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May 2019

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Isolation and Imaging of His- and RFP-tagged Amyloid-like Proteins from Caenorhabditis elegans by TEM and SIM
TEM和SIM技术用于秀丽隐杆线虫His-和RFP标记淀粉样蛋白的分离和成像   

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Abstract

In our recently published paper, we highlight that during normal aging of C. elegans age-dependent aggregates of proteins form and lead to functional decline of tissues. The protocol described here details the isolation of two proteins from C. elegans in their aggregated amyloid-like form, casein kinase I isoform alpha (KIN-19) and Ras-like GTP-binding protein rhoA (RHO-1). We used nickel beads to isolate His-tagged KIN-19 and RHO-1, and thus permitting the isolation of both small and large aggregated or fibrillary forms of the proteins. We characterized their morphology by transmission electron microscopy. We further expressed RFP-tagged proteins and stained them with a fluorescent molecule, thioflavin T, which identifies β-sheet structures, and which is a defining feature of amyloid fibrils. We further applied structured illumination microscopy to determine the level of colocalization between RFP and thioflavin T.

Keywords: His-tag (His标签), RFP-tag (RFP标签), Amyloid (淀粉样), Aggregates (聚集物), Structured illumination microscopy (结构光照明显微镜), Transmission electron microscopy (透射电子显微镜), RHO-1 (RHO-1), KIN-19 (KIN-19)

Background

During aging, there is often a dysregulation in protein homeostasis and therefore an accumulation of damaged or non-functional proteins. Protein damage can occur by reactive oxygen species or post-translational modifications, along with mutations and transcriptional changes that naturally occur with age. One of the most studied forms of protein malfunction during aging are protein aggregates referred to as amyloids. These amyloids are found in many age-associated diseases such as Alzheimer’s disease and Parkinson’s disease. Soluble monomeric protein converts into an insoluble state where structure alters and proteins aggregate to form β-sheet rich fibrils. It is not clear whether the body is sequestering damaged or misfolded proteins into these amyloid fibrils when protein homeostasis is dysregulated or whether the formation of amyloid fibrils further dysregulates protein homeostasis, or both. Screening for proteins most likely to become insoluble upon aging Caenorhabditis elegans (C. elegans) yielded two target proteins, casein kinase I isoform alpha (KIN-19) and Ras-like GTP-binding protein rhoA (RHO-1) (David et al., 2010). These two proteins were further studied to investigate their aggregation propensity during the ageing of C. elegans, whether they formed amyloid-like structures and whether they correlated with a functional decline (Huang et al., 2019). In this protocol we first express the amyloid-like proteins KIN-19 and RHO-1 with a 6xHis-tag, this permits us to isolate the proteins with nickel beads. There can often be fibrous structures within small organisms such as C. elegans, but isolating His-tagged proteins ascertains that we isolate the protein of interest. Use of nickel beads rather than a nickel chromatography column allowed us to isolate large structures such as fibrils and aggregates which can get stuck in nickel columns. Subsequent imaging of eluted His-tagged proteins on a transmission electron microscope (TEM) permitted us to investigate the morphology of potential amyloid fibrils due to the sub-nanometer resolution provided by the TEM. However, a defining feature of amyloid fibrils is the presence of highly structured β-sheets which cannot be determined using TEM. Thus, in order to determine the presence of β-sheet structures in our proteins of interest we used structure illumination microscopy (SIM) which has ~200 nm resolution. We expressed red fluorescent protein (RFP)-tagged proteins in C. elegans and concomitantly stained them with thioflavin-T (ThT), a molecule which fluoresces when it intercalates into β-sheet structures. We then consequently investigated the colocalization of RFP and ThT fluorescence.

