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Apr 2020

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Imaging Microtubules in vitro at High Resolution while Preserving their Structure
保留微管结构同时在体外对其进行高分辨率成像   

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Abstract

Microtubules (MT) are the most rigid component of the cytoskeleton. Nevertheless, they often appear highly curved in the cellular context and the mechanisms governing their overall shape are poorly understood. Currently, in vitro microtubule analysis relies primarily on electron microscopy for its high resolution and Total Internal Reflection Fluorescence (TIRF) microscopy for its ability to image live fluorescently-labelled microtubules and associated proteins. For three-dimensional analyses of microtubules with micrometer curvatures, we have developed an assay in which MTs are polymerized in vitro from MT seeds adhered to a glass slide in a manner similar to conventional TIRF microscopy protocols. Free fluorescent molecules are removed and the MTs are fixed by perfusion. The MTs can then be observed using a confocal microscope with an Airyscan module for higher resolution. This protocol allows the imaging of microtubules that have retained their original three-dimensional shape and is compatible with high-resolution immunofluorescence detection.

Keywords: Microtubules (微管), Microtubule-Associated Proteins (微管结合蛋白质), TIRF microscopy (全内反射荧光显微镜), High resolution imaging (高分辨率成像), Airyscan (Airyscan)

Background

Microtubules (MT) are polymers made by the combination of the heterodimers α- and β-tubulins and are a major component of the cell cytoskeleton. They are involved in fundamental mechanisms of cell function such as mitosis, intracellular transport, cytokinesis and maintenance of cell shape (Akhmanova and Steinmetz, 2015). Although inherently very rigid, MTs often appear curved in cells and few proteins have been described to bend microtubules (Brangwynne et al., 2006; Bechstedt et al., 2014; Leung et al., 2020; Cuveillier et al., 2020). Since the early 1970s (Weisenberg, 1972), the study of MTs in vitro has led to a better understanding of the molecular mechanism involved in the formation and dynamics of MTs. However, a detailed analysis of the shape of the microtubules remains technically difficult. Two main approaches are currently used: electron microscopy for the very detailed images obtained (up to a separation limit of a few Angstrom) (Alushin et al., 2014; Harris, 2015), and TIRF microscopy which allows the live observation of dynamic microtubules using fluorescently-labelled molecules (separation limit of about 200 nm) (Al-Bassam, 2014). However, these techniques are not suitable for the complete observation of microtubules with large three-dimensional curvatures such as the helical shape they adopt in the presence of MAP6 (Cuveillier et al., 2020). In order to obtain more clues on the structure of the MTs, we developed an assay that combines the use of fluorescent proteins and high-resolution imaging by Airyscan confocal microscopy (achievable resolution down to 120 nm in XY), while preserving the original shape of the MTs which is very sensitive to manipulation. This protocol has the advantage of avoiding unusual equipment and material. Moreover, it can be adapted to different super-resolution techniques such as expansion microscopy or stimulated-emission-depletion (STED) microscopy which would allow to increase the resolution up to 10 times (Blom and Brismar, 2014) and reach a separation limit of 50-20 nm.


Materials and Reagents

  1. Tubulin, Atto-565 labelled tubulin, and biotinylated tubulin are prepared as already fully described in Ramirez-Rios et al. (2017). Store up to 1 year in liquid nitrogen

  2. Cover glasses 26 × 76 mm #1 (VWR, catalog number: 630-2910)

  3. Double-face precut tape 70 µm thick, 3 mm wide (LIMA Company, catalog number: 0000P70PC3003)

  4. Siligum wax plate (VWR, MODU140013)

  5. 1.5 ml Eppendorf tubes (Fisher Scientific, catalog number: 11558232)

  6. 0.5 ml Eppendorf tubes (Fisher Scientific, catalog number: 10318661)

  7. Petri dishes (Greiner Bio-One, catalog number: 663102)

  8. Polycarbonate centrifuge tubes (Beckman, catalog number: 343775)

  9. 0.22 µm filters (Merck Millipore, catalog number: SLGP033RS)

  10. Silane-PEG (MW 30k) (Creative PEG-Works, catalog number: PSB-2014)

  11. Silane-PEG-biotin (MW 3,400) (LaysanBio, catalog number: BIOTIN-PEF-SIL, MW 3,400)

  12. NeutrAvidin (ThermoScientific, catalog number: 31000)

  13. PLL20K-G35-PEG2K (PLL-g-PEG) (Jenkem, catalog number: 13022755)

  14. Pluronic F-127 (Sigma-Aldrich, catalog number: P2443)

  15. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A7030)

  16. Acetone 100% (VWR, catalog number: 20066.321)

  17. Ethanol 96% (VWR, catalog number: 20823362)

  18. Hellmanex III (Sigma-Aldrich, catalog number: Z805939-1EA)

  19. Phosphate buffer saline (PBS) (Sigma-Aldrich, catalog number: P4417)

  20. 1,4-Piperazinediethanesulfonic acid (PIPES) (Sigma-Aldrich, catalog number: P6757)

  21. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 484016)

  22. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541)

  23. Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889)

  24. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: 2670)

  25. Sodium hydroxide (NaOH) (Carlo Erba, catalog number: 480507)

  26. DL-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632)

  27. Guanosine 5′-triphosphate (GTP) (Sigma-Aldrich, catalog number: G8877)

  28. Methyl cellulose 1,500 cP (Sigma-Aldrich, catalog number: M0387)

  29. GMPCPP (Euromedex, JE-NU-405S)

  30. HEPES (Sigma-Aldrich, catalog number: H3375)

  31. Glucose (Sigma-Aldrich, catalog number: G8270)

  32. Glucose oxidase (Sigma-Aldrich, catalog number: G6766-10KU)

  33. Catalase (Sigma-Aldrich, catalog number: C9322-1G)

  34. Diamond pencil (Agar Scientific, catalog number: AGT5347)

  35. Glutaraldehyde solution 25% (Sigma-Aldrich, catalog number: G5882)

  36. Clean nitrogen air flow (Air Liquide)

  37. Liquid nitrogen (Air Liquide)

  38. 5× BRB80 (see Recipes)

