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Aug 2019

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Single-Molecule Studies of Membrane Receptors from Brain Region Specific Nanovesicles
脑区特异性纳米泡膜受体的单分子研究   

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Abstract

Single molecule imaging and spectroscopy are powerful techniques for the study of a wide range of biological processes including protein assembly and trafficking. However, in vivo single molecule imaging of biomolecules has been challenging because of difficulties associated with sample preparation and technical challenges associated with isolating single proteins within a biological system. Here we provide a detailed protocol to conduct ex vivo single molecule imaging where single transmembrane proteins are isolated by rapidly extracting nanovesicles containing receptors of interest from different regions of the brain and subjecting them to single molecule study by using total internal reflection fluorescence (TIRF) microscopy. This protocol discusses the isolation and separation of brain region specific nanovesicles as well as a detailed method to perform TIRF microscopy with those nanovesicles at the single molecule level. This technique can be applied to study trafficking and stoichiometry of various transmembrane proteins from the central nervous system. This approach can be applied to a wide range of animals that are genetically modified to express a membrane protein-fluorescent protein fusion with a wide range of potential applications in many aspects of neurobiology.


Graphic abstract:



EX vivo single molecue imaging of membrane receptors


Keywords: Single molecule imaging (单分子成像), Nanovesicles (纳米囊泡), Nicotinic acetylcholine receptors (酰胺乙酰胆碱受体), Brain region specific imaging (脑区特异性成像), Membrane proteins (膜蛋白), TIRF imaging (TIRF成像)

Background

Single molecule imaging techniques have emerged as powerful methods to study the structure, function, and interactions of biomolecules (Joo et al., 2008; Choi et al., 2019). While ensemble measurements provide important information about the average population, single molecule methods can be used to evaluate the heterogeneity within this population revealing differences in structural and functional states (Lee et al., 2020). Single molecule methods have already been used in a diverse range of applications including measurements of protein and ligand interaction, conformational changes in proteins, and ion channel function (Xia et al., 2013; Martinac, 2017; Fu et al., 2019). These methods have provided powerful ways to characterize individual cellular constituents and single molecules in vitro.


Despite their successful implementations in vitro, single molecule methods for in vivo studies have been particularly challenging. Major challenges for in vivo studies include the heterogenous nature of cellular components, the difficulties associated with light penetration in thick tissues, and the need to isolate a single species from the complex environment within a living system (Fu et al., 2019). In addition, the concentration of protein of interest in some cases is much higher in the native environment than the optimum concentration required for single molecule imaging (Richards et al., 2012). Additionally, membrane proteins are not spatially isolated in a single location within the cellular environment and instead diffuse across a wide region, complicating single molecule studies. This makes isolation and separation of the molecule of interest a key aspect of any ex vivo single molecule imaging protocol. This protocol details an approach to address these challenges, by utilizing nanoscale vesicles generated from primary tissue as a way of isolating and immobilizing membrane proteins. We demonstrate this ex vivo single molecule imaging approach using knock-in mice containing a fluorescent protein labeled nicotinic acetylcholine receptor (nAChRs). Nicotinic acetylcholine receptors (nAChRs) are transmembrane proteins which respond to and can be upregulated during nicotine exposure (Cano et al., 2020). Their trafficking and stoichiometry have also shown to be altered by nicotine (Srinivasan et al., 2011). In this protocol, we used a mouse model where the nAChR α4 subunit is labelled with green fluorescence protein (GFP).


We believe this protocol can be widely applied to virtually any mouse model that focuses on a transmembrane protein that is genetically labeled with a fluorescent protein. This protocol will enable researchers to extend single molecule studies beyond common in vitro approaches by performing ex vivo characterization of physiological changes occurring in live animals at the individual protein level. Specifically, this protocol describes a detailed method to isolate nanovesicles from specific regions of the mouse brain in order to perform single molecule imaging to characterize membrane receptors. This technique can be used to understand brain region-specific structural and functional changes in various animal models. An advantage of this approach is that during the isolation of the vesicles, transmembrane receptors maintain their structural integrity because they remain in their endogenous membrane in the nanovesicles. This allows one to characterize their structural assembly as well as their functional properties as in their native physiological environment (Fu et al., 2019). This single molecule protocol which involves sample preparation and labelling along with Total Internal Reflection Fluorescence (TIRF) Microscopy should be immediately applicable to a wide range of existing mouse models already in use for other neuroscience applications.


 Materials and Reagents

  1. Tygon tubing (Garinger, Item number: 9MH86)

  2. 1 ml plastic syringe (Grainger, Item number: 19G334, Thermo Fisher Scientific, catalog number: NC0786233)

  3. Syringe needles (Air-Tite, catalog number: 161028)

  4. 50 ml centrifuge tubes (VWR, catalog number: 89039-658)

  5. 5 ml Serological pipettes (VWR, catalog number: 89130-896)

  6. 15 ml centrifuge tubes (VWR, catalog number: 89039-666)

  7. 1.5 ml Flex tubes Eppendorf (VWR, catalog number: 20901-421)

  8. Ultra-Clear ultracentrifuge tubes (Beckman Coulter, catalog number: 344061)

  9. Dounce Tissue Grinder 7ml (Omni International, catalog number: 07-357542)

  10. Glass bottom dish (MatTek, catalog number: P35G-1.5-14-C)

  11. Fatal Plus Solution (Drugs.com, product number: V.P.L. 9373, NDC number: 0298-9373-68)

  12. Protease inhibitor mini tablet (Thermo Fisher Scientific, catalog number: A32955)

  13. Sucrose (VWR, catalog number: 97063-788)

  14. HEPES (Fisher Scientific, catalog number: BP310-500)

  15. Ice

  16. Milli-Q Water (Fisher Scientific, catalog number: QGARD00D2)

  17. 1× PBS buffer (VWR, catalog number: 97062-948)

