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Oct 2015

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Analysis of TORC1-body Formation in Budding Yeast
芽殖酵母TORC1-body形成的分析   

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Abstract

The Target of Rapamycin kinase Complex I (TORC1) is the master regulator of cell growth and metabolism in eukaryotes. In the presence of pro-growth hormones and abundant nutrients, TORC1 is active and drives protein, lipid, and nucleotide synthesis by phosphorylating a wide range of proteins. In contrast, when nitrogen and/or glucose levels fall, TORC1 is inhibited, causing the cell to switch from anabolic to catabolic metabolism, and eventually enter a quiescent state. In the budding yeast Saccharomyces cerevisiae, TORC1 inhibition triggers the movement of TORC1 from its position around the vacuole to a single focus/body on the edge of the vacuolar membrane. This relocalization depends on the activity of numerous key TORC1 regulators and thus analysis of TORC1 localization can be used to follow signaling through the TORC1 pathway. Here we provide a detailed protocol for measuring TORC1 (specifically, Kog1-YFP) relocalization/signaling using fluorescence microscopy. Emphasis is placed on procedures that ensure: (1) TORC1-bodies are identified (and counted) correctly despite their relatively low fluorescence and the accumulation of autofluorescent foci during glucose and nitrogen starvation; (2) Cells are kept in log-phase growth at the start of each experiment so that the dynamics of TORC1-body formation are monitored correctly; (3) The appropriate fluorescent tags are used to avoid examining mislocalized TORC1.

Keywords: TORC1 (雷帕霉素激酶复合物I的靶点), Tor1 (Tor1), Kog1 (Kog1), TORC1-body (TORC1-body), Kog1-body (Kog1-body)

Background

The Target of Rapamycin kinase Complex I (TORC1) is the central hub in the cell growth control network of eukaryotes (Loewith and Hall, 2011; Gonzalez and Hall, 2017; Liu and Sabatini, 2020). In the presence of pro-growth hormones and abundant nutrients, TORC1 is active and phosphorylates a wide array of proteins to drive protein and ribosome synthesis, activate lipid and nucleotide synthesis, tune nitrogen and amino acid metabolism/transport, and repress autophagy (Kamada et al., 2000; Huber et al., 2009; Hsu et al., 2011; Kim et al., 2011; Loewith and Hall, 2011; Peterson et al., 2011; Robitaille et al., 2013; Ben-Sahra et al., 2016; Gonzalez and Hall, 2017; Ben-Sahra and Manning, 2017; Liu and Sabatini, 2020). In contrast, when cells are exposed to stress or starvation conditions, TORC1 is inactivated to limit cell growth and redirect available resources to the appropriate stress or starvation response (Barbet et al., 1996; Duvel et al., 2010).


Over the last 15 years, detailed analysis of TORC1 signaling has shed light on the protein network that transmits stress and starvation signals to TORC1, but many questions remain about how TORC1 is regulated in the wide range of stimuli that influence cell growth and survival (Loewith and Hall, 2011; Gonzalez and Hall, 2017; Liu and Sabatini, 2020). In the model organism Saccharomyces cerevisiae (where TORC1 was first discovered) (Loewith and Hall, 2011; Gonzalez and Hall, 2017), progress mapping the TORC1 regulatory circuit has been hindered by the absence of a rapid and scalable TORC1 signaling assay. Here we describe a protocol for monitoring the movement of TORC1 (made up of the TOR kinase Tor1, the essential regulatory protein Kog1, and two accessory proteins, Lst8 and Tco89) into a focus/body during glucose and/or nitrogen starvation. Since TORC1 relocalization is controlled by all known TORC1 regulators in yeast (Hughes Hallett et al., 2015; Sullivan et al., 2019), the assay can be used to (1) measure changes in TORC1 signaling at the single cell level and (2) rapidly test the impact that novel genes, mutations and/or drugs have on TORC1 activity.


