A Method for Extracting the Nuclear Scaffold from the Chromatin Network
一种从染色质网络中提取核骨架的方法   

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Manjula Mummadisetti Manjula Mummadisetti
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The Journal of Biological Chemistry
Feb 2018

Abstract

Each cell contains many large DNA polymers packed in a nucleus of approx. 10 μm in diameter. With histones, these DNA polymers are known to form chromatins. How chromatins further compact in the nucleus is unclear but it inevitably depends on an extensive non-chromatin nuclear scaffold. Imaging of endogenous chromatin network and the complementary scaffold that support this network has not been achieved but biochemical and proteomic investigations of the scaffold can still provide important insights into this chromatin-organizing network. However, this demands highly inclusive and reproducible extraction of the nuclear scaffold. We have recently developed a simple protocol for releasing the scaffold components from chromatins. The inclusiveness of the extract was testified by the observation that, upon its extraction from the nuclei, the remaining nuclear chromatins were liberated into extended and often parallel chromatin fibers. Basically, this protocol includes the generation of pure nuclei, treatment of the nuclei with Triton X-100 to generate envelope-depleted nuclei (TxN), and extraction of the nuclei at 500 mM NaCl in a sucrose-containing buffer. This combined extract of TxN is known as TxNE.

Keywords: Nuclei (核), Extract (提取), Scaffold (骨架), Nucleophosmin-1 (核仁磷酸蛋白-1), Chromatin (染色质)

Background

Chromatins are densely and dynamically compacted in the nuclei through a complex scaffold of proteins and ribonucleoproteins. Unlike the cytoskeletal networks (Fischer and Fowler, 2015), microscopic observation of this nuclear scaffold is technically challenging. This may reflect the dominance of chromatins inside each nucleus with which the scaffold mingles and negotiates in the nucleus. The spherical arrangement of the nucleus also contributes to challenges in imaging such scaffold structures. A major element of the nuclear scaffold is the nuclear lamina (NL) (Gruenbaum and Foisner, 2015). NL covers the surface of the entire nuclear chromatin mass on which the nuclear envelop (NE) attaches. The peripheral surface of the nuclear chromatin mass is characterized by dense heterochromatin (Gruenbaum and Foisner, 2015). Inside the nucleus, one or more nucleoli can be found that occupy significant nuclear regions (Jordan and McGovern, 1981; Shaw and Jordan, 1995; Pederson, 2011). The nucleolar surface is also surrounded by a rim of dense chromatins and potentially functions as an important interior scaffold to support the chromatins (Chen et al., 2018). The morphology of the nucleoli changes dynamically depending on the stage of the cell cycle which can significantly affect the organization of nuclear chromatins (Hernandez-Verdun, 2011; Chen et al., 2018).

The presence of such scaffold structures has been extensively investigated in 1980-90s although a direct visualization has not been achieved (Gasser, 2002; Laemmli et al., 1992). However, the composition of the nuclear scaffold and interactions among the scaffold elements could be learned through biochemical, proteomic and imaging studies. To this end, effective and reproducible extraction of the nuclear scaffold from the chromatin network is essential. We have recently developed a simple protocol for the extraction of nuclear scaffold proteins (Chen et al., 2018).

This protocol was initially developed based on a study showing that isolated nucleoli could be solubilized at 400 mM NaCl (Trimbur and Walsh, 1993). We adopted this condition to solubilize isolated nuclei. The inclusiveness of the extract was testified by the liberation of nuclear chromatins as extended parallel chromatin fibers (Chen et al., 2018). TxNE can be used to study many aspects of the nuclear scaffold.