  18. HCl (VWR, catalog number: 470301-260)

  19. NaOH ((VWR, catalog number: BDH7225-4)

  20. Biotin-PEG-Silane (Laysan Bio, Item number: Biotin-PEG-SIL-3400-500mg)

  21. Neutravidin protein (Thermo Fisher Scientific, catalog number: 31000)

  22. Biotinylated anti-GFP antibody (Rockland antibodies and assays, item number: 600-406-215)

  23. Sucrose buffer/Homogenization buffer, pH 7.4 (see Recipes)

  24. Fatal Plus Working Solution (see Recipes)

Equipment

  1. Smooth fine forceps (Electron Microscopy Sciences, 1209K43, catalog number: 72911-6)

  2. Surgical scissors (Medline, catalog number: MDS0838410)

  3. Operating scissors (Stoelting, catalog number: 52138-51)

  4. Flat tip forceps (Fisher Scientific, catalog number: 12-000-123)

  5. Ice bucket (VWR, catalog number: 10146-202)

  6. Adult Mouse Brain Slicer Matrix (Zivic Instruments, catalog number: BSMAS002-1)

  7. 1 ml pipettor (Gilson, catalog number: F123602)

  8. Pipet controller (VWR, catalog number: 613-4442)

  9. Autoclave (Steris Amsco Eagle, serial no. 096899 SAW)

  10. Perfusion pump (Grainger, catalog number: 2688)

  11. Centrifuge (Beckman Coulter, model: Allergra® X-22R; Rotor: Beckman Coulter, model: SX4250)

  12. Ultracentrifuge (Beckman Coulter, model: L-60)

  13. Swing bucket rotor (Beckman Coulter, model: SW 28)

  14. Fixed angle rotor (Beckman Coulter, model: 70 Ti)

  15. Belly dancer or orbital shaker (Fisher Scientific, catalog number: 15-453-211)

  16. Sonicator (Amsco Reliance Sonic 550)

  17. Fume Hood (Supreme Air LV, Kewaunee Scientific, VWR, catalog number: 97006-002) or an air compression system

  18. Oxygen plasma cleaner (Harrick Plasma, catalog number: PDC-32G)

  19. Vortex (VWR, catalog number: 10153-836)

  20. 4 °C fridge

  21. -20 °C freezer

  22. A TIRF-capable microscope (Olympus, model: IX2-ZDC2, serial number: 0F83094)

Software

  1. Metamorph (Available on Olympus Microscope)

  2. ImageJ/FIJI (Freely available from NIH)

Procedure

There are animal experiments involved in the following procedures. All animal experiments were conducted within the guidelines set forth by the National Institutes of Health (NIH) and were approved by the University of Kentucky’s Institutional Animal Care and Use Committee (IACUC). In our case, the α4-GFP knock-in mouse (2-4 months old) which has green fluorescence protein (GFP) expressed in the α4 subunit of α4 β2 nicotinic acetylcholine receptor (nAChR) were used throughout. The strains of α4-GFP knock-in mice were obtained from Dr. Jerry Stitzel’s Lab (Institute for Behavioral Genetics University of Colorado). These mice express a green fluorescent protein CHRNA4 fusion protein in replacement of the endogenous nicotinic receptor subunit.

  1. Perfusion and Brain Extraction

    1. Prepare the required buffers (see the Recipes section), rinse the surgical tools with water, dry them and autoclave in cycle 2 (Gravity cycle) prior to animal perfusion.

    2. Perform humane euthanasia to a mouse by injecting 200 μl of Fatal plus working solution (see Recipe 2). Wait until the animal becomes nonresponsive by checking foot pinch reflex. Usually, the mouse will become nonresponsive 2-3 min after the injection of fatal plus.

      Note: Intravenous injection is the most preferred method, however other injection method such as subcutaneous could be used when it is impractical to do so.

    3. Once nonresponsive, lay the mouse keeping ventral side up atop a foam piece and immobilize it with tape or metal pins. Using small scissors and forceps, pinch a piece of skin and make a small cut in the lower thorax. Make a parabolic, sagittal cut that runs up the length of each side of abdomen. Pull back the loosened tissue. Make a transverse cut across the pectoral region whilst tearing through the diaphragm. Anchor the newly loosened tissue to ensure it is out of the way (Gage et al., 2012).

      Note: You will require 3-4 mice for region specific nanovesicles preparation.

    4. Set the perfusion pump to Level 3. Run tygon tubing from the PBS buffer to the pump and then to a 1ml plastic syringe. Insert the needle into the left ventricle (LV). Turn the pump on to perfuse with PBS. Immediately make an incision in the aorta. Exsanguinate until the liver turns tan-colored and the blood is nearly translucent.

      Note: A flow rate of around 0.72 ml/min is good. It is important that all the blood is cleared, and liver is tan colored after perfusion otherwise there might still be some blood in the brain vasculature which can interfere with single molecule imaging. Usually, 100 ml PBS is enough to clear blood from a mouse.