Materials and Reagents

  1. 8-well micro-slide (Ibidi, catalog number: 80826)

  2. YEPD plates (VWR, catalog number: 10128-392)

  3. 28 ml test tube (VWR, catalog number: 47729-583)

  4. Wooden applicator sticks (Key scientific, catalog number: CA958701)

  5. BD Difco Yeast Nitrogen Base without Amino Acids (Fisher Scientific, BD Biosciences, catalog number: BD 291920)

  6. BD Difco Yeast Nitrogen Base without Amino Acids and Ammonium Sulfate (Fisher Scientific, BD Biosciences, catalog number: DF0335-15-9)

  7. Drop-out Mix Complete w/o Yeast Nitrogen Base (US Biological, catalog number: D9515)

  8. Dextrose Anhydrous Crystalline Granules (Fisher Scientific, Fisher BioReagents, catalog number: BP350-1)

  9. Ammonium sulfate (Fisher Scientific, catalog number: BP212-212)

  10. Glycerol (Sigma-Aldrich, catalog number: G7893)

  11. Concanavalin A (Fisher Scientific, MP Biomedicals, catalog number: ICN15071001)

  12. SD (see Recipes)

  13. S-glucose (see Recipes)

  14. S-nitrogen (see Recipes)

  15. 2 mg/ml Concanavalin A solution (see Recipes)

Equipment

  1. Drum style test-tube rotator (New Brunswick, TC-7 Roller Drum) at 30 °C

  2. 150 ml Erlenmeyer flasks (Fisher Scientific, catalog number: 50-172-0176)

  3. Platform Shaker (New Brunswick Innova 2300) at 30 °C

  4. Genesys 30 UV/Vis Spectrophotometer (ThermoFisher, catalog number: 840-297300)

  5. Nikon Eclipse Ti-E Microscope, 100× objective, Photometrics Prime 95-B Camera (or equivalent)

Software

  1. NIS-Elements (Nikon, https://www.microscope.healthcare.nikon.com/products/software/nis-elements)

  2. Fiji (free image analysis software that includes ImageJ and several useful plugins; https://imagej.net/Fiji); refer to Schindelin et al., (2012).

Procedure

This protocol accompanies Hughes Hallett et al. (2015) and Sullivan et al. (2019).

We generally follow movement of TORC1 on the vacuolar membrane in a standard W303 lab strain (trp1-1;can1-100;leu2-3,112;his3-11,15;ura3;GAL+;ADE+) carrying Kog1 with a yellow fluorescent protein tag at its native locus (Kog1-ECitrine, or Kog1-YFP for short). Tags on other TORC1 subunits (particularly Tco89) can also be used, but Kog1-YFP gives the strongest signal. It is worth noting fluorescent tags on Tor1 disrupt TORC1 localization and activity.


  1. Prepare an 8-well glass-bottom chamber slide by adding 200 μl of 2 mg/ml Concanavalin A (ConA) solution to each well (ensuring the bottom of each slide is completely coated). Incubate the slide for 10 min at 25 °C, and then remove the solution and allow the wells to air-dry overnight (in the dark) with the lid in place. The ConA treatment ensures that the yeast adheres to the coverslip at the bottom of each chamber, making medium swaps and high-quality imaging possible.

  2. Patch out the strains that are going to be examined onto fresh YEPD plates, starting from glycerol stocks (yeast in 15% glycerol and YEPD, stored at -80 °C) using sterile applicator sticks, and then incubate the plates at 30 °C overnight (or up to three days).

  3. Transfer approximately 5 μl of each strain/patch into a separate 28 ml tube containing 5 ml of SD medium (again using sterile applicator sticks).

  4. Grow the 5 ml starter cultures at 30 °C in a drum rotator (rotating at approximately 40 rpm) until they reach mid-log phase (OD600 between 0.5 and 0.7) – this usually takes 5 h.

  5. Transfer approximately 4 ml of each starter culture into a separate 150 ml Erlenmeyer flask containing 20 ml of SD medium, so that the final OD600 is 0.1.

  6. Grow the 20 ml cultures at 30 °C, shaking at 200 rpm, until they reach an OD600 ~0.4.

  7. While the cultures grow, heat the microscope chamber, 15 ml of fresh SD medium, and 15 ml of fresh starvation medium (e.g., S-glucose or S-nitrogen) to 30 °C.

  8. Pipet 300 μl of each culture into the wells of a ConA-treated chamber slide and then allow the cells to settle for 5 min at 30 °C.

