的发展。然而，朊病毒状扩频和细胞因子的许多方面，影响在后生动物细胞 – 细胞传输错误折叠的蛋白质都没有很好地理解，特别是在有机体的水平。
该线虫具有提供潜力几个优点 发现的朊病毒样spreadi新层面纳克在后生动物17。它是透明的，允许在体内跟踪的荧光标记的蛋白在活的有机体。此外，受疾病的许多细胞和生理过程是从蠕虫到人类的保守，和C.线虫也是适合于各种各样的遗传操作和分子和生物化学分析37-39的。正是959的体细胞组成成人雌雄同体用一个简单的身体的计划，仍然有几个不同的组织类型，包括肌肉，神经元和肠。
要建立C.一个新的朊病 毒模型线虫，我们选择了外源表达的良好表征谷氨酰胺/天冬酰胺（Q / N）的胞质酵母朊病毒Sup35的富朊病毒域NM，因为有在蠕虫4,40没有已知的内源性朊病毒蛋白。酵母朊病毒已经非常宝贵的阐明朊病毒复制41-44的基本机制。此外，NM是杉木已被证明扼要重述朊病毒的整个生命周期中的哺乳动物细胞培养45,46 ST胞质朊状蛋白质。同样地，在C时表示线虫 ，通过非常好，为在后生动物细胞繁殖的不同的要求相比，酵母细胞和朊病毒生物学表现关键特征40。NM聚集了深刻的毒性表型相关联，包括线粒体完整性和外观的破坏的Sup35朊病毒域各种自噬相关囊泡在细胞水平上，以及胚胎和幼虫逮捕，发育迟缓，并在有机体水平的蛋白质折叠的环境的一个普遍的干扰。引人注目的是，朊病毒域表现出细胞中自主和非细胞自主毒性，影响相邻的组织，其中，转基因不表达。此外，内和细胞间的朊病毒域的囊泡运输监测实时<EM>在体内40。
在这里，我们将介绍如何检查朊病毒般传播C.线虫 。我们将解释如何监控含使用时间推移荧光显微镜朊病毒域囊泡内和细胞间的运输。我们将强调使用组织特异性折叠传感器和遍在表达的应力记者，以评估对细胞的健身细胞中自主和非细胞自主效应。最后，我们将描述一个最近执行的全基因组RNA干扰（RNAi）屏幕的方法来识别朊病毒诱导的毒性的新调节剂。总之，这些方法可以帮助捉弄除了参与蛋白质的细胞间运动及其非细胞毒性自主遗传途径。
Protocol
Representative Results
Discussion
此处所描述的方法有助于说明扩频和朊病毒样蛋白的复杂细胞中自主和非细胞自主毒性。我们最近发现，一个聚集倾向的胞质朊病毒域被吸收到膜结合的囊泡中的自噬相关的过程。这些囊泡的特定子集运内和细胞和组织40之间的朊病 毒域。监测其在活的动物运动的关键是，该蛋白质必须具有标记MRFP，因为只有MRFP标签蛋白均在这些推测酸性囊泡可见。用同样的方法，我们目前正在研究…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Cindy Voisine and Yoko Shibata for helpful discussion and critical comments on the manuscript. We acknowledge the High Throughput Analysis Laboratory (HTAL) and the Biological Imaging Facility (BIF) at Northwestern University for their assistance. This work was funded by grants from the National Institutes of Health (NIGMS, NIA, NINDS), the Ellison Medical Foundation, and the Daniel F. and Ada L. Rice Foundation (to R.I.M.). C.I.N.-K. was supported by the Deutsche Forschungsgemeinschaft (KR 3726/1-1).
Materials
| Reagent | |||
| Nanosphere size standards 100 nm | ThermoScientific | 3100A | |
| Levamisole | Sigma | L-9756 | |
| IPTG | Sigma | 15502-10G | |
| Ahringer RNAi library | Source BioScience LifeSciences | http://www.lifesciences.sourcebioscience .com/clone-products/non-mammalian/c-elegans/c-elegans-rnai-library/ |
|
| Equipment | |||
| Sorvall Legend XTR Refrigerated Centrifuge, 120VAC | ThermoScientific | 75004521 | http://www.coleparmer.com/Product/Thermo_Scientific_Sorvall_Legend_ XTR_Refrigerated_Centrifuge_120 VAC/EW-17707-60 |
| 96 pin replicator | Scionomix | http://www.scinomix.com/all-products/96-pin-replicator/ | |
| HiGro high-capacity, incubating shaker | Digilab | http://www.digilabglobal.com/higro | |
| Multidrop Combi Reagent Dispenser | Titertrek | http://groups.molbiosci.northwestern.edu/hta/titertek.htm | |
| Biomek FX AP96 Automated Workstation | Beckman Coulter | http://groups.molbiosci.northwestern.edu/hta/biomek_multi.htm | |
| Innova44 shaker | New Brunswick | http://www.eppendorf.com/int///index.php?sitemap=2.3&pb=d78efbc05310ec 04&action=products&contentid=1& catalognode=83389 |
|
| M205 FA | Leica | http://www.leica-microsystems.com/de/produkte/stereomikroskope-makroskope/fluoreszenz/details/product/leica-m205-fa/ | |
| ORCA-R2 C10600-10BDigital CCD camera | Hamamatsu | http://www.hamamatsu.com/jp/en/community/life_science_camera/product/search/C10600-10B/index.html | |
| Spinning Disc AF Confocal Microscope | Leica | http://www.leica-microsystems.com/products/light-microscopes/life-science-research/fluorescence-microscopes/details/product/leica-sd-af/ | |
| Falcon 4M60 camera | Teledyne Dalsa | http://www.teledynedalsa.com/imaging/products/cameras/area-scan/falcon/PT-41-04M60/ | |
| Software | |||
| MetaMorph Microscopy Automation & Image Analysis Software | Molecular Devices | http://www.moleculardevices.com/products/software/meta-imaging-series/metamorph.html | |
| Hamamatsu SimplePCI Image Analysis Software | Meyer Instruments | http://meyerinst.com/imaging-software/hamamatsu/index.htm | |
| ImageJ | NIH | http://rsbweb.nih.gov/ij/download.html | |
| wrMTrck plugin for ImageJ | http://www.phage.dk/plugins/wrmtrck.html | ||
| C. elegans strains | |||
| N2 (WT) | Caenorhabditis Genetics Center (CGC) | http://www.cgc.cbs.umn.edu/strain.php?id=10570 | |
| AM815 rmIs323[myo-3p::sup35(r2e2)::rfp] | Morimoto lab | available from our laboratory | |
| See table 1 for a source for folding sensor and stress reporter strains |
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