    5. For the brain extraction, remove the head by decapitation using large, 6” scissors. Locate the spinal column and remove excess cervical vertebrae. Make an approximately 4 mm cut from the base of the magnum foramen (MF) towards the nose and from the MF laterally towards the ears. Using regular forceps, peel back loosened bone tissue. Make a midsagittal cut from the base of the skull all the way up to the nose being sure to insert the blunt edge of the scissors in first and dragging on the underside of the brain and then making the cut. Make a transverse cut from one eye to the other eye. Using tweezers peel back newly loosened skull tissue. Gently pull the brain out and put in a conical tube containing ice-cold homogenization buffer (Gage et al., 2012).


  2. Brain region specific nanovesicles isolation

    1. Put the brain in a 2 mm Zivic mouse brain matrix and slice the brain (Figure 1). Remove the olfactory bulb and the region of spinal cord if any is left. The first slice which comes after the olfactory bulb will contain the prefrontal cortex and striatum. Separate them and collect in 15 ml centrifuge tubes. The second slice will contain the cortex, hippocampus, thalamus, and hypothalamus. Isolate the regions and collect them in 15 ml centrifuge tubes. The slice posterior to the second slice will contain the cortex, hippocampus, and midbrain. Separate the regions and collect them in 15 ml centrifuge tubes. The remaining region will mostly contain cerebellum. Take out the slices and with the help of forceps, isolate the regions and combine the identical brain regions from multiple mice together. Be careful not to contaminate one region by another.



      Figure 1. Set up for Brain Slicing and Region Identification. A. A mouse brain kept in a 2 mm Zivic brain matrix. B. Blades kept on the brain matrix to make 2 mm thin mouse brain slices. C. An illustration of mouse brain to identify various brain regions from the slices. The anterior part contains the olfactory bulb, and the posterior part is the cerebellum attached to the spinal cord. Slices shown at the bottom contain parts between the olfactory bulb and the cerebellum.


    2. Use a Dounce homogenizer to homogenize the region-specific fresh tissues by using 2 ml of cold homogenization buffer (Recipe 1 below). Add additional 3 ml of buffer to the mixture and centrifuge at 200 × g for 15 min at 4 °C. Remove the pellet carefully and proceed to the next step with the supernatant.

    3. Centrifuge the supernatant brain lysate at 1,000 × g for 15 min at 4 °C to remove the pellet which contain nuclear fraction.

    4. Collect the supernatant and perform ultracentrifugation at 10,000 × g for 20 min at 4 °C to remove mitochondria. Remove the supernatant and centrifuge it at 100,000 × g for 2 h at 4 °C. This step will yield pellets containing vesicles. Resuspend the vesicles in 300-400 μl of 1× PBS buffer. The vesicles can be stored for few weeks at -80 °C.

      Note: Store 50-100 μl per vial to make more convenient while performing TIRF imaging.


  3. Single molecule labelling for TIRF imaging

    1. To perform glass cleaning, take 4-5 gamma irradiated glass bottom dishes and sonicate them in 5 M NaOH solution for 1 h at 45 °C. Rinse them 3 times with DI water and sonicate them again in 0.1 M HCl solution for 1 h at 45 °C. Finally rinse the dishes 3 times with water followed by rinsing with ethanol 3 times.

    2. Dry the cleaned dish by using compressed air and perform oxygen plasma cleaning. Oxygen plasma cleaning should be performed at high plasma level for 5 min. This helps to remove some organic impurities which are not removed from previous cleaning steps (Raiber et al., 2005).

      Note: It is strongly recommended to check background fluorescence of the dishes during this step. If the dishes have a large background characterized by the presence of >10-20 isolated background molecules in a field of view discard them or clean them again.

    3. Perform a functionalization of each dish using 1 mg/ml solution of Biotin-PEG-silane in 95% ethanol for 30 min. After 30 min, wash the dishes 3 times with DI water.

      Note: Functionalization of glass bottom dishes and immobilization of nanovesicles is carried out at room temperature.

    4. Add 0.1 mg/ml of NeutrAvidin solution in PBS to the dishes for 5 min. After 5 min, rinse the dishes with 1× PBS 3 times.

    5. Add 1 μg/ml of biotinylated anti-GFP antibody in 1× PBS and incubate the dishes for 30 min. Rinse the dishes with 1× PBS 3 times.

    6. The dishes should be imaged before adding vesicles, some dishes could also be kept filled with 1.5 ml of 1× PBS (without vesicles) to compare against the dishes where vesicles are added.

    7. Immobilize the spatially isolated nanovesicles on the functionalized dishes by incubating at room temperature for 30 min (The nanovesicles will be immobilized as shown in the Figure 2).

      Note: Usually 50 μl of nanovesicles in 200 μl of 1× PBS is good for TIRF imaging. If the vesicle concentration is too high (resulting in very bright fluorescence but no isolated molecules) or too low (resulting in very low or no single molecule fluorescence), change the concentration accordingly.

    8. Rinse the dish again with 1 ml of 1× PBS 3 times. Add 1 ml 1× PBS to the dish to perform TIRF imaging.



      Figure 2. Schematic Diagram of GFP containing vesicles immobilized in a dish. A clean glass bottom dish is coated with biotin-peg-silane, the silane will bind with the glass and biotin will be available to bind with neutravidin. Neutravidin has binding sites to bind with biotin. During washing steps, unbound neutravidin will be washed away while the leftover sites of the bound neutravidin will be used to bind biotinylated anti-GFP antibody. This antibody will bind to the GFP molecule present in the nicotinic acetylcholine receptor of the nanovesicles.


