JoVE Journal > Biology
Summary
Abstract
Introduction
Protocol
Résultats
Discussion
Divulgations
Acknowledgements
Materials
References
Traduction automatique
Please note that all translations are automatically generated. Click here for the English version.
Biologie

使用 卡诺哈布迪炎埃莱甘斯 筛选组织特异性查佩龙相互作用

Published: June 07, 2020
doi:
10.3791/61140
, , ,
1Department of Life Sciences,Ben-Gurion University of the Negev

Summary

为了研究伴奏和伴奏基底相互作用，我们使用RNA干扰与轻度突变或伴郎过度表达相结合，在有机体水平上监测组织特异性蛋白质功能障碍，在 卡诺哈布德炎中 执行合成相互作用屏幕。

Abstract

蛋白质和蛋白质复合物的正确折叠和组装对细胞功能至关重要。细胞采用质量控制途径，纠正、封存或消除受损蛋白质，以维持健康的蛋白质组，从而确保细胞蛋白石和防止进一步的蛋白质损伤。由于蛋白酶网络中的多余功能，使用多细胞生物 体Caenorhabdd炎的 伴随编码基因的敲击或突变来筛选可检测表型，在大多数情况下，可检测到小或无表型。我们制定了有针对性的筛选策略，以确定特定功能所需的陪护人员，从而弥合表型和功能之间的差距。具体来说，我们使用RNAi合成交互屏幕监测新的伴郎互动，敲击伴郎表情，一次一个伴郎，在携带伴郎编码基因突变或过度表达兴趣伴郎的动物中。通过破坏两个单独呈现无严重表型的陪护，我们可以识别在两者都扰动时加重或暴露特定表型的陪护。我们证明，这种方法可以识别出特定的伴郎组合，这些伴郎可以协同工作，调节与给定表型相关的蛋白质或蛋白质复合物的折叠。

Introduction

细胞通过使用质量控制机器来修复、封存或去除任何受损的蛋白质1，2来应对蛋白质的损害。蛋白质复合物的折叠和组装由分子伴奏支持，这是一组高度保存的蛋白质，可以修复或封存受损的蛋白质3，4，5，6，7。去除受损蛋白质由无处不在的蛋白系统（UPS）8或自噬机械9与伴奏10，11，12合作进行调解。因此，蛋白质平衡（蛋白石）是由折叠和降解机3，13组成的质量控制网络维持的。然而，了解体内蛋白石网络各组成部分之间的相互作用是一项重大挑战。虽然蛋白质-蛋白质相互作用屏幕有助于提供关于物理相互作用和伴郎复合物的重要信息14，15，了解组织特定的伴郎网络在体内的组织和补偿机制是缺乏的。

基因相互作用经常被用作一种强大的工具，用来研究参与共同或补偿性生物通路16、17、18的基因对之间的关系。这种关系可以通过结合一对突变和量化一个基因突变对第二个基因16突变引起的表型严重性的影响来测量。虽然大多数此类组合在表型方面没有表现出任何效果，但某些遗传相互作用可能加剧或减轻所测量表型的严重程度。当双删除突变体的表型比组合单个删除突变体时看到的预期表型更严重时，观察到加重突变，这意味着两个基因在平行通路中工作，共同影响给定函数。相比之下，当双删除突变体的表型不如单个删除突变体中出现的表型严重时，则观察到缓解突变，这意味着两个基因作为一个复合体协同作用或参与同一通路16、18。因此，可以量化的多种表型，包括广泛的表型，如致命性、生长率和育雏大小，以及特定表型（如转录记者）已用于识别遗传相互作用。例如，Jonikas等人依靠ER应激记者使用配对基因删除分析19来检查糖核酸细胞ER展开蛋白反应蛋白体网络的相互作用。