Materials and Reagents

  1. Microlance needle 30G x 0.5” (Fisher Scientific Lt, catalog number: 10442014)
  2. 1.5 ml microcentrifuge tubes (Fisher Brand, catalog number: FB74031)
  3. 1.5 ml plastic pestles (StarLab, catalog number: I1415-539)
  4. 400 mesh copper grids with carbon coat (EM Resolutions, catalog number: C400cu100)
  5. Transfer membrane PVDF 0.45 μm (Merck Millipore, catalog number: IVPH08100)
  6. HisTrap FF Crude column–extract the nickel beads (Merck, catalog number: GE11-0004-58)
  7. Nunc® Lab-Tek® II Chamber SlideTM system (Merck, catalog number: C7057-1PAK)
  8. 0.22 μm filter (Sartorius, catalog number: SAT16532K)
  9. C. elegans uqIs22 [Pkin19::kin19::hisavi + Pkin19::birAtagrfp] also called DCD242 in (Huang et al., 2019)
  10. C. elegans uqIS19 [Pmyo2::rho1::hisavi + Pmyo2::birAtagRFP] also called DCD243 in (Huang et al., 2019)
  11. C. elegans uqIs9 [Pmyo-2::rho-1::tagrfp + Ptph-1::gfp] also called DCD13 in (Huang et al., 2019)
  12. C. elegans fem-1(hc17ts) IV (Caenorhabditis genetic center, catalog number: BA17)
  13. Anti-6x His tag® [4D11] primary antibody (Abcam, catalog number: ab5000)
  14. Goat anti-Mouse IgG (H + L) Secondary Antibody, Alexa Fluor® 647 conjugate (Merck, catalog number: A21235)
  15. Amersham ECL Mouse IgG, HRP-linked whole Ab (from sheep) (GE Healthcare, catalog number: NA931-1ML)
  16. Bovine serum albumin (BSA) fraction V (Merck, catalog number: 9048-46-8)
  17. PBS tablets, Dulbeco A ,1 tablet per 100 ml H2O (Oxoid, catalog number: BR0014G)
  18. Radioimmunoprecipitation assay (RIPA) buffer (Merck, catalog number: R0278-50ML)
  19. Protease inhibitor tablets, Complete, Mini EDTA-free (Roche, catalog number: 11836170001)
  20. Imidazole (Merck, catalog number: I5513)
  21. HCl (Merck, catalog number: 84435)
  22. Thioflavin T (Abcam, catalog number: ab120751) -important to use within shelf life
  23. 4-12% Bis-Tris polyacrylamide gels (Invitrogen, catalog number: NP0323BOX)
  24. Supersignal West PicoPLUS Chemiluminescent Substrate (Thermo Fisher Scientific, catalog number: 34577)
  25. NuPAGETM LDS buffer (4x) (Thermo Fisher Scientific, catalog number: NP0007)
  26. NuPAGETM MES SDS Running buffer (20x) (Thermo Fisher Scientific, catalog number: NP0002)
  27. NuPAGETM Transfer Buffer (20x) (Thermo Fisher Scientific, catalog number: NP0006)
  28. Methanol (Alfa Aesar, catalog number: L123255.0F)
  29. Tween® 20 (Merck, catalog number: P1379)
  30. 0.1% (w/v) Poly-L-Lysine in H2O (Merck, catalog number: P8920-100ML)

Equipment

  1. Sonicator (SANYO, model: Soniprep 150)
  2. Benchtop cooled microcentrifuge (Thermo Scientific, model: Heraeus Fresco 21)
  3. Fixed speed rotator (Stuart, model: SB2)
  4. Microbalance (Sartorius, model: Secura 26-1S)
  5. pH meter (Mettler Toledo, model: Education line and inLab® Routine Pro probe)
  6. Dri-block Heat Block (Techne, model: DB-2TC)
  7. XCell SureLock® Mini-Cell and XCell IITM Blot module for running SDS-PAGE gels and transfer (Thermo Fisher Scientific)
  8. Power Pac (Bio-Rad, model: Power Pac 300)
  9. Roller mixer (Stuart, model: SRT1)
  10. G:Box Chemi imaging system (Syngene, model: XX6)
  11. Transmission electron microscope (TEM) (Thermo Fisher Scientific, model: Tecnai G2 80-200keV) 
  12. Glow discharge system with argon gas (Quorum Technologies, Model: EMITECH K100X) 
  13. Structured illumination microscope (SIM) (Custom built) (Muller et al., 2016)

Software

1. Fiji open source image processing program (https://fiji.sc)

Procedure

This protocol describes the preparation of worms for imaging techniques. For protocols on the number of worms to grow, growth conditions, collection and freezing of worms please refer to William B. W. (1988) and Groh et al. (2017).

  1. Lysis of C. elegans
    1. Defrost pellets of C. elegans expressing KIN-19::Hisavi and expressing RHO-1::Hisavi, these should ideally have been frozen in PBS with protease inhibitors (one protease inhibitor tablet per 10 ml of PBS, PBS is made with 1 tablet in 100 ml H2O). 
    2. To reduce C. elegans protein adherence to the microcentrifuge tubes, all tubes used in the experiments need to be incubated with 10% BSA in PBS for 10 min to coat the tubes.
    3. Using a pipette with the end cut off giving a wide pore to allow easy mixing of the suspension, resuspend the worm pellet mixture in a 1:1 ratio with radioimmunoprecipitation assay (RIPA) buffer with protease inhibitor tablets to a volume of 250 μl. 
    4. In a 1.7 ml microcentrifuge tube sonicate the suspension at an amplitude of 10 microns for 10 s on, 30 s off, 10 s on, on ice or in a cold room and further homogenise it manually with a plastic pestle for 10 strokes.
    5. Centrifuge the tubes for 1 min at 800 x g at 4 °C. The pellet contains the unwanted cuticle fragments and unlysed worms, carefully remove the supernatant. 
    6. Then further centrifuge the supernatant at 21,000 x g for 15 min at 4 °C. Carefully transfer the supernatant and resuspend the remaining pellet in 250 μl of PBS with protease inhibitors.
    7. Push and pull the resuspended pellet through a 30 G needle three times.