  4. TIRF Imaging of single molecules and data analysis

    1. Turn on the Microscope system including laser, stage controller and accompanying software (e.g., Metamorph for Olympus microscope). Wait for 30 min to allow software, camera, and other parts in the microscope system to equilibrate.

      Note: The microscope camera should be sufficiently cooled (~ -80 °C) before conducting the imaging. Please do not turn on the camera at higher temperatures.

    2. Enter into the epifluorescence mode by using the appropriate software in the microscope system (Metamorph software in the Olympus Microscope). Set the laser power to approximately 3 mW/cm2, and make sure the laser is focused and aligned. Ideally the laser should be focused on a point or a small circle at a distance from the objective while in epifluorescence mode. Set up an exposure time to 100 ms. Emission light is detected by an electron multiplying charge coupled device (EMCCD, Andor).

    3. Put a drop of oil on the 60× oil objective. Place a clean glass bottom dish containing PBS above the objective and use eyepiece and focus knob to bring the objective close to the bottom of the dish. Be careful not to hit the dish by objective. The oil drop should stay between the base of the dish and the objective.

      Note: You can visualize the fluorescence by using the eyepiece or the software accompanying the microscope. Once the molecules are in focus, you can enter into TIRF mode.

    4. Adjust the angle to gain total internal reflection. You can visualize the laser beam to ensure it is in TIRF mode (as shown in Figure 3). In addition, the nanovesicles will also be spatially isolated in TIRF mode (as shown in Figure 4A). Acquire 800 frames with 100 ms exposure time for each field of view. Store the collected frame by using the stream acquisition mode in the software.



      Figure 3. Schematic Diagram of TIRF Microscopy on a Glass Bottom Dish. An oil objective is used where the laser beam is adjusted such that it returns by total internal reflection. The bottom ~200 nm thickness of the dish is illuminated by the evanescent field that originates from total internal reflection on the glass side. This filed allows to image fluorescent single molecule nanovesicles on the bottom surface of the glass bottom dish.


    5. Use ImageJ/FIJI software to open the frames (Figure 4) and select regions of interest to perform analysis of change in fluorescence intensity (Rueden et al., 2017).



      Figure 4. Example data from the single molecule imaging of alpha-4 GFP nanovesicles . A. A TIRF image of spatially isolated nanovesicles containing GFP tethered membrane receptors (scale bar = 5 µm). B. Fluorescence intensity pattern of an individual single molecule from the nanovesicle showing two photobleaching steps (a single molecule region of interest (ROI) is shown in (A)). These photobleaching steps give information about how the alpha-4 beta-2 nAChRs are distributed in the brain.

Recipes

  1. Sucrose buffer/Homogenization buffer, pH 7.4

    0.32 M sucrose

    10 mM HEPES

    2 mM EDTA

    Protease inhibitor (One protease inhibitor tablet per 10 ml buffer)

  2. Fatal Plus Working Solution

    1 ml Fatal Plus

    10 ml PBS (1×)

Acknowledgments

This work was adapted from Fu et al. Anal. Chem. 2019, 91(15): 10125-10131. Biorender was used to construct most figures.

Competing interests

We declare no conflict of interest or competitive interest related to this publication.

Ethics

All the experiments were conducted within the guidelines set forth by the National Institutes of Health and were approved by the University of Kentucky’s Institutional Animal Care and Use Committee.

References

  1. Cano, M., Reynaga, D. D., Belluzzi, J. D., Loughlin, S. E. and Leslie, F. (2020). Chronic exposure to cigarette smoke extract upregulates nicotinic receptor binding in adult and adolescent rats. Neuropharmacology 181: 108308.
  2. Choi, J., Grosely, R., Puglisi, E. V. and Puglisi, J. D. (2019). Expanding single-molecule fluorescence spectroscopy to capture complexity in biology. Curr Opin Struct Biol 58: 233-240.
  3. Fu, X., Moonschi, F. H., Fox-Loe, A. M., Snell, A. A., Hopkins, D. M., Avelar, A. J., Henderson, B. J., Pauly, J. R. and Richards, C. I. (2019). Brain Region Specific Single-Molecule Fluorescence Imaging. Anal Chem 91(15): 10125-10131.
  4. Gage, G. J., Kipke, D. R. and Shain, W. (2012). Whole animal perfusion fixation for rodents. J Vis Exp(65): e3564.
  5. Joo, C., Balci, H., Ishitsuka, Y., Buranachai, C. and Ha, T. (2008). Advances in single-molecule fluorescence methods for molecular biology. Annu Rev Biochem 77: 51-76.
  6. Lee, H., Kang, H., Kang, M., Han, C., Yi, J., Kwon, Y. and Park, J. (2020). Heterogeneous Subcellular Origin of Exosome-Mimetic Nanovesicles Engineered from Cells. ACS Biomater Sci Eng 6(11): 6063-6068.
  7. Martinac, B. (2017). Single-molecule FRET studies of ion channels. Prog Biophys Mol Biol 130(Pt B): 192-197.
  8. Raiber, K., Terfort, A., Benndorf, C., Krings, N. and Strehblow, H. H. (2005). Removal of self-assembled monolayers of alkanethiolates on gold by plasma cleaning.Surface Science 595(1-3): 56-63.
  9. 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.
  10. Rueden, C. T., Schindelin, J., Hiner, M. C., DeZonia, B. E., Walter, A. E., Arena, E. T. and Eliceiri, K. W. (2017). ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics 18(1): 529.
  11. Srinivasan, R., Pantoja, R., Moss, F. J., Mackey, E. D., Son, C. D., Miwa, J. and Lester, H. A. (2011). Nicotine up-regulates α4β2 nicotinic receptors and ER exit sites via stoichiometry-dependent chaperoning. 137(1): 59-79.
  12. Xia, T., Yuan, J. and Fang, X. (2013). Conformational dynamics of an ATP-binding DNA aptamer: a single-molecule study. J Phys Chem B 117(48): 14994-15003.


