Xenopus laevis Oocytes Preparation for in-Cell EPR Spectroscopy
制备非洲爪蟾卵母细胞用于细胞内EPR光谱学分析   

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Physical Chemistry Chemical Physics
Jul 2017

Abstract

One of the most exciting perspectives for studying bio-macromolecules comes from the emerging field of in-cell spectroscopy, which enables to determine the structure and dynamics of bio-macromolecules in the cell. In-cell electron paramagnetic resonance (EPR) spectroscopy in combination with micro-injection of bio-macromolecules into Xenopus laevis oocytes is ideally suited for this purpose. Xenopus laevis oocytes are a commonly used eukaryotic cell model in different fields of biology, such as cell- and development-biology. For in-cell EPR, the bio-macromolecules of interest are microinjected into the Xenopus laevis oocytes upon site-directed spin labeling. The sample solution is filled into a thin glass capillary by means of Nanoliter Injector and after that microinjected into the dark animal part of the Xenopus laevis oocytes by puncturing the membrane cautiously. Afterwards, three or five microinjected Xenopus laevis oocytes, depending on the kind of the final in-cell EPR experiment, are loaded into a Q-band EPR sample tube followed by optional shock-freezing (for experiment in frozen solution) and measurement (either at cryogenic or physiological temperatures) after the desired incubation time. The incubation time is limited due to cytotoxic effects of the microinjected samples and the stability of the paramagnetic spin label in the reducing cellular environment. Both aspects are quantified by monitoring cell morphology and reduction kinetics.

Keywords: Xenopus laevis oocytes (非洲爪蟾卵母细胞), in-Cell EPR (细胞内EPR), in-Cell spectroscopy (细胞内光谱学分析), Site-directed spin labeling (位置定向自旋标记), Microinjection (显微注射), in vivo structure determination (体内结构测定), Dynamics of biomacromolecules (生物大分子动力学)

Background

Electron paramagnetic resonance (EPR) spectroscopy is the method of choice for characterization of paramagnetic systems (Atherton, 1993; Gerson et al., 1994; Jeschke and Schweiger, 2001). Diamagnetic bio-macromolecules can be made accessible for EPR spectroscopy by site-directed spin labeling (SDSL), commonly using nitroxides as spin labels (Hubbell and Altenbach, 1994; Feix and Klug, 2002; Likhtenshtein et al., 2008; Berliner and Reuben, 2012). The combination of SDSL with in-cell EPR spectroscopy is a powerful tool to gain information about structure and dynamics of bio-macromolecules such as proteins or nucleotides in their natural environment (Azarkh et al., 2013; Martorana et al., 2014; Qi et al., 2014; Cattani et al., 2017). The most common experimental procedure for the fledging technique of in-cell EPR is based on the microinjection of the target molecules into oocytes from the African frog Xenopus laevis, which are a widely used eukaryotic cell model (Kay, 1991; Barnard et al., 1982; Mishina et al., 1984; Dawid and Sargent, 1988; Richter, 1999).

The advantages of Xenopus laevis oocytes for in-cell EPR are the large size of approximately 1 mm in diameter (approximately 1 µl cell volume), the resulting easy handling and the fact that only three or five of them are required for an in-cell EPR sample (Qi et al., 2014; Cattani et al., 2017). Consequently, bio-macromolecules can be introduced relatively easily in the quantity required for EPR measurements into the Xenopus laevis oocyte by microinjection. Hence, there have been numerous intracellular distance measurements of spin labelled DNA and proteins performed by double electron-electron resonance (DEER) measurements after microinjection into Xenopus laevis oocytes (Igarashi et al., 2010; Azarkh et al., 2011; Krstic et al., 2011; Azarkh et al., 2013; Martorana et al., 2014; Wojciechowski et al., 2015; Cattani et al., 2017).

Materials and Reagents

  1. Glass capillaries (3.5 inch length, Drummond Scientific, catalog number: 3-000-203-G/X )
  2. Single-use syringe (Sigma-Aldrich, catalog number: Z230723 )
  3. Parafilm (Sigma-Aldrich, catalog number: P7793-1EA )
  4. Petri dish, size 60 x 15 mm (Corning, catalog number: 430166 )
  5. Razor blade (Plano, catalog number: T5016 )
  6. Pasteur capillary pipette (150 mm, WU Mainz)
  7. Brand pipette controller micro-classic (BRAND, catalog number: 25900 )
  8. Q-band sample tubes (quartz glass, 1 mm i.d., Bruker, catalog number: ER 221TUB-Q10 )
  9. Hamilton syringe (Hamilton, catalog number: 80500 )
  10. Petri dish, size 35 x 10 mm (Corning, catalog number: 430165 )
  11. Capillary tube sealing compound (Cha-seal, DWK Life Sciences, Kimble, catalog number: 43510 )
  12. Xenopus laevis oocytes on stage V/VI in MBS buffer (Ecocyte Bioscience, ecocyte-us.com/products/xenopus-oocyte-delivery-service/)
  13. Modified Barth’s Saline (MBS) buffer (1x) (Ecocyte Bioscience) (88 mM NaCl, 1 mM KCl, 1 mM MgSO4, 5 mM HEPES, 2.5 mM NaHCO3, 0.7 mM CaCl2)
  14. Mineral oil (Sigma-Aldrich, catalog number: M5904 )
  15. Liquid nitrogen
  16. 3-Maleimido-PROXYL (3-Maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxy) (Sigma-Aldrich, catalog number: 253375 )