  39. BSA 10% (see Recipes)

  40. Neutravidine (see Recipes)

  41. DTT, 200 mM (see Recipes)

  42. KCl, 500 mM (see Recipes)

  43. PLL-PEG (see Recipes)

  44. Silane-PEG or Silane-PEG-biotin (see Recipes)

  45. GTP, 20 mM (see Recipes)

  46. Glucose, 450 mg/ml (see Recipes)

  47. Deoxymix (catalase and glucose oxydase) (see Recipes)

  48. NaOH, 1 M (see Recipes)

  49. BSA 1%/BRB80 (see Recipes)

  50. Methyl cellulose 1,500 cP (see Recipes)

  51. HEPES, 10 mM (see Recipes)

  52. Pluronic F27, 10% (see Recipes)

  53. PBS (see Recipes)

  54. Neutravidin stock solution (see Recipes)

  55. Red tubulin mix (see Recipes)

  56. Imaging buffer (see Recipes)

Equipment

  1. Inverted Eclipse Ti microscope (Nikon) with PSF focus

  2. Apochromat 100×/1.49 N.A. oil immersion objective (Nikon) with heated objective (Okolab)

  3. Temperature chamber (Technico Plast)

  4. Ilas2 TIRF system (Roper Scientific)

  5. Cooled Charged-coupled device camera (Evolve 512, Photometrics)

  6. Temperature Stage Controller (Linkam Scientific)

  7. LSM 710 confocal (Zeiss) Airyscan

  8. Plan Apochromat 100×/1.4 N.A. oil immersion objective (Zeiss)

  9. Ultracentrifuge (Beckman Coulter Optima, Model Max-XP, catalog number: 393315)

  10. Rotor (Beckman, model: TLA100-1, catalog number: 343837)

  11. Plasma cleaner FEMTO Diener Electric (Germany) coupled to a vacuum pump Trivac D2.5E Oerliken (Germany)

  12. Sonicator (Elmasonic S30, Elma, Germany)

  13. Glass staining dishes and tray (VWR, catalog number: MARI4200004)

  14. Plastic box for glass microscopy (any brand)

  15. Diamond Pencil (Oxford Instruments, catalog number: T5347)

  16. Tweezers (Dutscher, catalog numbers: 076100 and 005093)

  17. Gloves powder-free (any brand)

  18. Water-bath (any brand)

Software

  1. MetaMorph 7.8.5 software (Molecular Devices, https://www.moleculardevices.com)

  2. Zen Black 2.1 (Zeiss)

Procedure

  1. Flow chamber preparation

    Note: This procedure is adapted from Portran et al. (2013) and Leslie et al. (2013). Cover glasses are used to make both sides of the perfusion chambers. Cover glasses are handled with tweezers or by hand with non-powdered gloves on the edges when drying with airflow so as not to damage their surface by more than 1 cm.

    1. Glass cleaning

      1. Immerge the cover glasses in a clean glass staining dish (105 × 85 × 70 mm) filled with 200-250 ml acetone and sonicate for 30 min in sweep mode.

        Note: 6 cover glasses are required to make 10 flow chambers, but some glass breakage may occur during the procedure, so it is advisable to start with a little more cover glasses.

      2. Replace acetone and incubate for an additional 30 min with orbital shaking (80 rpm).

        Note: Acetone from the second bath can be recycled for the first bath of the next cleaning procedure.

      3. Incubate 15 min with ethanol and rinse 10 times with deionized filtered water.

      4. Incubate 2 h in prewarmed (60 °C) 2% (v/v) Hellmanex on an orbital shaker (80 rpm), then rinse 10 times with deionized filtered water.

      5. Sonicate 15 min in 1 M NaOH solution.

      6. Rinse 10 times with deionized filtered water.

      7. Sonicate 15 min in ethanol and rinse each coverslip 10 times in a large volume (2 × 2 L) of deionized filtered water.

      8. Dry the cover glasses with a clean air flow (nitrogen gas).

      9. Dispense the glasses into two clean, dry glass staining tray for activation and silanization (see the next step below).

        Note: 5 silane-PEG-biotin-coated glasses and 1 silane-PEG-coated glass are used to prepare 10 perfusion chambers (see below paragraph 3).

    2. Glasses activation and silanization

      1. Plasma activate the glasses for 3 min at 80% of maximum power at a pressure of 0.7 mbar.

      2. Rapidly immerge the glasses in either silane-PEG-biotin or silane-PEG solutions and incubate overnight on an orbital shaker.

        Note: Identify the glass staining dishes containing either silane-PEG-biotin or silane-PEG to avoid mistakes.

      3. Recover the coating solutions (they can be reused for several months as long as they remain clean) and replace them with ethanol (Figure 1A).

      4. Wash each coating slide 10 times in a beaker containing ethanol (Figure 1B) and 10 times in a large volume (2 × 2 L) of water (Figure 1C, 1D).

        Note: We recommend to wash first the silane-PEG slides then the silane-PEG-biotin ones to avoid potential silane-PEG-biotin binding to silane-PEG slides.



        Figure 1. Washing of coated slides. In A we have coated slides in ethanol. B contains ethanol. C and D contain deionized filtered water. E containing deionized filtered water is used to keep rinsed slides before drying.


      5. Dry the coverglasses with a stream of nitrogen.

      6. Store the silanized lamellae at 4 °C in a plastic box sealed with plastic film for a maximum of one week.

    3. Construction of the flow chamber

      Note: When handling the silanized cover glasses, make sure that the face inside the chamber remains clean and un-touched.

      1. Transfer 1 silane-PEG-biotin-coated slide to a clean Petri dish and, using the diamond pencil, cut the glass into four parts (approx. 19/26 mm) (Figures 2A, 2B).

        Note: When cutting with the diamond pencil, the Petri dish bottom bends leading to slide breaking. To avoid this, we use a thin plastic that we put under the Petri dish (Figure 2A).

      2. Similarly, transfer the silane-PEG-coated slide to another Petri dish and cut them into 7 parts of about 11 mm width. Discard the two end parts and cut the others in half to make fourteen 11 × 13 mm pieces (Figures 2C, 2D).



        Figure 2. Cutting coated slides. A. From left to right, diamond pencil, plastic to avoid breaking, Petri dish containing silane-PEG-biotin slide, tweezers. B. The slide is cut in four equal parts. C. A silane-PEG slide is cut in 7 parts. D. Each part is cut in half.