简介

[摘要]单分子成像和光谱学是研究包括蛋白质组装和运输在内的广泛生物学过程的强大技术。然而,由于与样品制备相关的困难以及与在生物系统内分离单个蛋白质相关的技术挑战,生物分子的体内单分子成像一直具有挑战性。在这里,我们提供了进行体外实验的详细方案单分子成像,其中通过从大脑不同区域快速提取含有目标受体的纳米囊泡来分离单个跨膜蛋白,并使用全内反射荧光(TIRF)显微镜对它们进行单分子研究。该协议讨论了脑区域特异性纳米囊泡的分离和分离,以及在单个分子水平上用这些纳米囊泡进行TIRF显微镜检查的详细方法。该技术可用于研究来自中枢神经系统的各种跨膜蛋白的运输和化学计量。该方法可以应用于经过遗传修饰以表达膜蛋白-荧光蛋白融合物的多种动物,在神经生物学的许多方面具有广泛的潜在应用。

图形摘要:


膜受体的离体单分子成像


[背景]单分子成像技术已经成为有力的方法来研究的结构,功能,和生物分子的相互作用(珠等人,2008;彩。等人,2019) 。虽然总体测量提供了有关平均种群的重要信息,但单分子方法可用于评估该种群内的异质性,从而揭示结构和功能状态的差异(Lee等人,2020年)。单分子方法已经在多种应用中使用,包括蛋白质和配体相互作用的测量,蛋白质的构象变化以及离子通道功能(Xia等人,2013; Martinac,2017;Fu等人,2019)。这些方法提供了强大的方法来表征体外单个细胞成分和单分子。

尽管它们在体外已成功实施,但用于体内研究的单分子方法仍然具有很大的挑战性。体内研究的主要挑战包括细胞成分的异质性,与厚组织中的光穿透相关的困难以及需要将单一物种与生物系统中复杂环境隔离的需求(Fu et al。,2019)。另外,在某些情况下,天然环境中目标蛋白质的浓度比单分子成像所需的最佳浓度高得多(Richards等,2012)。此外,膜蛋白不是在细胞环境中的单个位置上被空间隔离,而是扩散到很宽的区域,这使单分子研究变得复杂。这使得目标分子的分离和分离成为任何离体单分子成像方案的关键方面。该协议详细介绍了一种通过利用从原发组织产生的纳米级囊泡作为分离和固定膜蛋白的方法来应对这些挑战的方法。我们展示了使用含有荧光蛋白标记的烟碱乙酰胆碱受体(nAChRs )的敲入小鼠的这种离体单分子成像方法。烟碱乙酰胆碱受体(nAChRs的)是跨膜蛋白,其响应并可以尼古丁曝光期间被上调(卡诺等人,2020) 。其贩卖和化学计量公顷S还示出由尼古丁被改变(斯里尼瓦桑等人,2011) 。在这个协议中,我们使用其中的小鼠模型的nAChR α4亚基标记有绿色荧光蛋白(GFP)。

我们相信,该协议实际上可以广泛应用于任何侧重于跨膜蛋白的小鼠模型,该跨膜蛋白已用荧光蛋白进行了遗传标记。该协议将使研究人员能够通过对活体动物在单个蛋白质水平上发生的生理变化进行体外表征,从而将单分子研究扩展到普通的体外方法之外。具体来说,该协议描述了一种从小鼠大脑特定区域分离纳米囊泡的详细方法,以执行单分子成像以表征膜受体。该技术可用于了解各种动物模型中特定于大脑区域的结构和功能的变化。该方法的优点是在囊泡分离期间,跨膜受体保持其结构完整性,因为它们保留在纳米囊泡的内源膜中。这使人们能够表征其在天然生理环境中的结构装配及其功能特性(Fu等人,2019)。这种涉及样品制备和标记以及全内反射荧光(TIRF)显微镜检查的单分子方案应立即适用于已经用于其他神经科学应用的多种现有小鼠模型。

关键字:单分子成像, 纳米囊泡, 酰胺乙酰胆碱受体, 脑区特异性成像, 膜蛋白, TIRF成像



材料和试剂


Tygon管(Garinger ,项目编号:9MH86)
1毫升塑料注射器(固安捷,商品编号:19G334,热连接SH呃科学,目录号:NC0786233)
注射器针头(Air- Tite ,目录号:161028)
50 ml离心管(VWR,目录号:89039-658)
5 ml血清移液器(VWR,目录号:89130-896)
15 ml离心管(VWR,目录号:89039-666)
1.5 ml微量离心管Eppendorf(VWR,目录号:20901-421)
超清晰超速离心管(Beckman Coulter,目录号:344061)
杜恩斯组织研磨7毫升(全向我国际电信联盟,目录号:07-357542)
玻璃底盘(MatTek ,目录号:P35G-1.5-14-C )
致命Plus解决方案(Drugs.com,p RODUCT号:VPL 9373,NDC号码:0298-9373-68)
蛋白酶抑制剂迷你片剂(Thermo Fisher Scientific,目录号:A32955)
蔗糖(VWR,目录号:97063-788)
HEPES(Fisher Scientific,目录号:BP310-500)