  2. Isolation of aggregated RHO-1 and KIN-19 by nickel beads
    1. By cutting the end of a HisTrap FF Crude column remove 20 µl of precharged Nickel Sepharose 6 Fast Flow beads and incubate them with the resuspended pellet overnight at 4 °C on a fixed speed rotator at 20 rpm.
      Note: Any nickel bead will likely work.
    2. The next day centrifuge the tubes at 800 x g for 1 min remove the supernatant (containing unbound proteins) to leave the His-tagged proteins, fibrils and beads.
    3. To remove the His-tagged aggregates and fibrils from the beads add 60 µl of 500 mM imidazole in PBS pH 8.0 (adjust pH with HCl) and incubate overnight at 4 °C on a fixed speed rotator at 20 rpm. 
    4. To isolate eluted aggregates and fibrils, centrifuge the beads at 800 x g for 1 min and collect the supernatant containing the aggregates and fibrils which are then used for imaging experiments.
      Note: We tried shorter times for His-tagged protein/fibril adherence to the beads and for elution, but this was not as successful as the overnight incubations. Unsuccessful elution protocol: incubate precharged Nickel Sepharose 6 Fast Flow beads with resuspended worm pellets for 15 min on a vortex mixer (Stuart, SA8) at 400 rpm. Then centrifuge the beads to pellet them at 800 x g for 1 min and remove the supernatant. Add 60 µl of 500 mM imidazole in PBS pH 8 (adjust pH with HCl) to the beads and incubated for 3 min on a vortex mixer at 400 rpm, but this was not long enough to successfully remove fibrils, shown by Western Blot and TEM in Figures 1A and 1B.

  3. Confirming protein capture and release with nickel beads by Western blot
    1. We tested the capture of protein aggregates and fibrils to the nickel beads and their successful elution by Western blot. Incubate 5 µl of crude worm pellet + 5 µl PBS, 3 µl of nickel beads after incubation + 7 µl PBS, 10 µl of supernatant after incubation and 10 µl of eluted aggregates with 4x LDS buffer at 100 °C in a heat block for 5 min. Use 10 µl of the sample to separate by 4-12% Bis-Tris SDS-PAGE gel and then transfer to a PVDF membrane for Western blotting. 
    2. Block the membrane with 5% BSA in PBS with 0.05% Tween-20 for 30 min to reduce background signal.
    3. Incubate the membrane with anti-6xHis tag primary antibody at 1:1000 with PBS and 0.05% Tween-20 on a roller for 1 h. Then wash the membrane three times in PBS and 0.05% Tween-20 for 5 min each time.
    4. Incubate the secondary antibody, anti-mouse-HRP, with the membrane at 1:1000 with PBS and 0.05% Tween-20 on a roller for 1 h. Wash the membrane five times in PBS and 0.05% Tween-20.
    5. Incubate the membrane with Supersignal West PicoPLUS Chemiluminescent Substrate for 5 min before imaging using the G:Box Chemi imager (Figure 1A).


      Figure 1. Representative Western blot and TEM images of successful and unsuccessful binding and elution of aggregated His-tagged proteins to nickel beads. A. Western blot using an α-6xHis primary antibody to show representative successful (lanes under blue line) elution of His-tagged RHO-1 and unsuccessful (lanes under black line) elution of KIN-19 from nickel beads. The α-6xHis has identified 6xHis-tagged RHO-1 and KIN-19 of varying sizes indicating the presence of aggregates as well as degraded protein. Negative-stain transmission electron micrographs showing (B) unsuccessful elution of KIN-19::His protein from nickel beads and (C) successful capture and elution of RHO-1::His (RHO-1 fibrils indicated by a blue arrow) and (D) KIN-19::His (KIN-19 fibrils indicated by a blue arrow). Grids are negatively stained using 2% uranyl acetate. Scale bar = 100 nm.

  4. Imaging isolated protein aggregates by TEM 
    1. Glow discharge the carbon coated copper grids using argon gas on a Quorum Technology K100X at 25mAmps for 2 min.
    2. Add 10 µl of protein sample to the grid and incubated for 1 min.
    3. Wash the grid twice for 5 min in dH2O by making a 200 μl droplet of dH2O and placing the grid in the droplet.
    4. Incubate the grid once in 10 μl 2% uranyl acetate for negative staining for 30 s before drying it by blotting with filter paper. 
    5. A Tecnai G2 80-200kv TEM at the Cambridge Advanced Imaging Centre was used for imaging the grids (Figures 1B-1D).
    6. The Tecnai imaging software allows you to do point-to-point measurements of fibril widths and lengths, the diameters of the RHO-1 and KIN-19 fibrils were ~15-20 nm. Point-to-point measurements can be used to determine periodicity/helicality of fibrils, although periodicity was not observed in the RHO-1 or KIN-19 fibrils.
      Note: We attempted to immunolabel the fibrils with the anti-6x His tag primary antibody and a secondary antibody conjugated to 10 nm gold beads, but the fibrils had been washed off the grids at the end of the staining process. It might be useful to not glow discharge the grids to keep it hydrophobic, allowing the fibrils to adhere better.

  5. Preparation of C. elegans protein extracts for imaging by SIM
    1. We initially tried to image RHO-1::His aggregates that were fluorescently labelled with antibodies, using the Anti-6x His tag primary antibody and the Goat anti-Mouse Alexa Fluor® 647 conjugate secondary antibody, but the extensive washing involved in antibody staining meant many aggregates were washed off the chamber slides and very few remained for imaging (Figure 2A). To obtain more aggregates and fibrils for fluorescence imaging, we instead used RHO-1::tagRFP worms which were already fluorescently labeled, and control worms which did not contain any amyloid aggregates as confirmed by the absence of ThT staining, fem-1(-). Process the worms as per Protocol A, Lysis of C.elegans, and use the crude worm pellet for imaging using SIM.
    2. Coat Nunc Lab-Tek II Chamber Slide wells with 0.1% poly-L-Lysine for 30 min at RT before incubating 200 μl of the resuspended crude worm pellets in the wells along with 50 µM of ThT in PBS for 1 h at RT, keep the chamber slide under foil to keep in the dark (ThT powder added to PBS and filtered through a 0.22 μm filter). 
    3. Remove the non-adhered pellet by pipetting and wash three times with 200 μl PBS by pipette before imaging (Figure 2B).
      Note: The ThT stock solution must be within its recommended usage period and freshly prepared for optimal binding and fluorescence.