  9. Gently aspirate the medium from each well and discard.

  10. Add 300 μl fresh SD medium at 30 °C.

  11. Capture log-growth (time 0) images in each well using a 100× objective and YFP excitation/emission filter (λEX 510/25; λEM 540/21) collecting a stack of sixteen images, each separated by 0.4 µm in the z-plane, with a 200 ms exposure per plane*. In general you need to capture images in 2-3 fields to ensure you have data for enough cells (>100) to get an accurate TORC1-body count. Once the fluorescence imaging is complete, capture a Differential Interference Contrast (DIC) or Brightfield reference image.

    *Note: The imaging is difficult since there are only ~100 TORC1 molecules per cell. First, you must have a high-quality microscope and a sensitive camera to see Kog1-YFP – particularly when the TORC1 molecules are spread across the vacuole. We currently use a Nikon Ti-E inverted microscope with a Photometrics Prime 95-B camera. Second, you can only image a given field once before significant photobleaching occurs. Third, you must take z-stacks (3D images) that span the entire depth of the cell to identify all of the TORC1 foci since the bodies form on the edge of the vacuole and are thus often at the top or bottom of the cell.

  12. Remove the chamber slide from the microscope, aspirate and discard medium from each well.

  13. Wash each well three times with synthetic medium missing glucose (S-glucose), or synthetic medium missing nitrogen (S-nitrogen), at 30 °C, using 350 μl, 400 μl, and then 450 μl of medium, by gently pipetting against the same corner of each well and then aspirating and discarding each wash except the last (which is left in the well during imaging).

  14. Start a timer after the first wash and capture z-stacks at each time-point in all wells as described in Step 11 (we typically take pictures every 10 min for 1 h), keeping the slide at 30 °C during the entire experiment.


In each experiment, a wild-type (or relevant mutant strain) missing a fluorescent (YFP) tag should also be imaged as a control. We have found that starvation (particularly glucose starvation) can trigger the formation of autofluorescent puncta. The intensity and number of puncta increase over time, and their appearance is highly dependent on the batch of medium being used. We discard data from experiments/time-points where a significant number of autofluorescent foci form in the control strain and rerun the experiment in a fresh batch of medium.

Data analysis

Compress the z-stack for each time-point and strain (composed of all 16 planes) into a maximum intensity projection, using Fiji or other software. Identify in focus cells using the DIC image, and then count the fraction of (in focus) cells that contain a TORC1 (Kog1-YFP) body. A mother cell with an attached bud should be counted as two individual cells due to the observation that a newly formed daughter cell can contain its own TORC1-body. To establish statistical significance, experiments must be completed in triplicate on three different days, and the average fraction of cells with a body at each time-point, and the corresponding standard deviation, were calculated.



Figure 1. Kog1-YFP (TORC1) foci in starvation conditions. Cells that are in the focal plane and used in the analysis are numbered and colored white if they contain a TORC1 body.


In Figure 1 there are 18 in-focus cells, including buds. The fluorescence (maximum projection) image shows the presence of 13 cells containing TORC1-bodies, and thus 72% of cells in this field contain a TORC1-body. For other sample images see Hughes Hallett et al. (2015) and Sullivan et al. (2019).

Recipes

Medium:

  1. SD

    200 ml of 5× amino acids stock (US Biological)

    100 ml of 10× YNB stock (BD Biosciences)

    50 ml of 40% glucose stock (Fisher Scientific)

    650 ml ddH2O

  2. S-glucose

    200 ml of 5× amino acids stock (US Biological)

    100 ml of 10× YNB stock (BD Biosciences)

    700 ml ddH2O

  3. S-nitrogen

    100 ml of 10× YNB stock w/o ammonium sulfate (BD Biosciences)

    50 ml of 40% glucose stock (Fisher Scientific)

    850 ml ddH2O

  4. 2 mg/ml Concanavalin A (ConA)

    Dissolve 20 mg of ConA (MP Biomedicals) into 10 ml ddH2O and aliquot into 1 ml tubes and stored at -20 °C

Acknowledgments

This work was supported by the National Institutes of Health (NIH) grants (R01GM097329 and T32GM008659). This protocol is based on our previous work, described in Hughes Hallett et al. (2015) and Sullivan et al. (2019).

Competing interests

No competing interests.

