Mammalian Cell-derived Vesicles for the Isolation of Organelle Specific Transmembrane Proteins to Conduct Single Molecule Studies
哺乳动物细胞源性囊泡用于分离细胞器特异性跨膜蛋白以进行单分子研究   

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Rahul  Srinivasan Rahul Srinivasan
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The Journal of Biological Chemistry
Dec 2017

 

Abstract

Cell-derived vesicles facilitate the isolation of transmembrane proteins in their physiological membrane maintaining their structural and functional integrity. These vesicles can be generated from different cellular organelles producing, housing, or transporting the proteins. Combined with single-molecule imaging, isolated organelle specific vesicles can be employed to study the trafficking and assembly of the embedded proteins. Here we present a method for organelle specific single molecule imaging via isolation of ER and plasma membrane vesicles from HEK293T cells by employing OptiPrep gradients and nitrogen cavitation. The isolation was validated through Western blotting, and the isolated vesicles were used to perform single molecule studies of oligomeric receptor assembly.

Keywords: Single molecule (单分子), Vesicles (囊泡), Stoichiometry (化学计量学), ER and plasma membrane protein separation (ER和质膜蛋白分离), Nicotinic receptor (烟碱受体), OptiPrep (OptiPrep), Photobleaching (光漂白), Protein trafficking (蛋白质转运)

Background

A large number of transmembrane proteins are formed through the assembly of multiple subunits leading to complicated oligomeric structures that can often exist in multiple stoichiometries. Understanding how changes in assembly alter trafficking and localization within different organelles is essential to determining a protein’s physiological role and the connection to diseases associated with maturation and transport. Single molecule approaches can provide a better understanding of the assembly of oligomeric proteins by directly measuring their stoichiometry (Ulbrich and Isacoff, 2007; Richards et al., 2012). This approach avoids ensemble averaging which provides the average state of all the stoichiometries (Walter and Bustamante, 2014). Single molecule studies have recently been employed to understand the structural and functional properties of macromolecules including conformational dynamics (Tan et al., 2014), ion channel gating (Wang et al., 2016), ligand-receptor interaction (Moonschi et al., 2015), and stoichiometric assembly (Ulbrich and Isacoff, 2007; Moonschi et al., 2015). To conduct single molecule studies, it is imperative to isolate single receptors in a supporting bilayer. Separation from the cellular environment is often necessary because the endogenous concentration of membrane proteins in mammalian cells is typically much higher than conditions compatible with single molecule studies (Richards et al., 2012). Additionally, live cell single molecule studies suffer from a number of disadvantages including high levels of auto-fluorescence, limited fluorophore brightness and the mobility of membrane proteins on the cell surface (Andersson et al., 1998; Lippincott-Schwartz et al., 1999). Isolation strategies using artificial bilayers such as liposomes are also problematic as they include an intermediate step where the protein is stabilized into a detergent solution which poses a threat to the structural integrity of the transmembrane protein. Membrane-derived vesicles enable the protein to remain embedded in its physiological membrane maintaining its structural and functional integrity. These nanoscale vesicles have been employed to study stoichiometric assembly of multimeric proteins, to probe ligand receptor interactions, and to understand the effect of ligands on the assembly of nicotinic receptors (Fox et al., 2015; Moonschi et al., 2015; Fox-Loe et al., 2017).

Transmembrane proteins are synthesized and assembled in the endoplasmic reticulum and then trafficked to the plasma membrane. Oligomeric proteins with multiple non-identical subunits can often be assembled with different subunit stoichiometries potentially leading to different trafficking and functional properties (Grady et al., 2010). For instance, α4β2 nicotinic receptors are pentameric receptor with two possible stoichiometric assemblies: (α4)2(β2)3 and (α4)3(β2)2. It has been hypothesized that cellular machinery preferentially traffics the high sensitivity isoform (α4)2(β2)3 over the other isoform from the ER to the plasma membrane. It has also been hypothesized that nicotine alters the assembly of this receptor from the low sensitivity to high sensitivity isoform in the ER leading to higher levels of the preferentially trafficked assembly in both the ER and plasma membrane (Lester et al., 2009; Henderson and Lester, 2015). Single molecule studies of proteins from specific organelles enable the correlation of changes in structural assembly of the nicotinic receptors to changes in trafficking and assembly.

Here we present a method which can isolate single transmembrane proteins into the ER and plasma membrane derived vesicles using nitrogen cavitation and an OptiPrep gradient. The ER and plasma membrane derived vesicles exhibit different densities because they contain different phospholipids and associated proteins. ER originated vesicles are much denser than those obtained from the plasma membrane. The OptiPrep gradient was selected because of its superior ability to maintain isosmotic pressure independent of the density of the gradient used to isolate cellular organelles and subcellular vesicles (Graham et al., 1994). We applied this method to study ligand induced changes in the assembly of nicotinic receptors in the ER as well as the plasma membrane and validated our method with Western blotting and single molecule step-wise photobleaching. We believe this method can be applied for virtually any type of transmembrane proteins to conduct single molecule studies and to understand organelle-specific structural and functional properties.

