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Biología

乳酸脱氢酶螯合测定--一种简便、可靠的测定哺乳动物细胞中自噬性螯合活性的方法

Published: July 27, 2018
doi:
10.3791/57971
, ,
1The Autophagy Team, Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership,University of Oslo

Summary

本文介绍了一种简便、有效的测量哺乳动物细胞中大块自噬性螯合活性的协议。该方法以定量测定乳酸脱氢酶 (ldh) 在 sedimentable 细胞分数与总细胞 LDH 水平的比例。

Abstract

大块自噬的特点是将大部分细胞质封存成双/多膜结构, 称为自噬体。这里介绍了一个用于监视此过程的简单协议。此外, 还提供了不同类型培养哺乳动物细胞在自噬诱导条件下的典型结果和实验验证。在大块自噬期间, 自噬体封存细胞质, 从而也可溶胞浆蛋白, 连同其他自噬性货物。乳酸脱氢酶是一种稳定、高度丰富的可溶性胞浆酵素, 无选择性地将其自噬体。因此, LDH 螯合的数量反映了散装自噬性封存的数量。为了有效、准确地测定细胞中 LDH 的封存, 我们采用了一种基于 electrodisruption 的分馏协议, 有效地将 sedimentable 与胞浆 ldh 分离, 其次是 sedimentable 酶活性的测定。分数与全细胞样本。自噬性螯合是通过从治疗细胞中减去未经处理细胞中 sedimentable LDH 的比例来确定的。LDH 螯合法的优点是它能定量测量内源性货物的自噬性封存, 而不是其他涉及螯合探针或半定量蛋白酶的异位表达的方法。自噬标记物或受体的保护分析。

Introduction

自噬 (希腊语为 “自我吃”) 是一个进化保守的过程, 泡/溶酶体降解细胞内物质。在发现自噬相关的 (“ATG”) 基因, 这是重要的是在酵母和人类的自噬, 并认识到自噬在人类健康和疾病中发挥重要作用 (承认2016诺贝尔医学或生理学奖, 以Ohsumi), 自噬已迅速成为细胞生物学1,2最强烈的研究过程之一。

Macroautophagy (以下简称 “自噬”) 的特点是将胞内膜池 (“phagophores”) 扩展和折叠成密封、双层或多膜结构 (“自噬体”), 有效地封存从细胞质的其余部分包裹材料。自噬体与溶酶体融合后, 内 autophagosomal 膜和螯合货物均被降解和回收。自噬体可以在随机 (非选择性自噬) 和选择性 (选择性自噬) 方式中封存细胞质材料。大块自噬很可能代表了非选择性和选择性自噬的混合。

在1960代和70代 (自噬研究的 “形态学时代”), 自噬性封存主要通过超微结构分析来评估。在1980代和开始的1990代 (“生物化学时代”) 每 Seglen 和同事-谁研究了自噬在主要鼠肝细胞-开发了第一种方法定量测量自噬性封存活动3。使用这些化验, Seglen 定义和描绘了自噬性溶酶通路4,5的不同步骤, 发现和铸造 amphisome6 (endosome autophagosome 融合的产物), 是第一个描述蛋白质磷酸化在自噬规则7中的作用。然而, 在 ATGs 的发现以后在1990代 (“分子时代”) 和第一个描述哺乳动物 ATG8 蛋白质, 微管伴生的蛋白质 1 a/1 b 光链子 3 (LC3) 在 2000年8, 使用 ATG 蛋白质作为标记为自噬性过程迅速获得普及, 并留下了更古老和更费力的生化方法。事实上, 在过去的18年中, LC3 的印迹和荧光显微分析已经成为目前为止最受欢迎的 (在许多情况下, 唯一的) 方法研究哺乳动物细胞的自噬。好处是这些方法的相对易用性可以进行。缺点是, 一个是研究车组件 (LC3) 而不是实际的自噬性货物。这是一个相当严重的劣势, 因为 LC3 的状态和/或通径与货物的封存和通量之间的关系是非常不清楚的。事实上, 我们已经表明, 尽管在细胞9中存在共轭 LC3, 但在没有 LC3 通量的情况下, 散装货物流量可以保持在高水平。此外, 我们证明了大体积自噬不受有效 LC3 损耗的影响, 因此很可能是 LC3-independent9。这一发现后来得到了10,11LC3 的研究证实, 这也表明, 依赖于 mitophagy (选择性自噬线粒体) 是独立的 LC310,11.

