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A Protocol of Using White/Red Color Assay to Measure Amyloid-induced Oxidative Stress in Saccharomyces cerevisiae
测定酿酒酵母中淀粉样蛋白诱导氧化应激的白/红色实验方案   

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Abstract

The yeast Saccharomyces cerevisiae (S. cerevisiae) harboring ade1 or ade2 mutations manifest red colony color phenotype on rich yeast medium YPD. In these mutants, intermediate metabolites of adenine biosynthesis pathway are accumulated. Accumulated intermediates, in the presence of reduced glutathione, are transported to the vacuoles, whereupon the development of the red color phenotype occurs. Here, we describe a method to score for presence of oxidative stress upon expression of amyloid-like proteins that would convert the red phenotype of ade1/ade2 mutant yeast to white. This assay could be a useful tool for screening for drugs with anti-amyloid aggregation or anti-oxidative stress potency.

Keywords: Amyloid(淀粉样蛋白), Oxidative stress(氧化应激), ROS(ROS), Yeast(酵母), ade1 mutant(ade1突变体), TDP-43(TDP-43), DCFDA(DCFDA)

Background

The yeast Saccharomyces cerevisiae cells mutant for ADE1 or ADE2 genes (e.g., ade1∆, ade2∆, ade1-14, ade2-1), when grown on YPD (Yeast Peptone Dextrose) medium, accumulate red pigment in the vacuole which is an intermediate metabolite of adenine biosynthesis pathway (Sharma et al., 2003). The suppressible allele ade1-14 which contains a premature stop codon, has been widely used to score for [PSI+] prion state of the translation termination factor Sup35 protein. In a [psi-] yeast, the Sup35p remains soluble and functional, therefore translation is terminated efficiently at the premature stop codon of the ade1-14 allele leading to synthesis of truncated and non-functional Ade1 protein. Thus, the adenine biosynthesis cascade remains incomplete leading to accumulation of intermediate metabolite yielding the red phenotype of the [psi-] yeast. In contrast, in a [PSI+] yeast, the Sup35p is aggregated and partially inactivated, thereby causing read-through of the premature non-sense codon of the ade1-14 allele that leads to functional Ade1 protein synthesis. Thus, adenine biosynthesis pathway is completed and no red intermediate metabolite is accumulated, consequently giving the [PSI+] cells, a white phenotype (Chernoff et al., 1993). In the view that the red color development from this adenine biosynthesis intermediate metabolite requires presence of reduced glutathione(Sharma et al., 2003), we reasoned that presence of oxidative stress which would oxidize glutathione, would also cause white color conversion alike to the white phenotype of the [PSI+] yeast. Additionally, it is known that the aggregations of several amyloid proteins cause cellular oxidative stress, therefore we attempted to develop a red/white reporter color assay for amyloid-induced oxidative stress in the ade1/ade2 mutant yeast background similar to the widely used red/white assay for the [psi-] to [PSI+] conversion (Bharathi et al., 2016). We present here red/white color switch assay using two amyloid-like proteins, TAR DNA binding protein 43 (TDP-43) and Fused in Sarcoma (FUS), both of which are implicated in the pathogenesis of the motor neuron disease, Amyotrophic Lateral Sclerosis (ALS) (Rossi et al., 2016). Such simplistic color assay for amyloid-induced oxidative stress in yeast has never been reported previous to our Bharathi et al., 2016 manuscript and has the potential to be a highly useful methodology to study for amyloid protein-induced toxicity in yeast.

Materials and Reagents

  1. Pipette tips (Tarsons products pvt ltd., India)
  2. Microcentrifuge tubes (Tarsons products pvt ltd., India)
  3. Petri dishes (Genaxy scientific pvt ltd., India)
  4. Sterile toothpick
  5. Yeast Saccharomyces cerevisiae strain: 74D-694 (MATa ade1-14, his3-200, ura3-52, leu2-3, 112, trp1-289, [psi-])
    Note: This assay can also be performed using an ade2-1 allele bearing mutant yeast or an ade2∆ mutant yeast as well as using an ade1∆ mutant yeast.
  6. Plasmids
    1. pRS416-GAL1p-FUS-YFP (URA3) (Addgene, catalog number: 29593 )
    2. pRS416-GAL1p-TDP43-YFP (URA3) (Addgene, catalog number: 27447 )
    3. pRS416 (URA3)
    4. pAG416 GAL1p-ccdB-EGFP (URA3) (Addgene, catalog number: 14195 )
  7. 0.1 M phosphate buffer (pH 7.4)
  8. 2’,7’-Dichlorofluoroscein diacetate [DCFDA] (Sigma-Aldrich, catalog number: D6883 )
  9. Lithium acetate (Sigma-Aldrich, catalog number: 517992 )
  10. Peptone (HiMedia Laboratories, catalog number: RM001 )
  11. D-glucose [Dextrose] (AMRESCO, catalog number: 0188 )
  12. Yeast extract (HiMedia Laboratories, catalog number: RM027 )
  13. Bacteriological agar (HiMedia Laboratories, catalog number: RM026 )
  14. D-raffinose (Sigma-Aldrich, catalog number: 83400 )
  15. D-galactose (Sigma-Aldrich, catalog number: 48260 )
  16. Yeast nitrogen base (HiMedia Laboratories, catalog number: G090 )
  17. Sodium dodecyl sulphate [SDS] (Sigma-Aldrich, catalog number: L3771 )
  18. Chloroform (Sigma-Aldrich, catalog number: 372978 )
  19. Ammonium sulfate (Sigma-Aldrich, catalog number: A2939 )
  20. Amino acids: Arginine, Histidine, Isoleucine, Valine, Lysine, Methionine, Adenine, Phenylalanine, and Tyrosine (HiMedia Laboratories, India); Leucine and Tryptophan (Sigma-Aldrich, USA)
  21. YPD medium (see Recipes)
  22. SRaf-Ura + 0.1% gal + ¼ YP plate (see Recipes)
  23. SRaf-Ura broth (see Recipes)
  24. SD-Ura + ¼ YP plate (see Recipes)
  25. SD-Ura + ⅓ YP plate (see Recipes)
  26. DCF extraction buffer (see Recipes)