Equipment

  1. Flaming/Brown Micropipette Puller (Sutter Instrument, model: P-97 )
  2. Nanoject II Auto-Nanoliter Injector (Drummond Scientific, catalog number: 3-000-205A )
  3. Micromanipulator MM33 (Drummond Scientific, catalog number: 3-000-024-R ) with Support Base (Drummond Scientific, catalog number: 3-000-025-SB )
  4. Binocular microscope (ZEISS, model: Stemi 2000-C , attended with an AxiaCam ERc 5s camera (ZEISS, model: AxiaCam ERc 5s ))
  5. Home-built polytetrafluoroethylene holder
  6. Dewar for liquid nitrogen (KGW-Isotherm, catalog number: 1021 )
  7. -80 °C freezer

Procedure

  1. Preparation of the injector glass capillary
    1. The desired thin top of the glass capillary is formed with the Flaming/Brown Micropipette Puller. The program parameters are chosen as follows: Pressure Setting (P) = 500, Heat = 558, Pull = 100, Velocity (VEL) = 120 and Time = 100 msec (agrees with 200 units).
    2. The exact position of the Nanoliter Injector can be adjusted with the micromanipulator. At the front of the Nanoliter Injector are a screw clip and a stamp, the position of the latter is controlled by the control computer (see Figure 1).


      Figure 1. Assembly of the microinjection equipment. A. Magnified view of the control computer; B. Binocular microscope and microinjection equipment; C. Magnified view of the top of the Nanoliter Injector.

    3. To avoid air pockets between the stamp and the injection solution, the sharpened glass capillary is filled up with mineral oil using a single-use syringe. Afterwards, the filled glass capillary is clamped over the stamp into the screw clip of the Nanoliter Injector. Exiting mineral oil is wiped away with a tissue.
    4. For the opening of the clamped glass capillary, Parafilm is stretched over the back of a Petri dish (size 60 x 15 mm). A small mark is pressed into the Parafilm at the margin of the Petri dish. The glass capillary is slowly moved towards the mark on the Petri dish with the micromanipulator under observation through the binocular microscope. When the glass capillary starts touching the Parafilm, it is put down horizontally on the Parafilm by simultaneously lowering the capillary with the micromanipulator and moving the Petri dish away from the latter. The laid down, forward piece of the glass capillary (around 2.5 mm) is cut carefully with a razor blade.
    5. Subsequently, the mineral oil is pressed out of the opened glass capillary up to around 2 cm with the stamp of the Nanoliter Injector. This run-off can take up to 15 min.
    6. The top of the prepared glass capillary is dipped into a drop of the sample solution (2-4 µl depending on the number of Xenopus laevis oocytes to be microinjected). The sample solution is soaked up into the capillary by using the Nanoliter Injector (see Figure 2 for prepared injector glass capillary). 


      Figure 2. Ready prepared Xenopus laevis oocytes and glass capillary. A. Magnified view of the self-made polytetrafluoroethylene holder with Xenopus laevis oocytes in MBS buffer. B. Prepared injector glass capillary clamped in the Nanoliter Injector with the self-made polytetrafluoroethylene holder.

  2. Microinjection of the Xenopus laevis oocytes
    1. The Xenopus laevis oocytes at stage V/VI in MBS buffer can be ordered from Ecocyte Bioscience and arrive cooled the next day. They are kept in MBS buffer at 18 °C (room temperature is set to 18 °C) and used for the sample preparation not later than the day of delivery.
    2. For the microinjection, 6-7 Xenopus laevis oocytes are prepared with a Pasteur capillary pipette on a self-made polytetrafluoroethylene (PTFE) holder (produced by PTFE milling, size 35 x 10 mm, with 1 mm wide and 2 mm high grooves at a distance of 4 mm) in MBS buffer, in which they are shipped, and visually inspected for their state with the binocular microscope (see Figure 2). Xenopus laevis oocytes with initial signs of apoptosis, such as flabby membrane or light discolorations in the dark animal hemisphere of the Xenopus laevis oocyte, are rejected (for comparison of healthy and damaged Xenopus laevis oocytes see Figure 3). 


      Figure 3. Micrographs of Xenopus laevis oocytes. A. Healthy Xenopus laevis oocytes without signs of apoptosis; B. and C. Light discolorations in the dark animal hemisphere of the Xenopus laevis oocytes as an initial sign of apoptosis are tagged by orange circles. C. Progressed apoptosis in the form of flabby membrane and cell deformation is tagged by a red ellipse. Scale bar = 1 mm.