      3. Place two pieces of double-sided adhesive tape, 5 mm apart, in the center of a piece of silane-PEG-biotin coated coverslips (Figure 3A).

      4. Tape one piece of PEG-silane-coated strips above the two pieces of tape (the volume of the chamber is about 5 µl) (Figure 3B).

        Note: Carefully press the silane-PEG-coated strip on the tape to ensure correct sealing. You might gently remove excess tape (Figure 3D).



        Figure 3. Construction of the flow chamber. A. One piece of silane-PEG-biotin is put in a Petri dish. B. Adhesive tape is stuck to the slide. C. Protective plastic above the tape is removed and one piece of silane-PEG slide is placed onto the tape. D. Excessive tape is cut and removed.


  2. Preparation of GMPCCP seeds

    Note: GMPCPP seeds tend to depolymerize at low temperatures, try to keep them warm all along the procedure.

    1. Prepare a mix of freshly thawed ATTO-565 tubulin and biotinylated tubulin at a 1:1 molar ratio for a final concentration of 10 µM tubulin in BRB80 supplemented with 0.5 mM GMPCPP.

      Note: Usually, 300 µl of seeds are necessary for at least 500 experiments.

    2. The mix is placed at 35 °C in a water-bath during 1 h to allow polymerization.

    3. Spin down the GMPCPP seeds at 130,000 × g using a TLA 100 rotor during 5 min at 35 °C.

    4. Discard supernatant and gently rinse with 2 × 100 µl of prewarmed BRB80.

      Note: After the centrifugation, the red seeds are visible in the pellet. Avoid resuspending the pellet during washing steps.

    5. Gently resuspend the pellet in prewarmed BRB80 containing 1 mM GMPCPP.

      Note: Resuspending might take some time. For 300 µl of polymerization solution, resuspend the pellet in 500 µl.

    6. Aliquot by 1-2 µl in 0.5 ml Eppendorf and quickly freeze in liquid nitrogen.

    7. Store in liquid nitrogen up to one year.


  3. Microtubule polymerization and fixation

    Note: The perfusion is performed by loading the solution into one side of the chamber while wiping the other side with a piece of paper (Figure 4).



    Figure 4. Perfusion in flow chamber. From A to D are sequential images of perfusion of the desired solution into the flow chamber.


    1. GMPCPP seeds immobilization in the flow chamber

      1. Perfuse 10 µl of Neutravidin (25 µg/ml in 1% (w/v) BSA/BRB80) in the flow chamber and wait for 2 min.

      2. Perfuse 30 µl of PLL-PEG (0.1 mg/ml in 10 mM HEPES, pH 7.4) and wait for 30 s.

      3. Wash by perfusing 60 µl of 1% (w/v) pluronic in 1% (w/v) BSA/BRB80.

      4. Wash by perfusing 2 times with 70 µl 1% (w/v) BSA/BRB80.

      5. Quickly thaw GMPCPP seeds and dilute in 1% (w/v) BSA/BRB80.

        Note: Adjust seeds dilution if needed, usually start with a 1/100 to 1/500 dilution.

      6. Perfuse 30 µl of diluted GMPCPP seeds and let them adhere for 5 min at RT.

      7. Wash unbound seeds 3 times with 70 µl 1% (w/v) BSA/BRB80.

    2. Microtubule polymerization and fixation

      1. Perfuse freshly prepared polymerization mix (Recipe 19).

        Note: Adjust tubulin/associated proteins concentration if needed. It depends on the affinity and effects of the associated proteins of interest. For tubulin, a concentration of 5 to 20 µM is a good start and for associated proteins from 5 nM to 500 nM should be informative.

      2. Put the flow chamber in a warm (32-35 °C) place with a water-saturated atmosphere to avoid desiccation for MT polymerization (usually at least 30 min).

      3. After polymerization, fix MTs by gently perfusing approximately 30 µl of 0.1% Methyl cellulose 1,500 cP, 0.5% (v/v) Glutaraldehyde/BRB80.

        Note: Critical step, fast perfusion will alter MT structure. Put the chamber inside a Petri dish (you might use tape to prevent it from moving). Then, slightly tilt the Petri dish and add drop by drop the fixing buffer at the top of the chamber. The solution will slowly flow down the chamber. You can use blotting paper to clean excess liquid. The fixation buffer should be at least 3 min inside the chamber for proper fixation. Even if the fixation occurs rapidly, dilution might induce MT depolymerization. To overcome this issue, use MT stabilizing compounds such as taxol (taxol affects MT persistence length) or grow MT longer.

      4. Wash fixation buffer with 50 µl of 0.1% (v/v) Methyl cellulose 1,500 cP/BRB80.

      5. Perfuse imaging buffer (BRB80 supplemented with 0.1% Methyl cellulose 1,500 cP, 2 mg/ml glucose, 1mg/ml glucose oxidase and 150 µg/ml catalase).

      6. Seal the flow chamber with the siligum wax.

        Note: Imaging of the chambers should be done within one week.


  4. Imaging of the microtubules

    1. Control fixation: TIRF Microscopy

      Note: To estimate the effect of the fixation process on the sample, it is necessary to compare the sample before/after fixation.

      1. Place the flow chamber on the inverted microscope.

        Note: For imaging before fixation, set the temperature of the stage at 35 °C to avoid MT depolymerization.

      2. Adjust laser intensity, exposure and focus in order to observe the microtubules.

      3. Acquire images both before (Figure 5A) and after fixation (Figure 5B) of the sample.



      Figure 5. Structure of helical microtubules is not altered by the fixation process. TIRFM image of a helical MT before (A) and after (B) fixation. MTs were grown from GMPCPP seeds with 12 µM tubulin (9:1 non-labeled tubulin:ATTO-565 labelled tubulin) with 200 nM MAP6-N-GFP. At this concentration, MAP6-N-GFP induces microtubule coiling. Scale bar: 5 µm.


    2. Confocal imaging with Airyscan processing

      1. Place the chamber above the objective.

      2. Adjust focus and laser intensity in order to observe MTs.

      3. Perform acquisition (z-stack series using 220 nm step-size).

      4. Use Zen built-in Airyscan processing. See Figure 6 for TIRF versus confocal imaging with Airyscan processing of helical MT. Figure 7 shows the advantage of performing z-stacks on MTs with a particular shape.