Materials and Reagents

  1. Cell culture flasks (Greiner Bio One International, catalog number: 658175 )
  2. 50 ml centrifuge tubes (VWR, catalog number: 89039-658 )
  3. Ultra-Clear ultracentrifuge tubes (Beckman Coulter, catalog number: 344061 )
  4. Four 5-ml Serological pipettes (VWR, catalog number: 89130-896 )
  5. One 9-inch-flint-glass Pasteur pipette (VWR, catalog number: 14672-380 )
  6. 15 ml centrifuge tubes (VWR, catalog number: 89039-666 )
  7. 1.5-ml tubes
  8. Wine cork
  9. Razor blades (VWR, catalog number: 55411-050 )
  10. Two ice buckets (VWR, catalog number: 10146-202 )
  11. HEK293T cells (Sigma-Aldrich, catalog number: 85120602-1VL )
  12. Deionized water
  13. PBS buffer (VWR, catalog number: 97062-948 )
  14. Versene solution (Thermo Fisher Scientific, GibcoTM, catalog number: 15040066 )
  15. Nitrogen gas
  16. OptiPrep (60% solution, Sigma-Aldrich, catalog number: D1556-250ML )
  17. Anti-calnexin (Abcam, catalog number: ab92573 )
  18. Anti-sodium potassium ATPase (Abcam, catalog number: ab76020 )
  19. Mouse anti-Rabbit secondary antibody (Santa Cruz Biotechnology, catalog number: sc-2357 )
  20. ClarityTM Western ECL Substrate (Bio-Rad Laboratories, catalog number: 1705060 )
  21. Tris-HCl (Sigma-Aldrich, Roche Diagnostics, catalog number: 10812846001 )
  22. Sodium chloride (NaCl) (Fisher Scientific , catalog number: BP358-1 )
  23. Magnesium chloride hexahydrate (MgCl2·6H2O) (Fisher Scientific, catalog number: BP214-500 )
  24. Calcium chloride (CaCl2) (Fisher Scientific, catalog number: C614-500 )
  25. Protease inhibitor mini tablet (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: A32955 )
  26. Sucrose (VWR, catalog number: 97063-788 )
  27. HEPES (Fisher Scientific, catalog number: BP310-500 )
  28. Hypotonic protease inhibitor solution (see Recipes)
  29. Sucrose buffer (see Recipes)

Equipment

  1. One 1 ml pipettor (Gilson, catalog number: F123602 )
  2. One pipet controller (VWR, catalog number: 613-4442 )
  3. One pair of scissors
  4. A set of stand (a stand and a clamp)
  5. Incubator
  6. Biosafety cabinet (Class II, Type A2)
  7. Centrifuge (Beckman Coulter, model: Allergra® X-22R ; Rotor: Beckman Coulter, model: SX4250 )
  8. Ultracentrifuge (Beckman Coulter, model: L-60 )
  9. Swing bucket rotor (Beckman Coulter, model: SW 28 )
  10. Fixed angle rotor (Beckman Coulter, model: 70 Ti )
  11. Belly dancer or orbital shaker
  12. Nitrogen cavitation chamber/vessel(Parr Instrument Company, catalog number: 4639 )
  13. Variable-Flow Peristaltic Pumps (Fisherbrand, Thermo Fisher)

Procedure

  1. Take two flasks (250 ml, 75 cm2) of confluent HEK293T cells (about 12 million cells per flask) that are expressing a fluorescently labeled membrane protein.
  2. Aspirate the media from the cells and rinse the cells with 5 ml 1x PBS buffer.
  3. Add 5 ml Versene solution and incubate at 37 °C for 5 min to detach the cells from the flasks.
  4. Collect the cell slurry in a 15 ml centrifuge tube.
  5. Centrifuge the slurry at 300 x g for 5 min at room temperature.
  6. Discard the supernatant (Versene solution) from the tube by careful aspiration and add 3 ml hypotonic protease inhibitor solution (Recipe 1) in the centrifuge tube. Resuspend the cells using pipetting in and out or swirling.
  7. Place the tube in an ice bucket and shake the bucket on an orbital shaker for 10 min to facilitate swelling of the cells.
  8. Transfer the cell slurry from the centrifuge tube into a pre-chilled (on ice) nitrogen cavitation chamber/vessel. Attach the vessel head and flow in nitrogen gas up to 600 psi. Incubate the cavitation chamber under pressure on ice for 20 min. Slowly open the valve to simultaneously release the pressure and collect the cell lysate into a 15 ml centrifuge tube (Figure 1). Keep the tube in ice until the cell lysate is added on top of the OptiPrep gradient.


    Figure 1. Instrumental setup used for nitrogen cavitation. A. Take the precooled nitrogen cavitation chamber, close the valve (red circle) and add the cell slurry into the chamber. B. Attach the vessel head and open valve B1 to flow nitrogen gas inside the chamber and close valve B1 once the pressure reaches 600 psi. Place this chamber into ice for 5 min. Take the chamber out from the ice and slowly open valve B2 to simultaneously release the pressure and to collect the cell lysate into a 15 ml centrifuge tube.