References

  1. Barbet, N. C., Schneider, U., Helliwell, S. B., Stansfield, I., Tuite, M. F. and Hall, M. N. (1996). TOR controls translation initiation and early G1 progression in yeast. Mol Biol Cell 7(1): 25-42.
  2. Ben-Sahra, I. and Manning, B. D. (2017). mTORC1 signaling and the metabolic control of cell growth. Curr Opin Cell Biol 45: 72-82.
  3. Ben-Sahra, I., Hoxhaj, G., Ricoult, S. J. H., Asara, J. M. and Manning, B. D. (2016). mTORC1 induces purine synthesis through control of the mitochondrial tetrahydrofolate cycle. Science 351(6274): 728-733.
  4. Duvel, K., Yecies, J. L., Menon, S., Raman, P., Lipovsky, A. I., Souza, A. L., Triantafellow, E., Ma, Q., Gorski, R., Cleaver, S., Vander Heiden, M. G., MacKeigan, J. P., Finan, P. M., Clish, C. B., Murphy, L. O. and Manning, B. D. (2010). Activation of a metabolic gene regulatory network downstream of mTOR complex 1. Mol Cell 39(2): 171-183.
  5. Gonzalez, A. and Hall, M. N. (2017). Nutrient sensing and TOR signaling in yeast and mammals. EMBO J 36(4): 397-408.
  6. Hsu, P. P., Kang, S. A., Rameseder, J., Zhang, Y., Ottina, K. A., Lim, D., Peterson, T. R., Choi, Y., Gray, N. S., Yaffe, M. B., Marto, J. A. and Sabatini, D. M. (2011). The mTOR-regulated phosphoproteome reveals a mechanism of mTORC1-mediated inhibition of growth factor signaling. Science 332(6035): 1317-1322.
  7. Huber, A., Bodenmiller, B., Uotila, A., Stahl, M., Wanka, S., Gerrits, B., Aebersold, R. and Loewith, R. (2009). Characterization of the rapamycin-sensitive phosphoproteome reveals that Sch9 is a central coordinator of protein synthesis. Genes Dev 23(16): 1929-1943.
  8. Hughes Hallett, J. E., Luo, X. and Capaldi, A. P. (2015). Snf1/AMPK promotes the formation of Kog1/Raptor-bodies to increase the activation threshold of TORC1 in budding yeast. Elife 4: e09181.
  9. Kamada, Y., Funakoshi, T., Shintani, T., Nagano, K., Ohsumi, M. and Ohsumi, Y. (2000). Tor-mediated induction of autophagy via an Apg1 protein kinase complex. J Cell Biol 150(6): 1507-1513.
  10. Kim, J., Kundu, M., Viollet, B. and Guan, K. L. (2011). AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol 13(2): 132-141.
  11. Liu, G. Y. and Sabatini, D. M. (2020). mTOR at the nexus of nutrition, growth, ageing and disease. Nat Rev Mol Cell Biol 21: 183-203.
  12. Loewith, R. and Hall, M. N. (2011). Target of rapamycin (TOR) in nutrient signaling and growth control. Genetics 189(4): 1177-1201.
  13. Peterson, T. R., Sengupta, S. S., Harris, T. E., Carmack, A. E., Kang, S. A., Balderas, E., Guertin, D. A., Madden, K. L., Carpenter, A. E., Finck, B. N. and Sabatini, D. M. (2011). mTOR complex 1 regulates lipin 1 localization to control the SREBP pathway. Cell 146(3): 408-420.
  14. Robitaille, A. M., Christen, S., Shimobayashi, M., Cornu, M., Fava, L. L., Moes, S., Prescianotto-Baschong, C., Sauer, U., Jenoe, P. and Hall, M. N. (2013). Quantitative phosphoproteomics reveal mTORC1 activates de novo pyrimidine synthesis. Science 339(6125): 1320-1323.
  15. Schindelin, J., Arganda-Carreras, I. and Frise, E. et al. (2012). Fiji: an open-source platform for biological-image analysis.Nature Methods 9(7): 676-682.
  16. Sullivan, A., Wallace, R. L., Wellington, R., Luo, X. and Capaldi, A. P. (2019). Multilayered regulation of TORC1-body formation in budding yeast. Mol Biol Cell 30(3): 400-410.