    3. The membrane of the Xenopus laevis oocytes is punctured carefully in the dark animal hemisphere, near the parting line, which separates the animal hemisphere from the light vegetal hemisphere of the Xenopus laevis oocyte. The penetrating glass capillary is moved in the direction of the parting line so that the glass capillary points at the parting line from diagonally above. The injection of the sample solution (typically 50 nl) is carried out in this position by the Nanoliter Injector so that the nucleus in the animal hemisphere will not be injected (see Figure 4).


      Figure 4. Process flow of the microinjection. A. Diagram of a Xenopus laevis oocyte; B. For the microinjection, the glass capillary penetrates the Xenopus laevis oocyte in the animal hemisphere, very close to the vegetal hemisphere, at an approximately 10° angle, tagged by a red arrow. C. Position of the glass capillary (shown in dark blue) during the injection.

    4. The glass capillary is removed by carefully pulling it off. Injured Xenopus laevis oocytes are thrown away, while successfully injected Xenopus laevis oocytes are washed carefully with MBS buffer. 

  3. Collecting in an EPR sample tube
    1. Upon microinjection, three (for pulsed Q-band EPR measurements at ~34 GHz) or five (for X-band continuous wave [cw] EPR measurements at ~9.5 GHz) Xenopus laevis oocytes are carefully transferred into a Q-band EPR sample tube. For this, a slight negative pressure is built up with a pipette controller on one end of the sample tube, which is moistened with MBS buffer.
    2. Initially a small volume of MBS buffer is collected, followed by the Xenopus laevis oocytes and again a small volume of MBS buffer. Care should be taken to ensure that there is no space between the Xenopus laevis oocytes within the sample tube. Furthermore, it must be ensured by visual inspection using the binocular microscope that the cell membrane of the Xenopus laevis oocytes is not damaged through the collection and that no air pockets are in the sample tube. Otherwise, the sample is unusable. This visual inspection must be performed again after the desired incubation time of the Xenopus laevis oocytes at 18 °C.
    3. The incubation time has to be selected in accordance with the morphological stability of the Xenopus laevis oocytes upon microinjection and the stability of the paramagnetic spin label in the reducing environment of the Xenopus laevis oocyte. Both are determined prior to the final in-cell EPR experiment (see Data analysis).
    4. After the incubation time at 18 °C, the supernatant MBS buffer in the sample tube is removed using a Hamilton syringe in such a way that the MBS buffer just covers the edge of the outer Xenopus laevis oocytes, maximum 1-2 mm distance from the edge of the Xenopus laevis oocytes (finished prepared sample tube see Figure 5).


      Figure 5. Prepared Q-band sample tube with three Xenopus laevis oocytes in MBS buffer immediately before shock-freezing for pulsed Q-band EPR measurements

    5. For pulsed Q-band EPR measurements (~34 GHz) the sample is subsequently shock-frozen in liquid nitrogen and stored in the freezer at -80 °C until measuring without thawing. In contrast, for X-band cw-EPR measurements (~9.5 GHz) at 20 °C the sample tube is sealed with the capillary tube sealing compound at the top to guarantee a fixed sample position within the tube. Furthermore, X-band cw-EPR measurements must be performed immediately after sample preparation because the stability of the paramagnetic spin label inside the cell and the morphological stability of the microinjected Xenopus laevis oocytes might be time-limited at 20 °C (see Data analysis).

Data analysis

  1. Investigating cytotoxic effects of injected samples: The morphological stability of the Xenopus laevis oocytes has to be checked upon microinjection of the paramagnetic sample solution to ensure that after the chosen incubation time all cells in the sample are still intact. This can be done with a morphological study over several hours, as suggested by Groß et al. (Qi et al., 2014; Wojciechowski et al., 2015).
  2. Investigating stability of paramagnetic spin label: The stability of the spin labels in the reducing environment of the Xenopus laevis oocytes is tested by measuring the X-band cw-EPR signal of the labeled macromolecules microinjected into Xenopus laevis oocytes at 18 °C. The decay of the signal intensity corresponds to a reduction of the paramagnetic spin labels under the applied conditions. There are different half-times of the signal decay for different spin labels at 18 °C in Xenopus laevis oocytes (Qi et al., 2014; Karthikeyan et al., 2017).
    For spin labels featuring sufficient stability, incubation times around 60 min at 18 °C are typically chosen (Qi et al., 2014; Wojciechowski et al., 2015). Using 3-maleimido-PROXYL (3-Maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxy) spin labels, typical incubation time at 18 °C is in the range of 15 min (Cattani et al., 2017).
  3. Endogenous paramagnetic species: Xenopus laevis oocytes contain endogenous paramagnetic Mn(II) species, which are also represented in EPR spectra (Qi et al., 2014). Thus, untreated Xenopus laevis oocytes are measured for background correction (Qi et al., 2014; Cattani et al., 2017).
  4. Spatial expansion of the injected volume by diffusion: In order to determine the effect of translational diffusion of the target molecules within the Xenopus laevis oocyte upon microinjection, the local concentration can be determined by double electron-electron resonance (DEER) measurements (Jeschke et al., 2006; Cattani et al., 2017).