Materials and Reagents

  1. 0.5-10 L pipette tips (Corning, Axygen®, catalog number: T-300 )
  2. 10-200 ml pipette tips (Greiner Bio One International, catalog number: 739290 )
  3. 100-1,000 ml pipette tip (Greiner Bio One International, catalog number: 686290 )
  4. T 175 cell culture flask (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 159910 )
  5. T 75 cell culture flasks (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 156499 )
  6. 150 mm circular tissue culture dishes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 168381 )
  7. 50 ml Falcon tubes (Greiner Bio One International, catalog number: 227261 )
  8. 5 ml blunt-ended syringes (5 ml/cc, Luer slip, Cellotron, https://dcellotron.en.ec21.com/)
  9. 15 ml Falcon tubes (Greiner Bio One International, catalog number: 188271 )
  10. Nalgene Oak Ridge High-Speed Centrifuge Tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3114-0030 )
  11. Regular 1.5 ml microcentrifuge tubes (Corning, Axygen®, catalog number: MCT-150-C )
  12. Low adhesion microcentrifuge tubes (Eppendorf, catalog number: 022431081 )
  13. 12 mm glass coverslips (Electron Microscopy Sciences, catalog number: 72231-10 )
  14. Glass slides (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 6776215 )
  15. 50 ml syringes (Terumo, catalog number: SS*50LE )
  16. 0.22 μm filters (Merck, catalog number: SLGP033RS )
  17. HeLa S3 human cervical carcinoma cells (ATCC, catalog number: CCL-2 )
  18. Trypsin solution (0.05% w/v, Capricorn scientific, catalog number: TRY-1B10 )
  19. 0.5% Trypsin (Trypsin-EDTA, 10x stock, Thermo Fisher Scientific, GibcoTM, catalog number: 15400054 )
  20. Protease inhibitors PMSF (BDH, catalog number: 442172C )
  21. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  22. 5 M NaCl
  23. 1% (w/v) paraformaldehyde
  24. Mouse anti-nucleophosmin-1 (NPM1) antibody (Sigma-Aldrich, catalog number: B0556 )
  25. Rabbit anti-lamin B1 (LB1) antibody (Abcam, catalog number: ab16048 )
  26. Rabbit anti-histone H1.2 (Abcam, catalog number: ab17677 )
  27. Rabbit anti-lamin A/C (Santa Cruz Biotechnology, catalog number: sc-7292 )
  28. Rabbit anti-H1.x antibody (Abcam, catalog number: ab31972 )
  29. Goat anti-mouse IgG (Cy3-conjugated) (Jackson ImmunoResearch Laboratories, catalog number: 115-165-003 )
  30. Goat anti-rabbit IgG (Alexa Fluor 488-conjugated) (Jackson ImmunoResearch Laboratories, catalog number: 115-545-003 )
  31. Mounting medium (Vector laboratories, catalog number: H-1200 )
  32. Any clear Nail polish (We use Daiso Japan Nail Top Coat Clear, 10 ml bottle, made in Taiwan)
  33. 12.5% (w/v) gels
  34. Sodium chloride (NaCl) (Merck)
  35. Potassium chloride GR (KCl)(Merck)
  36. Di-sodium hydrogen phosphate (Merck)
  37. Sucrose (First BASE Laboratories, catalog number: BIO-1090-1kg )
  38. Sucrose (Merck)
  39. Tris (Merck)
  40. Tris base (Avantor Performance Materials, J.T. Baker, catalog number: 4109-02 )
  41. Magnesium chloride hexahydrate (Merck, catalog number: 1058331000 )
  42. Magnesium chloride hexahydrate (MgCl2·H2O)( Merck)
  43. DMEM medium (Thermo Fisher Scientific, GibcoTM, catalog number: 12430054 )
  44. Fetal bovine serum (FBS, GE Healthcare, HyClone, catalog number: SV30160.03 )
    Note: We purchased FBS and generated heat-inactivated FBS by incubation at 56 °C for 30 min.
  45. Penicillin-Streptomycin (100x stock, PAN Biotech, catalog number: P06-07100 )
  46. L-glutamine (200 mM, 100x stock, Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  47. PBS, pH 7.4 (see Recipes)
  48. 0.25 M sucrose buffer, pH 7.4 (see Recipes)
  49. 2.2 M sucrose buffer, pH 7.4 (see Recipes)
  50. DMEM culture medium (see Recipes)

Equipment

  1. 1,000 L pipette (Eppendorf, model: Research® plus, catalog number: 3120000062 )
  2. 200 L pipette (Eppendorf, model: Research® plus, catalog number: 3120000054 )
  3. Humidified cell incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: FormaTM Steri-CycleTM CO2 incubator , catalog number: 371)
  4. Homogenizer (Isobiotec Precision Engineering, Heidelberg, Germany)
  5. Tabletop microcentrifuge (Beckman Coulter, model: Microfuge 22R centrifuge , catalog number: 368831)
  6. Tabletop swing bucket centrifuge (Eppendorf, model: 5810 R , catalog number: 5811000320)
  7. Beckman Coulter centrifuge with a JA-20 rotor (Beckman Coulter, model: Avanti J-25 , catalog number: 363102)
  8. BSL-2 cell culture cabinet (Gelman, model: Bioessential Class II series Type A2 Laminar flow BSC, http://gelmansingapore.com/product/bioessential-class-ii-type-a2-series-laminar-flow-biological-safety-cabinets/)
  9. FluoView FV1000 confocal microscope
  10. Cool/SNAP HQ2 image acquisition camera (Olympus)

Software

  1. FV-ASW 1.6b software
  2. Imaris software (Bitplane AG)

Procedure

  1. Generation of cell homogenate
    1. Culture HeLa cells in a T-75 flask in a humidified BSL-2 incubator at 37 °C containing 25 ml of DMEM culture medium to confluence.
    2. Divide the cells equally five 150 mm round culture dishes and culture in the DMEM culture medium (25 ml/plate) to confluence.
    3. Remove the media and wash each plate using 10 ml of PBS.
    4. Add 2 ml of 1x trypsin solution to cover the surface of each plate and incubate the plates for 2 min at 37 °C in a humidified cell incubator.
    5. Tap the plates horizontally to suspend the cells and add 6 ml of culture medium to each plate.
    6. Harvest cells from each plate and combine in a 50 ml Falcon tube.
    7. Harvest cells by centrifugation for 5 min at 400 x g.
    8. Re-suspend cells in 50 ml of PBS and wash by centrifugation for 5 min at 400 x g. Repeat this step once.
    9. Re-suspend cells in 4 ml of 0.25 M sucrose buffer containing the protease inhibitor PMSF (0.25 mM) and pipet to get a single cell suspension.
    10. Assemble an isobiotec cell homogenizer using a 10 μm metal ball and pre-chill the cell homogenizer on ice (Figure 1).


      Figure 1. Assembly of the Isobiotec cell homogenizer. A. Parts of the homogenizer including the metal ball (10 μm clearance) in the white tube; B. Side view; C. The metal ball is placed in the central passage. D. Completed assembly of the homogenizer. E. Each outlet is fitted with a 5 ml syringe. F. 2 ml of cell suspension is added to one syringe (water is used in this demonstration). G. Press to pass the cell suspension through the metal ball-fitted central passage into the other syringe. Repeat another 11 times before harvesting the cell homogenate.