      5. Analyse images using Zen Blue.



        Figure 6. Observation of helical MTs with TIRFM or confocal Airyscan. MTs were grown as in Figure 5 and fixed using the described protocol. A. Fixed helical MTs observed using TIRF microscopy. B. z-projection of a fixed helical MT observed using confocal microscope followed by Airyscan processing. Scale bar: 5 µm.



        Figure 7. z-stack of a helical MT observed using confocal imaging with Airyscan processing. Images of the z-stack of the same MT as in Figure 6B, with a step of 220 nm between each plane. Scale bar: 5 µm.

Recipes

All solutions were conserved at indicated temperature without any observable deterioration along time unless stated otherwise. Most solutions were aliquoted to avoid freezing-thawing cycles.

  1. 5× BRB80

    36.28 g PIPES

    1.5 ml of MgCl2 (1 M)

    570.6 mg EGTA

    Dissolve in 250 ml of deionized water and adjust pH to 6.85 with KOH

    Add deionized water to 300 ml, filtrate, aliquot (1 ml and 50 ml) and store at -20 °C

    Note: The 5× BRB80 is used to make the BRB80 solution and to adjust the final concentration of the reaction to 1× BRB80. Upon 5× dilution the pH drops to 6.75.

  2. BSA 10%

    Dissolve 2 g of BSA in 20 ml PBS

    Filter and store at -20 °C up to 1 year

  1. Neutravidine

    Dissolve 10 mg Neutravidine in 10 ml H2O, aliquot (5 and 100 µl) and store at -20 °C

    Before the experiment, predilute 5 µl with 195 µl of 1% BSA/BRB80 (final concentration 25 µg/ml)

  2. DTT, 200 mM

    Dissolve 617 mg DTT powder in 4 ml deionized H2O, filtrate, aliquot and store at -20 °C

    The day of use, dilute 1/5 in H2O or BRB80. Discard aliquot after each day of use

  3. KCl, 500 mM

    Dissolve 1 g KCl into 50 ml deionized H2O (-20 °C) filtrate, aliquot and store at -20 °C

  4. PLL-PEG

    1. Make aliquot of the powder of known weight (20-30 mg) under argon gas and store at -20 °C.

    2. To make stock solution, dissolve the powder in 10 mM HEPES (pH 7.4) at 1 mg/ml, make 50 µl aliquots and store at -20 °C.

    3. The day of use, dilute one 50 µl aliquot with 450 µl of 10 mM HEPES (pH 7.4). Diluted PLL-PEG can be stored at 4 °C for one week.

  5. Silane-PEG or Silane-PEG-biotin

    Notes:

    1. Silane-PEG or Silane-PEG-biotin powders are weighted and aliquoted in 200 mg per eppendorf tube under argon gas. The tubes are tightly sealed with parafilm and stored at -20 °C.

    2. Silane-PEG and Silane-PEG-biotin solutions must be kept anhydrous and in the dark.

      1. Dissolve 200 mg powder in 200 ml of 96% ethanol plus 0.4 ml of 37% HCl.

      2. To solubilize the Silane-PEG solution, heat to 50 °C in a water-bath. Keep the solution in a glass bottle.

      3. Silane-PEG and silane-PEG-biotin solution are stored at room temperature in the dark for up to 4 months or around 15 coating. If experiments look dirty in the background, use freshly prepared coating solutions.

  6. GTP 20 mM

    Dissolve 250 mg GTP in 5 ml H2O

    Make aliquots and store at -20 °C

    Before use, dilute the stock solution to 20 mM in H2O or BRB80

  7. Glucose 450 mg/ml

    Dissolve 2.250 g of glucose in 5 ml of BRB80

    Filtrate, aliquot and store at -80 °C

    The day of use pre-dilute 1/10 in BRB80 and keep on ice

  8. Deoxymix (catalase and glucose oxydase)

    Dissolve 35 mg of catalase plus 250 mg of glucose-oxydase in 10 ml of BRB80

    Filtrate, make 25 µl aliquots, freeze in liquid nitrogen and store at -80 °C

    Discard thawed aliquot after each day of use

  9. NaOH, 1 M

    Dissolve 8 g of NaOH in 200 ml deionized water

    Discard after each use

  10. BSA 1%/BRB80

    Dilute 500 µl of 10% BSA with 1 ml of BRB80 5× in 3.5 ml of filtrated deionized water

  11. Methyl cellulose 1,500 cP

    Dissolve 100 mg of methyl cellulose 1,500 cP in 10 ml of prewarm (60 °C) deionized water

    Gently shake on rotating wheel for 30 min

    Store at 4 °C for two weeks

  12. HEPES, 10 mM

    Dissolve 477 mg in 200 ml deionized water

    Adjust pH to 7.4 with KOH

    Store in aliquots at -20 °C

  13. Pluronic F27, 10%

    Dissolve 100 mg in 1 ml of deionized water

    Store at 4 °C up to 2 months

  14. PBS

    Dissolve 1 tablet in 200 ml of deionized water

    Store at -20 °C

  15. Neutravidin stock solution

    Dissolve Neutravidin in deionized water at a final concentration of 1 mg/ml

    Store at -20 °C

  16. Red tubulin mix

    Dilute purified non-labelled and ATTO-565 tubulin at a 9:1 molar ratio in BRB80

    Spin down aggregates at 100,000 × g, 4 °C using TLA100 rotor

    Estimate tubulin concentration by measuring the OD280nm (1OD280nm = 1 mg/ml = 10 µM of tubulin)

    Aliquot, quickly freeze and store in liquid nitrogen for up to 1 year

  17. Polymerization mix

    BRB80 supplemented with:

    50 mM KCl

    1% BSA

    4 mM DTT

    1 mM GTP

    1 mM glucose

    0.05% Methyl cellulose 1,500 cP

    1/50 deoxy mix (70 µg/ml catalase, 500 µg/ml glucose oxidase, 1 mg/ml glucose)

    12 µM red tubulin mix

    200 nM MAP6-N-GFP

Acknowledgments

Fundings: This work was supported by INSERM (Institut National de la Santé Et de la Recherche Médicale), CEA (Commissariat à l’Energie Atomique), CNRS (Centre National de la Recherche Scientifique), Université Grenoble Alpes, by grants from the Agence Nationale de la Recherche ANR MAMAs 2017-CE11-0026, ANR-15-IDEX-02 NeuroCoG in the framework of the “Investissements d’avenir” program and by fundings from the Ministère de l’enseignement supérieur et de la recherche. GIN is a member of the Grenoble Center of Excellence in Neurodegeneration (GREEN). The Photonic Imaging Center of Grenoble Institute Neuroscience (Univ. Grenoble Alpes–Inserm U1216) is part of the ISdV core facility and certified by the IBiSA label.