Notes

  1. Compressible air pockets in the glass capillary would lead to a wrong injection volume and injection velocity.
  2. It is important to neither cut too much nor too little of the glass capillary when you open it with the razor blade. If too much is cut off, the diameter of the glass capillary will be too big for microinjection without extensively damaging the Xenopus laevis oocytes. If too little is cut off, the stability of the class capillary will not suffice to penetrate Xenopus laevis oocytes.
  3. Ensure that the Xenopus laevis oocytes are always covered with MBS buffer solution.
  4. For a quick succession of the microinjections, it is useful to align the Xenopus laevis oocytes in the polytetrafluoroethylene holder in the same way by carefully turning them with the glass capillary clamped in the Nanoliter Injector.
  5. The loading of the Xenopus laevis oocytes into the Q-band sample tube is facilitated by a Parafilm-strip on a Petri dish (size 35 x 10 mm) forming an elevation in the middle of the dish (see Figure 6). The Xenopus laevis oocytes are given with a Pasteur pipette into a drop of MBS buffer onto the Petri dish directly next to the elevation modeled out of Parafilm. By slightly pushing the Xenopus laevis oocyte with the Q-band sample tube against the Parafilm-elevation, the Xenopus laevis oocytes can be brought into a good position for the collection of the Xenopus laevis oocytes into the sample tube without space between each of them.


    Figure 6. Assembly for an easy collection of the Xenopus laevis oocytes into a Q-band sample tube. A. Xenopus laevis oocytes positioned in a drop of MBS buffer on a Petri dish directly next to an elevation made out of Parafilm can be easily collected in a Q-band sample tube by means of a pipette controller. B. Magnified view of the positioning and collection of the Xenopus laevis oocytes.

Acknowledgments

The protocol described has been developed and improved by different members of the Research Group of Prof. Dr. Malte Drescher and was based on previous protocols of Dr. Andreas Groß (Qi et al., 2014; Wojciechowski et al., 2015) and Dr. Julia Cattani (Cattani et al., 2017). We thank Juliane Stehle and Anna Bieber for the pictures in Figure 6. Support by the Deutsche Forschungsgemeinschaft within the priority programme SPP 1601 is gratefully acknowledged. The authors declare no conflicts of interest or competing interests.