      Figure 2. SIM imaging to confirm the presence of RHO-1 positive amyloid structures. Extracts from C. elegans worms were stained for amyloid structures using ThT and examined for colocalization with RHO-1. A. SIM showed colocalization of ThT positive structures and His-tag immunostained RHO-1 proteins. B. We also observed colocalization of ThT positive structures with RHO-1::RFP and observed both fibrillar and inclusion body-like structures. Scale bar: 1 μm.

  6. Imaging protein aggregates by SIM
    1. To visualize amyloids from worm extracts, we used our custom-built SIM providing a spatial resolution approaching 90 nm at frame rates reaching 22 Hz3.
    2. Objective-60x water.
    3. Exposure time: 25 ms for each channel.
    4. Wavelengths used:
      561 channel for RFP-Rho1
      488 channel for ThT
      647 channel for immuno-labeling with secondary antibody conjugated to AF647 dye.
    5. Hardware control and image reconstruction were performed with software written in LabView and Matlab (Ströhl and Kaminski., 2015). For visualization, Fiji was used.

Data analysis

  1. Image reconstruction
    Raw images acquired by SIM were reconstructed using the Fiji plugin LAG-SIM based on fairSIM (Figure 2, Young et al., 2016) 
  2. Parameter setting in LAG fairSIM-dialog
    Slice for parameters: 1
    Correction illumination: yes
    Filter type: RL-in, RL-out
    Wiener Parameter: 2.05
    Apodisation cutoff: 2.9
    Richardson-Lucy steps: 6
    OTF attenuation: yes
    Attenuation strength: 0.995
    Attenuation FWHM: 1.2
    Register images: yes (if acquiring multiple channels)
  3. To determine size or colocalization between the two channels, software such as particle analysis plugin (Schindelin et al., 2012) and the Coloc 2 plugin (Manders et al., 1993), respectively, in the image analysis software FIJI can be used

Acknowledgments

G.S.K.S. acknowledges funding from the Wellcome Trust, the UK Medical Research Council (MRC), Alzheimer Research UK (ARUK), and Infinitus China Ltd. This protocol was based on the research paper (Huang et al., 2019) in collaboration with Dr Della David.

Competing interests

None.

References

  1. David D, C., Ollikainen, N., Trinidad J, C., Cary M, P., Burlingame A, L. and Kenyon C. (2010). Widespread protein aggregation as an inherent part of aging in C. elegans. PLoS Biol 8(8): e1000450.
  2. Groh, N., Gallotta, I., Lechler, M. C., Huang, C., Jung, R. and David, D. C. (2017). Methods to study changes in inherent protein aggregation with age in Caenorhabditis elegans. J Vis Exp (129).
  3. Huang, C., Wagner-Valladolid, S., Stephens, A. D., Jung, R., Poudel, C., Sinnige, T., Lechler, M. C., Schlorit, N., Lu, M., Laine, R. F., Michel, C. H., Vendruscolo, M., Kaminski, C. F., Kaminski Schierle, G. S. and David, D. C. (2019). Intrinsically aggregation-prone proteins form amyloid-like aggregates and contribute to tissue aging in Caenorhabditis elegans. Elife 8: e43059.
  4. Manders, E. M. M., Verbeek, F. J. and Aten, J. A. (1993). Measurement of co‐localization of objects in dual-colour confocal images. J. Microsc. 169(3): 375-382.
  5. Muller, M., Monkemoller, V., Hennig, S., Hubner, W. and Huser, T. (2016). Open-source image reconstruction of super-resolution structured illumination microscopy data in ImageJ. Nat Commun 7: 10980.
  6. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J. Y., White, D. J., Hartenstein, V., Eliceiri, K., Tomancak, P. and Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7): 676-682.
  7. Ströhl, F. and Kaminski, C. F. (2015). A joint Richardson-Lucy deconvolution algorithm for the reconstruction of multifocal structured illumination microscopy data. Methods Appl Fluoresc 3(1): 014002.
  8. Young, L. J., Strohl, F. and Kaminski, C. F. (2016). A guide to structured illumination tirf microscopy at high speed with multiple colors. J Vis Exp (111).
  9. William, B. W. (1988). The nematode Caenorhabditis elegans. Cold Spring Harbor: Cold Spring Harbor Laboratory Press. ISBN: 978-087969433-3.