    This protocol is derived from Cuveillier et al. (2020).

Competing interests

The authors declare no competing interests.

References

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  13. Weisenberg, R. C. (1972). Microtubule Formation in vitro in Solutions Containing Low Calcium Concentrations. Science 177(4054): 1104-1105.

简介

[摘要]微管(MT)是细胞骨架中最刚性的组成部分。然而,它们在细胞环境中经常显得高度弯曲,并且控制它们整体形状的机理了解甚少。当前,体外微管分析主要依靠电子显微镜进行高分辨率分析,而全内反射荧光(TIRF )显微镜则可以对活的荧光标记的微管和相关蛋白进行成像。为了对具有微米曲率的微管进行三维分析,我们开发了一种在体外聚合MT的检测方法 用类似于常规TIRF显微镜操作规程的方式将MT种子的MT粘附到载玻片上。除去游离的荧光分子,并通过灌注固定MTs。然后可以使用带有Airyscan模块的共聚焦显微镜观察MT,以获得更高的分辨率。该协议允许对保留其原始三维形状并与高分辨率免疫荧光检测兼容的微管进行成像。

[背景]微管(MT)是通过异源二聚体的组合制成的聚合物α和β微管蛋白,并且是细胞骨架的主要成分。他们参与了细胞功能的基本机制,如有丝分裂,细胞内转运,胞质分裂和细胞形态的维持(Akhmanova和Steinmetz,2015)。尽管MT本身具有很高的刚性,但它们通常会在细胞中弯曲并产生一些蛋白,从而弯曲微管(Brangwynne等人,2006; Bechstedt等人,2014; Leung等人,2020; Cuveillier等人,2020 )。自1970年代初以来(Weisenberg,1972),对MTs的体外研究使人们对MTs的形成和动力学涉及的分子机理有了更好的了解。然而,对微管形状的详细分析在技术上仍然困难。当前使用两种主要方法:电子显微镜获得非常详细的图像(分离极限可达几埃)(Alushin等,2014;哈里斯,2015),以及TIRF显微镜,可以实时观察动态微管。使用荧光标记的分子(分离极限约为200 nm )(Al-Bassam,2014 )。但是,这些技术不适用于完整的观察具有大三维曲率的微管,例如在存在MAP6的情况下采用的螺旋形微管(Cuveillier等,2020)。为了获得有关MT的结构的更多线索,我们开发了一种测定方法,该方法结合了荧光蛋白的使用和Airyscan共聚焦显微镜的高分辨率成像(在XY中可实现低至120 nm的分辨率),同时保留了MT的原始形状对操纵非常敏感的MT。该协议的优点是避免了异常的设备和材料。此外,它可以适应不同的超分辨率技术,例如扩展显微镜或激发发射耗尽(STED)显微镜,这将使分辨率提高多达10倍(Blom和Brismar,2014),并达到了分离极限50-20纳米。

关键字:微管, 微管结合蛋白质, 全内反射荧光显微镜, 高分辨率成像, Airyscan



材料和试剂


1.如Ramirez- R ios等人已充分描述的那样,制备微管蛋白,Atto-5 65标记的微管蛋白和生物素化的微管蛋白。(2017年)。在液氮中储存长达1年     

2.盖玻片26 × 76 mm#1(VWR ,目录号:630-2910)     

3.双面胶带预切为70μm厚,宽3毫米(LIMA ç ompany,目录号码:0000P70PC3003)     

4.西里贡蜡板(VWR,MODU140013)     

5. 1.5毫升Eppendorf管(Fisher Scientific,目录号:11558232)     

6. 0.5毫升Eppendorf管(Fisher Scientific,目录号:10318661)     

7.培养皿(Greiner Bio-One,目录号:663102)     

8.聚碳酸酯离心管(贝克曼,目录号:343775)     

9. 0.22 µm过滤器(Merck Millipore SLGP033RS)     

10.硅烷-PEG(MW 30k)(Creative PEG-Works ,目录号:PSB-2014) 

11.硅烷-PEG-生物素(MW 3,400)(LaysanBio ,目录号:BIOTIN-PEF-SIL,MW 3,400) 

12. NeutrAvidin(T hermoScientific,目录号:31000) 

13. PLL20K-G35-PEG2K(PLL-g-PEG)(詹肯,目录号:13022755) 

14. Pluronic F-127(Sigma- A ldrich,目录号:P2443) 

15.牛血清白蛋白(BSA)(Sigma- A ldrich,目录号:A7030) 

16.丙酮100%(VWR,目录号:20066.321) 

17. 96%乙醇(VWR,目录号:20823362) 

18. Hellmanex III(Sigma - Aldrich,目录号:Z805939-1EA) 

19.磷酸盐缓冲盐水(PBS)(Sigma - Aldrich,目录号:P4417) 

20. 1,4-哌嗪二乙烷磺酸(PIPES)(Sigma - Aldrich,目录号:P6757) 

21.氢氧化钾(KOH)(Sigma - Aldrich,目录号:484016) 

22.氯化钾(KCl)(Sigma - Aldrich,目录号:P9541) 

23.乙二醇-双(2-氨基乙基醚)-N,N,N',N'-四乙酸(EGTA)(Sigma - Aldrich,目录号:E3889) 

24.氯化镁(MgCl 2 )(Sigma - Aldrich,目录号:2670) 

25.氢氧化钠(Carlo Erba,目录号:480507) 

26. DL-二硫苏糖醇(DTT)(Sigma - Aldrich,目录号:D0632) 

27.鸟苷5'-三磷酸(GTP)(Sigma - Aldrich,目录号:G8877) 