    11. Fit the two outlets on the homogenizer each with a 5 ml syringe.
    12. Remove the plunger from one syringe to add 2 ml of the cell suspension.
    13. Pass the cell suspension 12 times through the homogenizer.
    14. Transfer the homogenized cell lysate into a 15 ml Falcon tube on ice and repeat homogenization with the rest of the cell suspension.
    15. Wash the homogenizer 2 times each with 1 ml of 0.25 M sucrose buffer and combine with the lysate.

  2. Isolation of nuclei
    1. Centrifuge the cell homogenate for 10 min at 600 x g and 4 °C to pellet the nuclei.
    2. Re-suspend the pellet in 5 ml of 2.2 M sucrose buffer in Nalgene Oak Ridge High-Speed Centrifuge Tubes.
    3. Centrifuge for 30 min at 50,000 x g and 4 °C.
    4. Remove supernatant and add 2 ml of 0.25 M sucrose buffer to rinse the tube area near the pellet.
    5. Remove the sucrose buffer without disturbing the pellet.
    6. Re-suspend the pellet in 4 ml of 0.25 M sucrose buffer and transfer to a 15 ml Falcon tube.
    7. Centrifuge for 5 min at 600 x g and 4 °C.

  3. Depletion of the nuclear envelope (NE)
    1. Preparation of Triton-containing sucrose buffer.
      1. Add 0.1 ml of Triton X-100 in a 15 ml tube along the tube wall.
      2. Add 10 ml of 0.25 M sucrose buffer to the tube and vortex till Triton is mixed thoroughly with the sucrose buffer.
    2. Re-suspend the nuclear pellet in 4 ml of Triton-containing sucrose buffer and incubate for 1 h on ice.
    3. Centrifuge for 5 min at 600 x g and 4 °C.
    4. Re-suspend the pellet in another 5 ml of Triton-containing sucrose buffer.
    5. Centrifuge for 5 min at 600 x g and 4 °C.
    6. Wash these Triton-extracted nuclei (TxN) 2 times using 0.25 M sucrose buffer.
    7. Then resuspend the pellet in 0.9 ml of 0.25 M sucrose buffer.

  4. Extraction of the nuclear scaffold (Figure 2)
    1. Add 0.1 ml 5 M NaCl solution to the nuclear suspension (colloidal gel forms immediately at the interface).
    2. Pipette vigorously 20-30 times using a 1,000 L Eppendorf pipette until the entire suspension turns into a uniform colloidal gel.
    3. Add 0.5 ml of 0.25 M sucrose buffer containing 0.5 M NaCl and pipette to homogeneity.
    4. Centrifuge for 10 min at 2,000 x g and recover a small volume of supernatant if it is clearly observed.
    5. Add 0.5 ml of 0.25 M sucrose buffer and the colloidal gel should shrink immediately.
    6. Poke the gel vigorously using a 0.2 ml pipette tip so that the gel continues to shrink.
    7. Centrifuge for 10 min at 2,000 x g and 4 °C and recover the clear supernatant.
    8. Add another 0.5 ml of 0.25 M sucrose buffer to the pellet and continue to poke the fragment until it ceases shrinking (It should look like a brown and non-transparent fragment).
    9. Centrifuge again and combine all the supernatants as the extract.
    10. Measure the A280 absorption of the combined TxN extracts or TxNE. It should range 1.0-2.0.
    11. Aliquot TxNE in non-adhesive 1.5-ml microtubes and store at -80 °C.


      Figure 2. Extraction of TxNE from the nuclei. A. Single cell suspension generated from 6 x 108 HeLa cells in 1.35 ml of 2.5 M sucrose buffer. B. After adding 0.15 ml of 5 M NaCl, the interphase moved rapidly upward giving a transparent lower phase in less than 30 sec. C. After vigorously pipetting for ~30 times in less than 1 min, the entire cell suspension became one single colloidal gel. D. 0.75 ml of the 0.25 M sucrose buffer containing 0.5 M NaCl was added to the gel and mixed by tapping for ~20 times. E. Centrifuge for 10 min at 2,000 x g and ~0.2 ml supernatant can be recovered from the top. F. Add 0.75 ml of the 0.25 sucrose buffer (without additional NaCl) and mix vigorously for 1-2 min to observe the gel shrinking. Poke the gel 20-50 times with a 10-200 L tip fitted on a pipette until the gel stop shrinking. Centrifuge for 10 min at 2,000 x g and harvest the supernatant. G. Add 0.75 ml of the 0.25 M sucrose buffer to the pellet. H. Continue to ~50 times until when the pellet shrunk into an irregular fragment. Some smaller fragments are also visible. I. Centrifuge for 10 min at 1,000 x g to recover the supernatant. J. The supernatants are combined as TxNE and the pellet is typically the extracted chromatins which retained the cellular DNA and core histones.
      Note: To show the sample status in each step more clearly, we have scaled up the solution volumes 1.5x of that described in the main text of the protocol for photograph.

Data analysis

  1. The usual protein profile of TxNE is highly consistent (Chen et al. 2018). During TxNE isolation, some steps can be monitored for consistency. Firstly, the nuclei isolated after centrifugation through 2.2 M sucrose can be examined by confocal microscopy by immuno-staining of the signature NL protein lamin B1 and the signature nucleolar protein nucleophosmin-1 (NPM1) (Figure 3). The isolated nuclei mostly remain continuous in the LB1-defined NL although the nucleoli can sometimes become enlarged. The nuclei mostly remain oval in morphology. These nuclei should be free of vimentin although it often associates significantly with the nuclear surface if the nuclei are not centrifuged through the 2.2 M sucrose buffer. TxN should remain individual although these NE-depleted nuclei tend to form clusters. The LB1-defined NL should be mostly continuous although the shapes of TxN can be less oval in morphology (Figure 3).