简介

在我们最近发表的论文中,我们着重指出了 C的正常老化过程中。 线虫的年龄依赖性蛋白质聚集体形成并导致组织功能下降。 这里描述的协议详细介绍了从 C中分离两种蛋白质的方法。 线虫聚集在一起的淀粉样蛋白形式,酪蛋白激酶I同工型α(KIN-19)和拉斯样GTP结合蛋白rhoA(RHO-1)。 我们使用镍珠分离出带有His标签的KIN-19和RHO-1,因此可以分离出大小的聚集或原纤维状蛋白质。 我们通过透射电子显微镜表征了它们的形态。 我们进一步表达了RFP标记的蛋白,并用荧光分子thioflavin T对其进行了染色,该蛋白可鉴定β-折叠结构,并且是淀粉样蛋白原纤维的定义特征。 我们进一步应用结构化照明显微镜来确定RFP和硫黄素T之间的共定位水平。
【背景】在衰老过程中,蛋白质体内平衡常常失调,因此积累了受损或无功能的蛋白质。蛋白质的破坏可能是由于活性氧或翻译后修饰,以及随着年龄的增长而自然发生的突变和转录变化。衰老过程中蛋白质功能障碍研究最多的形式之一是称为淀粉样蛋白的蛋白质聚集体。这些淀粉样蛋白存在于许多与年龄相关的疾病中,例如阿尔茨海默氏病和帕金森氏病。可溶性单体蛋白质转变为不溶状态,在该状态下结构发生变化,蛋白质聚集形成富含β-折叠的原纤维。尚不清楚当蛋白质稳态失调时,人体是否将被破坏或折叠错误的蛋白质螯合到这些淀粉样原纤维中,或者淀粉样原纤维的形成是否进一步失调了蛋白质稳态,或者两者都不清楚。筛选最容易因衰老的秀丽隐杆线虫( C。elegans )蛋白而产生的两种靶蛋白,酪蛋白激酶I亚型α(KIN-19)和Ras样GTP结合蛋白rhoA(RHO-1)(David et al。,2010年)。进一步研究了这两种蛋白质,以研究其在C老化过程中的聚集倾向。线虫,它们是否形成淀粉样结构,是否与功能下降相关(Huang et al。,2019)。在该协议中,我们首先表达带有6xHis标签的淀粉样蛋白KIN-19和RHO-1,这使我们可以分离带有镍珠的蛋白。小型生物(例如 C)中通常可以存在纤维结构。线虫,但分离出带有His标签的蛋白后,可以确定我们分离出了目标蛋白。使用镍珠而不是镍色谱柱可以使我们分离出可能卡在镍柱中的大型结构,例如原纤维和聚集体。随后在透射电子显微镜(TEM)上对洗脱的His标记蛋白进行成像,这使我们能够研究由于TEM提供的亚纳米级分辨率而导致的潜在淀粉样原纤维的形态。然而,淀粉样蛋白原纤维的定义特征是高度结构化的β-折叠的存在,其不能使用TEM确定。因此,为了确定我们感兴趣的蛋白质中β-折叠结构的存在,我们使用了结构照明显微镜(SIM),分辨率约为200 nm。我们在 C中表达了带有红色荧光蛋白(RFP)标签的蛋白。线虫并伴随着它们被硫黄素-T(ThT)染色,该分子在插入β-折叠结构时会发出荧光。因此,我们随后研究了RFP和ThT荧光的共定位。

关键字:His标签, RFP标签, 淀粉样, 聚集物, 结构光照明显微镜, 透射电子显微镜, RHO-1, KIN-19

材料和试剂

  1. Microlance针30G x 0.5英寸(Fisher Scientific Lt,目录号:10442014)
  2. 1.5 ml微量离心管(Fisher品牌,目录号:FB74031)
  3. 1.5毫升塑料杵(StarLab,目录号:I1415-539)
  4. 具有碳涂层的400目铜网格(EM决议,目录号:C400cu100)
  5. 转印膜PVDF 0.45μm(Merck Millipore,目录号:IVPH08100)
  6. HisTrap FF粗柱–提取镍珠(Merck,目录号:GE11-0004-58)
  7. Nunc ® Lab-Tek ® II Chamber Slide TM 系统(Merck,货号:C7057-1PAK)
  8. 0.22μm滤光片(Sartorius,目录号:SAT16532K)
  9. C。秀丽线虫 uqIs22 [Pkin19 :: kin19 :: hisavi + Pkin19 :: birAtagrfp]在(Huang et al。,2019)中也称为DCD242
  10. C。线虫 uqIS19 [Pmyo2 :: rho1 :: hisavi + Pmyo2 :: birAtagRFP]在(Huang et al。,2019)中也称为DCD243
  11. C。线虫 uqIs9 [Pmyo-2 :: rho-1 :: tagrfp + Ptph-1 :: gfp]在(Huang et al。,2019)中也称为DCD13
  12. C。秀丽线虫fem-1(hc17ts)IV(秀丽隐杆线虫遗传学中心,货号:BA17)
  13. 抗6x His标签® [4D11]一抗(Abcam,目录号:ab5000)
  14. 山羊抗小鼠IgG(H + L)二抗,Alexa Fluor ® 647共轭物(默克,目录号:A21235)
  15. Amersham ECL小鼠IgG,与HRP相连的完整抗体(来自绵羊)(GE Healthcare,目录号:NA931-1ML)
  16. 牛血清白蛋白(BSA)组分V(Merck,目录号:9048-46-8)
  17. PBS片剂,Dulbeco A,每100 ml H 2 O 1片(阿片类药物,目录号:BR0014G)
  18. 放射免疫沉淀测定(RIPA)缓冲液(默克(Merck),目录号:R0278-50ML)
  19. 蛋白酶抑制剂片剂,完全,不含EDTA的迷你制剂(Roche,目录号:11836170001)
  20. 咪唑(Merck,目录号:I5513)
  21. HCl(Merck,目录号:84435)
  22. 硫黄素T(Abcam,目录号:ab120751)-在保质期内重要使用
  23. 4-12%Bis-Tris聚丙烯酰胺凝胶(Invitrogen,目录号:NP0323BOX)
  24. Supersignal West PicoPLUS化学发光基质(Thermo Fisher Scientific,目录号:34577)
  25. NuPAGE TM LDS缓冲区(4x)(Thermo Fisher Scientific,目录号:NP0007)
  26. NuPAGE TM MES SDS运行缓冲区(20x)(Thermo Fisher Scientific,目录号:NP0002)
  27. NuPAGE TM 传输缓冲液(20x)(Thermo Fisher Scientific,目录号:NP0006)
  28. 甲醇(Alfa Aesar,货号:L123255.0F)
  29. Tween ® 20(默克,目录号:P1379)
  30. H 2 O中的0.1%(w / v)聚-L-赖氨酸(Merck,目录号:P8920-100ML)