简介

[摘要]雷帕霉素激酶复合体I(TORC1)的靶标是真核生物中细胞生长和代谢的主要调节剂。在促生长激素和丰富营养素的存在下,TORC1活跃并通过磷酸化多种蛋白质来驱动蛋白质,脂质和核苷酸合成。相反,当氮和/或葡萄糖水平下降时,TORC1被抑制,导致细胞从合成代谢转换为分解代谢,并最终进入静止状态。在发芽酵母中酿酒酵母 抑制TORC1会触发TORC1从其在液泡周围的位置移动到液泡膜边缘的单个焦点/物体。这种重新定位取决于许多关键的TORC1调节子的活动,因此可以使用TORC1定位分析来跟踪通过TORC1途径发出的信号。在这里,我们提供了使用荧光显微镜测量TORC1(特别是Kog1-YFP)重新定位/信号传递的详细协议。重点放在确保以下方面的程序上:(1)尽管葡萄糖和氮饥饿期间它们相对较低的荧光和自发荧光灶的积累,但仍正确地识别(并计数)TORC1抗体;(2)在每次实验开始时,使细胞保持对数生长期,以便正确监测TORC1体形成的动力学;(3)使用适当的荧光标签来避免检查错误定位的TORC1。

[背景]雷帕霉素的靶激酶复合物I(TORC1 )是真核生物的细胞生长控制网络中的中央轮毂(Loewith和Hall,2011;冈萨雷斯和Hall,2017; Liu和萨巴蒂尼,2020 )。在存在促生长激素和丰富营养的情况下,TORC1活跃并磷酸化多种蛋白质,以驱动蛋白质和核糖体合成,激活脂质和核苷酸合成,调节氮和氨基酸的代谢/转运,并抑制自噬(Kamada等等人,2000年; Huber等人,2009年; Hsu等人,2011年; Kim等人,2011年; Loewith和Hall,2011年; Peterson等人,2011年; Robitaille等人,2013年; Ben-Sahra等人等人,2016年;冈萨雷斯和霍尔,2017年;本·萨赫拉和曼宁,2017年;刘和萨巴蒂尼,2020年)。相反,当细胞暴露于应激或饥饿条件,TORC1被失活,以限制细胞生长和重定向可用resou ř CES到Appro公司p riate应力或饥饿反应(Barbet的等人,1996; Duvel 。等人,2010 ) 。

在过去的15年中,对TORC1信号转导的详细分析揭示了将压力和饥饿信号传递至TORC1的蛋白质网络,但是仍然存在许多问题,涉及如何在影响细胞生长和存活的各种刺激中调节TORC1 (Loewith和霍尔,2011年;冈萨雷斯和霍尔,2017年;刘和萨巴蒂尼,2020年)。在模式生物酿酒酵母(其中TORC1首先被发现)(Loewith和IIa LL,2011;冈萨雷斯和Hall,2017 ),进展映射TORC1调节电路由缺少快速,可扩展的信令TORC1测定受到阻碍。在这里,我们描述了一种在葡萄糖和/或氮饥饿期间监视TORC1(由TOR激酶Tor1,必需的调节蛋白Kog1和两个辅助蛋白Lst8和Tco89组成)向焦点/体内运动的协议。由于TORC1的重新定位受酵母中所有已知的TORC1调节剂控制(Hughes Hallett等人,2015 ; Sullivan等人,2019 ),因此该测定可用于(1)在单细胞水平上测量TORC1信号的变化,并且( 2)快速测试新型基因,突变和/或药物对TORC1活性的影响。

关键字:雷帕霉素激酶复合物I的靶点, Tor1, Kog1, TORC1-body, Kog1-body



材料和试剂


8 -孔微量滑动(Ibidi,目录号:80826)
YEPD板(VWR,目录号:10128-392)
28 ml试管(VWR,目录号:47729-583)
木制涂药棒(关键科学产品,目录号:CA958701)
不含氨基酸的BD Difco酵母氮基地(Fisher Scientific,BD Biosciences,目录号:BD 291920)
不含氨基酸和硫酸铵的BD Difco酵母氮基(Fisher Scientific,BD Biosciences,目录号:DF0335-15-9)
不含酵母氮基地的脱卸剂混合物(美国生物学,目录号:D9515)
无水葡萄糖结晶颗粒(Fisher Scientific,Fisher BioReagents,目录号:BP350-1)
硫酸铵(Fisher Scientific,目录号:BP212-212 )
甘油(Sigma - Aldrich,目录号:G7893)
伴刀豆球蛋白A(Fisher Scientific,MP生物医学,目录号:ICN15071001)
SD(请参阅食谱)
S-葡萄糖(请参阅食谱)
氮(请参阅食谱)
2 mg / m l伴刀豆球蛋白A溶液(请参阅食谱)