References

  1. Atherton, N. (1993). Principles of electron spin resonance. Ellis Horwood.
  2. Azarkh, M., Okle, O., Singh, V., Seemann, I. T., Hartig, J. S., Dietrich, D. R. and Drescher, M. (2011). Long-range distance determination in a DNA model system inside Xenopus laevis oocytes by in-cell spin-label EPR. Chembiochem 12(13): 1992-1995.
  3. Azarkh, M., Singh, V., Okle, O., Seemann, I. T., Dietrich, D. R., Hartig, J. S. and Drescher, M. (2013). Site-directed spin-labeling of nucleotides and the use of in-cell EPR to determine long-range distances in a biologically relevant environment. Nat Protoc 8(1): 131-147.
  4. Barnard, E. A., Miledi, R. and Sumikawa, K. (1982). Translation of exogenous messenger RNA coding for nicotinic acetylcholine receptors produces functional receptors in Xenopus oocytes. Proc R Soc Lond B Biol Sci 215(1199): 241-246.
  5. Berliner, L. J. and Reuben, J. (2012). Spin labeling: theory and applications (Vol. 8). Springer Science & Business Media.
  6. Cattani, J., Subramaniam, V. and Drescher, M. (2017). Room-temperature in-cell EPR spectroscopy: alpha-Synuclein disease variants remain intrinsically disordered in the cell. Phys Chem Chem Phys 19(28): 18147-18151.
  7. Dawid, I. B. and Sargent, T. D. (1988). Xenopus laevis in developmental and molecular biology. Science 240(4858): 1443-1448.
  8. Feix, J. B. and Klug, C. S. (2002). Site-directed spin labeling of membrane proteins and peptide-membrane interactions. In: Berliner, L. J. (Ed.). Biological Magnetic Resonance. Springer pp: 251-281.
  9. Gerson, F., Weil, J. A., Bolton, J. R. and Wertz, J. E. (1994). Electron paramagnetic resonance: Elementary theory and applications. Wiley‐interscience, New York.
  10. Hubbell, W. L. and Altenbach, C. (1994). Investigation of structure and dynamics in membrane proteins using site-directed spin labeling. Curr Opin Struct Biol 4(4): 566-573.
  11. Igarashi, R., Sakai, T., Hara, H., Tenno, T., Tanaka, T., Tochio, H. and Shirakawa, M. (2010). Distance determination in proteins inside Xenopus laevis oocytes by double electron-electron resonance experiments. J Am Chem Soc 132(24): 8228-8229.
  12. Jeschke, G., Chechik, V., Ionita, P., Godt, A., Zimmermann, H., Banham, J., Timmel, C. R., Hilger, D. and Jung, H. (2006). DeerAnalysis2006—a comprehensive software package for analyzing pulsed ELDOR data. Appl Magn Reson 30(3): 473-498.
  13. Jeschke, G. and Schweiger, A. (2001). Principles of pulse electron paramagnetic resonance. Oxford University Press, Oxford.
  14. Karthikeyan, G., Bonucci, A., Casano, G., Gerbaud, G., Abel, S., Thome, V., Kodjabachian, L., Magalon, A., Guigliarelli, B., Belle, V., Ouari, O. and Mileo, E. (2017). A bioresistant nitroxide spin label for in-cell EPR spectroscopy: in vitro and in oocytes protein structural dynamics studies. Angew Chem Int Ed Engl.
  15. Kay, B. K. (1991). Xenopus laevis: Practical uses in cell and molecular biology. Injections of oocytes and embryos. Methods Cell Biol 36: 663-669.
  16. Krstic, I., Hansel, R., Romainczyk, O., Engels, J. W., Dotsch, V. and Prisner, T. F. (2011). Long-range distance measurements on nucleic acids in cells by pulsed EPR spectroscopy. Angew Chem Int Ed Engl 50(22): 5070-5074.
  17. Likhtenshtein, G. I., Yamauchi, J., Nakatsuji, S. I., Smirnov, A. I. and Tamura, R. (2008). Nitroxides: applications in chemistry, biomedicine, and materials science. John Wiley & Sons.
  18. Martorana, A., Bellapadrona, G., Feintuch, A., Di Gregorio, E., Aime, S. and Goldfarb, D. (2014). Probing protein conformation in cells by EPR distance measurements using Gd3+ spin labeling. J Am Chem Soc 136(38): 13458-13465.
  19. Mishina, M., Kurosaki, T., Tobimatsu, T., Morimoto, Y., Noda, M., Yamamoto, T., Terao, M., Lindstrom, J., Takahashi, T., Kuno, M. et al. (1984). Expression of functional acetylcholine receptor from cloned cDNAs. Nature 307(5952): 604-608.
  20. Qi, M., Groß, A., Jeschke, G., Godt, A. and Drescher, M. (2014). Gd(III)-PyMTA label is suitable for in-cell EPR. J Am Chem Soc 136(43): 15366-15378.
  21. Richter, J. D. (1999). A comparative methods approach to the study of oocytes and embryos. Oxford University Press.
  22. Wojciechowski, F., Groß, A., Holder, I. T., Knorr, L., Drescher, M. and Hartig, J. S. (2015). Pulsed EPR spectroscopy distance measurements of DNA internally labelled with Gd3+-DOTA. Chem Commun (Camb) 51(72): 13850-13853.

简介

研究生物大分子的最令人兴奋的观点之一来自于新兴的细胞内光谱学领域,它能够确定细胞中生物大分子的结构和动力学。细胞内电子顺磁共振(EPR)光谱结合将生物大分子微注射到非洲爪蟾卵母细胞中非常适合于此目的。非洲爪蟾卵母细胞是生物学不同领域常用的真核细胞模型,如细胞和发育生物学。对于细胞内EPR,感兴趣的生物大分子通过定点自旋标记显微注射到非洲爪蟾卵母细胞中。通过Nanoliter注射器将样品溶液填充到薄玻璃毛细管中,然后通过小心地穿刺薄膜将其微注射入非洲爪蟾卵母细胞的黑色动物部分。之后,取决于最终的细胞内EPR实验的种类,将三个或五个显微注射的非洲爪蟾卵母细胞装载到Q波段EPR样品管中,随后进行任选的休克冷冻(用于实验冷冻溶液)并且在期望的温育时间之后测量(在低温或生理温度下)。由于显微注射样品的细胞毒性作用和顺磁性自旋标记在还原性细胞环境中的稳定性,孵育时间受到限制。通过监测细胞形态和减少动力学来量化这两个方面。

【背景】电子顺磁共振(EPR)光谱学是用于表征顺磁系统的选择方法(Atherton,1993; Gerson等人,1994; Jeschke和Schweiger,2001)。反磁性生物大分子可以通过定点自旋标记(SDSL)进行EPR光谱学分析,通常使用氮氧化物作为自旋标记(Hubbell和Altenbach,1994; Feix和Klug,2002; Likhtenshtein等人,2008; Berliner和Reuben,2012)。 SDSL与内嵌式EPR光谱学的结合是获取有关生物大分子(如蛋白质或核苷酸)在自然环境中的结构和动力学的有力工具(Azarkh et al。 ,2013; Martorana ,2014; Qi et al。,2014; Cattani et。,2017)。内嵌EPR的羽化技术最常用的实验方法是基于将目标分子微注射到非洲蛙非洲爪蟾卵母细胞中,这是一种广泛使用的真核细胞模型(Kay, 1991; Barnard等人,1982; Mishina等人,1984; Dawid和Sargent,1988; Richter,1999)。