    Figure 3. Integrity of isolated and Triton-extracted nuclei. HeLa cells homogenized and nuclei were isolated by centrifugation through the 2.2-M sucrose buffer. The nuclei were incubated with 12-mm glass coverslips for 5 min on ice and then fixed for 30 min in 1% (w/v) paraformaldehyde. The nuclei were then incubated for 30 min in 1% (v/v) Triton X-100 in PBS. The nuclei were also re-suspended in the 0.25-M sucrose buffer containing 1% (v/v) Triton X-100 and incubated for 1 h on ice to generate Triton-extracted nuclei (TxN) (right panel). TxN was washed in 0.25 M sucrose buffer and then incubated for 5 min on ice with 12-mm glass coverslips. The coverslips were fixed for 30 min in 1% (w/v) paraformaldehyde. All coverslips were first incubated for 30 min with mouse anti-NPM1 and rabbit anti-LB1 antibodies and were, after washing, incubated for 30 min with goat anti-rabbit (Alexa Fluor 488, green) and anti-mouse (Cy3, red) IgG. After washing, coverslips were mounted using DAPI-containing VectorShield medium and viewed using a FluoView FV1000 confocal microscope equipped with a 100x oil objective (aperture 1.45) and Cool/SNAP HQ2 image acquisition camera (Olympus). Images are captured with the FV-ASW 1.6b software and analyzed using the Imaris software (Bitplane AG). Scale bars = 5 μm.

  2. TxNE does not contain noticeable levels of core histones (Chen et al., 2018). However, the linker H1 histone is included in this extract (Figure 4). Little LB1 is extracted in TxNE (Figure 4). LB1 remains associated with the extracted chromatins although it is dispersed among the extended chromatins as fragments (Chen et al., 2018). In contrast, lamin A/C is effectively extracted with TxNE (Figure 4).


    Figure 4. Selective elution of nuclear proteins from the chromatin network. Nuclei are isolated and depleted of NE with Triton X-100. These NE-depleted nuclei (TxN) were extracted with 0.25 M sucrose buffer containing 100, 200, 300, 400 or 500 mM of NaCl. Equal volumes of the supernatants were subjected to SDS-PAGE on 12.5% (w/v) gels and analyzed by Western blotting. The cytosol fraction and TxN used in the extraction were also included as controls. All antibodies used are affinity-purified polyclonal rabbit antibodies obtained from Abcam.

Notes

  1. This protocol is highly reproducible as stated. However, researchers may encounter difficulties during cell homogenization if single cell suspension is not achieved.
  2. This protocol is optimized for HeLa cells. For other cell types, the metal ball diameter needs to be optimized. The quality of nuclei thus generated can be assessed by simple immuno-staining for LB1, chromatins, and NPM1.
  3. TxNE should be examined by SDS-PAGE and Coomassie Blue staining to assess consistency in protein profiles (Chen et al., 2018).

Recipes

  1. PBS (pH 7.4)
    137 mM NaCl
    2.7 mM KCl
    10 mM disodium hydrogen phosphate
  2. 0.25 M sucrose buffer (pH 7.4)
    0.25 M sucrose
    10 mM Tris
    5.0 mM MgCl2
    Filter through a 0.22 μm filter and store at 4 °C
  3. 2.2 M sucrose buffer (pH 7.4)
    2.2 M sucrose
    10 mM Tris
    5.0 mM MgCl2
    Filter through a 0.22 μm filter and store at 4 °C
  4. DMEM culture medium
    450 ml of DMEM medium
    50 ml of fetal bovine serum (FBS)
    5 ml of penicillin-streptomycin (100x stock)
    5 ml of L-glutamine (200 mM, 100x stock)

Acknowledgments

The authors thank Shu Ying Lee for assistance in confocal microscopy. This project was supported by a Singapore National University Health System Basic Research grant (NUHSRO/2011/005/STB/B2B-01) and a National Medical Research Council grant (NMRC/OFIRG/0013/2016). Authors declare no conflict of interest or competing interests.

References

  1. Chen, J., Teo, B. H. D., Cai, Y., Wee, S. Y. K. and Lu, J. (2018). The linker histone H1.2 is a novel component of the nucleolar organizer regions. J Biol Chem 293(7): 2358-2369.
  2. Fischer, R. S. and Fowler, V. M. (2015). Thematic minireview series: The state of the cytoskeleton in 2015. J Biol Chem 290(28): 17133-17136.
  3. Gasser, S. M. (2002). Visualizing chromatin dynamics in interphase nuclei. Science 296(5572): 1412-1416.
  4. Gruenbaum, Y., and Foisner, R. (2015). Lamins: nuclear intermediate filament proteins with fundamental functions in nuclear mechanics and genome regulation. Annu Rev Biochem 84: 131-164.
  5. Hernandez-Verdun, D. (2011). Assembly and disassembly of the nucleolus during the cell cycle. Nucleus 2(3): 189-194.
  6. Jordan, E. G. and McGovern, J. H. (1981). The quantitative relationship of the fibrillar centres and other nucleolar components to changes in growth conditions, serum deprivation and low doses of actinomycin D in cultured diploid human fibroblasts (strain MRC-5). J Cell Sci 52: 373-389.
  7. Laemmli, U. K., Kas, E., Poljak, L. and Adachi, Y. (1992). Scaffold-associated regions: cis-acting determinants of chromatin structural loops and functional domains. Curr Opin Genet Dev 2(2): 275-285.
  8. Pederson, T. (2011). The nucleolus. Cold Spring Harb Perspect Biol 3(3).
  9. Shaw, P. J. and Jordan, E. G. (1995). The nucleolus. Annu Rev Cell Dev Biol 11: 93-121.
  10. Trimbur, G. M. and Walsh, C. J. (1993). Nucleolus-like morphology produced during the in vitro reassociation of nucleolar components. J Cell Biol 122(4): 753-766.