  9. Make the OptiPrep gradient in an Ultra-Clear ultracentrifuge tube as follows:
    1. Cut a wine cork into ~2 mm thick circular slice using a razor blade (Figure 2A). Cut along the perimeter of the cork slice using a pair of scissors so that it can easily fit into the Ultra-clear ultracentrifuge tube. Wash the cork slice with water to remove debris. Leave it in sterile water until adding it to the ultracentrifuge tube.


      Figure 2. Pictorial presentation of the collection steps for ER and plasma membrane vesicles. A. A wine cork (A1) is cut into a slice of about 2 mm thickness (A2). This slice (A3) is cut along the circumference to discard the outer area (A4) and keep a small circular slice (A5) which can easily fit into an Ultra-Clear ultracentrifuge tube. A scale (A6) is placed on the image for comparison of sizes. B. The small slice of cork is inserted into the tube and floats on the solution. C. As additional solution (20%, and 10% OptiPrep) is added the cork rises to the top. After the addition of all layers, the cork floats atop the solution. D. The cork slice is removed using a forceps without disturbing the gradient. E. A Pasteur pipette is broken to obtain the narrow tube which is inserted in the inlet tubing of a peristaltic pump. F. The resultant inlet tubing of the pump is inserted at the bottom-center of the Ultra-Clear tube containing the gradient solution.

    2. Prepare 30%, 20%, and 10% OptiPrep solutions by diluting the original 60% OptiPrep solution with sucrose buffer (Recipe 2) into three 50 ml tubes. Calculations associated with the preparation of 30 ml of each solution can be found in Table 1.

      Table 1. Preparation of 30, 20 and 10% OptiPrep solution from 60% OptiPrep solution with sucrose buffer. The original OptiPrep solution obtained from the supplier contains 60% OptiPrep.


    3. In an Ultra-Clear ultracentrifuge tube, using a 5-ml Serological pipette and a pipette controller, add 3 ml 30% OptiPrep solution, then add the cork slice on top of the solution using a forceps (Figure 2B). Now add dropwise 3 ml 20% OptiPrep, then 3 ml 10% OptiPrep and finally 3 ml cell lysate on top of the cork slice. With the dropwise addition of solution in the tube, the cork slice stays floating on top of the solution and thus helps to avoid mixing of the added solution with the solution already present in the tube (Figure 2C).
      Note: Three distinct interfaces should be visible indicating 4 layers of solutions of different densities. The interfaces are clearly visible when the tube is placed against a light source.
    4. Carefully remove the wine cork using a forceps without disturbing the gradient (Figure 2D).
  10. Centrifuge the ultracentrifuge tube containing OptiPrep gradient in a swing bucket rotor at 25,000 rpm (112,000 x g) for 90 min at 4 °C.
  11. After the centrifugation, three layers of vesicles should be observable: between cell lysate and 10% OptiPrep, 10% and 20% OptiPrep, and 20% and 30% OptiPrep (as shown in Figure 3).


    Figure 3. Presence of vesicles between the interfaces of OptiPrep solutions and the relative positions of different fractions collected. A. Three layers of vesicles are visible: on the interface of 0% and 10% OptiPrep, 10% and 20% OptiPrep and 20% and 30% OptiPrep. This image was taken with a mobile phone camera. B. By inserting the inlet tubing of a peristaltic pump in the bottom-center of the tube, collect fractions 1 and 2 in series, 1.0-ml each, then fractions 3-8 in a series, 1.5 ml each.

  12. Use a peristaltic pump to collect ER and PM fractions as follows:
    1. Place the Ultra-Clear centrifuge tube vertically using a stand and a clamp.
    2. Break the glass Pasteur pipette at the junction of the small-diameter tube and large diameter tube and insert the small-diameter tube inside the inlet tubing of the peristaltic pump (Figure 2E).
    3. Carefully, insert the inlet tube of the peristaltic pump in the ultracentrifuge tube (fixed with a clamp and stand) until it touches the bottom-center of the centrifuge tube (Figure 2F). It is necessary to make sure that the gradient is not disturbed by the insertion process.
    4. Start the peristaltic pump to collect different fractions keeping the interfaces in the same fraction (Figure 3B). To do so, collect fractions 1 and 2, 1 ml each and fractions 3 to 8, 1.5 ml each.
    5. According to our Western blot results with anti-calnexin as an ER marker and anti-sodium potassium ATPase as a plasma membrane marker, fractions 3 and 4 contain only the ER and fractions 7 and 8 include only the plasma membrane vesicles (Figure 4). However, we have selected fractions 3 and 7 as the ER and plasma membrane vesicles respectively for our experiments.