28.甲基纤维素1,500 cP(Sigma - Aldrich,目录号:M0387) 

29. GMPCPP(Euromedex,JE-NU-405S) 

30. HEPES(Sigma - Aldric h ,目录号:H3375) 

31.葡萄糖(Sigma-Aldrich,目录号:G8270) 

32.葡萄糖氧化酶(Sigma-Aldrich,目录号:G6766-10KU) 

33.过氧化氢酶(Sigma-Aldrich,目录号:C9322-1G) 

34.钻石铅笔(琼脂小号系统求解,目录号:甲GT5347) 

35.戊二醛溶液25%(Sigma - Aldrich,目录号:G5882) 

36.清洁氮气流(液化空气) 

37.液氮(液化空气) 

38. 5 × BRB80(请参阅食谱) 

39. BSA 10%(请参阅食谱) 

40.中性核苷(参见食谱) 

41. DTT ,200 mM (请参阅食谱) 

42. KCl ,500 mM (请参阅食谱) 

43. PLL-PEG (请参阅食谱) 

44.硅烷-PEG或硅烷-PEG-生物素(请参见食谱) 

45. GTP ,20 mM (请参阅食谱) 

46.葡萄糖,450毫克/毫升(请参阅食谱) 

47.脱氧混合物(过氧化氢酶和葡萄糖氧化酶)(请参阅食谱) 

48. NaOH ,1M (请参阅食谱) 

49. BSA 1%/ BRB80 (请参阅食谱) 

50.甲基纤维素1 ,500厘泊(见配方) 

51. HEPES ,10 mM (请参阅食谱) 

52.普朗尼克F27 ,10% (见配方) 

53. PBS (请参阅食谱) 

54.中性亲和素储备溶液(请参阅食谱) 

55.红色微管蛋白混合物(请参阅食谱) 

56.成像缓冲液(请参见食谱) 



设备


带有PSF聚焦的Eclipse Ti倒置显微镜(Nikon)
带加热物镜(Okolab)的Apochromat 100 ×/ 1.49 NA油浸物镜(Nikon)
温度室(Technico Plast)
Ilas2 TIRF系统(Roper Scientific)
冷却的带电耦合设备相机(Evolve 512,光度法)
温度平台控制器(Linkam Scientific)
LSM 710共焦(蔡司)Airyscan
Plan Apochromat 100 × /1.4 NA油浸物镜(Zeiss)
超速离心机(Bekman Coulter Optima,型号Max-XP,目录号:393315)
转子(Bekman ,型号:TLA100-1 ,目录号:343837)
等离子清洁剂FEMTO Diener Electric(德国)与真空泵Trivac D2.5E Oerliken(德国)连接
超声仪(Elmasonic S30,埃尔马,德国)
玻璃染色皿和托盘(VWR,目录号:MARI4200004)
玻璃显微镜用塑料盒(任何品牌)
钻石笔(牛津仪器(Oxford Instruments),目录号:T5347)
镊子(D utscher ,目录号s :076100和005093)
无粉手套(任何品牌)
水浴(任何品牌)


软件


7.8.5的MetaMorph软件(Molecular d evices,https://www.moleculardevices.com)
Zen Black 2.1(蔡司)


程序


流动室准备
注意:此程序改编自Portran等。(2013 )和L esli e等。(2013年)。盖玻片用于制作灌注腔的两侧。用气流吹干时,用镊子或用边缘未磨细的手套用手操作防护玻璃,以免损坏其表面超过1厘米。


玻璃清洁
将盖玻片浸入装有200-250 ml丙酮的干净玻璃染色皿(105 × 85 × 70 mm)中,并在扫频模式下超声处理30分钟。
注意:制作10个流通室需要6个防护玻璃罩,但是在操作过程中可能会发生玻璃破损的情况,因此建议您多加一些防护玻璃罩。


更换丙酮,并在轨道摇动(80 rpm)下再孵育30分钟。
注意:第二个浴槽中的丙酮可在下一个清洁程序的第一个浴槽中回收。


用乙醇孵育15分钟,然后用去离子滤水冲洗10次。
孵育2小时在预热(60 ℃)2%(V / V)Hellmanex在定轨振荡器(80 RPM) ,吨母鸡漂洗10次与去离子过滤水。
在1 M NaOH溶液中超声处理15分钟。
用去离子过滤水冲洗10次。
在乙醇中超声处理15分钟,然后在大量(2 × 2 L)去离子过滤水中冲洗每张盖玻片10次。
用干净的空气流(氮气)干燥防护玻璃。
将玻璃粉分配到两个干净,干燥的玻璃染色托盘中,以进行活化和硅烷化(请参阅下面的下一章)。
注意:使用5个硅烷-PEG-生物素涂层玻璃和1个硅烷-PEG涂层玻璃制备10个灌注室(请参阅下面的第3段)。


玻璃活化和硅烷化
等离子在0.7毫巴的压力下以最大功率的80%激活眼镜3分钟。
将玻璃杯快速浸入硅烷-PEG-生物素或硅烷-PEG溶液中,并在定轨振荡器上孵育过夜。
注意:确定含有硅烷-PEG-生物素或硅烷-PEG的玻璃染色皿,以免出错。


恢复的涂层溶液(它们可以被重复使用好几个月,只要它们保持清洁),并用乙醇(图替换它们URE 1A)。
清洗每个涂层载玻片在含有乙醇的烧杯中(图10倍URE 1B)和10倍大的体积(2 ×水(图2L)URE 1C,1 d)。
注意:我们建议先洗硅烷-PEG玻片,然后再洗涤硅烷-PEG-生物素,以避免潜在的硅烷-PEG-生物素与硅烷-PEG玻片结合。


图1.清洗带涂层的载玻片。在A中,我们将载玻片涂在乙醇中。B含有乙醇。C和D含有去离子过滤水。含有去离子过滤水的E用于干燥前保持漂洗过的载玻片。


用氮气流干燥防护玻璃。
将硅烷化的层状胶条在4°C的塑料盒中保存,最多可保存一周。
流动室的构造
注意:在处理硅烷化的盖玻片时,请确保腔室内的表面保持清洁且未触碰。