设备

  1. Sonicator(SANYO,型号:Soniprep 150)
  2. 台式冷却微量离心机(Thermo Scientific,型号:Heraeus Fresco 21)
  3. 定速旋转器(Stuart,型号:SB2)
  4. 微量天平(Sartorius,型号:Secura 26-1S)
  5. pH计(Mettler Toledo,型号:Education line和inLab ® Routine Pro探针)
  6. Dri-block加热块(技术,型号:DB-2TC)
  7. XCell SureLock ® Mini-Cell和XCell II TM 印迹模块,用于运行SDS-PAGE凝胶和转移(Thermo Fisher Scientific)
  8. Power Pac(Bio-Rad,型号:Power Pac 300)
  9. 滚筒式搅拌机(Stuart,型号:SRT1)
  10. G:Box Chemi成像系统(Syngene,型号:XX6)
  11. 透射电子显微镜(TEM)(Thermo Fisher Scientific,型号:Tecnai G2 80-200keV)
  12. 带氩气的辉光放电系统(Quorum Technologies,型号:EMITECH K100X)
  13. 结构照明显微镜(SIM)(定制)(Muller 等人,2016年)

软件

1.斐济开源图像处理程序( https://fiji.sc )

程序

该协议描述了用于成像技术的蠕虫的制备。有关蠕虫的数量,生长条件,蠕虫的收集和冻结的协议,请参阅William B. W.(1988)和Groh et al。(2017)。

  1. C的裂解。线虫
    1. 为 C除霜。代表KIN-19 :: Hisavi和RHO-1 :: Hisavi的秀丽线虫,理想情况下应将它们与蛋白酶抑制剂一起冷冻在PBS中(每10毫升PBS含一片蛋白酶抑制剂片,PBS制成1片100 ml H 2 O)。
    2. 降低 C。线虫蛋白质附着在微量离心管上,实验中使用的所有试管均需与PBS中的10%BSA孵育10分钟,以覆盖试管。
    3. 使用移液器将末端切除以提供宽阔的孔,以使悬浮液易于混合,然后将蠕虫沉淀混合物以1:1的比例用放射免疫沉淀测定(RIPA)缓冲液和蛋白酶抑制剂片剂重悬至250μl的体积。 ;
    4. 在1.7 ml微量离心管中,在冰上或冷藏室中,以10微米的振幅对悬浮液进行10 s开,30 s关,10 s开的声处理,在冰上或在冷室中进行超声处理,然后用塑料杵将其手动匀浆10次。
    5. 在4°C下以800 x g 的速度离心管1分钟。沉淀中含有多余的表皮碎片和未溶解的蠕虫,请小心除去上清液。
    6. 然后在4°C下以21,000 x g 进一步离心上清液15分钟。小心地转移上清液,并将剩余的沉淀物重悬于250μl含蛋白酶抑制剂的PBS中。
    7. 通过30 G针将重悬的沉淀物推入并拉出3次。

  2. 镍珠分离聚集的RHO-1和KIN-19
    1. 通过切下HisTrap FF粗柱的末端,取出20 µl预装的镍Sepharose 6 Fast Flow珠,然后将其与重悬浮的沉淀物在4°C的恒速旋转器上以20 rpm孵育过夜。
      注意:任何镍珠都可能起作用。
    2. 第二天,将离心管以800 xem离心1分钟,除去上清液(含有未结合的蛋白质),留下带有His标记的蛋白质,原纤维和珠子。
    3. 为了从珠子上除去带有His标签的聚集物和原纤维,在PBS pH 8.0中加入60 µl 500 mM咪唑(用HCl调节pH),并在固定转速下以20 rpm在4°C下孵育过夜。
    4. 为了分离洗脱的聚集体和原纤维,将珠子在800 x g 下离心1分钟,收集含有聚集体和原纤维的上清液,然后将其用于成像实验。
      注意:我们尝试过较短的时间以使His标记的蛋白/原纤维粘附于珠子上并进行洗脱,但这并不像过夜孵育那样成功。洗脱方案未成功:将预充电的镍琼脂糖6快速流动珠与重悬浮的蠕虫沉淀在涡旋混合器(Stuart,SA8)上以400 rpm孵育15分钟。然后将珠子离心,以800 x g 的速度沉淀1分钟,然后除去上清液。向微珠中加入60 µl PBS pH 8中的500 mM咪唑(用HCl调节pH),并在涡旋混合器上以400 rpm孵育3分钟,但这时间不足以成功去除原纤维,如Western Blot和TEM所示在图1A和1B中。

