监测内质网钙稳态使用<em> Gaussia</em>荧光素酶SERCaMP
Summary
Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.
Abstract
内质网（ER）中含有的细胞内钙的最高水平，其浓度大约5000倍大于细胞质水平。严格控制ER钙是必不可少的蛋白质折叠，修改和贩运。扰动ER钙可导致解折叠蛋白质效应，一个三脚ER应激反应机制的激活，和在各种疾病有助于发病机理。在疾病发作和进展并监控ER钙改变的能力是在实践中在原则上重要，但具有挑战性。用于监测的ER钙目前可用的方法，如钙依赖性荧光染料和蛋白质，都提供了深入了解细胞中的ER钙动力学，但是这些工具不适合用于体内研究 。我们的实验室已经证明，一个修改Gaussia萤光素酶的羧基末端赋予记者的分泌响应于ER钙枯竭。该方法使用荧光素酶基，分泌的ER钙监测蛋白（SERCaMP），用于在体外和体内应用如本文所述。该视频强调了ER钙纵向监测肝注射，药理学处理GLUC-SERCaMP，血液采集和加工，检测参数。
Introduction
内质网（ER）中的许多细胞的能力的功能，包括蛋白质折叠，蛋白质分泌，脂质稳态，和细胞内信号1。中央对正常的雌激素受体 的功能是保持管腔钙离子浓度在约5000倍于细胞质2-4找到。这种能量密集型过程由萨尔科/内质网钙ATP酶（SERCA），一个泵，移动钙离子进入内质网调节。从ER钙的流出主要由兰尼（碱受体）和肌醇三磷酸（IP3受体）受体介导的。由于许多ER过程是依赖于钙，破坏商店会导致内质网应激和最终的细胞死亡。
ER钙失调已经在疾病，包括心肌病，糖尿病，阿尔茨海默氏观察到的，和帕金森氏5。由于这些疾病的进步性，已经挑战划定因果重发病机制和改建在ER钙库之间lationship。多种技术已经允许在我们的ER钙动力学，包括染料和遗传编码钙指标（GECIs）的理解显著进展。低亲和性钙染料，当结合到钙离子在荧光其中增加，可以装载到细胞以检查亚细胞区室的高浓度的钙6。 GECIs，如D1ER和捕手允许与亚细胞定位7-9的更精确的控制监测钙的波动。近日，又有类GECIs称为钙测定细胞器包埋蛋白质指标（CEPIA）已被描述10。第三种方法结合遗传学和小分子化学是靶向酯酶染料加载（TED），其利用遗传编码羧酸酯酶（靶向ER）与基于酯的钙染料11。
虽然AFORementioned方法都有内在的优势和弱点，他们可以通过荧光急性测量提供了有价值的见解ER钙的动态。然而，它们不是最佳的为经常需要调查疾病进展的纵向研究。以制定一个方法过度延长的时期以监测钙动力学的目标，我们确定并开发出一种蛋白修饰以创建分泌的ER钙监测蛋白（SERCaMPs）12。
SERCaMP规避与其他方法相关的，通过提供一种微创方法反复询问ER钙库一些局限性。我们已经证实了羧基末端肽ASARTDL（丙氨酸 – 丝氨酸 – 丙氨酸 – 精氨酸 – 苏氨酸 – 天冬氨酸 – 亮氨酸）足以促进ER滞留;然而，导致减少在ER钙的条件下，肽序列是不再能保留ER localization和蛋白质分泌13。所述SERCaMP技术的基础是ASARTDL到一个分泌蛋白的羧基末端（例如Gaussia萤光素酶，或GLUC），使得分泌受ER钙耗竭触发，从而产生的ER钙失调12健壮记者的附属物。 GLUC-SERCaMP通过转基因方法中的表达使得生物流体包括细胞培养基和等离子体对作为内质网钙稳态的指标的变化GLUC活性进行分析。该方法具有渐进的改变在ER钙库在体外和体内的纵向研究应用。以下协议被写为用于使用GLUC基于SERCaMP研究ER钙稳态的概要，但协议可以作为替代记者SERCaMPs的指南。
Protocol
Representative Results
Discussion
这个协议强调了在体外和 GLUC-SERCaMP的体内效用并监控ER钙耗竭。虽然蛋白修饰产生SERCaMP似乎推广到其他记者蛋白12，我们选择了Gaussia荧光素酶是因为其强健（200-1000倍以上）生物发光相对于其他的荧光素酶18。我们证明跨越递送至原代大鼠皮质神经元，SH-SY5Y细胞，和大鼠肝100倍的剂量范围GLUC-SERCaMP病毒的检测的毒胡萝卜素诱导的GLUC-SERCaMP释放（图3…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Intramural Research Program at the National Institute on Drug Abuse. We thank Doug Howard, Chris Richie, Lowella Fortuno, and Josh Hinkle for their contributions to developing this method.