传送1个硅烷-PEG-生物素涂覆的滑动到一个干净的培养皿中,并使用金刚石笔,切玻璃分为四个部分(约19/26毫米)(图URE小号2A,2 B)。
注意:用金刚石铅笔切割时,培养皿的底部弯曲会导致滑动断裂。为了避免THI S,我们使用一个薄的塑料,我们把培养皿(图下URE 2A)。


同样,将涂有硅烷PEG的玻片转移到另一个培养皿中,并将其切成7个约11毫米宽的部分。丢弃这两个端部和切断其余的一半,使14 11 × 13毫米件(图URES 2C,2 d)。




图2.切割镀膜的幻灯片。一。˚F ROM从左到右,金刚石铅笔,塑料,以避免破坏含硅烷的PEG-生物素滑动,镊子,陪替氏培养皿。乙。Ť他幻灯片是切四个等份。Ç 。将硅烷-PEG玻片切成7份。d 。Ë ACH部分被削减了一半。


地方的两片双面粘合带,除了5mm时,在一块硅烷-PEG-生物素涂覆的盖玻片(图的中心URE 3A)。
带一条PEG-硅烷涂布的胶带(该腔室的体积为约5μl)(图2的两片上述带状URE 3B)。
注意:小心地将胶带上的硅烷-PEG涂层条按在胶带上,以确保正确密封。你


可能轻轻除去过量的胶带(图URE 3D)。




图3.流动槽的构造。一。将一块硅烷-PEG-生物素放入培养皿中。乙。一个dhesive胶带粘在幻灯片。Ç 。P rotective塑料胶带以上被去除,硅烷-PEG滑动的一个片放置在磁带上。d 。Ë XC essive带切断除去。


GMPCCP种子的制备
注意:GMPCPP种子倾向于在低温下解聚,请在整个过程中尽量使其变热。


1.以1:1的摩尔比制备新鲜融化的ATTO-565微管蛋白和生物素化微管蛋白的混合物,使BRB80中的终浓度为10 µM微管蛋白,并添加0.5 mM GMPCPP。     

注意:通常,至少500次实验需要300 µl种子。


2.将混合物在35℃的水浴中放置1小时以允许聚合。     

3.使用TLA 100转子在35°C下旋转5分钟,使GMPCPP种子旋转t 130,000 × g 。     

4.弃去上清液,并用2 × 100 µl预热的BRB80轻轻冲洗。     

注意:离心后,沉淀物中可见红色种子。避免在洗涤步骤中重悬沉淀。


5.轻轻地将沉淀重悬在预热的含有1 mM GMPCPP的BR B80中。     

注意:重悬可能需要一些时间。对于300 µl的聚合溶液,将沉淀重新悬浮在500 µl中。


6.在0.5 ml Eppendorf中以1-2 µl等分,然后在液氮中快速冷冻。     

7.储存在液氮中最多一年。     



微管聚合和固定
注意:灌注是通过将溶液加载到腔室的一侧,同时用一张纸擦拭另一侧来进行的(图4)。




图4.流动室中的灌注。从A到D是将所需溶液灌注到流动室中的顺序图像。


GMPCPP种子固定在流动室中
在流动室中灌注10 µl Neutravidin(25 µg / ml,在1%(w / v)BSA / BRB80中的溶液中),等待2分钟。
灌注30 µl PLL-PEG(在10 mM HEPES中,0.1 mg / ml,pH 7.4)并等待30 s。
通过在1%(w / v)BSA / BRB80中灌注60 µl 1%(w / v)普朗尼克来洗涤。
通过用70 µl 1%(w / v)BSA / BRB80灌注2次进行洗涤。
快速解冻GMPCPP种子,并用1%(w / v)BSA / BRB80稀释。
注意:根据需要调整种子稀释度,通常从1/100到1/500稀释度开始。


灌注30 µl稀释的GMPCPP种子,使其在室温下粘附5分钟。
用70 µl 1%(w / v)BSA / BRB80将未结合的种子洗涤3次。
微管聚合和固定
灌注新鲜制备的聚合混合物(配方19)。
注意:如果需要,可调整微管蛋白/相关蛋白的浓度。它取决于相关目的蛋白的亲和力和作用。对于微管蛋白,浓度从5到20 µM是一个好的开始,而从5 nM到500 nM的相关蛋白应该是有益的。


将流动室放置在水饱和的温暖环境中(32-35°C),以避免MT聚合干燥(通常至少30分钟)。
聚合后,通过轻轻甲基灌注大约30微升0.1%的MT修复纤维素1 ,500厘泊,0.5%(V / V)戊二醛/ BRB80。
注意:关键步骤是快速灌注会改变MT结构。把室内insid E中的切赫我d ISH(你可以用胶带,以防止其移动)。然后,稍微倾斜培养皿,并逐滴添加位于腔室顶部的固定缓冲液。溶液将缓慢流向腔室。您可以使用吸墨纸清洁多余的液体。固定缓冲液应在腔室内至少3分钟以正确固定。即使固定发生迅速,稀释也可能引起MT解聚。为了克服此问题,使用MT稳定如紫杉酚化合物(紫杉醇影响小号MT持续长度)或生长MT更长。


用50μl0.1%洗涤固定缓冲液(V / V)甲基纤维素1 ,500厘泊/ BRB80。
灌注成像缓冲液(BRB80补充有0.1%甲基纤维素1 ,500厘泊,2mg / ml的葡萄糖,1mg / ml的葡萄糖氧化酶和150微克/毫升过氧化氢酶)。
用硅蜡密封流室。
注意:应在一周之内对腔室进行成像。


微管成像
对照固定:TIRF显微镜
注意:要估计固定过程对样品的影响,有必要在固定之前/之后比较样品。           

将流动室放在倒置显微镜上。
注意:对于在固定之前进行成像,请将载物台的温度设置为35°C,以避免MT发生解聚。


调整激光强度,曝光和聚焦以观察微管。
获取图像两者之前(图URE 5A)和固定后(图URE 5B)的样品。




图5.固定过程不会改变螺旋微管的结构。固定前(A)和之后(B)的螺旋MT的TIRFM图像。MTs从含有12 µM微管蛋白(9:1非标记微管蛋白:ATTO-565标记微管蛋白)和200 nM MAP6-N-GFP的GMPCPP种子中生长。在该浓度下,MAP6-N-GFP诱导小号微管卷取。比例尺:5 µm。