    Figure 4. Western blot of the different fractions collected from OptiPrep gradient to isolate single receptors into the ER and plasma membrane specific vesicles. Anti-sodium potassium ATPase antibody (1:200 dilution) was used to bind with the plasma membrane marker, sodium potassium ATPase, in the OptiPrep gradient (upper panel). An anti-calnexin antibody (1:200 dilution) was employed to bind with the ER marker, calnexin, in the gradients (lower panel). An HRP conjugated mouse anti-Rabbit secondary antibody (1:5,000 dilution) and ClarityTM Western ECL Substrate on a ChemiDoc imaging system (Bio-Rad) were utilized to locate the ER and Plasma membrane markers on the gels. The plasma membrane marker was found in the cell lysate and fractions 5 to 8 and the ER marker was located in the cell lysate and fractions 3 to 6. Therefore, fractions 3 and 4 contain only the ER vesicles; fractions 7 and 8 contain only the plasma membrane vesicles. We selected fraction 3 for ER vesicles and fraction 7 for the plasma membrane vesicles.

  13. Dilute the ER and the plasma membrane fractions with 1x PBS buffer to 1:3 and centrifuge at 30,000 rpm (100,000 x g) for 1 h at 4 °C with the fixed angle rotor.
  14. Resuspend the pellets each in 600 μl 1x PBS buffer or sucrose buffer to obtain ER and plasma membrane derived vesicles. We usually aliquot the vesicle solution into two or three 1.5-ml tubes and store at -80 °C. We use the vesicles within 7 days.
    Note: If a fraction contains no vesicles or a very small amount of vesicles, there will be no visible pellet on the surface of the tube after centrifugation. In this case, we collected the sample from the area where the vesicles might have present. It can be noted that a pellet is formed on the surface of the tube furthest from the center of the rotor and the possible location of the pellet can easily be assessed based on the location of the visible pellets in the other tubes.
    At this point, a fraction of the ER and plasma membrane derived vesicles will contain the protein of interest. For single molecule fluorescence studies, it is necessary to start with cells expressing the target protein conjugated with a fluorescent protein. Single receptor containing vesicles can be employed to study the stoichiometric assembly of the receptors by step-wise photobleaching technique, to probe ligand-receptor interaction using fluorescence correlation spectroscopy and to understand dynamics and conformational changes of the receptor using single molecule Forster resonance energy transfer (smFRET).

Recipes

  1. Hypotonic protease inhibitor solution
    10 mM Tris-HCl
    10 mM NaCl
    1.5 mM MgCl2
    0.2 mM CaCl2
    Adjust pH to 7.4
    One protease inhibitor tablet per 10 ml buffer
  2. Sucrose buffer
    250 mM sucrose
    10 mM HEPES
    Adjust pH to 7.4

Acknowledgments

This work was supported in part by the NIDA training grant DA016176 (AMF) and NIDA DA038817. This work was adapted from Fox-Loe et al., 2017, J Biol Chem 292(51): 21159-21169. We declare no conflict of interest or competitive interest related to this publication.

References

  1. Andersson, H., Baechi, T., Hoechl, M. and Richter, C. (1998). Autofluorescence of living cells. J Microsc 191(Pt 1): 1-7.
  2. Grady, S. R., Drenan, R. M., Breining, S. R., Yohannes, D., Wageman, C. R., Fedorov, N. B., McKinney, S., Whiteaker, P., Bencherif, M., Lester, H. A. and Marks, M. J. (2010). Structural differences determine the relative selectivity of nicotinic compounds for native α4β2*-, α6β2*-, α3β4*- and α7-nicotine acetylcholine receptors. Neuropharmacology 58(7): 1054-1066.
  3. Graham, J., Ford, T. and Rickwood, D. (1994). The preparation of subcellular organelles from mouse liver in self-generated gradients of iodixanol. Anal Biochem 220(2): 367-373.
  4. Fox, A. M., Moonschi, F. H. and Richards, C. I. (2015). The nicotine metabolite, cotinine, alters the assembly and trafficking of a subset of nicotinic acetylcholine receptors. J Biol Chem 290(40): 24403-24412.
  5. Fox-Loe, A. M., Moonschi, F. H. and Richards, C. I. (2017). Organelle-specific single-molecule imaging of α4β2 nicotinic receptors reveals the effect of nicotine on receptor assembly and cell-surface trafficking. J Biol Chem 292(51): 21159-21169.
  6. Henderson, B. J. and Lester, H. A. (2015). Inside-out neuropharmacology of nicotinic drugs. Neuropharmacology 96(Pt B): 178-193.
  7. Lester, H. A., Xiao, C., Srinivasan, R., Son, C. D., Miwa, J., Pantoja, R., Banghart, M. R., Dougherty, D. A., Goate, A. M. and Wang, J. C. (2009). Nicotine is a selective pharmacological chaperone of acetylcholine receptor number and stoichiometry. Implications for drug discovery. AAPS J 11(1): 167-177.
  8. Lippincott-Schwartz, J., Presley, J. F., Zaal, K. J., Hirschberg, K., Miller, C. D. and Ellenberg, J. (1999). Chapter 16: Monitoring the dynamics and mobility of membrane proteins tagged with green fluorescent protein. In: Sullivan, K. F. and Kay, S. A. (Eds.). Methods in Cell Biology. Academic Press 261-281.
  9. Moonschi, F. H., Effinger, A. K., Zhang, X., Martin, W. E., Fox, A. M., Heidary, D. K., DeRouchey, J. E. and Richards, C. I. (2015). Cell-derived vesicles for single-molecule imaging of membrane proteins. Angew Chem Int Ed Engl 54(2): 481-484.
  10. Richards, C. I., Luong, K., Srinivasan, R., Turner, S. W., Dougherty, D. A., Korlach, J. and Lester, H. A. (2012). Live-cell imaging of single receptor composition using zero-mode waveguide nanostructures. Nano Lett 12(7): 3690-3694.
  11. Tan, Y. W., Hanson, J. A., Chu, J. W. and Yang, H. (2014). Confocal single-molecule FRET for protein conformational dynamics. Methods Mol Biol 1084: 51-62
  12. Ulbrich, M. H. and Isacoff, E. Y. (2007). Subunit counting in membrane-bound proteins. Nat Methods 4(4): 319-321.
  13. Wang, S., Vafabakhsh, R., Borschel, W. F., Ha, T. and Nichols, C. G. (2016) Structural dynamics of potassium-channel gating revealed by single-molecule FRET. Nat Struct Mol Biol 23: 31
  14. Walter, N. G. and Bustamante, C. (2014) Introduction to single molecule imaging and mechanics: seeing and touching molecules one at a time. Chem Rev 114(6): 3069-71.