基因相互作用屏幕涉及系统地交叉配对删除突变，以产生一套全面的双突变体20。然而，在动物模型中，特别是在C.elegans中，这种大规模的方法是不可行的。相反，突变菌株可以通过使用RNA干扰（RNAi）21对基因表达进行向下调节来测试其遗传相互作用模式。C. elegans是一个强大的系统，基于 RNAi22，23屏幕。在C.elegans中，双链RNA（dsRNA）的传递是通过细菌喂养实现的，导致dsRNA分子扩散到许多组织。通过这种方式，引入的dsRNA分子通过快速和简单的程序21影响动物。因此，使用RNAi的基因相互作用屏幕可以揭示使用RNAi库24对一组基因或大多数C.elegans编码基因进行下调节的影响。在这样的屏幕上，影响感兴趣的突变体的行为，但不是野生类型菌株的命中是被监测的表型的潜在修饰剂25。在这里，我们应用突变和RNAi筛选的组合，系统地绘制C.elegans组织特异性伴郎相互作用的地图。

Protocol

1. 为 RNAi 准备线虫生长介质板 在 1 L 瓶中，加入 3 克 NaCl、2.5 克巴克托-佩普顿、17 克醋和蒸馏水至 1 L 和高压灭菌液。 冷瓶至 55 °C。 加入 25 mL 的 1 M KH2PO4、 pH 6.0、 1 mL 的 1 M CaCl2、 1 mL 的 1 M MgSO4和 1 mL 的胆固醇溶液 （表 1）来制作线虫生长介质 （NGM）。 加入1 mL的安培素（100毫克/毫升）和0.5兆升的1M IPTG（表1），…

Representative Results

分别使用 UNC-45 中的温度敏感突变来筛选在允许或限制条件下加重或减轻相互作用的情况肌肉组装和维护提供了一个有效的系统来研究组织特定的伴郎相互作用。收缩肌肉的功能单元，肉瘤，呈现一个水晶般的结构和调节蛋白排列。运动蛋白肌素的稳定性及其融入收缩肌瘤的厚丝取决于伴郎和UPS组件30的合作。一个例子，这样的陪护是保存和专门肌素伴奏UNC-45，主…

Discussion

一个反映它是如何组织起来的，并在不同的元子细胞和组织中发挥作用的蛋白石网络的综合图片仍然缺乏。为了解决这一缺陷，需要提供关于该网络各组成部分（如分子伴郎）在发育和老化过程中特定组织中相互作用的具体信息。在这里，我们展示了组织特异性扰动的使用如何使我们能够检查给定组织中的伴郎网络。为了探索组织特异性伴郎遗传相互作用，考虑了三种不同的方法。在第一种方法?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

我们感谢由NIH国家研究中心（NCRR）资助的卡诺哈布迪炎遗传学中心，感谢其一些线虫菌株。爱泼斯坦H.F.开发的单克隆抗体来自由NICHD主持、由爱荷华大学生物系维护的发展研究杂交瘤库。这项研究得到了以色列科学基金会的赠款（第278/18号赠款）和以色列科技部以及意大利共和国国家促进总局外交和国际合作部的赠款（第3-14337号赠款）的支持。我们感谢本-兹维实验室的成员帮助编写这份手稿。

Materials

12-well-plates SPL BA3D16B
40 mm plates Greiner Bio-one 627160
60 mm plates Greiner Bio-one 628102
6-well plates Thermo Scientific 140675
96 well 2 mL 128.0/85mm Greiner Bio-one 780278
Agar Formedium AGA03
Ampicillin Formedium 69-52-3
bromophenol blue Sigma BO126-25G
CaCl2 Merck 1.02382.0500
Camera Qimaging q30548
Cholesterol Amresco 0433-250G
Confocal Leica DM5500
Filter (0.22 µm) Sigma SCGPUO2RE
Fluorescent stereomicroscope Leica MZ165FC
Glycerol Frutarom 2355519000024
IPTG Formedium 367-93-1
KCl Merck 104936
KH2PO4 Merck 1.04873.1000
KOH Bio-Lab 001649029100
MgSO4 Fisher 22189-08-8 Gift from the Morimoto laboratory
Myosin MHC A (MYO-3) antibody Hybridoma Bank 5-6
Na2HPO4·7H2O Sigma s-0751
NaCl Bio-Lab 001903029100
Peptone Merck 61930705001730
Plate pouring pump Integra does it p920
RNAi Chaperone library NA NA
SDS VWR Life Science 0837-500
ß-mercaptoethanol Bio world 41300000-1
stereomicroscope Leica MZ6
Tetracycline Duchefa Biochemie 64-75-5
Tris Bio-Lab 002009239100
Tween-20 Fisher BP337-500
DOWNLOAD MATERIALS LIST