总而言之, 显然需要以货物为基础的化验来监测自噬性活动。最佳的这种化验应广泛适用, 定义明确, 易于执行。在过去的几年中, 我们对 LDH 螯合法的研究特别感兴趣, 这是在1980的12中由每 Seglen 开发的, 并以测量胞浆 ldh 为 sedimentable、自噬性液泡细胞的方法为基础。分数。乳酸脱氢酶是一种稳定的可溶性胞浆蛋白, 在 phagophores 包裹细胞质货物时易于相互隔离。因此, LDH 的封存是自噬性封存的一般措施。LDH 是完全退化的自噬性溶酶通路12。因此, 在酶抑制剂的存在,例如, bafilomycin A1 (生物滤池)13, 实验治疗效果直接反映了自噬性螯合活动的变化。在没有降解抑制剂的情况下, 可以测量 LDH 螯合和降解的改变的净效应。

乳酸脱氢酶螯合的测定是广泛适用的, 因为 ldh 是高度和无所不在表达的所有细胞类型, 和 LDH 水平可以准确地量化的酵素化验14,15。然而, 最初的协议12 -建立在原发鼠肝细胞-是相当耗时, 需要大量的启动材料以及一个定制的放电电容器。以循序渐进的方式, 我们已逐步将该检测转化为一种简便、通用的方法。首先, 原始的协议被适应了用于哺乳动物细胞线16。第二, 该方法实质上是缩小3,9。第三, 消除了协议中的几个步骤, 包括一个费力的密度缓冲步骤17。这同时使方法的进一步降尺度, 从最初的起点使用 10 cm 板材每样品16使用一个井从12井板材每样品 (即, 大约15倍较少开始材料)17。第四, 我们确定了一种商用电穿孔装置, 可取代自定义的放电电容器17。

在这里, 我们最新的乳酸脱氢酶封存试验的协议, 其中包括一些进一步简化的方法与以前发表的17相比, 提出。同时, 给出了一系列不同细胞类型的典型结果, 并给出了利用药理学和遗传击倒和剔除方法进行多行实验验证的方法。有关整个协议的总体流程方案, 请参见图 1。

Protocol

1. 细胞播种和治疗 培养黏附细胞在 75 cm2组织培养烧瓶在一个湿式孵化器与 5% CO2在37°c, 使用优选的培养基为有关的细胞类型。允许细胞生长, 直到它们到达接近汇合的细胞层。注: 使用 RPMI 1640 培养基辅以10% 胎牛血清 (血清), 用于 LNCaP、HEK293、小鼠胚成纤维细胞 (MEFs)、BJ、MCF-7 和 RPE-1 细胞。 用3毫升37摄氏度磷酸盐缓冲的生理盐水 (PBS) 冲洗细胞, pH 值?…

Representative Results

使用这里描述的协议, 大量自噬性螯合活动在许多不同的哺乳动物细胞系, 包括 LAPC4, DU145, Huh7, PNT2A, HeLa, VCaP, H3122, Hec1A, MCF-7, 小鼠胚胎成纤维细胞 (T47D), U2OS,HEK293、BJ 和 LNCaP 细胞进行了测量。在基本条件下 (在完全营养丰富的培养基中), 或者在急性饥饿的血清和氨基酸细胞 (一种真正的自噬诱导条件22) 下, 对螯合进行评估。结果表明, 在饥饿条件下?…