用于细胞内EPR的非洲爪蟾卵母细胞的优点是直径大约1mm(大约1μl细胞体积)的大尺寸,由此产生的易处理性以及仅三种或五种对于电池内EPR样品是必需的(Qi等人,2014; Cattani等人,2017)。因此,可以通过显微注射将生物大分子相对容易地引入到EPR测量到爪蟾卵母细胞中所需的量中。因此,在微注射入非洲爪蟾卵母细胞后(Igarashi等人),通过双电子电子共振(DEER)测量进行了自旋标记的DNA和蛋白质的许多细胞内距离测量,2010; Azarkh等人,2011; Krstic等人,2011; Azarkh等人,2013; Martorana等人, 2014年; Wojciechowski等人,2015年; Cattani等人,2017年)。

关键字:非洲爪蟾卵母细胞, 细胞内EPR, 细胞内光谱学分析, 位置定向自旋标记, 显微注射, 体内结构测定, 生物大分子动力学

材料和试剂

  1. 玻璃毛细管(3.5英寸长,Drummond Scientific,目录号:3-000-203-G / X)
  2. 一次性注射器(西格玛奥德里奇公司,产品目录号:Z230723)
  3. Parafilm(Sigma-Aldrich,目录号:P7793-1EA)
  4. 培养皿,尺寸60 x 15毫米(Corning,目录号:430166)
  5. 剃刀刀片(普莱诺,目录号:T5016)
  6. 巴斯德毛细吸管(150毫米,美因茨)
  7. 品牌移液器控制器微型经典(品牌,目录号:25900)
  8. Q波段样品管(石英玻璃,1 mm i.d.,Bruker,目录号:ER 221TUB-Q10)
  9. 汉密尔顿注射器(汉密尔顿,目录号:80500)
  10. 培养皿,大小35 x 10毫米(Corning,目录号:430165)
  11. 毛细管密封剂(Cha-seal,DWK Life Sciences,Kimble,目录号:43510)
  12. 在MBS缓冲液中的阶段V / VI上的非洲爪蟾卵母细胞(Ecocyte Bioscience, ecocyte-us.com/products/xenopus-oocyte-delivery-service/
  13. (1x)(Ecocyte Bioscience)(88mM NaCl,1mM KCl,1mM MgSO 4,5mM HEPES,2.5mM NaHCO 3)的改良Barth's盐水(MBS)缓冲液,0.7mM CaCl 2)
  14. 矿物油(Sigma-Aldrich,目录号:M5904)
  15. 液氮
  16. (马来酰亚胺基-2,2,5,5-四甲基-1-吡咯烷氧基)(Sigma-Aldrich,目录号:253375)

设备

  1. Flaming / Brown Micropipette Puller(Sutter Instrument,型号:P-97)
  2. Nanoject II Auto-Nanoliter注射器(Drummond Scientific,目录号:3-000-205A)
  3. 带支持基地(Drummond Scientific,目录号:3-000-025-SB)的微操作器MM33(Drummond Scientific,目录号:3-000-024-R)
  4. 双目显微镜(ZEISS,型号:Stemi 2000-C,参加了AxiaCam ERc 5s相机(蔡司,型号:AxiaCam ERc 5s))
  5. 自制聚四氟乙烯支架
  6. 杜瓦液氮(KGW-Isotherm,目录号:1021)
  7. -80°C冰箱

程序

  1. 注射器玻璃毛细管的制备
    1. 玻璃毛细管所需的薄顶部由Flaming / Brown Micropipette拉拔器形成。程序参数选择如下:压力设置(P)= 500,加热= 558,拉= 100,速度(VEL)= 120和时间= 100毫秒(与200单位一致)。
    2. Nanoliter注射器的确切位置可以通过显微操作器进行调整。 Nanoliter注射器的前端有一个螺丝夹和一个印章,后者的位置由控制计算机控制(见图1)。


      图1.显微注射设备的组装。 :一种。控制计算机的放大视图; B.双目显微镜和显微注射设备; C. Nanoliter注射器顶部的放大视图。

    3. 为避免印模与注射溶液之间产生气泡,使用一次性注射器将尖锐的玻璃毛细管充满矿物油。之后,将填充好的玻璃毛细管夹在邮票上,插入Nanoliter注射器的螺丝夹中。离开矿物油用纸巾擦掉。
    4. 为了打开夹住的玻璃毛细管,将Parafilm拉伸到培养皿(尺寸60×15mm)的背面。一个小标记被压入培养皿边缘的Parafilm中。通过双目显微镜观察显微操纵器,玻璃毛细管缓慢移向皮氏培养皿上的标记。当玻璃毛细管开始接触石蜡膜时,通过同时降低显微操作器的毛细管并将培养皿从后者上移开,将其水平放置在石蜡膜上。用剃刀刀片仔细切割玻璃毛细管(约2.5毫米)的放下的前片。
    5. 随后,将矿物油用打开的玻璃毛细管从Nanoliter注射器的印模上压出约2cm。这个流失可能需要15分钟。
    6. 准备好的玻璃毛细管的顶部浸入一滴样品溶液(取决于要显微注射的爪蟾卵母细胞的数量,2-4微升)。使用Nanoliter注射器将样品溶液吸入毛细管中(见图2,制备的注射器玻璃毛细管)。 