  3. 通过蛋白质印迹法确认蛋白质的捕获和镍珠释放
    1. 我们测试了蛋白质聚集体和原纤维捕获到镍珠中的情况,并通过蛋白质印迹法成功地洗脱了它们。将5 µl蠕虫粗粒沉淀+ 5 µl PBS,3 µl孵育后的镍珠+ 7 µl PBS,10 µl孵育后的上清液和10 µl洗脱的聚集体与4x LDS缓冲液一起在100℃下于加热块中孵育5分钟。分钟使用10 µl样品通过4-12%Bis-Tris SDS-PAGE凝胶分离,然后转移到PVDF膜上进行蛋白质印迹。
    2. 用5%BSA的PBS和0.05%Tween-20的PBS封闭膜30分钟以减少背景信号。
    3. 用PBS和0.05%Tween-20在滚轮上以1:1000将抗6xHis标签一抗的膜孵育1小时。然后将膜在PBS和0.05%Tween-20中清洗3次,每次5分钟。
    4. 用PBS和0.05%Tween-20在滚筒上以1:1000将二抗anti-mouse-HRP孵育1小时。用PBS和0.05%Tween-20清洗膜五次。
    5. 在使用G:Box Chemi成像仪进行成像之前,将膜与Supersignal West PicoPLUS化学发光底物一起孵育5分钟(图1A)。


      图1.成功的和未成功结合的His标签蛋白与镍珠的结合和洗脱的代表性Western印迹和TEM图像。 A.使用α-6xHis一抗显示代表性成功(泳道)的Western印迹蓝色标记)洗脱带His标签的RHO-1,镍珠中KIN-19洗脱不成功(黑色标记带)。 α-6xHis已鉴定出6xHis标记的RHO-1和KIN-19,大小不同,表明存在聚集体和降解的蛋白质。负染色透射电子显微照片,显示(B)从镍珠上洗脱KIN-19 :: His蛋白失败,以及(C)成功捕获和洗脱RHO-1 :: His(蓝色箭头指示的RHO-1原纤维)和(D)KIN-19 :: His(蓝色箭头指示KIN-19原纤维)。使用2%乙酸铀酰对网格进行负染色。比例尺= 100 nm。

  4. 通过TEM对分离的蛋白质聚集体进行成像
    1. 使用Quorum Technology K100X上的氩气以25mAmps的辉光将碳涂层的铜栅格放电2分钟。
    2. 将10 µl蛋白质样品添加到网格中,并孵育1分钟。
    3. 通过在dH 2 O中洗涤200升dH 2 O液滴两次,将网格洗涤5分钟,然后将网格放在液滴中。
    4. 将其在10μl2%乙酸铀酰中孵育一次,以进行30 s负染色,然后通过滤纸吸干将其干燥。
    5. 剑桥高级成像中心的Tecnai G2 80-200kv TEM用于对网格成像(图1B-1D)。
    6. Tecnai成像软件允许您对原纤维的宽度和长度进行点对点测量,RHO-1和KIN-19的原纤维直径约为15-20 nm。点对点测量可用于确定原纤维的周期性/螺旋性,尽管在RHO-1或KIN-19原纤维中未观察到周期性。
      注意:我们试图用抗6x His标签的一抗和与10 nm金珠缀合的二抗对纤维进行免疫标记,但是在染色过程结束时,将纤维从网格上洗了下来。最好不要通过辉光放电使网格保持疏水性,从而使原纤维更好地粘附。

  5. 制备 C。线虫蛋白提取物用于SIM成像
    1. 我们最初尝试使用Anti-6x His标签一抗和Goat抗小鼠Alexa Fluor ® 647共轭二抗,对用抗体进行荧光标记的RHO-1 :: His成像,但是抗体染色所涉及的大量洗涤意味着从室载玻片上洗去了许多聚集体,几乎没有剩余的用于成像(图2A)。为了获得更多的聚集体和原纤维用于荧光成像,我们改为使用已经进行了荧光标记的RHO-1 :: tagRFP蠕虫,以及不含ThT染色, fem- 1(-)。按照协议A,C.elegans 的裂解方法处理蠕虫,并使用粗蠕虫颗粒通过SIM进行成像。
    2. 在室温下将含有0.1%聚L-赖氨酸的Nunc Lab-Tek II腔室玻片孔在室温下孵育30分钟,然后将200μl重悬浮的粗蠕虫颗粒与50μMThT的PBS溶液一起在室温下孵育1 h,保持室温腔室在箔片下方滑动以保持黑暗(将ThT粉末添加到PBS中并通过0.22μm过滤器过滤)。
    3. 通过移液除去未粘附的沉淀,并在成像之前用移液器用200μlPBS洗涤3次(图2B)。
      注意:ThT储备溶液必须在建议的使用期限内,并且应新鲜制备,以实现最佳结合和荧光。


