隔离细胞脂滴：两个纯化技术从酵母细胞与人类的胎盘开始
Summary
两种技术从1分离细胞的脂滴），酵母细胞和2）人类胎盘介绍。这两个程序的核心是密度梯度离心法，在这里可以用肉眼很容易地可视化含有液滴产生的浮层，提取，并通过Western印迹分析纯度量化。
Abstract
脂滴是可以在大多数真核细胞和某些原核细胞中找到动态的细胞器。在结构上，所述液滴包括由磷脂单分子层所包围的中性脂质的核心。一种在确定液滴的细胞作用最有用的技术一直是蛋白质组学鉴定结合的蛋白，它可以随着液滴被分离的。在这里，两种方法都描述隔离脂滴和他们结合蛋白从两个广泛的真核生物：裂殖酵母和人类胎盘绒毛细胞。虽然这两种技术有差异，主要方法 – 密度梯度离心 – 是由两个准备共享。这显示了所提出的液滴分离技术的广泛适用性。
在第一协议，酵母细胞是将它们的细胞壁消化酶转化成原生质球。由此产生的原生质球，然后GENTLY溶解在宽松的均质机。聚蔗糖加入到裂解物中，以提供密度梯度，将混合物离心分离3次。第一自旋后，脂质微滴被定位于离心管中的白色的浮动层沿与内质网（ER），质膜，并且空泡。随后的两个自旋是用来消除这些其他三个细胞器。其结果是，仅具有小滴和结合蛋白的层。
在第二协议，胎盘绒毛细胞是从人的足月胎盘酶消化，用胰蛋白酶和DNA酶I。将细胞匀浆在一个宽松的匀浆中分离。低速和中速离心步骤用于去除完整的细胞，细胞碎片，细胞核，线粒体和。蔗糖加入到匀浆，以提供密度梯度，将混合物离心，以脂质小滴从其他cellula分开ř分数。
在这两种协议的脂滴的纯度是通过Western印迹分析来证实。从双方的PREPS液滴部分适用于后续的蛋白质和脂质组分析。
Introduction
细胞脂滴是具有多种功能的细胞活力的细胞器。它们是存储集线器为中性脂质，可被转化成能量，或用于磷脂的合成。液滴发挥中心作用在生理和病理条件，包括动脉粥样硬化，肥胖和相关代谢性疾病，并且还传染病1,2。此外，他们是耐人寻味的来源生物柴油燃料。
对脂滴的细胞作用的许多内容已经从广泛的生物纯化3滴的蛋白质和脂质组分析中得到。这些生物已包括4,5细菌，酵母6-11，12,13植物，线虫14，和苍蝇15,16。由于在人类的代谢性疾病的脂滴的作用的兴趣，液滴也被分离自培养的动物细胞和nimal组织。培养的细胞系已列出的3T3-L1脂肪细胞17，中国仓鼠卵巢（CHO）细胞中的K2 18，人hepatocyes 19,20和上皮细胞系21。动物组织从液滴被分离已包括小鼠骨骼肌22，肝脏23，和乳腺增生病23。如上所述，大多数液滴隔离研究的目的是执行蛋白质组分析的约束因素，并在中性和磷脂脂质组分析。
由于中性脂肪 – 脂滴的最多的部件 – 比大多数其他细胞材料密度较小，传统上一直采用密度梯度离心法进行飞沫隔离。该技术是在这里提出两个的PREPS的核心。以前的技 术6,24相结合，并修改成液滴从培养裂殖酵母细胞的分离的视觉呈现以及从胎盘组织获得的未培养的人类细胞。我们的目标是通过选择两个截然不同的细胞类型作为起始，在液滴分离点，以显示该技术的广泛适用性。这种技术应该是为那些希望以隔离大多数生物滴有用。
方案1描述了从裂殖酵母， 粟酒裂殖酵母 ，它已被用来作为一个模型用于在真核细胞分裂25观察液滴形成脂滴的分离。芽殖酵母酿酒酵母中已被广泛地用来作为模式生物研究脂滴生物学。方案1是适用于两种生物体和在制剂的差异突出显示。
方案2描述了从胎盘绒毛细胞，这是在从人足月胎盘获得的转脂滴的分离。该收集足月胎盘的提供了一个独特的机会，安全和道德得到200-250克现成的人体组织26，其中包含显著数字脂滴的。这是相对于在高等真核生物中，其中液滴从培养的细胞起源最脂滴隔离工作。在这些研究中，脂肪酸，通常加入到培养以促进中性脂质，因而液滴的生长的合成。这是相对于这里的工作，其中脂质小滴在天然条件下在胎盘组织形成。
在脂滴组分的纯度是通过蛋白质印迹分析用细胞器标记的抗体来确定。这两个协议将产生脂滴部分，适合在随后的蛋白质和脂质组分析。
Protocol
1。从（裂变）酵母细胞中分离脂滴芽殖酵母酿酒酵母从流行的模式生物滴的隔离几乎是相同的以下协议6。在筹备的差异说明。 1。培养酵母细胞准备媒体。在玻璃瓶或培养瓶中结合36克YE5S粉每升DH 2 O的。约2升的介质是必要的。高压灭菌介质在121℃下20分钟。允许介质冷却至室温。对于S。酵母与YPD代替YE5S。 …
Representative Results
如果密度梯度离心和预期一样，浮层应含有脂滴和在整个的高速旋转的进展耗尽其他细胞器。 对于方案1，西方印迹与标记抗体的脂滴（Erg6p），并且已经发现，与在酵母中，ER（Dpm1p），线粒体（Por1p）脂滴的细胞器相互作用进行的，质膜（Pma1p）和液泡（Vma1p）。 每3自旋的浮动层（步骤3.7，3.10，和3.13）收集等量，沉淀用三氯乙酸（15％终浓度），并溶解…
Discussion
本协议中的关键步骤
确保培养细胞的生长过程中以与介质和细胞密度是一致的。细胞脂滴是独特的，因为它们相关的蛋白是高度依赖于在其中培养细胞17的环境。因此，其中的细胞生长介质和细胞的密度应密切裂解之前进行监测。
脂滴的蛋白质组合物是酵母细胞的生长阶段的功能。较少的蛋白质将被绑定到液滴在?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作是由美国心脏协会奖13SDG14500046帕金森病，可持续能源教育与研究中心奖（田纳西大学）PD的支持，并由医师的医学教育和研究基金会（田纳西大学）颁发给JM的作者感谢卡罗琳Leplante（耶鲁大学）的协议转换裂殖酵母原生质球;埃里克T. BODER（田纳西大学）为利用他颤抖孵化器，台式离心机，和Western blot分析设备;和中心环境生物技术（Univ.田纳西州）的使用他们的超离心机;半滑舌鳎道姆（科技，奥地利格拉茨大学）酵母抗体;妇产科（Univ.田纳西州医学中心）的技术援助部门的人员。
Materials
| PROTOCOL #1: | |||
| 1.Growing yeast cells and converting to spheroplasts | |||
| Edinburgh Minimal Media (EMM) | Sunrise Science Products | 2005 | |