Equipment

  1. Pipettes (Corning, USA)
  2. 250 ml conical flasks (Borosil Glass Works ltd., India)
  3. Microcentrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: MicroCL 21R )
  4. Fluorescence microscope (Leica Microsystems, model: Leica DM2500 )
  5. Laminar flow biosafety cabinet (Esco Micro, model: ACB-4A1 )
  6. Temperature controlled incubator (JS Research, model: JSGI-100T )
  7. Temperature controlled orbital shaker (Eppendorf, New BrunswickTM, model: Excella® E24 )
  8. UV-Vis spectrophotometer (Hitachi High-Technologies, model: U-3900 )
  9. Vortex mixer (Remi, model: CM-101 )
  10. Autoclave sterilizer (JS Research, model: JSAC-100 )
  11. Multimode microplate reader (Molecular Devices, model: Spectramax M5e )
  12. Camera

Software

  1. GraphPad QuickCalcs software (from GraphPad Software Inc., USA)
    https://www.graphpad.com/quickcalcs/ttest1.cfm

Procedure

  1. Yeast cells bearing ade1-14 allele (or those with ade1∆, or ade2∆ or ade2-1 allele) are pre-transformed with URA3 marked, galactose-inducible GAL1 promoter driven plasmid encoding amyloid forming proteins (for example: TDP-43-YFP or FUS-YFP). These cells are spread on plasmid selective SD-Ura + ¼ YP plates (see Recipes) and incubated at 30 °C for 4-5 days to obtain independent single colonies. Please note that the presence of ¼ YP in the medium allows for the red colony color development even on the drop-out medium, which otherwise is normally scored on YPD medium (see Recipes).
  2. Subsequently, a red color phenotype manifesting single yeast colony from the SD-Ura + ¼ YP plates (Figure 1A) is picked and grown overnight in 5 ml of SRaf-Ura broth in a shaking incubator maintained at 200 rpm and 30 °C to overcome glucose repression.


    Figure 1. Assay of amyloid protein-induced oxidative stress in S. cerevisiae yeast model. A. Red phenotype bearing 74-D694 [psi-] yeast S. cerevisiae cells were pre-transformed either with empty vector plasmid pRS416 or those encoding EGFP, TDP-43-YFP or FUS-YFP proteins driven by GAL1 promoter. Overexpression of these proteins was induced with 2% galactose and cells were then plated on SRaf-Ura + 0.1% gal + ¼ YP plates to obtain single colonies. All the obtained colonies of empty vector and EGFP expressing cells were red, whereas ~17% FUS-YFP expressing colonies and 100% TDP-43-YFP expressing colonies displayed white phenotype. Images were taken by using a smartphone camera (13 MP). B. White phenotype displaying colonies obtained from FUS-YFP and TDP-43-YFP expressions on the SRaf-Ura + 0.1% gal + ¼ YP plates were sequentially passaged as patches on SD-Ura + ¼ YP plates. After the third passage, the TDP-43-YFP plasmid-bearing patches were found to turn red. However, the white patches of FUS-YFP plasmid-bearing cells remained white even after three passages on SD-Ura + ¼ YP plates but swiftly turned red on SD-Ura + ⅓ YP plates. The white patches from both TDP-43-YFP and FUS-YFP converted to red phenotype even after the first passage on the rich complex medium containing YPD plates. Patches from empty vector or EGFP expressing plasmid transformants remained red on SRaf-Ura + 0.1% gal + ¼ YP, SD-Ura + ¼ YP, SD-Ura + ⅓ YP as well as YPD plates. Images were taken by using a smartphone camera (13 MP). C. Relative ROS levels between the red and white phenotype manifesting yeast transformants were examined using DCFDA assay (Eruslanov and Kusmartsev, 2010; James et al., 2015). The yeast cells from TDP-43-YFP white patches (from first passage on SD-Ura + ¼ YP) yielded statistically significant [P = 0.0148, n = 3] increase in DCF fluorescence compared to the TDP-43-YFP red patches (from third passage on SD-Ura + ¼ YP plate) suggesting higher ROS in the white patches. Likewise, when white and red colonies from FUS-YFP expression from Figure 1A were passaged once as patches on SD-Ura + ¼YP plate (where they still retained their respective original phenotypes) and then assayed for ROS levels, the white patches yielded statistically significant [P = 0.0133, n = 3] higher DCF fluorescence compared to the red patches. As expected, the DCF fluorescence levels from white patches of TDP-43-YFP or FUS-YFP expressing cells were also significantly higher when compared with the cells transformed with the empty vector [P = 0.0072, n = 3; P = 0.0031, n = 3]. In all cases, unpaired t-test was used for the statistical comparisons.