Milli-Q Water (Fisher Scientific,目录号:QGARD00D2)
1 × PBS缓冲液(VWR,目录号:97062-948)
HCl(VWR,目录号:470301-260)
NaOH((VWR,目录号:BDH7225-4)
生物素-PEG-硅烷(Laysan Bio,项目号:生物素-PEG-SIL-3400-500mg)
中性蛋白(热网络SH呃科学,Ç atalog数:31000)
生物素化的抗GFP抗体(Rockland的抗体和测定法,我TEM号:600-406-215)
蔗糖缓冲液/均质缓冲液,pH 7.4 (请参见配方)
致命加工作解决方案(请参阅食谱)


设备


光滑的精细镊子(Electron Microscopy Sciences,1209 K43,货号:72911-6 )
手术剪(Medline,目录号:MDS0838410)
手术剪(Stoelting ,目录号:52138-51)
平头镊子(Fisher Scientific,目录号:12-000-123)
冰桶(VWR,货号:10146-202)
成年小鼠脑切片矩阵(Zivic我nstruments,目录号:BSMAS002-1)
1 ml移液器(Gilson,目录号:F123602)
移液器控制器(VWR,目录号:613-4442)
高压釜(Steris的AMSCO鹰,š erial号096899 SAW)
灌注泵(固安捷,ç atalog Ñ棕土:2688)
离心机(Beckman Coulter公司,型号:Allergra ® X-22R;转子:Beckman Coulter公司,型号:SX4250)
超速离心机(贝克曼库尔特公司,型号:L-60)
摆斗式转子(贝克曼库尔特(Beckman Coulter),型号:SW 28)
固定角转子(贝克曼库尔特(Beckman Coulter),型号70 Ti )
肚皮舞者或轨道振动器(Fisher Scientific,目录号:15-453-211)
超声波仪(Amsco Reliance Sonic 550)
通风橱(Supreme Air LV,Kewaunee Scientific,VWR ,目录号:97006-002)或空气压缩系统
氧气等离子清洁器(Harrick等离子,目录号:PDC-32G)
涡流(VWR,货号:10153-836)
4 °C冰箱
-20 °C冷冻室
具有TIRF功能的显微镜(奥林巴斯,型号:IX2-ZDC2 ,序列号:0F83094)


软件


变形(在奥林巴斯显微镜上可用)
ImageJ / FIJI(可从NIH免费获得)


程序


以下过程涉及动物实验。所有动物实验均在美国国立卫生研究院(NIH)制定的指导原则下进行,并得到肯塔基大学机构动物护理和使用委员会(IACUC)的批准。在我们的案例中,始终使用α4-GFP敲入小鼠(2-4个月大),该小鼠的绿色荧光蛋白(GFP)在α4β2烟碱乙酰胆碱受体(nAChR )的α4亚基中表达。α4-GFP敲入小鼠品系是从Jerry Stitzel博士实验室(科罗拉多州行为遗传学研究所)获得的。这些小鼠表达绿色荧光蛋白CHRNA4融合蛋白替代内源烟碱受体亚基。


灌注和脑提取
准备所需的缓冲器(参照ř ecipe小号部分),用清水冲洗手术工具,干燥,并在高压灭菌循环2(循环重力)之前的动物灌注。
通过注入200执行人道的安乐死于鼠标微升致命加工作溶液(见ř ecipe 2 )。等待直到动物不再无反应为止,方法是检查脚捏反射。通常,在注射致命性加号后2-3分钟,鼠标将变得无反应。
注意:静脉注射是最优选的方法,但是在不可行的情况下,可以使用其他注射方法,例如皮下注射。


一旦无反应,将鼠标保持腹侧朝上放在一块泡沫片上,并用胶带或金属销钉将其固定。用小剪刀和镊子捏一块皮肤,然后在下胸部做一个小切口。进行抛物线状,矢状切开,延伸至腹部各侧的长度。向后拉松动的纸巾。穿过胸膜横切,同时撕开横the膜。锚定新松动的组织,以确保其不会妨碍操作(Gage等,2012)。
注意:您将需要3-4只小鼠来准备区域特定的纳米囊泡。


将灌注泵设置为Level3。将tygon管从PBS缓冲液运至泵,然后运至1ml塑料注射器。将针头插入左心室(LV)。打开泵,用PBS灌注。立即在主动脉上切开切口。放血直到肝脏变成棕褐色并且血液几乎是半透明的。
注意:约0.72 ml / min的流速是好的。重要的是清除所有血液,并在灌注后使肝脏染成棕褐色,否则脑血管中仍可能存在一些血液,可能会干扰单分子成像。通常,100 ml PBS足以清除小鼠的血液。


对于脑部提取,请使用6英寸大剪刀通过头皮下垂来取下头部。找到脊柱并去除多余的颈椎。从大瓶孔(MF)的底部朝鼻子,从MF的侧面朝耳朵切大约4毫米。用常规镊子将松动的骨组织剥下。从头骨的底部一直到鼻子进行矢状切开,确保先将剪刀的钝边插入,然后在脑底拖动,然后再进行切开。从一只眼睛到另一只眼睛进行横向切割。用镊子将刚松开的颅骨组织剥开。轻轻拉出大脑,并把我含N冰冷均质化缓冲液的圆锥形管(盖奇等人,2012) 。