简介

细胞衍生的囊泡促进跨膜蛋白在其生理膜中的分离,从而维持其结构和功能完整性。 这些囊泡可以由产生,容纳或运输蛋白质的不同细胞器产生。 结合单分子成像,可以使用分离的细胞器特异性囊泡来研究嵌入蛋白质的运输和组装。 在这里,我们提出了一种通过使用OptiPrep梯度和氮气穴通过从HEK293T细胞中分离ER和质膜囊泡来进行细胞器特异性单分子成像的方法。 通过Western印迹验证分离,并使用分离的囊泡进行寡聚受体组装的单分子研究。

【背景】大量的跨膜蛋白通过多个亚基的组装形成,导致复杂的寡聚结构,其可以通常以多种化学计量存在。了解组装中的变化如何改变在不同细胞器中的贩运和本地化对于确定蛋白质的生理作用以及与成熟和运输相关的疾病的连接至关重要。单分子方法可以通过直接测量其化学计量比来更好地理解寡聚蛋白的组装(Ulbrich和Isacoff,2007; Richards等人,2012)。这种方法避免了整体平均,从而提供了所有化学计量的平均状态(Walter and Bustamante,2014)。单分子研究近来已被用于理解大分子的结构和功能特性,包括构象动力学(Tan等人,2014),离子通道门控(Wang等人 ,2016),配体 - 受体相互作用(Moonschi等人,2015)和化学计量组装(Ulbrich和Isacoff,2007; Moonschi等人,2015)。进行单分子研究时,必须在支持双层中分离单个受体。从细胞环境中分离通常是必需的,因为哺乳动物细胞中膜蛋白的内源浓度通常比与单分子研究相容的条件高得多(Richards等人,2012)。此外,活细胞单分子研究存在许多缺点,包括高水平的自体荧光,有限的荧光团亮度和细胞表面上膜蛋白的迁移率(Andersson等,1998; Lippincott -Schwartz等人,1999)。使用人造双层如脂质体的分离策略也是有问题的,因为它们包括中间步骤,其中蛋白质稳定在对跨膜蛋白质的结构完整性构成威胁的洗涤剂溶液中。膜衍生的囊泡能够使蛋白质保持嵌入其生理膜中,保持其结构和功能的完整性。这些纳米囊泡已被用于研究多聚蛋白的化学计量组装,探测配体受体相互作用,并理解配体对烟碱受体组装的影响(Fox等人,2015; Moonschi等,等,2015; Fox-Loe等,<2017>)。

跨膜蛋白在内质网中合成并组装,然后转运至质膜。具有多个不同亚基的寡聚蛋白通常可以用不同的亚基化学计量法组装,这可能导致不同的运输和功能特性(Grady等,,2010)。例如,α4β2烟碱受体是具有两种可能的化学计量组装的五聚体受体:(α4)2(β2)3和(α4)3( β2)<子> 2 。据推测,细胞机器优先从ER到质膜上运送高灵敏度异构体(α4)2(β2)3→另一种异构体。还假设尼古丁改变了该受体从ER中的低敏感性到高敏感性异构体的组装,导致更高水平的ER和质膜中优先贩卖的组装(Lester等人, em>,2009; Henderson和Lester,2015)。来自特定细胞器的蛋白质的单分子研究使得烟碱受体的结构组装变化与贩运和装配中的变化相关。

在这里我们提出一种方法,可以使用氮空化和OptiPrep梯度将单个跨膜蛋白分离成ER和质膜衍生的囊泡。 ER和质膜衍生的囊泡表现出不同的密度,因为它们含有不同的磷脂和相关蛋白。 ER起源的囊泡比从质膜获得的囊泡更密集。选择OptiPrep梯度是因为其优异的维持等渗压力的能力,而与用于分离细胞器和亚细胞囊泡的梯度密度无关(Graham等,1994)。我们应用这种方法来研究配体诱导的内质网中烟碱受体组装以及质膜上的变化,并通过蛋白质印迹和单分子逐步光漂白验证了我们的方法。我们相信这种方法几乎可以应用于任何类型的跨膜蛋白进行单分子研究和了解细胞器特定的结构和功能特性。