简介

每个细胞都含有许多大型DNA聚合物,其中包含大约一个核。直径10微米。用组蛋白,已知这些DNA聚合物形成染色质。染色质在核中如何进一步致密还不清楚,但它不可避免地依赖于广泛的非染色质核支架。内源性染色质网络的成像和支持该网络的互补支架尚未实现,但支架的生化和蛋白质组学研究仍然可以提供关于该染色质组织网络的重要见解。但是,这需要高度包容和可重复的提取核支架。我们最近开发了一个简单的协议,用于从染色质中释放脚手架组件。提取物的包容性由以下观察结果证实:当从核中提取时,剩余的核染色质被释放为延伸且通常平行的染色质纤维。基本上,该方案包括纯核的产生,用Triton X-100处理细胞核以产生包膜消耗的细胞核(TxN),并在含蔗糖的缓冲液中在500mM NaCl中提取细胞核。 TxN的这个组合提取被称为TxNE。

【背景】通过蛋白质和核糖核蛋白的复杂支架,染色质在细胞核中密集并动态地压缩。与细胞骨架网络不同(Fischer和Fowler,2015),对这种核支架的显微观察在技术上是具有挑战性的。这可能反映了每个细胞核内染色质的主导地位,支架与细胞核交织在一起。核的球形排列也对成像这种支架结构造成挑战。核支架的主要元素是核层(NL)(Gruenbaum和Foisner,2015)。 NL覆盖核包膜(NE)附着的整个核染色质块的表面。核染色质的外围表面具有致密的异染色质特征(Gruenbaum和Foisner,2015)。在细胞核内,可以发现一个或多个占据重要核区的核仁(Jordan and McGovern,1981; Shaw and Jordan,1995; Pederson,2011)。核仁表面也被稠密的染色质边缘包围,并且可能作为支持染色质的重要内部支架起作用(Chen等人,2018)。核仁的形态根据可显着影响核染色质组织的细胞周期阶段而动态变化(Hernandez-Verdun,2011; Chen等人,<2018>)。

尽管尚未实现直接可视化(Gasser,2002; Laemmli等人,1992),但是这种支架结构的存在在1980-90年代被广泛研究。然而,可以通过生物化学,蛋白质组学和成像研究来了解核支架的构成和支架元件之间的相互作用。为此,从染色质网络有效和可重复地提取核支架是至关重要的。我们最近开发了用于提取核支架蛋白的简单方案(Chen等人,2018)。

该协议最初是基于一项研究开发的,该研究显示分离的核仁可以在400mM NaCl溶解(Trimbur和Walsh,1993)。我们采用这个条件来溶解孤立的细胞核。提取物的包容性通过作为延伸平行染色质纤维的核染色质的释放来证明(Chen等人,2018)。 TxNE可用于研究核支架的许多方面。

关键字:核, 提取, 骨架, 核仁磷酸蛋白-1, 染色质

材料和试剂

  1. 0.5-10 L移液器吸头(Corning,Axygen ®,目录号:T-300)

  2. 10-200 ml移液枪头(Greiner Bio One International,目录号:739290)

  3. 100-1,000毫升枪头(Greiner Bio One International,目录号:686290)
  4. T 175细胞培养瓶(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:159910)
  5. T75细胞培养瓶(Thermo Fisher Scientific,Thermo Scientific TM,目录号:156499)
  6. 150mm圆形组织培养皿(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:168381)

  7. 50 ml Falcon管(Greiner Bio One International,目录号:227261)
  8. 5毫升平头注射器(5毫升/立方厘米,鲁尔滑,Cellotron,https://dcellotron.en.ec21.com/)