脑区域特异的纳米囊泡分离
把大脑在2毫米Zivic小鼠脑基质和切片脑(˚F igure 1)。除去嗅球和剩余的脊髓区域。嗅球后的第一个切片将包含前额叶皮层和纹状体。分离它们并收集在15 ml离心管中。第二个切片将包含皮质,海马,丘脑和下丘脑。隔离区域并将其收集在15 ml离心管中。第二个切片后的切片将包含皮质,海马和中脑。分离区域并收集在15 ml离心管中。其余区域将主要包含小脑。取出切片,并在镊子的帮助下,将这些区域隔离,并将来自多个小鼠的相同大脑区域组合在一起。注意不要污染一个区域。




图1.进行脑切片和区域识别的设置。A.将老鼠的大脑保存在2毫米的Zivic脑矩阵中。B.刀片保留在脑基质上,可制成2毫米薄的小鼠脑切片。C.从切片中识别各种大脑区域的小鼠大脑的图解。前部包含嗅球,后部是附着在脊髓上的小脑。底部显示的切片包含嗅球和小脑之间的部分。


使用Dounce均质器,通过使用2 ml冷均质缓冲液来均质化特定于区域的新鲜组织(下面的R ecipe 1 )。向混合物中再添加3 ml缓冲液,并在4 °C下以200 ×g离心15分钟。小心取出沉淀物,然后将上清液用于下一步。
在4°C下以1,000 ×g离心上清液的脑裂解液15分钟,以除去含有核仁级分的沉淀。
收集上清液,并在4°C下以10,000 ×g超速离心20分钟以去除线粒体。除去上清液,在4 °C下以100,000 ×g离心2小时。该步骤将产生包含囊泡的丸粒。重悬在囊泡300-400微升的1 × PBS缓冲液中。囊泡可以在-80 °C下保存数周。
注:存储50-100微升每瓶使在执行全内反射荧光成像更加方便。


TIR F成像的单分子标记
要进行玻璃清洁,请使用4-5个经γ射线辐照的玻璃底盘,并在45°C下于5 M NaOH溶液中超声处理1小时。将它们用去离子水冲洗3次,然后在45°C下在0.1 M HCl溶液中再次超声处理1小时。最后用水冲洗餐具3次,然后用乙醇冲洗3次。
用压缩空气擦干干净的碟子,然后进行氧等离子体清洗。氧气血浆清洁应在高血浆水平下进行5分钟。这有助于去除一些先前的清洁步骤未除去的有机杂质(Raiber等,2005)。
注意:强烈建议在此步骤中检查餐具的背景荧光。如果洗碗具有大背景,以视野中存在> 10-20个分离的背景分子为特征,则将其丢弃或再次清洗。


执行一个有趣的使用在95%乙醇1毫克/毫升溶液生物素PEG-硅烷的30分钟每道菜的ctionalization。30分钟后,用去离子水洗碗3次。
注意:玻璃底盘的功能化和纳米囊泡的固定在室温下进行。


向培养皿中加入0.1 mg / ml的NeutrAvidin PBS溶液5分钟。5分钟后,用1 × PBS冲洗餐具3次。
加入1 μ克/ ml生物素化抗GFP抗体的1 × PBS中并孵育菜30分钟。用1 × PBS冲洗餐具3次。
在添加囊泡之前,应先对培养皿进行成像,有些培养皿中也可以装满1.5 ml的1 × PBS(不含囊泡),以与添加囊泡的培养皿进行比较。
IMMO通过在室温下孵育30分钟bilize上官能菜空间分离纳米囊泡(如图中的纳米囊泡将被固定化˚F igure 2) 。
注意:通常为50微升的纳米囊泡在200微升1 × PBS为TIRF成像良好。如果囊泡浓度太高(导致非常明亮的荧光但没有孤立的分子)或太低(导致非常低的荧光或没有单分子的荧光),请相应地更改浓度。


再次用1 ml的1 × PBS冲洗培养皿3次。向培养皿中加入1 ml 1 × PBS进行TIRF成像。




图2.固定在培养皿中的含有GFP的囊泡的示意图。干净的玻璃底盘涂有生物素-聚硅烷,硅烷将与玻璃结合,生物素可与中性亲和素结合。中性亲和素具有结合位点以与生物素结合。在洗涤步骤s中,未结合的中性亲和素将被洗掉,而结合的中性亲和素的剩余位点将用于结合生物素化的抗GFP抗体。该抗体将与纳米囊泡的烟碱乙酰胆碱受体中存在的GFP分子结合。


单分子的TIRF成像和数据分析
打开显微镜系统,包括激光,平台控制器和随附的软件(例如,奥林巴斯显微镜的Metamorph )。等待30分钟,以使显微镜系统中的软件,相机和其他部件达到平衡。
注意:在进行成像之前,应将显微镜照相机充分冷却(〜- 80 °C)。请不要把在相机在更高的温度。


使用显微镜系统中的适当软件(Olympus Microscope中的Metamorph软件)进入落荧光模式。将激光功率设置为大约3 mW / cm 2 ,并确保激光聚焦并对准。理想情况下,在落射荧光模式下,激光应聚焦在距物镜一定距离的一点或小圆圈上。将曝光时间设置为100毫秒。发射光由电子倍增电荷耦合器件(EMCCD,Andor )检测。
在60 ×油镜上放一滴油。将一个装有PBS的干净玻璃底部皿放在物镜上方,并使用目镜和聚焦旋钮将物镜靠近皿底部。小心不要被客观击中。所述OI升降应留盘和物镜的基极之间。
注意:您可以通过可视化的荧光在目镜或随附的显微镜软件。分子聚焦后,您可以进入TIRF模式。