关键字:单分子, 囊泡, 化学计量学, ER和质膜蛋白分离, 烟碱受体, OptiPrep, 光漂白, 蛋白质转运

材料和试剂

  1. 细胞培养瓶(Greiner Bio One International,目录号:658175)

  2. 50 ml离心管(VWR,目录号:89039-658)
  3. 超清晰超速离心管(Beckman Coulter,目录号:344061)
  4. 四个5毫升血清移液器(VWR,目录号:89130-896)
  5. 一个9英寸的火石玻璃巴斯德吸管(VWR,目录号:14672-380)

  6. 15 ml离心管(VWR,目录号:89039-666)
  7. 1.5毫升管
  8. 葡萄酒软木塞
  9. 剃须刀刀片(VWR,目录号:55411-050)
  10. 两个冰桶(VWR,目录号:10146-202)
  11. HEK293T细胞(Sigma-Aldrich,目录号:85120602-1VL)
  12. 去离子水
  13. PBS缓冲液(VWR,目录号:97062-948)
  14. Versene解决方案(Thermo Fisher Scientific,Gibco TM,目录号:15040066)
  15. 氮气
  16. OptiPrep(60%溶液,Sigma-Aldrich,目录号:D1556-250ML)
  17. 抗calnexin(Abcam,目录号:ab92573)
  18. 抗钠钾ATP酶(Abcam,目录号:ab76020)
  19. 小鼠抗兔二抗(Santa Cruz Biotechnology,目录号:sc-2357)
  20. Clarity TM Western ECL底物(Bio-Rad Laboratories,目录号:1705060)
  21. Tris-HCl(Sigma-Aldrich,Roche Diagnostics,目录号:10812846001)
  22. 氯化钠(NaCl)(Fisher Scientific,目录号:BP358-1)
  23. 氯化镁六水合物(MgCl 2·6H 2 O)(Fisher Scientific,目录号:BP214-500)
  24. 氯化钙(CaCl 2 2)(Fisher Scientific,目录号:C614-500)
  25. 蛋白酶抑制剂迷你片(Thermo Fisher Scientific,Thermo Scientific TM,目录号:A32955)
  26. 蔗糖(VWR,目录号:97063-788)
  27. HEPES(Fisher Scientific,目录号:BP310-500)
  28. 低渗蛋白酶抑制剂溶液(见食谱)
  29. 蔗糖缓冲液(见食谱)

设备

  1. 一个1毫升移液器(吉尔森,目录号:F123602)
  2. 一个移液器控制器(VWR,目录号:613-4442)
  3. 一把剪刀
  4. 一套立场(立场和夹子)
  5. 孵化器
  6. 生物安全柜(Class II,Type A2)
  7. 离心机(Beckman Coulter,型号:Allergra X-22R;转子:Beckman Coulter,型号:SX4250)
  8. 超速离心机(Beckman Coulter,型号:L-60)
  9. 摆臂式转子(Beckman Coulter,型号:SW 28)
  10. 固定角转子(Beckman Coulter,型号:70 Ti)
  11. 肚皮舞者或轨道摇床
  12. 氮空化室/容器(帕尔仪器公司,目录号:4639)
  13. 变流量蠕动泵(Fisherbrand,Thermo Fisher)

程序

  1. 取两个表达荧光标记膜蛋白的融合HEK293T细胞(每个瓶约1200万个细胞)的两个烧瓶(250ml,75cm2)。
  2. 从细胞中吸出培养基并用5ml 1x PBS缓冲液冲洗细胞。
  3. 加入5 ml Versene溶液并在37°C孵育5分钟以从培养瓶中分离细胞。
  4. 将细胞浆收集在15ml离心管中。

  5. 在室温下将浆液在300×g下离心5分钟。
  6. 小心吸出后弃去试管中的上清液(Versene溶液),并在离心管中加入3 ml低渗蛋白酶抑制剂溶液(配方1)。
    使用移液器进出或旋转重新悬浮细胞。
  7. 将试管置于冰桶中,摇动摇床上的桶10分钟以促进细胞膨胀。
  8. 将细胞浆从离心管转移到预冷的(冰上的)氮空化室/容器中。连接容器头,并在氮气中流动至600 psi。在冰上压力下孵育空化室20分钟。缓慢打开阀门同时释放压力,并将细胞裂解液收集到15 ml离心管中(图1)。将试管置于冰上,直至细胞裂解液加入OptiPrep梯度液上。


    图1.用于氮空化的仪器设置A.取预冷的氮空化室,关闭阀(红圈)并将细胞浆加入室中。 B.连接容器头部并打开阀门B1,使氮气在腔室内流动,并在压力达到600psi时关闭阀门B1。放入冰箱5分钟。从冰中取出腔室并缓慢打开阀门B2,同时释放压力并将细胞裂解液收集到15ml离心管中。