Discussion

总之, 这里描述的协议是一种可靠和广泛适用的方法来监测哺乳动物细胞中的大块自噬性封存活动。与原来的方法12,16相比, 我们已经删除了一些不必要的步骤, 简化了几个剩余的步骤, 并引入了一个实质性的降尺度。因此, 该协议在成本和时间效率方面有了很大的改进, 与原始协议相比, 现在可以在不到一半的时间内处理相同数量的样本。对于24个样本, 步…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

这项工作得到了挪威研究委员会、奥斯陆大学、安德斯 Jahre 基金会、南森基金会的资助, 以及纪念亨利霍曼的遗产。我们感谢郎水岛博士 ATG5+/+ MEFs 和 ATG5-/MEFs, 为 ATG7+/+ MEFs 和 ATG7-/MEFs, 并 Shizuo 博士为 ATG9A+/+ MEFs 和 ATG9A-/MEFs。我们感谢弗兰克 Sætre 的技术援助, 以及每 Seglen 博士为建设性的方法讨论。

Materials

1.5 ml and 2 ml microcentrifuge tubes  Eppendorf 211-2130 and 211-2120
12-well plates  Falcon 353043
Accumax  cell detachment solution Innovative Cell Technologies A7089 Keep aliquots at -20 °C for years, and in fridge for a few months
Bafilomycin A1 Enzo BML-CM110-0100 Dissolve in DMSO
BJ cells ATCC CRL-2522 use at passage <30
Bovine serum albumin (BSA) VWR 422361V
Burker counting chamber Fisher Scientific 139-658585
Countess Cell Counting Chamber Slides ThermoFisher Scientfic C10228
Countess II Automated Cell Counter ThermoFisher Scientfic AMQAX1000
Cover glass for the Burker counting chamber Fisher Scientific 139-658586
Criterion Tris-HCl Gel, 4–20%, 26-well, 15 µl, 13.3 x 8.7 cm (W x L)  Bio-Rad 3450034
DTT Sigma-Aldrich D0632
Earle's balanced salt solution (EBSS) Gibco 24010-043 conatains 0.1% glucose
EDTA Sigma-Aldrich E7889
Electroporation cuvette (4 mm) Bio-Rad 1652088
Exponential decay wave electroporator BTX Harvard Apparatus  EMC 630
Fetal bovine serum (FBS) Sigma F7524 10% final concentration in RPMI 1640 medium
HEK293 cells ATCC CRL-1573
Imidazole Sigma-Aldrich 56750 Autoclave a 65 mM solution and keep in fridge for months
Incubator; Autoflow IR Direct Heat CO2 incubator NuAire NU-5510E
Lipofectamine RNAiMAX Transfection Reagent ThermoFisher 13778150
LNCaP cells ATCC CRL-1740 use at passage <30
3-Methyl Adenine (3MA) Sigma-Aldrich M9281 Stock 100 mM in RPMI in -20 °C.  Heat stock to 65 °C for 10 minutes, and use at 10 mM final concentration
Refridgerated Microcentrifuge Beckman Coulter Life Sciences 368831
Refridgerated Microcentrifuge with soft-mode function Eppendorf  Eppendorf 5417R 