References

  1. Gomez-Pastor, R., Burchfiel, E. T., Thiele, D. J. Regulation of heat shock transcription factors and their roles in physiology and disease. Nature Reviews Molecular Cell Biology. 19 (1), 4-19 (2018).
  2. Dubnikov, T., Ben-Gedalya, T., Cohen, E. Protein quality control in health and disease. Cold Spring Harbor Perspectives in Biology. 9 (3), (2017).
  3. Bar-Lavan, Y., Shemesh, N., Ben-Zvi, A. Chaperone families and interactions in metazoa. Essays in Biochemistry. 60 (2), 237-253 (2016).
  4. Schopf, F. H., Biebl, M. M., Buchner, J. The HSP90 chaperone machinery. Nature Reviews Molecular Cell Biology. 18 (6), 345-360 (2017).
  5. Carra, S., et al. The growing world of small heat shock proteins: from structure to functions. Cell Stress Chaperones. 22 (4), 601-611 (2017).
  6. Rosenzweig, R., Nillegoda, N. B., Mayer, M. P., Bukau, B. The Hsp70 chaperone network. Nature Reviews Molecular Cell Biology. , (2019).
  7. Gestaut, D., Limatola, A., Joachimiak, L., Frydman, J. The ATP-powered gymnastics of TRiC/CCT: an asymmetric protein folding machine with a symmetric origin story. Current Opinion in Structural Biology. 55, 50-58 (2019).
  8. Bett, J. S. Proteostasis regulation by the ubiquitin system. Essays in Biochemistry. 60 (2), 143-151 (2016).
  9. Jackson, M. P., Hewitt, E. W. Cellular proteostasis: degradation of misfolded proteins by lysosomes. Essays in Biochemistry. 60 (2), 173-180 (2016).
  10. Kettern, N., Dreiseidler, M., Tawo, R., Hohfeld, J. Chaperone-assisted degradation: multiple paths to destruction. Biological Chemistry. 391 (5), 481-489 (2010).
  11. Cuervo, A. M., Wong, E. Chaperone-mediated autophagy: roles in disease and aging. Cell Research. 24 (1), 92-104 (2014).
  12. Kevei, E., Pokrzywa, W., Hoppe, T. Repair or destruction-an intimate liaison between ubiquitin ligases and molecular chaperones in proteostasis. FEBS Letters. 591 (17), 2616-2635 (2017).
  13. O’Brien, D., van Oosten-Hawle, P. Regulation of cell-non-autonomous proteostasis in metazoans. Essays in Biochemistry. 60 (2), 133-142 (2016).
  14. Taipale, M., et al. Quantitative analysis of HSP90-client interactions reveals principles of substrate recognition. Cell. 150 (5), 987-1001 (2012).
  15. Taipale, M., et al. A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways. Cell. 158 (2), 434-448 (2014).
  16. Baryshnikova, A., Costanzo, M., Myers, C. L., Andrews, B., Boone, C. Genetic interaction networks: toward an understanding of heritability. Annual Review of Genomics and Human Genetics. 14, 111-133 (2013).
  17. Costanzo, M., et al. Global Genetic Networks and the Genotype-to-Phenotype Relationship. Cell. 177 (1), 85-100 (2019).
  18. Domingo, J., Baeza-Centurion, P., Lehner, B. The Causes and Consequences of Genetic Interactions (Epistasis). Annual Review of Genomics and Human Genetics. 20, 433-460 (2019).
  19. Jonikas, M. C., et al. Comprehensive characterization of genes required for protein folding in the endoplasmic reticulum. Science. 323 (5922), 1693-1697 (2009).
  20. Schuldiner, M., et al. Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile. Cell. 123 (3), 507-519 (2005).
  21. Billi, A. C., Fischer, S. E., Kim, J. K. Endogenous RNAi pathways in C. elegans. WormBook. , 1-49 (2014).
  22. Kamath, R. S., et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature. 421 (6920), 231-237 (2003).
  23. Rual, J. F., et al. Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library. Genome Research. 14 (10), 2162-2168 (2004).