设备


鼓式试管旋转器(新不伦瑞克省,TC-7滚筒鼓),温度30 °C
150 ml锥形瓶(Fisher Scientific,目录号:50-172-0176)
摇床(New Brunswick Innova 2300)在30 °C
Genesys 30紫外/可见分光光度计(Thermo F垫圈,目录号:840-297300)
尼康Eclipse Ti-E显微镜,100 ×物镜,Photometrics Prime 95-B相机(或等效相机)


软件


NIS-Elements(Nikon,https://www.microscope.healthcare.nikon.com/products/software/nis-elements)
斐济(包含ImageJ和一些有用插件的免费图像分析软件; https://imagej.net/Fiji); 参见Schindelin等。,(2012年)。


程序


该协议伴随Hughes Hallett等。(2015年)和Sullivan等人。(2019)。

我们通常跟踪携带带有黄色荧光蛋白标签的Kog1的标准W303实验室菌株(trp1-1; can1-100; leu2-3,112; his3-11,15; ura3; GAL +; ADE + )中TORC1在液泡膜上的运动。在其天然位点(Kog1-ECitrine或较短的Kog1-YFP )。也可以使用其他TORC1亚基(尤其是Tco89)上的标签,但Kog1-YFP提供最强的信号。值得注意的是Tor1上的荧光标记破坏了TORC1的定位和活性。


加入200μ制备的8孔玻璃底室滑动升的2毫克/米升伴刀豆球蛋白A(刀豆)溶液至每孔(确保每张幻灯片的底部完全涂覆)。将载玻片在25°C下孵育10分钟,然后取出溶液,并在有盖的情况下,将孔在阴暗处风干过夜(在黑暗中)。而ConA处理确保了酵母坚持š在每个腔室的底部的盖玻片,使得介质互换和高-品质成像成为可能。
使用无菌涂药棒将要检查的菌株从甘油库存(酵母中含15%甘油和YEPD中的酵母,在-80°C下储存)上修补到新鲜的YEPD板上,然后将其在30°C下孵育过夜(或最多三天)。
输送约5μ升每种菌株/补丁的到一个单独的28米升含有5M管升SD培养基(再次使用无菌涂布器棒)。
在30°C的转鼓旋转器中(以大约40 rpm的转速旋转)生长5 ml发酵剂,直到达到对数中期(OD 600在0.5到0.7之间)–通常需要5个小时。
将大约4 ml的每种发酵剂培养物转移到一个单独的150 ml的锥形瓶中,该烧瓶中装有20 ml的SD培养基,因此最终的OD 600为0.1。
生长20米升培养物在30℃下,以200rpm振荡,直到它们达到的OD 600 〜0.4。
虽然培养物生长,加热显微镜室15 ml的新鲜的SD培养基中,和15 ml的新鲜饥饿培养基(的例如,S-葡萄糖或S-氮)至30℃。
吸管300μ升每种培养成的ConA处理的腔室滑动件的孔中,然后使细胞沉降5分钟,在30℃。
轻轻地从每个孔中吸出培养基并丢弃。
添加300 μ升新鲜的SD培养基在30℃下。
捕获数生长(时间0)在每孔用100张图片×物镜和YFP的激发/发射过滤器(λ EX 25分之510; λ EM 21分之540)收集16个图像的堆叠,每个通过0.4μm的处于分离z平面,每个平面曝光200毫秒*。通常,您需要在2-3个字段中捕获图像,以确保拥有足够的细胞数据(> 100)才能获得准确的TORC1抗体计数。荧光成像完成后,捕获微分干涉对比(DIC)或明场参考图像。
*注:牛逼他IMAGIN g是很难罪CE只有100〜每个细胞TORC1分子。首先,您必须拥有高质量的显微镜和灵敏的摄像头才能看到Kog1-YFP –特别是当TORC1分子散布在液泡中时。我们目前使用带有Photometrics Prime 95-B相机的尼康Ti-E倒置显微镜。其次,您只能在给定的场成像一次,然后再进行显着的光致漂白。第三,您必须拍摄横跨细胞整个深度的z堆栈(3D图像),以识别所有TORC1病灶,因为这些体形成在液泡的边缘,因此通常位于细胞的顶部或底部。