  9. 在Ultra-Clear超速离心管中制备OptiPrep梯度,如下所示:
    1. 用刀片将葡萄酒瓶塞切成厚约2毫米的圆形切片(图2A)。用一把剪刀沿着软木切片的周边切割,以便它可以很容易地装入超清晰的超速离心管中。用水清洗软木切片以除去碎片。将其放入无菌水中,直至将其加入超速离心管。


      图2. ER和质膜囊泡收集步骤的图片展示A.将葡萄酒软木塞(A1)切成约2mm厚的切片(A2)。该切片(A3)沿圆周切割以丢弃外部区域(A4)并保持可容易地装入Ultra-Clear超速离心管中的小圆片(A5)。比例尺(A6)放置在图像上进行尺寸比较。 B.将小软木塞插入管中并漂浮在溶液上。 C.随着附加解决方案(20%和10%OptiPrep)的添加,软木塞升至顶部。在添加所有层之后,软木浮在溶液顶上。 D.使用镊子去除软木切片而不干扰梯度。 E.将巴斯德移液管破碎以获得插入蠕动泵入口管中的窄管。 F.泵的最终入口管插入含有梯度溶液的Ultra-Clear管的底部中心。

    2. 准备30%,20%和10%的OptiPrep解决方案,通过用蔗糖缓冲液(配方2)将最初的60%OptiPrep溶液稀释到三个50 ml试管中。
      计算与制备30毫升每种溶液相关的参数见表1
      表1.用60%OptiPrep溶液和蔗糖缓冲液制备30%,20%和10%OptiPrep溶液从供应商处获得的原始OptiPrep溶液含有60%OptiPrep。


    3. 在Ultra-Clear超速离心管中,使用5ml血清移液管和移液管控制器,加入3ml 30%OptiPrep溶液,然后使用镊子在溶液顶部添加软木切片(图2B)。现在滴加3毫升20%OptiPrep,然后3毫升10%OptiPrep,最后3毫升细胞裂解物顶部的软木切片。随着溶液滴入管中,软木切片保持漂浮在溶液顶部,从而有助于避免添加的溶液与管中已经存在的溶液混合(图2C)。
      注意:应该可见三个不同的接口,指示不同密度的4层解决方案。当管子靠着光源放置时,接口清晰可见。

    4. 使用镊子小心地取出葡萄酒软木塞而不干扰梯度(图2D)。
  10. 在25,000 rpm(112,000×g g)下,在4°C下将装有OptiPrep梯度的超速离心管在摇摆转子转子中离心90分钟。
  11. 离心后,应观察到三层囊泡:细胞裂解物与10%OptiPrep,10%和20%OptiPrep,以及20%和30%OptiPrep之间(如图3所示)。


    图3. OptiPrep解决方案界面之间囊泡的存在与收集的不同馏分的相对位置A.三层囊泡可见:0%和10%OptiPrep界面,10%和20%OptiPrep和20%和30%OptiPrep。这张照片是用手机拍摄的。 B.通过将蠕动泵的入口管插入管底部中心,收集串联的馏分1和2,每个1.0ml,然后收集串联的3-8个馏分,每个1.5ml。

  12. 使用蠕动泵收集ER和PM部分如下:
    1. 使用支架和夹子垂直放置Ultra-Clear离心管。
    2. 将玻璃巴斯德吸管分离到小直径管和大直径管的连接处,然后将小直径管插入蠕动泵的进样管内(图2E)。
    3. 小心地将蠕动泵的入口管插入超速离心管(用夹子和支架固定),直到其接触离心管的底部中心(图2F)。有必要确保梯度不受插入过程的干扰。
    4. 启动蠕动泵收集不同的馏分,保持界面处于相同的分数(图3B)。为此,收集馏分1和2,每个1毫升,馏分3至8,每个1.5毫升。
    5. 根据我们用抗钙联接蛋白作为ER标记和抗钠钾ATP酶作为质膜标记的Western印迹结果,组分3和组分4仅含有ER,组分7和组分8仅包含质膜囊泡(图4)。然而,我们已经选择分数3和7作为ER和质膜囊泡分别用于我们的实验。


    图4.从OptiPrep梯度收集的不同级分的蛋白质印迹,以将单个受体分离成ER和质膜特异性囊泡。使用抗钠钾ATP酶抗体(1:200稀释度)与在OptiPrep梯度(上图)中的质膜标记,钠钾ATP酶。采用抗钙联接蛋白抗体(1:200稀释)与梯度物质中的ER标记calnexin结合(下图)。利用ChemiDoc成像系统(Bio-Rad)上的HRP缀合的小鼠抗兔二级抗体(1:5,000稀释)和Clarity TM Western ECL底物以将ER和质膜标记定位于凝胶。在细胞裂解物中发现质膜标记,并且部分5-8和ER标记位于细胞裂解物中并且部分3-6。因此,部分3和4仅包含ER囊泡;级分7和8只含有质膜囊泡。我们选择了第3部分的ER囊泡和第7部分的质膜囊泡。