  3. Next morning, suitable aliquots of yeast cells from the overnight culture are inoculated to 5 ml of fresh SRaf-Ura broth to obtain starting OD of A600 nm = 0.2 and induced with 2% galactose final concentration to overexpress the GAL1 promoter driven genes and then allowed to grow at 30 °C for 8 h for the expressions of the FUS-YFP or TDP-43-YFP proteins (or until the amyloid aggregation is observed, which can vary for different amyloid proteins). Amyloid-like aggregation is scored by examining the yeast cells in fluorescence microscope for formation of punctate fluorescent foci of the FUS-YFP or TDP-43-YFP proteins.
  4. Next, the cells are harvested by centrifugation at 3,000 x g at 25 °C in a table-top microcentrifuge and re-suspended in sterile water and then appropriately diluted, after recording A600 nm in a spectrophotometer, to spread ~200 cells per plate evenly on SRaf-Ura + 0.1% gal + ¼ YP plates (see Recipes) (1 OD600 nm = 1 x 107 cells/ml). Plates are then incubated at 30 °C for 4-5 days to grow single yeast colonies.
  5. After four days, plates are examined for colony color and the appearance of white colonies due to oxidative-stress induced by the prior over-expression of the amyloid proteins (TDP-43-YFP and FUS-YFP) using 2% galactose (step 3) and the continued moderate expression of the amyloid proteins on the SRaf-Ura + 0.1% gal + ¼ YP plate (step 4) (Figure 1A). Please note that the percentage of white colonies obtained may vary for different amyloid proteins. When TDP-43-YFP was expressed, 100% white colonies were observed whereas for FUS-YFP, only 17% of the colonies were white and the remaining were red color. In controls (carrying only empty vector plasmid or any non-amyloid protein expressing plasmid like EGFP), only red color colonies were observed on SRaf-Ura + 0.1% gal + ¼ YP plate as amyloid-induced oxidative stress is not present.
  6. Next, using sterile toothpick, small amount of cells from the white colonies on SRaf-Ura + 0.1% gal + ¼ YP plates are patched to SRaf-Ura + 0.1% gal + ¼ YP, SD-Ura + ¼ YP and YPD plates. Yeast cells continue to look white on the SRaf-Ura + 0.1% gal + ¼ YP and SD-Ura + ¼ YP plates but turn red on YPD plates (Figure 1B).
    Note: Normalizations of cells are not required while passaging.
  7. Subsequently, the cells from the white patches are sequentially passaged from the SD-Ura + ¼ YP plates to the same SD-Ura + ¼ YP medium plates as patches to observe the yeast color switch from white to red. This indicates that in the absence of protein expression and aggregate formation, the oxidative stress in the yeast cells gradually ceases and causes the white to red color switch. Please note that the amount of added YP required for obtaining red phenotype may vary for different amyloid proteins. For example, the white colonies of TDP-43-YFP turn red after the three passages on SD-Ura + ¼ YP plate, whereas white colonies of FUS-YFP require additional passage on SD-Ura + ⅓ YP plate (see Recipes) for conversion to the red phenotype (Figure 1B).
  8. To ascertain that the white color conversion is indeed due to oxidative stress, presence of reactive oxygen species (ROS) is subsequently analyzed. For this quantification of the ROS levels in the white and red color colonies, the yeast cells from the plates (or even from same liquid broth) are first washed twice with 0.1 M phosphate buffer (pH 7.4, temperature 25 °C) using centrifugation at 3,000 x g at 25 °C in a table-top microcentrifuge and then re-suspended in 450 µl of the phosphate buffer. To this cell suspension, 50 µl of 2’,7’-dichlorofluoroscein diacetate (DCFDA) dye is then added from a 100 µM stock (stored at -20 °C) to obtain 10 µM final DCFDA followed by incubation in the dark for 30 min at 25 °C. Then, the cells are centrifuged at 3,000 x g for 5 min at 25 °C.
  9. Resulting pellet is re-suspended in 500 µl of 2 M lithium acetate (temperature 25 °C) and agitated gently on vortex mixer for 2 min. Then cells are pelleted by centrifugation at 3,000 x g at 25 °C for 5 min. Next, the DCF molecules, produced due to cleavage of DCFDA by cellular esterases followed by oxidation by cellular ROS, are extracted from the cells by re-suspending the pellet in 500 µl of DCF extraction buffer (see Recipes) and agitating vigorously for 2 min on vortex mixer. Finally, samples are centrifuged at 3,000 x g for 5 min at 25 °C and 300 µl of supernatant is used for DCF fluorescence measurement.
  10. The DCF fluorescence is recorded using Spectramax M5e multimode microplate reader by recording emission at 524 nm after excitation at 485 nm (Eruslanov and Kusmartsev, 2010; James et al., 2015). The presence of elevated ROS levels is correlated by comparing the DCF fluorescence of appropriate control samples assayed similarly and simultaneously (Figure 1C).