从显微镜上取下载玻片,从每个孔中吸出并丢弃培养基。
洗涤用合成培养基缺少葡萄糖(S-葡萄糖),或合成的培养基缺少氮(S-氮),每个孔三次在30℃下,使用350μ升,400μ升,然后450μ升培养基中,由轻轻移液到每个孔的同一角,然后抽吸并丢弃除最后一个(在成像过程中留在孔中)以外的所有洗液。
第一次清洗后启动计时器,并按照步骤11所述在所有孔中的每个时间点捕获z堆栈(我们通常每10分钟拍照1小时),在整个实验过程中将玻片保持在30°C 。


在每个实验中,也应将缺少荧光(YFP)标签的野生型(或相关突变株)成像作为对照。我们发现饥饿(尤其是葡萄糖饥饿)可以触发自发性荧光点的形成。泪点的强度和数目随时间增加,并且它们的出现高度依赖于所用培养基的批次。我们从实验/时间点丢弃了在对照菌株中形成大量自发荧光病灶的数据,并在新一批培养基中重新进行了实验。


数据分析


使用斐济或其他软件,将每个时间点和应变(由所有16个平面组成)的z堆栈压缩为最大强度投影。使用DIC图像识别焦点细胞,然后计算包含TORC1(Kog1-YFP)物体的(焦点)细胞的分数。由于观察到新形成的子细胞可以包含其自己的TORC1体,因此带有芽芽的母细胞应计为两个单独的细胞。为了建立统计学意义,必须在三个不同的天重复三次以完成实验,并计算每个时间点上带有主体的细胞的平均分数以及相应的标准差。





图1.饥饿条件下的Kog1-YFP (TORC1)病灶。ç ELL小号是在焦平面上,并在分析中使用的编号,如果它们包含3个颜色的白色N A TORC1体。


在图URE 1有18个对焦细胞,包括芽。荧光(最大投影)图像显示存在13个含有TORC1抗体的细胞,因此该领域中有72%的细胞含有TORC1抗体。有关其他示例图像,请参见Hughes Hallett等。(2015年)和Sullivan等人。(2019)。


菜谱


中等的:

标清
200米升的5 ×氨基酸库存(US生物)

百米升的10 × YNB股票(BD Biosciences)中

50米升的40%葡萄糖的库存(Fisher Scientific公司)

650米升的DDH 2 ö

葡萄糖
200米升的5 ×氨基酸库存(US生物)

百米升的10 × YNB股票(BD Biosciences)中

700百万升ddH 2 O


百米升的10 × YNB股票的w / o硫酸铵(BD Biosciences)中

50米升的40%葡萄糖的库存(Fisher Scientific公司)

850百万升ddH 2 O

2 mg / m l伴刀豆球蛋白A(ConA)
溶解20毫克的ConA(MP Biomedicals公司)的成10米升的DDH 2 O和等分入1M升管中并储存在-20℃下


致谢


这项工作得到了美国国立卫生研究院(NIH)赠款(R01GM097329和T32GM008659 )的支持。该协议基于我们先前的工作,如Hughes Hallett等人所述。(2015年)和Sullivan等人。(2019 )。