| Yeast extract with 5 supplements (YE5S) | Sunrise Science Products | 2011 | YE5S media with 225 mg/ml of each supplement: adeninie, histidine, leucine, lysine, uracil. The equivalent for budding yeast would be YPD. |
| YPD powder | Sunrise Science Products | 1875 | For S. cerevisiae |
| Sorbitol | Fisher Scientific | BP439 | |
| Yeast Lytic Enzyme | MP Biomedicals | 215352610 | |
| Lysing Enzymes from Trichoderma harzianum | Sigma-Aldrich | L1412 | |
| Zymolayse-20T | Sunrise Science Products | N0766391 | For S. cerevisiae |
| BODIPY 493/503 | Invitrogen | D-3922 | |
| Microscope Slides | Fisher Scientific | 12-544-7 | |
| Microscope Cover Glass | Fisher Scientific | 12-542-B | |
| Plastic transfer pipette | Fisher Scientific | 137115AM | |
| 1 liter glass bottle | |||
| 250 ml flask | |||
| 2.8 liter flasks | |||
| 2. Yeast lipid droplet isolation | |||
| Tris-HCl | Fisher Scientific | BP153 | |
| EDTA | Fisher Scientific | BP120 | |
| Ficoll 400 | Fisher Scientific | BP525 | |
| 12-14k Spectra/Por Dialysis Membrane | SpectrumLabs | 132680 | |
| EDTA-free Protease Inhibitor Cocktail Tablets | Roche Diagnostics | 11873580001 | irritant |
| Dounce Homogenizer | Sigma-Aldrich | D9938 | |
| Ultracentrifuge Tubes 25x89mm (for SW28) | Beckman-Coulter | 355642 | |
| 12-14k Spectra/Por Dialysis Membrane | SpectrumLabs | 132680 | |
| Name of Equipment | Company | Catalog Number | Comments/Description |
| Temperature-controlled shaker | New Brunswick Scientific | C25KC | |
| Thermo Sorvall Legend XTR centrifuge | Thermo-Scientific | 75004521 | |
| Swinging Bucket Centrifuge Rotor | Thermo-Scientific | 75003607 | |
| Fiberlite* F15-6x100y Fixed-Angle Rotor | Thermo-Scientific | 75003698 | |
| Ultracentrifuge LB-M | Beckman-Coulter | ||
| SW28 Ultracentrifuge Rotor | Beckman-Coulter | 342204 | |
| PROTOCOL #2 | |||
| 1. Placental villous cells isolation | |||
| Disposable underpads | Fisher Scientific | 23666062 | |
| Autoclavable pan (container), 3L | Fisher Scientific | 1336110 | |
| Fine scissors, sharp-sharp, straight | Fine science tools | 1406011 | |
| London Forceps | Fine science tools | 1108002 | |
| Dumont #7b Forceps | Fine science tools | 1127020 | |
| Razor blades | Fisher Scientific | S65921 | |
| Screen cup for CD-1 | Fisher Scientific | S1145 | |
| 40 mesh screen | Fisher Scientific | S0770 | |
| Fisherbrand cell stainers 100μm | Fisher Scientific | 22363549 | |
| 150 mm Petri Dishes | Fisher Scientific | NC9054771 | |
| NaCl | Fisher Scientific | S642 | |
| KCl | Fisher Scientific | P333 | |
| KH2PO4 | Fisher Scientific | P386 | |
| Na2HPO4 | Fisher Scientific | S374 | |
| D-glucose | Fisher Scientific | D16 | |