Data analysis

DCF fluorescence was used for relative ROS level measurements in the red versus white phenotype manifesting yeast cells. To compare the statistical significance of the observed DCF fluorescence (and hence the relative ROS levels) between the red and white yeast cells after expression of FUS-YFP or TDP-43-YFP, data from three independent colonies of red and white phenotype each, were used and the P values were obtained by unpaired t-test using GraphPad QuickCalcs software (from GraphPad Software Inc., USA).

Notes

DCFDA assay should always be performed in the dark and amber colored vials should be used to store the stock solution in the view that DCFDA is light-sensitive and can readily auto-oxidize when directly exposed to light.

Recipes

  1. YPD plate (100 ml)
    2 g peptone
    2 g dextrose
    1 g yeast extract
    2 g agar
  2. SRaf-Ura + 0.1% gal + ¼ YP plate (100 ml)
    0.7 g yeast nitrogen base with ammonium sulfate and amino acids but lacking uracil
    1 g raffinose
    0.1 g galactose
    0.5 g peptone
    0.25 g yeast extract
    2 g agar
  3. SRaf-Ura broth (100 ml)
    0.7 g yeast nitrogen base with ammonium sulfate and amino acids but lacking uracil
    1 g raffinose
  4. SD-Ura + ¼ YP plate (100 ml)
    0.7 g yeast nitrogen base with ammonium sulfate and amino acids but lacking uracil
    2 g dextrose
    0.5 g peptone
    0.25 g yeast extract
    2 g agar
  5. SD-Ura + ⅓ YP plate (100 ml)
    0.7 g yeast nitrogen base with ammonium sulfate and amino acids but lacking uracil
    2 g dextrose
    0.66 g peptone
    0.33 g yeast extract
    2 g agar
  6. DCF extraction buffer
    5 µl 10% SDS
    5 ml sterile distilled water
    100 µl chloroform

Acknowledgments

This protocol is adapted from our original manuscript published previously as Bharathi et al. (2016). We thank Prof. Susan Liebman, University of Nevada Reno, USA, for yeast strains and plasmids. We thank IIT-Hyderabad, funded by MHRD, Government of India, for consumable support and research infrastructure. Junior Research Fellowship to Vidhya Bharathi from DBT, Government of India, and Senior Research Fellowship to Amandeep Girdhar from MHRD, Government of India, are also being duly acknowledged.

References

  1. Bharathi, V., Girdhar, A., Prasad, A., Verma, M., Taneja, V. and Patel, B. K. (2016). Use of ade1 and ade2 mutations for development of a versatile red/white colour assay of amyloid-induced oxidative stress in Saccharomyces cerevisiae. Yeast 33: 607-620.
  2. Chernoff, Y. O., Derkach, I. L. and Inge-Vechtomov, S. G. (1993). Multicopy SUP35 gene induces de-novo appearance of psi-like factors in the yeast Saccharomyces cerevisiae. Curr Genet 24(3): 268-270.
  3. Eruslanov, E. and Kusmartsev, S. (2010). Advanced Protocols in Oxidative Stress II. In: Armstrong, D. (Ed.). Humana Press 57-72.
  4. James, J., Fiji, N., Roy, D., Andrew, MG. D., Shihabudeen, M. S., Chattopadhyay, D. and Thirumurugan, K. (2015). A rapid method to assess reactive oxygen species in yeast using H2DCF-DA. Analytical Methods 7: 8572-8575.
  5. Rossi, S., Cozzolino. M., and Carri, M. T. (2016). Old versus new mechanisms in the pathogenesis of ALS. Brain Pathol 26: 276-286.
  6. Sharma, K. G., Kaur, R. and Bachhawat, A. K. (2003). The glutathione-mediated detoxification pathway in yeast: an analysis using the red pigment that accumulates in certain adenine biosynthetic mutants of yeasts reveals the involvement of novel genes. Arch Microbiol 180(2): 108-117.