  24. Lehner, B., Crombie, C., Tischler, J., Fortunato, A., Fraser, A. G. Systematic mapping of genetic interactions in Caenorhabditis elegans identifies common modifiers of diverse signaling pathways. Nature Genetics. 38 (8), 896-903 (2006).
  25. Frumkin, A., et al. Challenging muscle homeostasis uncovers novel chaperone interactions in Caenorhabditis elegans. Frontiers in Molecular Biosciences. 1, 21 (2014).
  26. Brehme, M., et al. A chaperome subnetwork safeguards proteostasis in aging and neurodegenerative disease. Cell Reports. 9 (3), 1135-1150 (2014).
  27. Shpigel, N., Shemesh, N., Kishner, M., Ben-Zvi, A. Dietary restriction and gonadal signaling differentially regulate post-development quality control functions in Caenorhabditis elegans. Aging Cell. 18 (2), 12891 (2019).
  28. Shai, N., Shemesh, N., Ben-Zvi, A. Remodeling of proteostasis upon transition to adulthood is linked to reproduction onset. Current Genomics. 15 (2), 122-129 (2014).
  29. Trent, C., Tsuing, N., Horvitz, H. R. Egg-laying defective mutants of the nematode Caenorhabditis elegans. Génétique. 104 (4), 619-647 (1983).
  30. Pokrzywa, W., Hoppe, T. Chaperoning myosin assembly in muscle formation and aging. Worm. 2 (3), 25644 (2013).
  31. Barral, J. M., Bauer, C. C., Ortiz, I., Epstein, H. F. Unc-45 mutations in Caenorhabditis elegans implicate a CRO1/She4p-like domain in myosin assembly. Journal of Cell Biology. 143 (5), 1215-1225 (1998).
  32. Venolia, L., Ao, W., Kim, S., Kim, C., Pilgrim, D. unc-45 gene of Caenorhabditis elegans encodes a muscle-specific tetratricopeptide repeat-containing protein. Cell Motility and the Cytoskeleton. 42 (3), 163-177 (1999).
  33. Hoppe, T., et al. Regulation of the myosin-directed chaperone UNC-45 by a novel E3/E4-multiubiquitylation complex in C. elegans. Cell. 118 (3), 337-349 (2004).
  34. Etard, C., et al. The UCS factor Steif/Unc-45b interacts with the heat shock protein Hsp90a during myofibrillogenesis. Biologie du développement. 308 (1), 133-143 (2007).
  35. Wohlgemuth, S. L., Crawford, B. D., Pilgrim, D. B. The myosin co-chaperone UNC-45 is required for skeletal and cardiac muscle function in zebrafish. Biologie du développement. 303 (2), 483-492 (2007).
  36. Barral, J. M., Hutagalung, A. H., Brinker, A., Hartl, F. U., Epstein, H. F. Role of the myosin assembly protein UNC-45 as a molecular chaperone for myosin. Science. 295 (5555), 669-671 (2002).
  37. Gazda, L., et al. The myosin chaperone UNC-45 is organized in tandem modules to support myofilament formation in C. elegans. Cell. 152 (1-2), 183-195 (2013).
  38. Gaiser, A. M., Kaiser, C. J., Haslbeck, V., Richter, K. Downregulation of the Hsp90 system causes defects in muscle cells of Caenorhabditis elegans. PLoS One. 6 (9), 25485 (2011).
  39. Karady, I., et al. Using Caenorhabditis elegans as a model system to study protein homeostasis in a multicellular organism. Journal of Visualized Experiments. (82), e50840 (2013).
  40. Bar-Lavan, Y., et al. A Differentiation Transcription Factor Establishes Muscle-Specific Proteostasis in Caenorhabditis elegans. PLoS Genetics. 12 (12), 1006531 (2016).
  41. Qadota, H., et al. Establishment of a tissue-specific RNAi system in C. elegans. Gene. 400 (1-2), 166-173 (2007).
  42. Firnhaber, C., Hammarlund, M. Neuron-specific feeding RNAi in C. elegans and its use in a screen for essential genes required for GABA neuron function. PLoS Genet. 9 (11), 1003921 (2013).
  43. Fisher, A. L. Of worms and women: sarcopenia and its role in disability and mortality. Journal of the American Geriatrics Society. 52 (7), 1185-1190 (2004).