  13. 用1x PBS缓冲液稀释ER和质膜部分至1:3,并在4°C用固定角转子以30,000rpm(100,000×g g)离心1小时。
  14. 用600μl1x PBS缓冲液或蔗糖缓冲液重悬沉淀,以获得ER和质膜衍生的囊泡。我们通常将囊泡溶液等分到两个或三个1.5-ml管中,并储存在-80°C。我们在7天内使用囊泡。
    注意:如果一个部分不包含囊泡或非常少量的囊泡,离心后管道表面没有可见的沉淀。在这种情况下,我们从囊泡可能存在的区域收集样品。可以注意到,在离转子中心最远的管的表面上形成颗粒,并且可以基于其他管中可见颗粒的位置容易地评估颗粒的可能位置。
    此时,一部分ER和质膜衍生的囊泡将含有感兴趣的蛋白质。对于单分子荧光研究,有必要从表达与荧光蛋白结合的靶蛋白的细胞开始。可以使用含有单个受体的囊泡通过逐步光漂白技术来研究受体的化学计量组装,使用荧光相关光谱学来探测配体 - 受体相互作用,并且使用单分子Forster共振能量转移来理解受体的动力学和构象变化( smFRET)。

食谱

  1. 低渗蛋白酶抑制剂溶液
    10 mM Tris-HCl
    10 mM NaCl
    1.5mM MgCl 2 2/2 0.2mM CaCl 2 2/2 调整pH值到7.4
    每10ml缓冲液一种蛋白酶抑制剂片剂
  2. 蔗糖缓冲液
    250 mM蔗糖
    10 mM HEPES
    调整pH值到7.4

致谢

这项工作部分得到了NIDA培训资助DA016176(AMF)和NIDA DA038817的支持。这项工作改编自Fox-Loe等人,2017,J Biol Chem 292(51):21159-21169。我们声明与本出版物无关的利益冲突或竞争利益。

参考

  1. Andersson,H.,Baechi,T.,Hoechl,M.和Richter,C。(1998)。 活细胞的自体荧光。 J Microsc 191(Pt 1):1-7。
  2. Grady,SR,Drenan,RM,Breining,SR,Yohannes,D.,Wageman,CR,Fedorov,NB,McKinney,S.,Whiteaker,P.,Bencherif,M.,Lester,HA and Marks,MJ(2010) 。 结构差异决定烟碱类化合物对天然α4β2* - ,α6β2* - ,α3β4* - 和α7-尼古丁乙酰胆碱受体。
  3. Graham,J.,Ford,T.和Rickwood,D。(1994)。 从自生梯度的碘克沙醇中制备小鼠肝脏中的亚细胞器 Anal Biochem 220(2):367-373。
  4. Fox,A.M。,Moonschi,F.H。和Richards,C.I。(2015)。 尼古丁代谢物可替宁改变烟碱型乙酰胆碱受体子集的组装和运输。 J Biol Chem 290(40):24403-24412。
  5. Fox-Loe,A.M.,Moonschi,F.H。和Richards,C.I。(2017)。 细胞器特异性α4β2烟碱受体单分子成像揭示了尼古丁对受体组装和细胞的影响 - 表面交易。 J Biol Chem 292(51):21159-21169。
  6. Henderson,B.J。和Lester,H.A。(2015)。 内向外神经药理学烟碱药物 神经药理学 96 (铂B):178-193。
  7. Lester,H.A.,Xiao,C.,Srinivasan,R.,Son,C.D。,Miwa,J.,Pantoja,R.,Banghart,M.R.,Dougherty,D.A.,Goate,A.M。和Wang,J.C。(2009)。 尼古丁是一种乙酰胆碱受体数量和化学计量比的选择性药理学分子伴侣。对药物发现的启示。 AAPS J 11(1):167-177。
  8. Lippincott-Schwartz,J.,Presley,J.F.,Zaal,K.J.,Hirschberg,K.,Miller,C.D。和Ellenberg,J。(1999)。 第16章:监测用绿色荧光蛋白标记的膜蛋白的动态和移动性。在:Sullivan,KF和Kay,SA(Eds。)。细胞生物学方法。 学术新闻 261-281。
  9. Moonschi,F. H.,Effinger,A. K.,Zhang,X.,Martin,W. E.,Fox,A. M.,Heidary,D. K.,DeRouchey,J. E.和Richards,C. I.(2015)。 细胞衍生的囊泡用于膜蛋白的单分子成像 Angew Chem Int Ed Engl 54(2):481-484。
  10. Richards,C.I.,Luong,K.,Srinivasan,R.,Turner,S. W.,Dougherty,D. A.,Korlach,J.和Lester,H.A。(2012)。 使用零模式波导纳米结构的单个受体组合物的活细胞成像 Nano Lett 12(7):3690-3694。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Moonschi, F. H., Fox-Loe, A. M., Fu, X. and Richards, C. I. (2018). Mammalian Cell-derived Vesicles for the Isolation of Organelle Specific Transmembrane Proteins to Conduct Single Molecule Studies. Bio-protocol 8(9): e2825. DOI: 10.21769/BioProtoc.2825.
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