Materials
| 1.5mL tubes | Fisher | 02-682-550 | |
| 10% NP-40 solution | Pierce | 28324 | for intracellular GLuc assays |
| 1mL luer-lok syringes | Fisher | 14-823-30 | |
| 200uL filter tips | Rainin | RT-L200F | |
| 3-0 surgical sutures | Fisher | NC9598192 | |
| 30g needles | Fisher Scientific | 14-821-13A | |
| Adhesive microplate sealing sheets | Thermo | AB-0558 | |
| Alcohol prep pads | Fisher | 22-246-073 | |
| Anesthesia Auto Flow System | E-Z Anesthesia | EZ-AF9000 | |
| Animal recovery chamber | Lyon Vet | ICU-912-004 | |
| B27 supplement | Life Technologies | 17504-044 | |
| Betadine solution | Fisher | NC9386574 | |
| Bleach | Clorox | n/a | |
| Bovine growth serum | Thermo | SH30541.03 | |
| Coelenterazine, Native | Regis Technologies | 1-361204-200 | |
| Cotton tipped applicators | Puritan | 806-WC | |
| Cutting needles 3/8 circle sutures | WPI | 501803 | |
| Digital ultrasconic cleaner | Fisher Scientific | FS60D | |
| DMEM high glucose, GlutaMAX, pyruvate | Life Technologies | 10569-010 | |
| DNA mass ladder | Life Technologies | 10496-016 | |
| Gaussia luciferase (recombinant protein) | Nanolight | 321-100 | |
| Gaussia luciferase antibody (for WB, ICC, or IHC) | New England Biolabs | E8023S | 1:2000 (WB) |
| Germinator 500 | CellPoint Scientific | DS-401 | |
| Gluc assay plates (96 well, opaque) | Fisher | 07-200-589 | |
| Hank's balanced salt solution | Life Technologies | 14175-095 | |
| Heparin | Allmedtech | 63323-276-02 | |
| Isoflurane | Butler Schein | 29404 | |
| Ketamine | Henry Schein | 995-2949 | |
| Kwik Stop Styptic powder | Butler Schein | 5867 | |
| L-glutamine | Sigma | G8540 | |
| Methanol | Fisher | a452-4 | |
| Microfuge 22R Centrifuge | Bekman Colter | 368831 | |
| Neosporin | Fisher | 19-898-143 | |
| Neurobasal medium | Life Technologies | 21103049 | |
| Nikon Stereoscope | Nikon | SMZ745T | |
| Nucleospin Gel and PCR Cleanup | Machery-Nagel | 740609 | |
| P200 pipet | Rainin | L-200XLS+ | |
| p24 Lenti-X rapid titer kit | Clontech | 632200 | |
| PCR film seal | Fisher | AB0558 | |
| Penicillin/streptomycin | Life Technologies | 15140-122 | |
| Protease inhibitor cocktail | Sigma | P8340 | |
| ReFresh Charcoal Filter canister | E-Z Anesthesia | EZ-258 | |
| Scalpel blades, #10 | Fine Science tools Inc | 10010-00 | |
| SD rats 150-200g | Charles River | Rats | rats ordered at 150-200g. Surgery 5 days after arrival |
| Small animal ear tags | National Band and Tag co | 1005-1 | |
| Sterile surgical drapes | Braintree Scientific | SP-MPS | |
| Synergy 2 plate reader | BioTek | n/a | |
| TaqMan Universal PCR Master Mix | Applied Biosystems | 4304437 | |
| Thapsigargin | Sigma | T9033 | harmful to human health |
| Virapower lentiviral packaging mix | Life Technologies | K4975-00 | |
| Xfect Transfection reagent | Clontech | 631318 | |
| Xylazine | Valley Vet | 468RX |
References
- Sitia, R., Braakman, I. Quality control in the endoplasmic reticulum protein factory. Nature. 426 (6968), 891-894 (2003).