简介

携带 ade1 或 ade2 突变体的酵母酿酒酵母( S。cerevisiae )在富酵母上显示红色菌落色表型 中等YPD。 在这些突变体中,积累了腺嘌呤生物合成途径的中间代谢物。 在还原型谷胱甘肽存在下,累积的中间体被转移到空泡中,由此发生红色表型的发生。 在这里,我们描述了一种通过淀粉样样蛋白的表达来评估氧化应激存在的方法,其将将ade1 / ade2突变体酵母的红色表型转化为白色。 该测定可能是用于筛选具有抗淀粉样蛋白聚集或抗氧化应激效力的药物的有用工具。
【背景】ADE1或ADE2基因的酵母(Saccharomyces cerevisiae)突变体(例如,,ade1Δ,ade2Δ ade1-14 ade2-),当在YPD(酵母蛋白胨葡萄糖)培养基上生长时,作为腺嘌呤生物合成途径的中间代谢物的液泡(Sharma等人,2003)。包含早熟终止密码子的可抑制等位基因ade1-14 已被广泛用于评价翻译的朊病毒状态终止因子Sup35蛋白。在[ psi - ]酵母中,Sup35p保持可溶性和功能性,因此翻译在有效地终止于ade1-14的早熟终止密码子>等位基因导致截短和非功能性Ade1蛋白的合成。因此,腺嘌呤生物合成级联保持不完整,导致中间体代谢产物的积累,产生酵母的红色表型。相比之下,在[PSI + ]酵母中,Sup35p被聚集并部分失活,从而导致了早期非感知密码子的读取, ade1-14等位基因导致功能性Ade1蛋白质合成。因此,腺嘌呤生物合成途径完成,并且没有红色的中间体代谢物被积累,从而得到白色表型的细胞(Chernoff等人,J.Immun- 1993)。鉴于来自该腺嘌呤生物合成中间体代谢物的红色发育需要还原型谷胱甘肽的存在(Sharma等,2003),我们推断存在氧化谷胱甘肽的氧化应激也会引起白色转换与[PSI +]酵母的白色表型相似。另外,已知几种淀粉样蛋白的聚集引起细胞氧化应激,因此,我们尝试开发了在ade1 / ade2突变体酵母背景中的淀粉样蛋白诱导的氧化应激的红/白报告色彩测定类似于广泛使用的用于[]到[ PSI + ]转换的红/ Bharathi等人,2016)。我们在这里使用两种淀粉样蛋白,TAR DNA结合蛋白43(TDP-43)和融合在肉瘤(FUS)中的两个这些参与运动神经元疾病,肌萎缩性侧索的发病机制的红/白色开关测定硬化症(ALS)(Rossi等人,2016)。对酵母中淀粉样蛋白诱导的氧化应激的这种简单的颜色测定在我们的Bharathi等人,2016手稿之前从未有报道,并且有可能是研究淀粉样蛋白诱导的非常有用的方法酵母中的毒性

关键字:淀粉样蛋白, 氧化应激, ROS, 酵母, ade1突变体, TDP-43, DCFDA

材料和试剂

  1. 移液器提示(Tarsons products pvt ltd。,India)
  2. 微量离心管(Tarsons products pvt ltd。,India)
  3. 培养皿(Genaxy science pvt ltd。,印度)
  4. 无菌牙签
  5. 酵母酿酒酵母菌株:74D-694(MATa ade1-14,his3-200,ura3-52,leu2-3,112,trp1-289,[ psi - ])
    注意:该测定还可以使用携带突变体酵母或ade2Δ突变酵母的ade2-1等位基因以及使用ade1Δ突变体酵母进行。
  6. 质粒
    1. pRS416-GAL1p-FUS-YFP(URA3)(Addgene,目录号:29593)
    2. pRS416-GAL1p-TDP43-YFP(URA3)(Addgene,目录号:27447)