      图2. SIM成像以确认RHO-1阳性淀粉样蛋白结构的存在。 C的提取物。线虫使用ThT对淀粉样结构进行染色,并与RHO-1进行共定位。 A. SIM显示ThT阳性结构与His-tag免疫染色的RHO-1蛋白共定位。 B.我们还观察到ThT阳性结构与RHO-1 :: RFP的共定位,并观察到纤维状和包涵体样结构。规模
      条:1μm。

  6. 通过SIM成像蛋白质聚集体
    1. 为了可视化蠕虫提取物中的淀粉样蛋白,我们使用了定制的SIM,在帧速率达到22 Hz 3 时提供了接近90 nm的空间分辨率。
    2. 物镜-60x水。
    3. 曝光时间:每个通道25毫秒。
    4. 使用的波长:
      RFP-Rho1的561通道
      488通道用于ThT
      647通道,用于与AF647染料偶联的二抗进行免疫标记。
    5. 硬件控制和图像重建使用LabView和Matlab(Ströhland Kaminski。,2015)编写的软件进行。为了进行可视化,使用了斐济。

数据分析

  1. 图像重建
    通过使用基于fairSIM的斐济插件LAG-SIM重建了通过SIM获取的原始图像(图2,Young et al。,2016)。
  2. LAG fairSIM-dialog中的参数设置
    参数切片:1
    校正照明:是
    过滤器类型:RL-in,RL-out
    维纳参数:2.05
    切趾截止:2.9
    理查森-露西步骤:6
    OTF衰减:是
    衰减强度:0.995
    衰减FWHM:1.2
    注册图像:是(如果获取多个通道)
  3. 为了确定两个通道之间的大小或共定位,可以使用粒子分析插件(Schindelin等,2012)和Coloc 2插件(Manders等,1993)之类的软件。 ),分别可以在图像分析软件FIJI中使用

致谢

G.S.K.S.感谢Wellcome Trust,英国医学研究理事会(MRC),英国阿尔茨海默病研究(ARUK)和Infinitus China Ltd.的资助。该协议基于研究论文(Huang et al。,2019 )与Della David博士合作。

利益争夺

没有。

参考文献

  1. David D.C.,Ollikainen,N.,Trinidad J.C.,Cary M.P.,Burlingame A.L.和Kenyon C.(2010年)。 广泛传播的蛋白质聚集是 C衰老的固有部分。线虫。 PLoS Biol 8(8):e1000450。
  2. Groh,N.,Gallotta,I.,Lechler,M. C.,Huang,C.,Jung,R.和David,D.C.(2017)。 研究秀丽隐杆线虫中固有蛋白质聚集随年龄的变化的方法。 J Vis Exp (129)。
  3. Huang,C.,Wagner-Valladolid,S.,Stephens,AD,Jung,R.,Poudel,C.,Sinnige,T.,Lechler,MC,Schlorit,N.,Lu,M.,Laine,RF,Michel ,CH,Vendruscolo,M.,Kaminski,CF,Kaminski Schierle,GS和David,DC(2019)。 固有地,易于聚集的蛋白形成淀粉样样聚集体,并导致秀丽隐杆线虫的组织衰老。 。 Elife 8:e43059。
  4. Manders,E.M.M.,Verbeek,F.J。和Aten,J.A。(1993)。 双色共焦图像中对象共定位的测量 J。 169(3):375-382。
  5. M.Muller,V.Monkemoller,S.Hennig,W.Hubner和T.Huser(2016)。 ImageJ中超分辨率结构化照明显微镜数据的开源图像重建。 Nat Commun 7:10980。
  6. Schindelin,J.,Arganda-Carreras,I.,Frise,E.,Kaynig,V.,Longair,M.,Pietzsch,T.,Preibisch,S.,Rueden,C.,Saalfeld,S.,Schmid,B ,Tinevez,JY,White,DJ,Hartenstein,V.,Eliceiri,K.,Tomancak,P.和Cardona,A.(2012)。 斐济:用于生物图像分析的开源平台。 Nat方法 9(7):676-682。
  7. Ströhl,F.和Kaminski,C.(2015)。 Richardson-Lucy联合反卷积算法,用于重建多焦点结构照明显微镜数据。 Methods Appl Fluoresc 3(1):014002。
  8. Young,L.J.,Strohl和F.Kaminski,C.(2016年)。 结构化照明Tirf显微镜高速使用多种颜色的指南。 J Vis Exp (111)。
  9. 威廉·B·W·(1988)。 线虫秀丽隐杆线虫。冷泉港:冷泉港实验室出版社。 ISBN:978-087969433-3。
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Copyright Stephens 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. Stephens, A. D., Lu, M. and Kaminski Schierle, G. S. (2019). Isolation and Imaging of His- and RFP-tagged Amyloid-like Proteins from Caenorhabditis elegans by TEM and SIM. Bio-protocol 9(21): e3408. DOI: 10.21769/BioProtoc.3408.
  2. Huang, C., Wagner-Valladolid, S., Stephens, A. D., Jung, R., Poudel, C., Sinnige, T., Lechler, M. C., Schlorit, N., Lu, M., Laine, R. F., Michel, C. H., Vendruscolo, M., Kaminski, C. F., Kaminski Schierle, G. S. and David, D. C. (2019). Intrinsically aggregation-prone proteins form amyloid-like aggregates and contribute to tissue aging in Caenorhabditis elegans. Elife 8: e43059.
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