      图2.准备好的非洲爪蟾卵母细胞和玻璃毛细管。 :一种。在MBS缓冲液中用非洲爪蟾卵母细胞自制聚四氟乙烯支架的放大图。 B.使用自制的聚四氟乙烯支架,将制备好的注射器玻璃毛细管夹在Nanoliter注射器中。

  2. 显微注射非洲爪蟾卵母细胞
    1. 在MBS缓冲液中的V / VI阶段的非洲爪蟾卵母细胞可以从Ecocyte Bioscience订购并在第二天冷却。将它们保存在18℃的MBS缓冲液中(室温设定为18℃),并在不迟于分娩当天用于样品制备。
    2. 对于显微注射,用自制聚四氟乙烯(PTFE)支架上的巴斯德毛细管移液管制备6-7非洲爪蟾卵母细胞(通过PTFE研磨生产,尺寸为35×10mm,宽度为1mm和2毫米高的凹槽距离为4毫米)放入MBS缓冲液中,并用双目显微镜目视检查它们的状态(见图2)。在非洲爪蟾卵母细胞的黑色动物半球中具有凋亡初始迹象(例如松弛膜或轻度变色)的非洲爪蟾卵母细胞被拒绝(用于比较健康和受损< em>爪蟾卵母细胞见图3)。 


      图3.非洲爪蟾卵母细胞的显微照片A.健康非洲爪蟾卵母细胞没有细胞凋亡迹象; B.和C.在非洲爪蟾卵母细胞的黑色动物半球中的光变色作为凋亡的初始迹象用橙色圆圈标记。 C.以松弛膜和细胞变形形式的进展性细胞凋亡由红色椭圆标记。比例尺= 1毫米。

    3. 非洲爪蟾卵母细胞的细胞膜在黑暗的动物半球中,在分离线附近仔细刺破,分离线将动物半球与非洲爪蟾卵母细胞的轻质植物半球分开。穿透玻璃毛细管沿着分型线的方向移动,使得玻璃毛细管从斜上方指向分型线。样品溶液的注射(通常为50nl)在纳洛特注射器的这个位置进行,以便动物半球内的细胞核不会被注射(见图4)。


      图4.显微注射的工艺流程。 :一种。非洲爪蟾卵母细胞的图解; B.对于显微注射,玻璃毛细管穿过动物半球中非常接近植物半球的非洲爪蟾卵母细胞,大约10°角,用红色箭头标记。 C.注射期间玻璃毛细管的位置(以深蓝色显示)。

    4. 将玻璃毛细管小心拔出即可取出。受伤的爪蟾卵母细胞被丢弃,而成功注射的非洲爪蟾卵母细胞用MBS缓冲液仔细洗涤。

  3. 收集在EPR样品管中
    1. 在显微注射时,三个(用于约34GHz的脉冲Q波段EPR测量)或五个(用于约9.5GHz的X波段连续波[cw] EPR测量)非洲爪蟾卵母细胞仔细转移到Q波段EPR样品管。为此,在样品管的一端用移液器控制器构建轻微的负压,该样品管用MBS缓冲液润湿。
    2. 最初收集少量MBS缓冲液,然后收集非洲爪蟾卵母细胞,并再次收集少量MBS缓冲液。应注意确保样品管内非洲爪蟾卵母细胞之间没有空间。此外,必须通过使用双目显微镜进行肉眼观察来确保非洲爪蟾卵母细胞的细胞膜未被收集损坏,并且样品管中没有气泡。否则,该样本不可用。在18℃,在非洲爪蟾卵母细胞所需的孵育时间后,必须再次进行这种视觉检查。
    3. 孵育时间必须根据显微注射时非洲爪蟾卵母细胞的形态学稳定性和非还原性非洲爪蟾还原环境中顺磁性自旋标记的稳定性来选择,卵母细胞。两者都是在最终的单元格内EPR实验之前确定的(请参阅数据分析)。
    4. 在18℃温育时间之后,使用Hamilton注射器移除样品管中的上清液MBS缓冲液,使得MBS缓冲液刚好覆盖外部非洲爪蟾卵母细胞的边缘,最大值距离非洲爪蟾卵母细胞边缘1-2毫米的距离(完成准备的样品管参见图5)。