调整角度以获得全内反射。可以想像的激光束,以确保它处于TIRF模式(如图˚F igure 3)。此外,纳米囊泡还将在TIRF模式下进行空间隔离(如图4A所示)。对于每个视野,以100毫秒的曝光时间获取800帧。通过使用存储所收集的框架中的软件流采集模式。




TIRF显微镜图3.示意图上一个玻璃底菜。在调整激光束使其通过全内反射返回的情况下,可以使用油镜。底部200〜纳米厚的菜被照亮的渐逝场的是源自小号从玻璃侧的全内反射。该领域允许在玻璃底盘的底表面上成像荧光单分子纳米囊泡。


使用ImageJ的/斐济软件打开帧(˚F igure 4),并选择感兴趣的区域进行在荧光强度变化的分析(Rueden酒店等人,2017) 。




图4。来自α-4GFP纳米囊泡的单分子成像的示例数据。A.含有GFP系膜受体的空间隔离纳米囊泡的TIRF图像(比例尺= 5 µm)。从示出了两个光漂白步骤的纳米囊泡单个单分子B.荧光强度图案(关注区域(ROI的单个分子的区域)示出在(甲))。这些光漂白步骤提供了有关alpha-4 beta-2 nAChRs如何在大脑中分布的信息。


菜谱


蔗糖缓冲液/均质缓冲液,pH 7.4
0.32 M蔗糖             

10毫米HEPES


2毫米EDTA


蛋白酶抑制剂(每10毫升缓冲液含一片蛋白酶抑制剂)


致命加工作解决方案                                                       
                                                                      1毫升致命加


10毫升PBS(1 × )


致谢


这项工作改编自Fu等。肛门 化学 2019 ,91 (15 ):10125 - 10131. Biorender用于构建最数字。


利益争夺


我们声明与本出版物无关的利益冲突或竞争利益。


伦理


所有实验均在美国国立卫生研究院(National Institutes of Health)规定的指导下进行,并得到肯塔基大学机构动物护理和使用委员会的批准。


参考


Cano,M.,Reynaga,DD,Belluzzi,JD,Loughlin,SE和Leslie F.(2020年)。长期接触香烟烟雾提取物会上调成年和青春期大鼠的烟碱样受体结合。神经药理学181:108308。
Choi,J.,Grosely,R.,Puglisi,EV和Puglisi,JD(2019)。扩展单分子荧光光谱法以捕获生物学的复杂性。Curr Opin Struct Biol 58:233-240。
Fu X.,Moonschi,FH,Fox-Loe,AM,Snell,AA,Hopkins,DM,Avelar,AJ,Henderson,BJ,Pauly,JR和Richards,CI(2019)。脑区域特定的单分子荧光成像。Anal Chem 91(15):10125-10131。
Gage,GJ,Kipke,DR和Shain,W.(2012年)。啮齿动物的全动物灌注固定。J Vis Exp (65):e3564。
Joo C.,Balci H.,Ishitsuka Y.,Buranachai C.和Ha T.(2008年)。单分子荧光方法在分子生物学中的进展。生物化学年鉴(Annu Rev Biochem)77:51-76。
Lee,H.,Kang,H.,Kang,M.,Han,C.,Yi,J.,Kwon,Y.和Park,J.(2020年)。从细胞工程改造的外泌体模拟纳米囊泡的异质亚细胞起源。ACS Biomater Sci Eng 6(11):6063-6068。
Martinac,B.(2017年)。离子通道的单分子FRET研究。Prog Biophys分子生物学130(Pt B):192-197。
Raiber,K.,Terfort,A.,Benndorf,C.,Krings,N.和Strehblow,HH(2005)。通过等离子清洗去除金上链烷硫醇盐的自组装单分子层。表面科学595 (1-3):56-63。
理查兹(Richards)CI,Luong K.,Srinivasan R.,Turner SW,Dougherty DA,Korlach J.和Lester HA(2012)。使用零模式波导纳米结构对单个受体成分进行活细胞成像。Nano Lett 12(7):3690-3694。
康涅狄格州鲁登(Rueden),辛德林(Schindelin),辛格(Hiner),MC,德佐尼亚(DeZonia),比利时沃尔特(AE),阿里纳特(Arena),ET和埃里切里(KW)(2017)。ImageJ2:用于下一代科学图像数据的ImageJ。BMC生物信息学18(1):529。
Srinivasan,R.,Pantoja,R.,Moss,FJ,Mackey,ED,Son,CD,Miwa,J.和Lester,HA(2011)。尼古丁通过化学计量依赖的伴侣分子上调α4β2烟碱受体和ER出口位点。137 (1):59-79。
夏婷,袁洁和方兴(2013)。ATP结合DNA适体的构象动力学:单分子研究。J Phys Chem B 117(48):14994-15003。
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引用:Aryal, S. P., Fu, X., Masud, A. A., Neupane, K. R. and Richards, C. I. (2021). Single-Molecule Studies of Membrane Receptors from Brain Region Specific Nanovesicles. Bio-protocol 11(10): e4018. DOI: 10.21769/BioProtoc.4018.
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