  9. 15 ml Falcon管(Greiner Bio One International,目录号:188271)
  10. Nalgene Oak Ridge高速离心管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:3114-0030)
  11. 常规1.5ml微量离心管(Corning,Axygen ,目录号:MCT-150-C)
  12. 低粘附微量离心管(Eppendorf,目录号:022431081)
  13. 12毫米玻璃盖玻片(电子显微镜科学,目录号:72231-10)
  14. 玻璃载玻片(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:6776215)
  15. 50毫升注射器(Terumo,目录号:SS * 50LE)
  16. 0.22μm过滤器(Merck,目录号:SLGP033RS)
  17. HeLa S3人宫颈癌细胞(ATCC,目录号:CCL-2)
  18. 胰蛋白酶溶液(0.05%w / v,摩羯座科学,目录号:TRY-1B10)
  19. 0.5%胰蛋白酶(胰蛋白酶-EDTA,10×储备,Thermo Fisher Scientific,Gibco TM,目录号:15400054)
  20. 蛋白酶抑制剂PMSF(BDH,目录号:442172C)
  21. Triton X-100(Sigma-Aldrich,目录号:T8787)
  22. 5 M NaCl
  23. 1%(w / v)多聚甲醛
  24. 小鼠抗核磷蛋白-1(NPM1)抗体(Sigma-Aldrich,目录号:B0556)
  25. 兔抗纤维蛋白B1(LB1)抗体(Abcam,目录号:ab16048)
  26. 兔抗组蛋白H1.2(Abcam,目录号:ab17677)
  27. (Santa Cruz Biotechnology,目录号:sc-7292)
  28. 兔抗H1.x抗体(Abcam,目录号:ab31972)
  29. 山羊抗小鼠IgG(Cy3-缀合的)(Jackson ImmunoResearch Laboratories,目录号:115-165-003)
  30. 山羊抗兔IgG(Alexa Fluor 488-结合的)(Jackson ImmunoResearch Laboratories,目录号:115-545-003)
  31. 安装介质(Vector实验室,目录号:H-1200)
  32. 任何透明指甲油(我们使用Daiso日本指甲油透明,10毫升瓶,台湾制造)
  33. 12.5%(w / v)凝胶
  34. 氯化钠(NaCl)(Merck)
  35. 氯化钾GR(KCl)(默克)
  36. 磷酸氢二钠(Merck)
  37. 蔗糖(第一BASE实验室,目录号:BIO-1090-1kg)
  38. 蔗糖(默克)
  39. Tris(默克)
  40. Tris碱(Avantor Performance Materials,J.T.Baker,目录号:4109-02)
  41. 氯化镁六水合物(Merck,目录号:1058331000)
  42. 氯化镁六水合物(MgCl 2·2H 2 O)(Merck)
  43. DMEM培养基(Thermo Fisher Scientific,Gibco TM,目录号:12430054)
  44. 胎牛血清(FBS,GE Healthcare,HyClone,目录号:SV30160.03)
    注:我们购买了FBS,并通过在56℃孵育30分钟产生热灭活的FBS。
  45. 青霉素 - 链霉素(100x原液,PAN Biotech,目录号:P06-07100)
  46. L-谷氨酰胺(200mM,100x储备液,Thermo Fisher Scientific,Gibco TM,目录号:25030081)。
  47. PBS,pH 7.4(见食谱)
  48. 0.25M蔗糖缓冲液,pH7.4(见食谱)
  49. 2.2 M蔗糖缓冲液,pH 7.4(见食谱)
  50. DMEM培养基(见食谱)

设备

  1. 1000升移液器(Eppendorf,型号:Research plus,产品目录号:3120000062)
  2. 200升移液器(Eppendorf,型号:Research ®plus,产品目录号:3120000054)
  3. 加湿的细胞培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:Forma TM Steri-Cycle TM CO 2,培养箱,目录编号:371)
  4. 均质机(Isobiotec Precision Engineering,德国海德堡)
  5. 台式微量离心机(Beckman Coulter,型号:Microfuge 22R离心机,目录号:368831)
  6. 台式摆动桶式离心机(Eppendorf,型号:5810 R,目录号:5811000320)
  7. 具有JA-20转子的Beckman Coulter离心机(Beckman Coulter,型号:Avanti J-25,目录号:363102)
  8. BSL-2细胞培养箱(Gelman,型号:Bioessential Class II系列A2型层流BSC,http://gelmansingapore.com/product/bioessential-class-ii-type-a2-series-laminar-flow-biological-safety -cabinets /)
  9. FluoView FV1000共聚焦显微镜
  10. 酷/ SNAP HQ2图像采集相机(奥林巴斯)

软件

  1. FV-ASW 1.6b软件
  2. Imaris软件(Bitplane AG)

程序

  1. 细胞匀浆的产生
    1. 培养HeLa细胞在T-75烧瓶中,在37℃潮湿的BSL-2培养箱中,含有25ml DMEM培养基进行融合。
    2. 将细胞等分为5个150mm圆形培养皿,并在DMEM培养基(25ml /平板)中培养至汇合。
    3. 取出培养基并用10 ml PBS清洗每个培养板。
    4. 加入2ml 1x胰蛋白酶溶液以覆盖每个平板的表面,并在37℃下在湿润细胞培养箱中孵育平板2分钟。

    5. 水平轻敲平板以悬浮细胞,并向每个平板添加6毫升培养基。
    6. 从每个板收获细胞,并结合在50ml Falcon管中。

    7. 在400×g离心5分钟收获细胞。
    8. 将细胞重新悬浮在50ml PBS中并通过在400×g下离心5分钟进行洗涤。重复此步骤一次。
    9. 将细胞重悬于4ml含有蛋白酶抑制剂PMSF(0.25mM)的0.25M蔗糖缓冲液中并移液以获得单细胞悬液。
    10. 使用10μm金属球组装一个isobiotec细胞匀浆器,并在冰上预冷细胞匀浆器(图1)。


      图1. Isobiotec细胞匀浆器的组装A. A.包括金属球(10μm间隙)的均化器的部分在白色管中; B.侧视图; C.金属球被放置在中央通道中。 D.完成均质器的组装。 E.每个出口配有5毫升注射器。 F.将2ml细胞悬液加入到一个注射器中(在此示例中使用水)。 G.按压将细胞悬液通过装有金属球的中央通道送入另一个注射器。
      在收集细胞匀浆前再重复11次

    11. 在匀浆器上安装两个出口,每个用5毫升注射器。
    12. 从一个注射器上卸下柱塞以添加2毫升细胞悬液。

    13. 通过细胞悬液12次通过均质器
    14. 将匀浆的细胞裂解物转移到15ml Falcon管中并用剩余的细胞悬液重复均质化。
    15. 用1ml的0.25M蔗糖缓冲液将均质器各洗涤2次,并与裂解物混合。

  2. 核的分离
    1. 在600×g和4℃下离心细胞匀浆10分钟以沉淀细胞核。
    2. 将沉淀重新悬浮在Nalgene Oak Ridge高速离心管中的5 ml 2.2 M蔗糖缓冲液中。