MRT67307 hydrochloride (ULKi) Sigma-Aldrich SML0702 Inhibits ULK kinase activity. Dissolve in DMSO.
MaxMat Multianalyzer instrument Erba Diagnostics PL-II
MCF7 cells ATCC HTB-22
NADH Merck-Millipore 1.24644.001
Nycodenz Axis-Shield 1002424
Opti-MEM Reduced Serum Medium ThermoFisher 31985062
Phosphate-buffered saline (PBS) Gibco 20012-019
Pipette tips 3 (0.5-20 µl) VWR 732-2223 Thermo Fischer ART Barrier tips
Pipette tips (1-200 µl) VWR 732-2207  Thermo Fischer ART Barrier tips
Pipette tips (100-1000µl) VWR 732-2355  Thermo Fischer ART Barrier tips
Pipettes ThermoFisher 4701070 Finnpipette F2 GLP Kit
Poly-D-lysine Sigma-Aldrich P6407-10X5MG Make a 1 mg/ml stock solution in sterile H2O. This solution is stable at -20 °C for at least 1 year.
Pyruvate Merck-Millipore 1066190050
RPE-1 cells (hTERT RPE-1) ATCC CRL-4000
RPMI 1640 Gibco 21875-037
SAR-405 ApexBio  A8883 Inhibits phosphoinositide 3-kinase class III (PIK3C3). Dissolve in DMSO.
Silencer Select Negative Control #1 (siCtrl) ThermoFisher/Ambion 4390843
Silencer Select ATG9-targeting siRNA (siATG9A) ThermoFisher/Ambion s35504
Silencer Select FIP200-targeting siRNA (siFIP200) ThermoFisher/Ambion s18995
Silencer Select ULK1-targeting siRNA (siULK1) ThermoFisher/Ambion s15964
Silencer Select ULK2-targeting siRNA (siULK2) ThermoFisher/Ambion s18705
Silencer Select GABARAP-targeting siRNA (siGABARAP) ThermoFisher/Ambion s22362
Silencer Select GABARAPL1-targeting siRNA (siGABARAPL1) ThermoFisher/Ambion s24333
Silencer Select GABARAPL2-targeting siRNA (siGABARAPL2) ThermoFisher/Ambion s22387
Sodium phosphate monobasic dihydrate (NaH2PO4 • 2H2O)  Merck-Millipore 1.06580.1000
Sodium phosphate dibasic dihydrate (Na2HPO4 • 2H2O )  Prolabo 28014.291
Sucrose VWR 443816T 10% final concentration in water; filter through 0.45 µm filter and keep in fridge for months
Thapsigargin Sigma-Aldrich T9033 Inhibits the SERCA ER Ca2+ pump. Dissolve in DMSO.
Triton X-405  Sigma-Aldrich X405 1% final
Trypan Blue stain 0.4% Molecular Probes T10282
Trypsin-EDTA (0.25% w/v Trypsin) Gibco 25200-056
Tween-20 Sigma-Aldrich P2287 0.01% final
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Referencias