  44. Herndon, L. A., et al. Stochastic and genetic factors influence tissue-specific decline in ageing C. elegans. Nature. 419 (6909), 808-814 (2002).
  45. Ben-Zvi, A., Miller, E. A., Morimoto, R. I. Collapse of proteostasis represents an early molecular event in Caenorhabditis elegans aging. Proceedings of the National Academy of Sciences of the United States of America. 106 (35), 14914-14919 (2009).
  46. Janiesch, P. C., et al. The ubiquitin-selective chaperone CDC-48/p97 links myosin assembly to human myopathy. Nature Cell Biology. 9 (4), 379-390 (2007).
  47. Shemesh, N., Shai, N., Ben-Zvi, A. Germline stem cell arrest inhibits the collapse of somatic proteostasis early in Caenorhabditis elegans adulthood. Aging Cell. 12 (5), 814-822 (2013).
  48. Vilchez, D., et al. RPN-6 determines C. elegans longevity under proteotoxic stress conditions. Nature. 489 (7415), 263-268 (2012).
  49. Liu, G., Rogers, J., Murphy, C. T., Rongo, C. EGF signalling activates the ubiquitin proteasome system to modulate C. elegans lifespan. EMBO Journal. 30 (15), 2990-3003 (2011).
  50. Asikainen, S., Vartiainen, S., Lakso, M., Nass, R., Wong, G. Selective sensitivity of Caenorhabditis elegans neurons to RNA interference. Neuroreport. 16 (18), 1995-1999 (2005).
  51. Calixto, A., Chelur, D., Topalidou, I., Chen, X., Chalfie, M. Enhanced neuronal RNAi in C. elegans using SID-1. Nature Methods. 7 (7), 554-559 (2010).
  52. Eremenko, E., Ben-Zvi, A., Morozova-Roche, L. A., Raveh, D. Aggregation of human S100A8 and S100A9 amyloidogenic proteins perturbs proteostasis in a yeast model. PLoS One. 8 (3), 58218 (2013).
  53. Gidalevitz, T., Ben-Zvi, A., Ho, K. H., Brignull, H. R., Morimoto, R. I. Progressive disruption of cellular protein folding in models of polyglutamine diseases. Science. 311 (5766), 1471-1474 (2006).
  54. Yu, A., et al. Tau protein aggregates inhibit the protein-folding and vesicular trafficking arms of the cellular proteostasis network. Journal of Biological Chemistry. , (2019).
  55. Yu, A., et al. Protein aggregation can inhibit clathrin-mediated endocytosis by chaperone competition. Proceedings of the National Academy of Sciences of the United States of America. 111 (15), 1481-1490 (2014).
  56. Parker-Thornburg, J., Bonner, J. J. Mutations that induce the heat shock response of Drosophila. Cell. 51 (5), 763-772 (1987).
  57. Boutros, M., Ahringer, J. The art and design of genetic screens: RNA interference. Nature Reviews Genetics. 9 (7), 554-566 (2008).
  58. Jorgensen, E. M., Mango, S. E. The art and design of genetic screens: caenorhabditis elegans. Nature Reviews Genetics. 3 (5), 356-369 (2002).
  59. Wang, Z., Sherwood, D. R. Dissection of genetic pathways in C. elegans. Methods in Cell Biology. 106, 113-157 (2011).
  60. Rohl, A., Rohrberg, J., Buchner, J. The chaperone Hsp90: changing partners for demanding clients. Trends in Biochemical Sciences. 38 (5), 253-262 (2013).

Tags

Keywords: Caenorhabditis ElegansEmbryo SynchronizationM9 BufferNGM PlateCell ScraperCentrifugationSynchronized NematodesRNAiEmbryonic Lethality
check_url/fr/61140?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citer Cet Article
Dror, S., Meidan, T. D., Karady, I., Ben-Zvi, A. Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions. J. Vis. Exp. (160), e61140, doi:10.3791/61140 (2020).
Télécharger la Citation
Réimpressions et Autorisations

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image