    3. pRS416(URA3)
    4. pAG416 GAL1p-ccdB-EGFP(URA3)(Addgene,目录号:14195)
  7. 0.1M磷酸盐缓冲液(pH 7.4)
  8. 2',7'-二氟荧光素二乙酸酯[DCFDA](Sigma-Aldrich,目录号:D6883)
  9. 乙酸锂(Sigma-Aldrich,目录号:517992)
  10. 胨(HiMedia Laboratories,目录号:RM001)
  11. D-葡萄糖[葡萄糖](AMRESCO,目录号:0188)
  12. 酵母提取物(HiMedia Laboratories,目录号:RM027)
  13. 细菌琼脂(HiMedia Laboratories,目录号:RM026)
  14. D-棉子糖(Sigma-Aldrich,目录号:83400)
  15. D-半乳糖(Sigma-Aldrich,目录号:48260)
  16. 酵母氮基(HiMedia Laboratories,目录号:G090)
  17. 十二烷基硫酸钠[SDS](Sigma-Aldrich,目录号:L3771)
  18. 氯仿(Sigma-Aldrich,目录号:372978)
  19. 硫酸铵(Sigma-Aldrich,目录号:A2939)
  20. 氨基酸:精氨酸,组氨酸,异亮氨酸,缬氨酸,赖氨酸,甲硫氨酸,腺嘌呤,苯丙氨酸和酪氨酸(HiMedia Laboratories,India);亮氨酸和色氨酸(Sigma-Aldrich,USA)
  21. YPD培养基(见食谱)
  22. SRaf-Ura + 0.1%gal +¼YP板(参见食谱)
  23. SRaf-Ura肉汤(见食谱)
  24. SD-Ura +¼YP板(参见食谱)
  25. SD-Ura + 1/3的平板(参见食谱)
  26. DCF提取缓冲液(见配方)

设备

  1. 移液器(康宁,美国)
  2. 250毫升锥形瓶(Borosil Glass Works ltd。,印度)
  3. 微量离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:MicroCL 21R)
  4. 荧光显微镜(Leica Microsystems,型号:Leica DM2500)
  5. 层流生物安全柜(Esco Micro,型号:ACB-4A1)
  6. 温度控制孵化器(JS研究,型号:JSGI-100T)
  7. 温控轨道摇床(Eppendorf,New Brunswick TM ,型号:Excella ® E24)
  8. UV-Vis分光光度计(日立高科技,型号:U-3900)
  9. 涡街搅拌机(Remi,型号:CM-101)
  10. 高压灭菌器(JS研究,型号:JSAC-100)
  11. 多模微板读数器(Molecular Devices,型号:Spectramax M5e)
  12. 相机

软件

  1. GraphPad QuickCalcs软件(来自GraphPad Software Inc.,USA)
    https://www.graphpad.com/quickcalcs/ttest1.cfm < / a>

程序

  1. 具有ade1-14等位基因的酵母细胞(或具有ade1Δ或ade2Δ或ade2的Ema细胞 > 1 等位基因)用编码淀粉样蛋白形成蛋白的标记的半乳糖诱导型GAL1启动子驱动的质粒预先转化(例如:TDP-43-YFP或FUS-YFP)。将这些细胞扩散在质粒选择性SD-Ura +¼YP平板上(参见食谱),并在30℃温育4-5天以获得独立的单个菌落。请注意,在介质中存在¼YP即使在辍学介质上也可以进行红色菌落颜色显影,否则通常在YPD培养基上得分(参见食谱)。
  2. 随后,挑选出来自SD-Ura +¼YP板(图1A)的单个酵母菌落的红色表型,并在保持在200rpm和30℃的振荡培养箱中在5ml SRaf-Ura肉汤中生长过夜以克服葡萄糖镇压

    图1.淀粉样蛋白诱导的氧化应激在S中的测定。酿酒酵母模型。 :一种。具有74-D694 [...]的红色表型将酵母酿酒酵母细胞用空白载体质粒pRS416或编码EGFP,TDP-43-YFP或由GAL1启动子驱动的FUS-YFP蛋白的那些。用2%半乳糖诱导这些蛋白质的过度表达,然后将细胞接种在SRaf-Ura + 0.1%gal +¼YP平板上以获得单个菌落。所有获得的空载体和EGFP表达细胞的菌落都是红色的,而表达17%FUS-YFP的菌落和100%TDP-43-YFP表达菌落显示白色表型。使用智能手机相机拍摄图像(13 MP)。将显示从FUS-YFP获得的菌落的白色表型和SRaf-Ura + 0.1%gal +¼YP板上的TDP-43-YFP表达依次传代为SD-Ura +¼YP平板上的贴片。第三次传代后,发现携带TDP-43-YFP质粒的斑块变红。然而,即使在SD-Ura +¼YP板上三次传代之后,FUS-YFP质粒携带细胞的白色斑块也保持白色,但在SD-Ura + 1 / YP平板上迅速变红。来自TDP-43-YFP和FUS-YFP的白色斑块即使在含有YPD平板的富含复合培养基上的第一次通过后也转变为红色表型。来自空载体或EGFP表达的质粒转化体的片段在SRaf-Ura + 0.1%gal +¼YP,SD-Ura +¼YP,SD-Ura + 1/3的YP以及YPD平板上保持红色。使用智能手机相机拍摄图像(13 MP)。使用DCFDA测定(Eruslanov和Kusmartsev,2010; James et al。,2015)检查了表现酵母转化体的红色和白色表型之间的相对ROS水平。与TDP相比,来自TDP-43-YFP白色斑块的酵母细胞(从SD-Ura +¼YP上的第一次传代)产生DCF荧光的统计学显着性差异[P = 0.0148,n = 3] -43-YFP红色斑块(来自SD-Ura +¼YP板上的第三代)表明白色斑块中较高的ROS。同样地,当来自图1A的FUS-YFP表达的白色和红色菌落被传代一次作为SD-Ura +¼YP平板上的斑块(仍然保留其相应的原始表型),然后测定ROS水平时,白色斑块产生统计学显着性与红色斑块相比,较高的DCF荧光[ P = 0.0133,n = 3]如所预期的,与用空载体转化的细胞相比,来自TDP-43-YFP或FUS-YFP表达细胞的白色斑块的DCF荧光水平也显着高于用<! - SIPO < 3; P = 0.0031,n = 3]。在所有情况下,使用非配对t检验进行统计比较。