  1. Rubinsztein, D. C., Frake, R. A. Yoshinori Ohsumi’s Nobel Prize for mechanisms of autophagy: from basic yeast biology to therapeutic potential. J R Coll Physicians Edinb. 46 (4), 228-233 (2016).
  2. Mizushima, N. The exponential growth of autophagy-related research: from the humble yeast to the Nobel Prize. FEBS Lett. 591 (5), 681-689 (2017).
  3. Seglen, P. O., et al. Macroautophagic cargo sequestration assays. Methods. 75, 25-36 (2015).
  4. Hoyvik, H., Gordon, P. B., Seglen, P. O. Use of a hydrolysable probe, [14C]lactose, to distinguish between pre-lysosomal and lysosomal steps in the autophagic pathway. Exp Cell Res. 166 (1), 1-14 (1986).
  5. Plomp, P. J., Gordon, P. B., Meijer, A. J., Hoyvik, H., Seglen, P. O. Energy dependence of different steps in the autophagic-lysosomal pathway. J Biol Chem. 264 (12), 6699-6704 (1989).
  6. Gordon, P. B., Seglen, P. O. Prelysosomal convergence of autophagic and endocytic pathways. Biochem Biophys Res Commun. 151 (1), 40-47 (1988).
  7. Holen, I., Gordon, P. B., Seglen, P. O. Protein kinase-dependent effects of okadaic acid on hepatocytic autophagy and cytoskeletal integrity. Biochem J. 284, 633-636 (1992).
  8. Kabeya, Y., et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. Embo j. 19 (21), 5720-5728 (2000).
  9. Szalai, P., et al. Autophagic bulk sequestration of cytosolic cargo is independent of LC3, but requires GABARAPs. Exp Cell Res. 333 (1), 21-38 (2015).
  10. Nguyen, T. N., et al. Atg8 family LC3/GABARAP proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation. J Cell Biol. , (2016).
  11. Pontano Vaites, L., Paulo, J. A., Huttlin, E. L., Harper, J. W. Systematic analysis of human cells lacking ATG8 proteins uncovers roles for GABARAPs and the CCZ1/MON1 regulator C18orf8/RMC1 in macro and selective autophagic flux. Mol Cell Biol. , (2017).
  12. Kopitz, J., Kisen, G. O., Gordon, P. B., Bohley, P., Seglen, P. O. Nonselective autophagy of cytosolic enzymes by isolated rat hepatocytes. J Cell Biol. 111 (3), 941-953 (1990).
  13. Bowman, E. J., Siebers, A., Altendorf, K. Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc Natl Acad Sci U S A. 85 (21), 7972-7976 (1988).
  14. Hadjivassiliou, A. G., Rieder, S. V. The enzymatic assay of pyruvic and lactic acids. A definitive procedure. Clin Chim Acta. 19 (3), 357-361 (1968).
  15. Bergmeyer, H. U., Bernt, E., Bergmeyer, H. U. . Methods of enzymatic analysis (2nd English ed). 2, 574-579 (1974).
  16. Engedal, N., et al. Modulation of intracellular calcium homeostasis blocks autophagosome formation. Autophagy. 9 (10), 1475-1490 (2013).
  17. Luhr, M., et al. A Simple Cargo Sequestration Assay for Quantitative Measurement of Nonselective Autophagy in Cultured Cells. Methods Enzymol. 587, 351-364 (2017).
  18. Mousavi, S. A., et al. Effects of inhibitors of the vacuolar proton pump on hepatic heterophagy and autophagy. Biochim Biophys Acta. 1510 (1-2), 243-257 (2001).
  19. Seglen, P. O., Gordon, P. B. 3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Natl Acad Sci U S A. 79 (6), 1889-1892 (1982).
  20. Ronan, B., et al. A highly potent and selective Vps34 inhibitor alters vesicle trafficking and autophagy. Nat Chem Biol. 10 (12), 1013-1019 (2014).
  21. Saetre, F., Hagen, L. K., Engedal, N., Seglen, P. O. Novel steps in the autophagic-lysosomal pathway. Febs j. 282 (11), 2202-2214 (2015).
  22. Klionsky, D. J., et al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy. 12 (1), 1-222 (2016).
  23. Seglen, P. O., Overbye, A., Saetre, F. Sequestration assays for mammalian autophagy. Methods Enzymol. 452, 63-83 (2009).
  24. Hurley, J. H., Young, L. N. Mechanisms of Autophagy Initiation. Annu Rev Biochem. 86, 225-244 (2017).
  25. Thastrup, O., Cullen, P. J., Drobak, B. K., Hanley, M. R., Dawson, A. P. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. Proc Natl Acad Sci U S A. 87 (7), 2466-2470 (1990).
  26. Kuma, A., et al. The role of autophagy during the early neonatal starvation period. Nature. 432 (7020), 1032-1036 (2004).