    3. 在50,000×g和4℃下离心30分钟。
    4. 去除上清液并加入2ml 0.25M蔗糖缓冲液以冲洗沉淀物附近的管道区域。

    5. 除去蔗糖缓冲液而不影响颗粒。
    6. 将沉淀重新悬浮于4ml 0.25M蔗糖缓冲液中并转移至15ml Falcon管中。
    7. 在600×g和4℃下离心5分钟。

  3. 核信封(NE)的消耗
    1. 制备含Triton的蔗糖缓冲液。

      1. 在15毫升管中沿管壁添加0.1毫升Triton X-100。
      2. 加入10毫升0.25M蔗糖缓冲液至管中,涡旋至Triton与蔗糖缓冲液充分混合。
    2. 重悬于4毫升含Triton的蔗糖缓冲液中的核沉淀,并在冰上孵育1小时。
    3. 在600×g和4℃下离心5分钟。

    4. 在另一个5毫升含有Triton的蔗糖缓冲液中重悬沉淀。
    5. 在600×g和4℃下离心5分钟。

    6. 用0.25M蔗糖缓冲液洗涤这些Triton提取的细胞核(TxN)2次
    7. 然后将沉淀重悬于0.9ml 0.25M蔗糖缓冲液中。

  4. 核支架的提取(图2)

    1. 添加0.1毫升5 M NaCl溶液到核悬浮液中(立即在界面处形成胶体凝胶)。
    2. 使用1,000 L Eppendorf移液管强力吸取20-30次,直到整个悬浮液变成均匀的胶体凝胶。

    3. 加0.5 ml含0.5 M NaCl的0.25 M蔗糖缓冲液和移液管使其均匀
    4. 如果清楚地观察到的话,以2000μgxg离心10分钟并回收少量上清液。
    5. 加入0.5 ml 0.25 M蔗糖缓冲液,胶体凝胶应立即收缩。

    6. 。使用0.2毫升移液管吸头强力打凝胶,使凝胶继续收缩。
    7. 在2000gxg和4℃下离心10分钟并回收澄清的上清液。
    8. 再加入0.5 ml的0.25 M蔗糖缓冲液,继续捅碎,直至其停止收缩(它应该看起来像一个棕色和不透明的碎片)。
    9. 再次离心并将所有上清液合并为提取物。
    10. 测量组合的TxN提取物或TxNE的A 280吸收。它应该在1.0-2.0范围内。
    11. 将TxNE分装在无粘性1.5-ml微管中,并保存在-80°C。


      图2.从细胞核中提取TxNE A.由6×10 8个HeLa细胞在1.35ml 2.5M蔗糖缓冲液中产生的单细胞悬液。 B.加入0.15ml 5M NaCl后,界面快速向上移动,在30秒内得到透明的较低相。 C.在不到1分钟的时间内剧烈吹打约30次后,整个细胞悬液变成单胶体凝胶。 D.将0.75ml含有0.5M NaCl的0.25M蔗糖缓冲液加入到凝胶中并通过轻敲约20次进行混合。 E.在2,000×g下离心10分钟,从顶部回收〜0.2ml上清液。 F.加入0.75ml的0.25蔗糖缓冲液(不含额外的NaCl)并剧烈混合1-2分钟以观察凝胶收缩。用吸管上的10-200μL吸头将凝胶打出20-50次,直到凝胶停止收缩。在2,000×g下离心10分钟并收集上清液。 G.将0.75ml的0.25M蔗糖缓冲液加入到沉淀中。 H.继续〜50次,直到颗粒收缩成不规则碎片。一些较小的碎片也是可见的。 I.在1,000×g下离心10分钟以回收上清液。 J.上清液合并为TxNE,沉淀通常是提取的染色质,保留细胞DNA和核心组蛋白。
      注意:为了更清楚地显示每个步骤中的样品状态,我们已经将方案主体文本中描述的溶液体积扩大了1.5倍以用于照片。 br />

数据分析

  1. TxNE的通常蛋白质谱高度一致(Chen等人,2018)。在TxNE隔离期间,可以监视某些步骤的一致性。首先,通过2.2 M蔗糖离心后分离的细胞核可以通过共聚焦显微镜检查免疫染色特征性NL蛋白lamin B1和特征性核仁蛋白核磷蛋白-1(NPM1)(图3)。尽管核仁有时可能变大,但分离的细胞核在LB1定义的NL中大部分保持连续。细胞核在形态上大部分保持椭圆形。这些细胞核应该没有波形蛋白,尽管如果细胞核未通过2.2M蔗糖缓冲液离心,它通常与核表面显着关联。尽管这些NE耗尽的核倾向于形成簇,但TxN应保持个体。 LB1定义的NL应该大部分是连续的,尽管TxN的形状在形态上可以不是椭圆形(图3)。