| HEPES | Fisher Scientific | BP310 | |
| 2.5% trypsin 10x | Invitrogen | 15090046 | |
| DNase I grade II, from bovine pancreas | Roche Applied Science | 10104159001 | |
| Sodium bicarbonate solution | Sigma-aldrich | S8761 | |
| 500 ml Erlenmeyer flasks | |||
| 250 ml beakers | |||
| 15 ml centrifuge tubes | |||
| 10 ml serological pipettes | |||
| 50 ml centrifuge tubes | |||
| DMEM | Invitrogen | 11965084 | |
| 2. Lipid droplets isolation from villous placental cells | |||
| Tris-HCl | Fisher Scientific | BP153 | |
| EDTA | Fisher Scientific | BP120 | |
| D-Sucrose | Fisher Scientific | BP220 | |
| Sodium Carbonate | Fisher Scientific | BP357 | |
| EDTA-free protease inhibitor cocktail tablets | Roche Diagnostics | 11873580001 | irritant |
| Dounce homogenizer | Sigma-Aldrich | D9938 | |
| Ultracentrifuge tubes 25x89mm (for SW28) | Beckman-Coulter | 355642 | |
| Ultra-Clear centrifuge tubes 14x89mm (for SW41) | Beckman-Coulter | 344059 | |
| Disposable borosilicate glass pasteur pipets | Fisher Scientific | 1367820C | |
| Name of Equipment | Company | Catalog Number | Comments/Description |
| Biological safety hood | Thermo-Scientific | ||
| Waterbath | Fisher Scientific | ||
| Temperature-controlled shaker | New Brunswick Scientific | C25KC | |
| Thermo Sorvall Legend XTR centrifuge | Thermo-Scientific | 75004521 | |
| Swinging Bucket Centrifuge Rotor | Thermo-Scientific | 75003607 | |
| Ultracentrifuge LB-M | Beckman-Coulter | ||
| SW28 Ultracentrifuge Rotor | Beckman-Coulter | 342204 | |
| SW41 Ti Ultracentrifuge Rotor | Beckman-Coulter | 331336 | |
| Western blot | |||
| IRDye 680 Goat Anti-Rabbit IgG | LI-COR | 926-68071 | dilution 1:15000 |
| IRDye 800CW Goat Anti-Mouse IgG | LI-COR | 926-32210 | dilution 1:5000 |
| NuPAGE® Novex® 12% Bis-Tris gels | Invitrogen | NP0341 | |
| primary antibodies for PROTOCOL #1 | |||
| Erg6p | gift from Dr. G. Daum | Graz University of Technology, Austria | dilution 1:5000 |
| Dpm1p | Abcam | ab113686 | 4 μg/ml |
| Por1p | gift from Dr. G. Daum | Graz University of Technology, Austria | dilution 1:5000 |
| Pma1p | gift from Dr. G. Daum | Graz University of Technology, Austria | dilution 1:10000 |
| Vma1p (anti-ATP6V1A) | Abcam | ab113745 | 0.5 μg/ml |
| primary antibodies for PROTOCOL #2 | |||
| perilipin 2 (anti-ADFP) | Abcam | ab52355 | 2 μg/ml |
| calnexin | Cell Signaling technology | 2679 | dilution 1:1000 |
| GM130 | Biorbyt | orb40533 | dilution 1:25 |
| COX IV | Cell Signaling technology | 4850 | dilution 1:1000 |
| MEK1 | Biorbyt | orb38775 | dilution 1:50 |
Referências
- Ploegh, H. L. A lipid-based model for the creation of an escape hatch from the endoplasmic reticulum. Nature. 448, 435-438 (2007).