  3. 第二天早晨,将来自过夜培养物的合适的酵母细胞等分试样接种到5ml新鲜SRaf-Ura肉汤中以获得A 600nm±0.2的起始OD,并用2%半乳糖终浓度诱导过表达GAL1启动子驱动的基因,然后在30℃下生长8小时用于表达FUS-YFP或TDP-43-YFP蛋白(或直到观察到淀粉样蛋白聚集,其可以不同的淀粉样蛋白不同)。通过在荧光显微镜下检查酵母细胞以形成FUS-YFP或TDP-43-YFP蛋白质的点状荧光病灶来评估淀粉样样聚集。
  4. 接下来,通过在25℃下在台式微量离心机中以3,000xg离心收获细胞,并重新悬浮于无菌水中,然后在记录A 600nm后进行适当稀释, sub>在分光光度计中,每片平板上扩散约200个细胞至SRaf-Ura + 0.1%gal +¼YP平板(参见食谱)(1 OD 600nm 1 = 10×10 7 细胞/ ml)。然后将板在30℃温育4-5天以培养单个酵母菌落。
  5. 四天后,使用2%半乳糖(TDP-43-YFP和FUS-YFP),通过使用2%半乳糖(TDP-43-YFP和FUS-YFP)预先过表达诱导的氧化应激,检查培养板的菌落颜色和白色菌落的出现(步骤3 )和SRaf-Ura + 0.1%gal +¼YP平板上的淀粉样蛋白的连续中度表达(步骤4)(图1A)。请注意,获得的白色菌落的百分比可能因不同的淀粉样蛋白而异。当表达TDP-43-YFP时,观察到100%的白色菌落,而对于FUS-YFP,只有17%的菌落是白色的,剩余的是红色的。在对照组(仅携带空载体质粒或任何非淀粉样蛋白质表达质粒如EGFP)中,仅在SRaf-Ura + 0.1%gal + 1 YP板上观察到红色菌落,因为淀粉样蛋白诱导的氧化应激不存在。 />
  6. 接下来,使用无菌牙签,将来自SRaf-Ura + 0.1%gal +¼YP板上的白色菌落的少量细胞修补到SRaf-Ura + 0.1%gal +¼YP,SD-Ura +¼YP和YPD平板。酵母细胞在SRaf-Ura + 0.1%gal +¼YP和SD-Ura +¼YP板上继续看起来白色,但在YPD平板上变红(图1B)。
    注意:传递时不需要对单元格进行规范化。
  7. 随后,将来自白色斑块的细胞从SD-Ura +¼YP平板顺序传代到作为斑块的相同的SD-Ura +¼YP培养基板上,以观察酵母颜色从白色变为红色。这表明在没有蛋白质表达和聚集体形成的情况下,酵母细胞中的氧化应激逐渐停止并导致白色至红色的切换。请注意,获得红色表型所需的添加YP量可能因不同的淀粉样蛋白而异。例如,在SD-Ura +¼YP板上三次传代之后,TDP-43-YFP的白色菌落变成红色,而FUS-YFP的白色菌落需要在SD-Ura + 1 / YP板上额外通过(参见食谱)转化为红色表型(图1B)
  8. 为了确定白色转换确实是由于氧化应激,随后分析了活性氧(ROS)的存在。为了对白色和红色菌落的ROS水平进行定量,首先用0.1M磷酸盐缓冲液(pH 7.4,温度25℃)将来自平板(或甚至来自相同液体培养液)的酵母细胞洗涤两次,离心在25℃下在台式微量离心机中重新悬浮在450μl磷酸盐缓冲液中。然后向该细胞悬浮液中加入50μl2',7'-二氯荧光素二乙酸酯(DCFDA)染料,从100μM储备液(储存在-20℃)中得到10μM最终的DCFDA,然后在黑暗中孵育30 min。然后,将细胞在25℃下以3,000×g离心5分钟。
  9. 将所得颗粒重新悬浮于500μl的2M乙酸锂(温度25℃)中,并在涡旋混合器上轻轻搅拌2分钟。然后通过在25℃下以3,000×g离心5分钟使细胞沉淀。接下来,通过将细胞重新悬浮在500μlDCF提取缓冲液(参见食谱)中并且剧烈搅拌2分钟,从细胞中提取由细胞酯酶切割DCFDA而后被细胞ROS氧化产生的DCF分子在涡旋混合器上。最后,将样品在25℃下以3,000×g离心5分钟,并将300μl上清液用于DCF荧光测量。
  10. 使用Spectramax M5e多模微板读数器记录DCF荧光,通过在485nm下激发后在524nm处记录发射(Eruslanov和Kusmartsev,2010; James等人,2015)。通过比较类似和同时测定的适当对照样品的DCF荧光来比较升高的ROS水平的存在(图1C)。