    图3.分离的和Triton提取的细胞核的完整性将HeLa细胞匀浆并通过2.2-M蔗糖缓冲液离心分离细胞核。将核用12-mm玻璃盖玻片在冰上孵育5分钟,然后在1%(w / v)多聚甲醛中固定30分钟。然后将细胞核在PBS中的1%(v / v)Triton X-100中温育30分钟。将细胞核再悬浮于含有1%(v / v)Triton X-100的0.25M蔗糖缓冲液中并在冰上温育1小时以产生Triton提取的细胞核(TxN)(右图)。 TxN在0.25M蔗糖缓冲液中洗涤,然后在冰上用12-mm玻璃盖玻片孵育5分钟。将盖玻片在1%(w / v)多聚甲醛中固定30分钟。首先将所有盖玻片与小鼠抗NPM1和兔抗LB1抗体孵育30分钟,洗涤后用山羊抗兔(Alexa Fluor 488,绿色)和抗小鼠(Cy3,红色)孵育30分钟, IgG抗体。洗涤后,使用含DAPI的VectorShield培养基安装盖玻片,并使用配备100x油物镜(孔径1.45)和Cool / SNAP HQ2图像采集相机(Olympus)的FluoView FV1000共焦显微镜观察。使用FV-ASW 1.6b软件捕获图像,并使用Imaris软件(Bitplane AG)进行分析。比例尺= 5微米。

  2. TxNE不包含显着水平的核心组蛋白(Chen等,<2018年)。然而,连接子H1组蛋白包含在此提取物中(图4)。小的LB1在TxNE中被提取(图4)。尽管LB1作为片段分散在扩展的染色质中,但它仍与提取的染色质相关(Chen等人,2018)。相反,lamin A / C用TxNE有效提取(图4)。


    图4.核染色质网络中核蛋白的选择性洗脱使用Triton X-100分离核并去除NE。用含有100,200,300,400或500mM NaCl的0.25M蔗糖缓冲液提取这些去除NE的细胞核(TxN)。等体积的上清液在12.5%(w / v)凝胶上进行SDS-PAGE并通过Western印迹分析。还包括用于提取的胞质溶胶部分和TxN作为对照。使用的所有抗体都是从Abcam获得的亲和纯化的多克隆兔抗体。

笔记

  1. 如上所述,该协议具有高度的可重复性。然而,如果单细胞悬液未达到,研究人员在细胞匀浆过程中可能会遇到困难。
  2. 该协议针对HeLa细胞进行了优化。对于其他细胞类型,需要优化金属球直径。通过对LB1,染色质和NPM1的简单免疫染色可以评估由此产生的细胞核的质量。
  3. 应通过SDS-PAGE和考马斯蓝染色检查TxNE以评估蛋白质谱中的一致性(Chen等人,2018)。

食谱

  1. PBS(pH 7.4)
    137mM NaCl
    2.7 mM KCl
    10 mM磷酸氢二钠
  2. 0.25 M蔗糖缓冲液(pH 7.4)
    0.25 M蔗糖
    10 mM Tris
    5.0mM MgCl 2


    通过0.22μm过滤器进行过滤并保存在4°C
  3. 2.2 M蔗糖缓冲液(pH 7.4)
    2.2 M蔗糖
    10 mM Tris
    5.0mM MgCl 2


    通过0.22μm过滤器进行过滤并保存在4°C
  4. DMEM培养基

    450毫升的DMEM培养基 50毫升胎牛血清(FBS)
    5毫升的青霉素 - 链霉素(100倍股票)
    5毫升的L-谷氨酰胺(200毫米,100倍股票)

致谢

作者感谢Shu Ying Lee在共聚焦显微镜中的帮助。该项目得到了新加坡国立大学卫生系统基础研究基金(NUHSRO / 2011/005 / STB / B2B-01)和国家医学研究委员会拨款(NMRC / OFIRG / 0013/2016)的支持。作者声明不存在利益冲突或利益冲突。

参考

  1. Chen,J.,Teo,B.H.D.,Cai,Y.,Wee,S.Y.K。和Lu,J。(2018)。 连接组蛋白H1.2是核仁组织区域的一个新组分。 < J Biol Chem 293(7):2358-2369。
  2. Fischer,R.S。和Fowler,V.M。(2015)。 主题minireview系列:2015年细胞骨架的状态。 J Biol Chem 290(28):17133-17136。
  3. Gasser,S.M。(2002)。 可视化间期细胞核中的染色质动力学 Science 296( 5572):1412-1416。
  4. Gruenbaum,Y.和Foisner,R.(2015)。 Lamins:在核力学和基因组调控中具有基本功能的核中间丝蛋白 Annu Rev Biochem 84:131-164。
  5. Hernandez-Verdun,D.(2011)。 细胞周期中核仁的装配和拆卸 Nucleus 2(3):189-194。
  6. Jordan,E.G。和McGovern,J.H。(1981)。 纤维中心和其他核仁成分与生长条件变化,血清剥夺和低剂量的放线菌素D在培养的二倍体人成纤维细胞(菌株MRC-5)中。 J Cell Sci 52:373-389。
  7. Laemmli,U.K。,Kas,E.,Poljak,L。和Adachi,Y。(1992)。 支架相关区域:染色质结构环和功能区的顺式作用决定簇。 Curr Opin Genet Dev 2(2):275-285。
  8. Pederson,T。(2011)。 核仁。 Cold Spring Harb Perspect Biol 3( 3)。
  9. Shaw,P.J。和Jordan,E.G。(1995)。 核仁。
  10. Trimbur,G.M。和Walsh,C.J。(1993)。 核仁组分在体外重新缔合过程中产生的类核仁形态 J细胞生物学 122(4):753-766。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Chen, J., Teo, B. H. and Lu, J. (2018). A Method for Extracting the Nuclear Scaffold from the Chromatin Network. Bio-protocol 8(8): e2821. DOI: 10.21769/BioProtoc.2821.
  2. Chen, J., Teo, B. H. D., Cai, Y., Wee, S. Y. K. and Lu, J. (2018). The linker histone H1.2 is a novel component of the nucleolar organizer regions. J Biol Chem 293(7): 2358-2369.
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