      图5.准备Q波段样品管,其中有三个非洲爪蟾卵母细胞在冲击冷冻前用于MBS缓冲液中脉冲Q波段EPR测量

    5. 对于脉冲Q波段EPR测量(约34 GHz),样品随后在液氮中冷冻并在-80°C冷冻箱中储存,直至测量而不解冻。相反,对于20°C下的X波段cw-EPR测量(〜9.5 GHz),样品管在顶部用毛细管密封剂密封,以确保管内固定样品位置。此外,X-band cw-EPR测量必须在样品制备后立即进行,因为细胞内顺磁性自旋标记的稳定性和微注射非洲爪蟾卵母细胞的形态稳定性可能受时间限制20°C(见数据分析)。

数据分析

  1. 研究注射样品的细胞毒性效应:必须在显微注射顺磁性样品溶液时检查非洲爪蟾卵母细胞的形态学稳定性,以确保在选择的温育时间之后样品中的所有细胞仍然完整。正如Groß等人所建议的,这可以在数小时内进行形态学研究。 (Qi等人,2014; Wojciechowski等人,2015)。
  2. 研究顺磁性自旋标记的稳定性:非洲爪蟾卵母细胞还原环境中自旋标记的稳定性通过测量标记的大分子的X带cw-EPR信号来测试,所述标记的大分子微量注射到非洲爪蟾卵母细胞在18°C。信号强度的衰减对应于在所施加的条件下顺磁性自旋标记的减少。 18℃下,爪蟾卵母细胞中不同半衰期的信号衰减(Qi等人,2014; Karthikeyan等人, 。,2017)。
    对于具有足够稳定性的自旋标记,通常选择在18℃约60分钟的孵育时间(Qi等人,2014; Wojciechowski等人,2015)。使用3-马来酰亚胺基-PROXYL(3-马来酰亚胺-2,2,5,5-四甲基-1-吡咯烷基氧基)自旋标记,18℃下的典型温育时间在15分钟的范围内(Cattani等人, ,2017)。
  3. 内源性顺磁性物种:非洲爪蟾卵母细胞含有内源性顺磁性Mn(II)物质,这些物质也在EPR谱中表示(Qi等人,2014)。因此,测量未处理的非洲爪蟾卵母细胞的背景校正(Qi等人,2014; Cattani等人,2017)。
  4. 通过扩散进行注射体积的空间扩张:为了确定在显微注射时爪蟾卵母细胞内靶分子的平移扩散的影响,局部浓度可以通过双电子电子共振来确定( DEER)测量值(Jeschke et al。,2006; Cattani em et al。,2017)。

笔记


  1. 玻璃毛细管中的可压缩气囊会导致错误的注射量和注射速度。
  2. 用剃刀刀片打开玻璃毛细管时,切割太多或太少的玻璃毛细管都很重要。如果太多被切断,玻璃毛细管的直径将太大而不能显微注射,而不会广泛地破坏非洲爪蟾卵母细胞。如果切断太少,类毛细管的稳定性将不足以穿透非洲爪蟾卵母细胞。
  3. 确保非洲爪蟾卵母细胞总是被MBS缓冲溶液覆盖。
  4. 对于快速连续的显微注射,将聚合四氟乙烯支架中的非洲爪蟾卵母细胞以相同的方式对齐,方法是用纳升注射器中夹住的玻璃毛细管仔细转动它们。
  5. 在培养皿(尺寸为35×10mm)上的Parafilm条带促进了非洲爪蟾卵母细胞在Q带样品管中的装载,从而在培养皿中间形成了一个升高点(参见图6)。将非洲爪蟾卵母细胞用巴斯德移液管加入一滴MBS缓冲液中,直接在模拟出石蜡膜的海拔旁边的陪替氏培养皿中。通过用Q波段样品管轻轻推动非洲爪蟾卵母细胞抵抗Parafilm提升,可以使非洲爪蟾卵母细胞进入适当位置收集非洲爪蟾卵母细胞进入样品管,它们之间没有空间。


    图6.将非洲爪蟾卵母细胞轻松收集到Q带样品管中的装配A.非洲爪蟾卵母细胞置于通过移液器控制器可以很容易地将一个MBS缓冲液滴在培养皿旁边的石油培养皿旁边,通过Parafilm制成的标高可以很容易地收集在Q波段样品管中。 B.非洲爪蟾卵母细胞定位和收集的放大视图。

致谢

所描述的方案已由Malte Drescher教授研究小组的不同成员开发和改进,并基于AndreasGroß博士的先前方案(Qi等人,2014; Wojciechowski,等),以及Julia Cattani博士(Cattani等人,2017)。我们感谢Juliane Stehle和Anna Bieber提供了图6中的图片。我们非常感谢Deutsche Forschungsgemeinschaft在SPP 1601优先计划中提供的支持。作者声明不存在利益冲突或利益冲突。

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引用:John, L. and Drescher, M. (2018). Xenopus laevis Oocytes Preparation for in-Cell EPR Spectroscopy. Bio-protocol 8(7): e2798. DOI: 10.21769/BioProtoc.2798.
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