- Herker, E. Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1. Nat. Med. 16, 1295-1298 (2010).
- Ding, Y. F., et al. Isolating lipid droplets from multiple species. Nat. Protoc. 8, 43-51 (2013).
- Kalscheuer, R., et al. Preparative isolation of lipid inclusions from Rhodococcus opacus and Rhodococcus rubber and identification of granule-associated proteins. Arch. Microbiol. 177, 20-28 (2001).
- Low, K. L., et al. Lipid droplet-associated proteins are involved in the biosynthesis and hydrolysis of triacylglycerol in mycobacterium bovis bacillus calmette-guerin. J. Biol. Chem. 285, 21662-21670 (2010).
- Leber, R., Zinser, E., Zellnig, G., Paltauf, F., Daum, G. Characterization of lipid particles of the yeast, Saccharomyces cerevisiae. Yeast. 10, 1421-1428 (1994).
- Grillitsch, K., et al. Lipid particles/droplets of the yeast Saccharomyces cerevisiae revisitied: lipidome meets proteome. Biochim. Biophys. Acta. , 1165-1176 (2011).
- Binns, D., et al. An intimate collaboration between peroxisomes and lipid bodies. J. Cell Biol. 173, 719-731 (2006).
- Ivashov, V. A., et al. Lipidome and proteome of lipid droplets from the methylotropic yeast Pichia pastoris. Biochim. Biophys. Acta. 1831, 282-290 (2013).
- Connerth, M., Grillitsch, K., Kofeler, H., Daum, G. Analysis of lipid particles from yeast. Lipidomics: Vol. 1: Methods and Protocols. 579, 359-374 (2009).
- Wolinski, H., Kohlwein, S. D. Microscopic analysis of lipid droplet metabolism and dynamics in yeast. Methods Mol. Biol. 457, 151-163 (2008).
- Jolivet, P., et al. Protein composition of oil bodies in Arabidopsis thaliana ecotype WS. Plant Physiol. Biochem. 42, 501-509 (2004).
- Katavic, V., Agrawal, G. K., Hajduch, M., Harris, S. L., Thelen, J. J. Protein and lipid composition analysis of oil bodies from two Brassica napus cultivers. Proteomics. 6, 4586-4598 (2006).
- Zhang, P., et al. Proteomic study and marker protein identification of Caenorhabditis elegans lipid droplets. Mol. Cell. Proteomics. 11, 317-328 (2012).
- Krahmer, N., Hilger, M., Kory, N., Wilfling, F., Stoehr, G., Mann, M., Farese, R. V., Walther, T. C. Protein correlation profiles identify lipid droplet proteins with high confidence. Mol. Cell Proteomics. 12, 1115-1126 (2013).
- Cermelli, S., Gou, Y., Gross, S. P., Welte, M. A. The lipid-droplet proteome reveals that droplets are a protein-storage depot. Curr. Biol. 16, 1783-1795 (2006).
- Brasaemle, D. L., Dolios, G., Shapiro, L., Wang, R. Proteomic analysis of proteins associated with lipid droplets of basal and lipolytically stimulated 3T3-L1 adipocytes. J. Biol. Chem. 279, 46835-46842 (2004).