利益争夺


没有竞争利益。


参考


北卡罗来纳州的巴贝特,美国的施奈德,南卡罗来纳州的赫利威尔,伊利诺伊州斯坦斯菲尔德,MF的图伊特和明尼苏达州的霍尔(1996)。TOR控制酵母中的翻译起始和早期G1进程。分子生物学细胞7(1):25-42。
I.Ben-Sahra和BD曼宁(2017)。mTORC1信号传导和细胞生长的代谢控制。Curr Opin Cell Biol 45:72-82。
Ben-Sahra,I.,Hoxhaj,G.,Ricoult,SJH,Asara,JM和Manning,BD(2016)。mTORC1通过控制线粒体四氢叶酸周期诱导嘌呤合成。科学351(6274):728-733。
Duvel,K.,Yecies,JL,Menon,S.,Raman,P.,Lipovsky,AI,Souza,AL,Triantafellow,E.,Ma,Q.,Gorski,R.,Cleaver,S.,Vander Heiden, MG,MacKeigan,JP,Finan,PM,Clish,CB,Murphy,LO和Manning,BD(2010)。mTOR复合物1下游的代谢基因调控网络的激活。Mol Cell 39(2):171-183。
Gonzalez,A.和Hall,MN(2017)。酵母和哺乳动物中的营养感测和TOR信号传导。EMBO J 36(4):397-408。
许(Hsu),PP,康(Kang),SA,拉美瑟(Rameseder),J。,张扬(Y.),奥蒂娜(Ottina),卡尔(Kim),林(L.马克(2011)。mTOR调节的磷酸化蛋白质组揭示了mTORC1介导的生长因子信号传导抑制的机制。科学332(6035):1317-1322。
A. Huber,B. Bodenmiller,B.Uotila,M.,Stahl,M.,Wanka,S.,Gerrits,B.,Aebersold,R.和Loewith,R.(2009)。雷帕霉素敏感的磷酸化蛋白质组的表征表明,Sch9是蛋白质合成的核心协调员。基因发展23(16):1929-1943。
Hughes Hallett,JE,Luo,X.和Capaldi,AP(2015)。Snf1 / AMPK促进Kog1 / Raptor抗体的形成,从而增加发芽酵母中TORC1的激活阈值。Elife 4:e09181。
Y. Kamada,T. Funakoshi,T. Shintani,T. Nagano,K.,Ohsumi,M. and Ohsumi,Y.(2000)。Tor通过Apg1蛋白激酶复合物介导的自噬诱导。细胞生物学杂志(J Cell Biol)150(6):1507-1513。
Kim,J.,Kundu,M.,Viollet,B. and Guan,KL(2011)。AMPK和mTOR通过直接将Ulk1磷酸化来调节自噬。Nat Cell Biol 13(2):132-141。
Liu,GY和Sabatini,DM(2020)。mTOR与营养,生长,衰老和疾病息息相关。Nat Rev Mol Cell Biol 21 :183-203。
Loewith,R.和Hall,MN(2011)。雷帕霉素(TOR)在营养信号传递和生长控制中的目标。遗传学189(4):1177-1201。
Peterson,TR,Sengupta,SS,Harris,TE,Carmack,AE,Kang,SA,Balderas,E.,Guertin,DA,Madden,KL,Carpenter,AE,Finck,BN和Sabatini,DM(2011)。mTOR复合物1调节脂蛋白1的定位,以控制SREBP途径。单元格146(3):408-420。
Robitaille,AM,Christen,S.,Shimobayashi,M.,Cornu,M.,Fava,LL,Moes,S.,Prescianotto-Baschong,C.,Sauer,U.,Jenoe,P.和Hall,MN(2013 )。定量磷酸化蛋白质组学揭示了mTORC1可以从头激活嘧啶的合成。科学339(6125):1320-1323。
Schindelin,J.,Arganda-Carreras,I。和Frise,E。等。(2012)。斐济:一个用于生物图像分析的开源平台。自然方法9(7):676-682。
Sullivan,A.,Wallace,RL,Wellington,R.,Luo,X.和Capaldi,AP(2019)。萌芽酵母中TORC1体形成的多层调节。分子生物学细胞30(3):400-410。
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Copyright Wallace et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Wallace, R. L., Lu, E., Sullivan, A., Hughes Hallett, J. E. and Capaldi, A. P. (2021). Analysis of TORC1-body Formation in Budding Yeast. Bio-protocol 11(7): e3975. DOI: 10.21769/BioProtoc.3975.
  2. Hughes Hallett, J. E., Luo, X. and Capaldi, A. P. (2015). Snf1/AMPK promotes the formation of Kog1/Raptor-bodies to increase the activation threshold of TORC1 in budding yeast. Elife 4: e09181.
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