  27. Komatsu, M., et al. Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice. J Cell Biol. 169 (3), 425-434 (2005).
  28. Saitoh, T., et al. Atg9a controls dsDNA-driven dynamic translocation of STING and the innate immune response. Proc Natl Acad Sci U S A. 106 (49), 20842-20846 (2009).
  29. Gordon, P. B., Seglen, P. O. Autophagic sequestration of [14C]sucrose, introduced into rat hepatocytes by reversible electro-permeabilization. Exp Cell Res. 142 (1), 1-14 (1982).
  30. An, H., Harper, J. W. Systematic analysis of ribophagy in human cells reveals bystander flux during selective autophagy. Nat Cell Biol. 20 (2), 135-143 (2018).
  31. Fass, E., Shvets, E., Degani, I., Hirschberg, K., Elazar, Z. Microtubules support production of starvation-induced autophagosomes but not their targeting and fusion with lysosomes. J Biol Chem. 281 (47), 36303-36316 (2006).
  32. Velikkakath, A. K., Nishimura, T., Oita, E., Ishihara, N., Mizushima, N. Mammalian Atg2 proteins are essential for autophagosome formation and important for regulation of size and distribution of lipid droplets. Mol Biol Cell. 23 (5), 896-909 (2012).
  33. Øverbye, A., Sætre, F., Hagen, L. K., Johansen, H. T., Seglen, P. O. Autophagic activity measured in whole rat hepatocytes as the accumulation of a novel BHMT fragment (p10), generated in amphisomes by the asparaginyl proteinase, legumain. Autophagy. 7 (9), 1011-1027 (2011).
  34. Kominami, E., Hashida, S., Khairallah, E. A., Katunuma, N. Sequestration of cytoplasmic enzymes in an autophagic vacuole-lysosomal system induced by injection of leupeptin. J Biol Chem. 258 (10), 6093-6100 (1983).
  35. Rosado, C. J., Mijaljica, D., Hatzinisiriou, I., Prescott, M., Devenish, R. J. Rosella: a fluorescent pH-biosensor for reporting vacuolar turnover of cytosol and organelles in yeast. Autophagy. 4 (2), 205-213 (2008).
  36. Katayama, H., Kogure, T., Mizushima, N., Yoshimori, T., Miyawaki, A. A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery. Chem Biol. 18 (8), 1042-1052 (2011).
  37. Ogier-Denis, E., et al. A heterotrimeric Gi3-protein controls autophagic sequestration in the human colon cancer cell line HT-29. J Biol Chem. 270 (1), 13-16 (1995).
  38. Seglen, P. O., Gordon, P. B., Tolleshaug, H., Hoyvik, H. Use of [3H]raffinose as a specific probe of autophagic sequestration. Exp Cell Res. 162 (1), 273-277 (1986).
  39. Luhr, M., Sætre, F., Engedal, N. The Long-lived Protein Degradation Assay: an Efficient Method for Quantitative Determination of the Autophagic Flux of Endogenous Proteins in Adherent Cell Lines. Bio-protocol. 8 (9), e2836 (2018).
  40. Ronning, O. W., Pettersen, E. O., Seglen, P. O. Protein synthesis and protein degradation through the cell cycle of human NHIK 3025 cells in vitro. Exp Cell Res. 123 (1), 63-72 (1979).
  41. Seglen, P. O., Grinde, B., Solheim, A. E. Inhibition of the lysosomal pathway of protein degradation in isolated rat hepatocytes by ammonia, methylamine, chloroquine and leupeptin. Eur J Biochem. 95 (2), 215-225 (1979).
  42. Seglen, P. O., Solheim, A. E. Valine uptake and incorporation into protein in isolated rat hepatocytes. Nature of the precursor pool for protein synthesis. Eur J Biochem. 85 (1), 15-25 (1978).
  43. Bauvy, C., Meijer, A. J., Codogno, P. Assaying of autophagic protein degradation. Methods Enzymol. 452, 47-61 (2009).
  44. Engedal, N., Seglen, P. O. Autophagy of cytoplasmic bulk cargo does not require LC3. Autophagy. 12 (2), 1-3 (2016).

Tags

Keywords: Lactate Dehydrogenase Sequestration AssayAutophagyAutophagic SequestrationCell CultureTrypsinCell CountingCell SeedingBafilomycin A1Cell Detachment
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Luhr, M., Szalai, P., Engedal, N. The Lactate Dehydrogenase Sequestration Assay — A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells. J. Vis. Exp. (137), e57971, doi:10.3791/57971 (2018).
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