- Liu, P. S., Ying, Y. S., Zhao, Y. M., Mundy, D. I., Zhu, M. F., Anderson, R. G. W. Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic. J. Biol. Chem. 279, 3787-3792 (2004).
- Fujimoto, Y., et al. Identification of major proteins in the lipid droplet-enriched fraction isolated from the human hepatocyte cell line HuH7. Biochim. Biophys. Acta. , 47-59 (2004).
- Sato, S., et al. Proteomic profiling of lipid droplet proteins in hepatoma cell lines expressing hepatitis C virus core protein. J. Biochem. 139, 921-930 (2006).
- Umlauf, E., Csaszar, E., Moertelmaier, M., Schuetz, G. J., Parton, R. G., Prohaska, R. Association of stomatin with lipid bodies. J. Biol. Chem. 279, 23699-23709 (2004).
- Zhang, H. N., et al. Proteome of skeletal muscle lipid droplet reveals association with mitochondria and apolipoprotein A-I. J. Proteome Res. 10, 4757-4768 (2011).
- Wu, C. C., Howell, K. E., Neville, M. C., Yates, J. R., McManaman, J. L. Proteomics reveal a link between the endoplasmic reticulum and lipid secretory mechanisms in mammary epithelial cells. Electrophoresis. 21, 3470-3482 (2000).
- Brasaemle, D. L., Wolins, N. E. Isolation of lipid droplets from cells by density gradient centrifugation. Curr. Protoc. Cell Biol. , 3.15.1-3.15.12 (2005).
- Long, A. P., et al. Lipid droplet de novo formation and fission are linked to the cell cycle in fission yeast. Traffic. 13, 705-714 (2012).
- Petroff, M. G., Phillips, T. A., Ka, H., Pace, J. L., Hunt, J. S. Isolation and culture of term human trophoblast cells. Methods Mol. Med. 121, 203-217 (2006).
- Cho, S. Y., et al. Identification of mouse Prp19p as a lipid droplet-associated protein and its possible involvement in the biogenesis of lipid droplets. J. Biol. Chem. 282, 2456-2465 (2007).
- Ding, Y. B., Wu, Y. B., Zeng, R., Liao, K. Proteomic profiling of lipid droplet-associated proteins in primary adipocytes of normal and obese mouse. Biochim. Biophys. Acta. 44, 394-406 (2012).
- Jagerstrom, S., et al. Lipid droplets interact with mitochondria using SNAP23. Cell Biol. Int. 33, 934-940 (2009).
- Xu, N. Y., et al. The FATP1-DGAT2 complex facilitates lipid droplet expansion at the ER-lipid droplet interface. J. Cell Biol. 198, 895-911 (2012).
- Ozeki, S., Cheng, J. L., Tauchi-Sato, K., Hatano, N., Taniguchi, H., Fujimoto, T. Rab18 localizes to lipid droplets and induces their close apposition to the endoplasmic reticulum-derived membrane. J. Cell Sci. 118, 2601-2611 (2005).
- Yang, H. Y., Galea, A., Sytnyk, V., Crossley, M. Controlling the size of lipid droplets: lipid and protein factors. Curr. Opin. Cell Biol. 24, 509-516 (2012).
- Jacquier, N., Choudhary, V., Mari, M., Toulmay, A., Reggiori, F., Schneiter, R. Lipid droplets are functionally connected to the endoplasmic reticulum in Saccharomyces cerevisiae. J. Cell Sci. 124, 2424-2437 (2011).
- Pu, J., Ha, C. W., Zhang, S., Jung, J. P., Huh, W. K., Liu, P. Interactomic study on interaction between lipid droplets and mitochondria. Protein Cell. 2, 487-496 (2011).
- Shaw, C. S., Jones, D. A., Wagemakers, A. J. M. Network distribution of mitochondria and lipid droplets in human muscle fibres. Histochem. Cell Biol. 129, 65-72 (2008).
- Goodman, J. M. The gregarious lipid droplet. J. Biol. Chem. 283, 28005-28009 (2008).
- Brasaemle, D. L., Wolins, N. E. Packaging of fat: an evolving model of lipid droplet assembly and expansion. J. Biol. Chem. 287, 2273-2279 (2012).
Citar este artigo
Mannik, J., Meyers, A., Dalhaimer, P. Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas. J. Vis. Exp. (86), e50981, doi:10.3791/50981 (2014).