- Burdakov, D., Petersen, O. H., Verkhratsky, A. Intraluminal calcium as a primary regulator of endoplasmic reticulum function. Cell Calcium. 38 (3-4), 303-310 (2005).
- Fu, S., et al. Aberrant lipid metabolism disrupts calcium homeostasis causing liver endoplasmic reticulum stress in obesity. Nature. 473 (7348), 528-531 (2011).
- Micaroni, M. The role of calcium in intracellular trafficking. Curr Mol Med. 10 (8), 763-773 (2010).
- Mekahli, D., Bultynck, G., Parys, J. B., De Smedt, H., Missiaen, L. Endoplasmic-reticulum calcium depletion and disease. Cold Spring Harb Perspect Biol. 3 (6), (2011).
- Paredes, R. M., Etzler, J. C., Watts, L. T., Zheng, W., Lechleiter, J. D. Chemical calcium indicators. Methods. 46 (3), 143-151 (2008).
- Whitaker, M. Genetically encoded probes for measurement of intracellular calcium. Methods Cell Biol. 99, 153-182 (2010).
- Tang, S., et al. Design and application of a class of sensors to monitor Ca2+ dynamics in high Ca2+ concentration cellular compartments. Proc Natl Acad Sci U S A. 108 (39), 16265-16270 (2011).
- Palmer, A. E., Jin, C., Reed, J. C., Tsien, R. Y. Bcl-2-mediated alterations in endoplasmic reticulum Ca2+ analyzed with an improved genetically encoded fluorescent sensor. Proc Natl Acad Sci U S A. 101 (50), 17404-17409 (2004).
- Suzuki, J., et al. Imaging intraorganellar Ca2+ at subcellular resolution using CEPIA. Nat Commun. 5, 4153 (2014).
- Rehberg, M., Lepier, A., Solchenberger, B., Osten, P., Blum, R. A new non-disruptive strategy to target calcium indicator dyes to the endoplasmic reticulum. Cell Calcium. 44 (4), 386-399 (2008).
- Henderson, M. J., Wires, E. S., Trychta, K. A., Richie, C. T., Harvey, B. K. SERCaMP: a carboxy-terminal protein modification that enables monitoring of ER calcium homeostasis. Mol Biol Cell. 25 (18), 2828-2839 (2014).
- Henderson, M. J., Richie, C. T., Airavaara, M., Wang, Y., Harvey, B. K. Mesencephalic astrocyte-derived neurotrophic factor (MANF) secretion and cell surface binding are modulated by KDEL receptors. J Biol Chem. 288 (6), 4209-4225 (2013).
- Shipman, C. Evaluation of 4-(2-hydroxyethyl)-1-piperazineëthanesulfonic acid (HEPES) as a tissue culture buffer. Proc Soc Exp Biol Med. 130 (1), 305-310 (1969).
- Zigler, J. S., Lepe-Zuniga, J. L., Vistica, B., Gery, I. Analysis of the cytotoxic effects of light-exposed HEPES-containing culture medium. In Vitro Cell Dev Biol. 21 (5), 282-287 (1985).
- Howard, D. B., Powers, K., Wang, Y., Harvey, B. K. Tropism and toxicity of adeno-associated viral vector serotypes 1, 2, 5, 6, 7, 8, and 9 in rat neurons and glia in vitro. Virology. 372 (1), 24-34 (2008).
- Smeets, E. F., Heemskerk, J. W., Comfurius, P., Bevers, E. M., Zwaal, R. F. Thapsigargin amplifies the platelet procoagulant response caused by thrombin. Thromb Haemost. 70 (6), 1024-1029 (1993).
- Tannous, B. A. Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo. Nat Protoc. 4 (4), 582-591 (2009).
- Sobrevals, L., et al. AAV vectors transduce hepatocytes in vivo as efficiently in cirrhotic as in healthy rat livers. Gene Ther. 19 (4), 411-417 (2012).
- Wang, Z., et al. Rapid and highly efficient transduction by double-stranded adeno-associated virus vectors in vitro and in vivo. Gene Ther. 10 (26), 2105-2111 (2003).
- Seppen, J., et al. Adeno-associated virus vector serotypes mediate sustained correction of bilirubin UDP glucuronosyltransferase deficiency in rats. Mol Ther. 13 (6), 1085-1092 (2006).
- Hareendran, S., et al. Adeno-associated virus (AAV) vectors in gene therapy: immune challenges and strategies to circumvent them. Rev Med Virol. 23 (6), 399-413 (2013).