数据分析

DCF荧光用于表现酵母细胞的红色与白色表型中的相对ROS水平测定。为了比较FUS-YFP或TDP-43-YFP表达后红色和白色酵母细胞之间观察到的DCF荧光(因此相对ROS水平)的统计学显着性,来自三个独立的红色和白色表型集落的数据,并且通过使用GraphPad QuickCalcs软件(来自GraphPad Software Inc.,USA)的非配对测试获得 P 值。

笔记

DCFDA测定应始终在黑暗中进行,琥珀色的小瓶应用于储存溶液,因为DCFDA对光敏感,并且在直接曝光时可以自动氧化。

食谱

  1. YPD板(100毫升)
    2克蛋白胨
    2 g葡萄糖
    1克酵母提取物
    2 g琼脂
  2. SRaf-Ura + 0.1%gal +¼YP板(100ml)
    0.7克酵母氮碱与硫酸铵和氨基酸但缺乏尿嘧啶
    1克棉子糖
    0.1克半乳糖
    0.5克蛋白胨
    0.25克酵母提取物
    2 g琼脂
  3. SRaf-Ura肉汤(100毫升)
    0.7克酵母氮碱与硫酸铵和氨基酸但缺乏尿嘧啶
    1克棉子糖
  4. SD-Ura +¼YP板(100 ml)
    0.7克酵母氮碱与硫酸铵和氨基酸但缺乏尿嘧啶
    2 g葡萄糖
    0.5克蛋白胨
    0.25克酵母提取物
    2 g琼脂
  5. SD-Ura + 1/3的板(100毫升)
    0.7克酵母氮碱与硫酸铵和氨基酸但缺乏尿嘧啶
    2 g葡萄糖
    0.66克蛋白胨
    0.33克酵母提取物
    2 g琼脂
  6. DCF提取缓冲液
    5μl10%SDS
    5毫升无菌蒸馏水
    100μl氯仿

致谢

这个协议是从我们原来的手稿改编为Bharathi等人。(2016)。感谢美国内华达里诺大学的Susan Liebman教授用于酵母菌株和质粒。我们感谢由印度政府MHRD资助的IIT海得拉巴用于消耗性支持和研究基础设施。印度政府DBT的Vidhya Bharathi初级研究奖学金和印度政府MHRD的Amandeep Girdhar高级研究奖学金也得到了正式的承认。

参考

  1. Bharathi,V.,Girdhar,A.,Prasad,A.,Verma,M.,Taneja,V.and Patel,BK(2016)。&nbsp; 使用 ade1 和 ade2 突变,用于开发多功能红/白色测定淀粉样蛋白诱导的酿酒酵母中的氧化应激 酵母 33:607-620。
  2. Chernoff,YO,Derkach,IL和Inge-Vechtomov,SG(1993)。多重拷贝SUP35 基因在酵母酿酒酵母中诱导了psi样因子的脱钙出现。 > 24(3):268-270。
  3. Eruslanov,E.和Kusmartsev,S.(2010)。&lt; a class =“ke-insertfile”href =“https://link.springer.com/book/10.1007%2F978-1-60761-411-1 “target =”_ blank“>氧化应激的高级方案II。在:Armstrong,D.(Ed。)。人文出版社 57-72。
  4. James,J.,斐济,N.,Roy,D.,Andrew,MG。 D.,Shihabudeen,MS,Chattopadhyay,D.和Thirumurugan,K.(2015)。&nbsp; 使用H 2 DCF-DA评估酵母中的活性氧物质的快速方法分析方法 7:8572-8575。
  5. Rossi,S.,Cozzolino。 M.和Carri,MT(2016)。&nbsp; 旧与ALS发病机制中的新机制相比较。脑部Pathol 26:276-286。
  6. Sharma,KG,Kaur,R。和Bachhawat,AK(2003)。酵母中的谷胱甘肽介导的解毒途径:使用积累在酵母的某些腺嘌呤生物合成突变体中的红色素的分析揭示了新基因的参与。 180微量元素 180 2):108-117。
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引用:Bharathi, V., Girdhar, A. and Patel, B. K. (2017). A Protocol of Using White/Red Color Assay to Measure Amyloid-induced Oxidative Stress in Saccharomyces cerevisiae. Bio-protocol 7(15): e2440. DOI: 10.21769/BioProtoc.2440.
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