使用Airyscan处理的共焦成像
将腔室放置在物镜上方。
调整聚焦和激光强度以观察MT。
执行采集(z堆栈系列使用220 nm步长)。
使用Zen内置的Airyscan处理。借助Airyscan处理螺旋MT的TIRF与共聚焦成像,请参见图6。图7显示了在具有特定形状的MT上执行z堆栈的优势。
使用Zen Blue分析图像。




图6.使用TIRFM或共聚焦Airescan观察螺旋MT。MT的生长如图URE 5和使用所描述的协议固定的。A.使用TIRF显微镜观察到的固定螺旋MT。B.使用共聚焦显微镜观察到的固定螺旋MT的z投影,然后进行Airyscan处理。比例尺:5 µm。




图7.使用共焦成像和Airyscan处理观察到的螺旋MT的z- stack。相同的MT的Z堆叠图像如在图6乙,用的各平面之间220nm处的步骤。比例尺:5 µm。


菜谱


除非另有说明,否则所有溶液均在指定的温度下保存,并且随时间推移没有任何可观察到的劣化。等分大多数溶液以避免冻融循环。


5 × BRB80
36.28克PIPES


1.5 ml的MgCl 2 (1 M )


570.6毫克EGTA


溶于250毫升去离子水中,并用KOH调节pH到6.85


添加去离子水至300 ml,滤液,等分试样(1 ml和50 ml)并储存在-20°C


注意:5 × BRB80用于制备BRB80溶液,并将反应的最终浓度调整为1 × BRB80。稀释5倍后,pH降至6.75。


BSA 10%
在20 ml PBS中溶解2 g BSA


过滤并在-20°C下储存长达1年


中性苷
将10 mg Neutravidine溶于10 ml H 2 O,等分试样(5和100 µl)中,并储存在-20°C下           

实验前,用195 µl 1%BSA / BRB80(终浓度25 µg / ml)预先稀释5 µl。


DTT ,200毫米
将617 mg DTT粉末溶于4 ml去离子H 2 O中,滤液,等分试样并储存在-20°C下


使用当天,用H 2 O或BRB80稀释1/5 。每天使用后丢弃等分试样


氯化钾,500mM的
将1 g KCl溶于50 ml去离子H 2 O(-20°C)滤液中,等分并保存在-20°C


锁相环
在氩气中分装已知重量的粉末(20-30毫克),并储存在-20°C下。
要制成储备溶液,请将粉末以1 mg / ml的浓度溶于10 mM HEPES (pH 7.4 )中,制成50 µl等分试样并在-20°C下储存。
使用当天,用450 µl的10 mM HEPES (pH 7.4 )稀释一份50 µl的等分试样。稀释的PLL-PEG可以在4 °C下保存1周。
硅烷-PEG或硅烷-PEG-生物素
笔记:


称重硅烷-PEG或硅烷-PEG-生物素粉末,并在氩气下在每支eppendorf管中以200mg等分。将试管用石蜡膜密封,并在-20°C下保存。
硅烷-PEG和硅烷-PEG-生物素溶液必须保持无水且在黑暗中。
在200毫升溶解200毫克粉末的96%乙醇加0.4毫升的37%的HCl 。
要溶解硅烷-PEG溶液,请在水浴中加热至50 °C。将溶液保存在玻璃瓶中。
硅烷-PEG和硅烷-PEG-生物素溶液在室温下于黑暗中保存长达4个月或约15次涂层。如果实验在背景中看起来很脏,请使用新鲜制备的涂料溶液。
GTP 20毫米
将250 mg GTP溶于5 ml H 2 O


分装并储存在-20°C


使用前,将原液在H 2 O或BRB80中稀释至20 mM


葡萄糖450 mg / ml
将2.250克葡萄糖溶解在5毫升BRB80中


滤液,等分试样并储存在-80°C


使用当天在BRB80中稀释1/10并保持在冰上


脱氧混合物(过氧化氢酶和葡萄糖氧化酶)
将35 mg过氧化氢酶和250 mg葡萄糖氧化酶溶解在10 ml BRB80中


过滤,制成25 µl等分试样,在液氮中冷冻并储存于-80°C


每天使用后丢弃解冻的等分试样


的NaOH ,1M的
将8 g NaOH溶于200 ml去离子水中


每次使用后丢弃


BSA 1%/ BRB80
用1 ml的BRB80 5 ×在3.5 ml的过滤去离子水中稀释500 µl的10%BSA。


甲基纤维素1 ,500厘泊
溶解100毫克甲基纤维素1 ,500厘泊在10毫升prewarm(60℃)去离子水的


在旋转轮上轻轻摇动30分钟


存放在4°C下两周


HEPES ,10mM的
将477 mg溶于200 ml去离子水


用KOH调节pH值至7.4


分装在-20°C下


普朗尼克F27 ,10%
将100毫克溶于1毫升去离子水中


储存在4 °C最多2个月


PBS
将1片药片溶于200毫升去离子水中


储存在-20°C


中性亲和素储备液
将中性亲和素以1 mg / ml的最终浓度溶于去离子水中


储存在-20 °C


红微管蛋白混合
在BRB80中以9:1的摩尔比稀释纯化的未标记和ATTO-565微管蛋白


使用TLA100转子以100,000 × g (4 °C)的转速旋转骨料


通过测量OD 280nm (1OD 280nm = 1 mg / ml = 10 µM微管蛋白)来估计微管蛋白浓度。


等分试样,迅速冷冻并储存在液氮中长达1年


聚合混合物
BRB80补充:


50毫米氯化钾


1%牛血清白蛋白


4毫米DTT


1毫米GTP


1 mM葡萄糖


0.05%甲基纤维素1 ,500厘泊


1/50脱氧混合物(70 µg / ml过氧化氢酶,500 µg / ml葡萄糖氧化酶,1 mg / ml葡萄糖)


12 µM红色微管蛋白混合物


200 nM MAP6-N-GFP


致谢


资金来源:这项工作得到了法国国家医学研究所和法国国家原子能研究中心(INSERM),欧洲原子能研究中心(CEA),法国科学技术中心(CNRS),格勒诺布尔阿尔卑斯大学的资助,并得到了法新社的资助。国家研究中心的ANR MAMAs 2017-CE11-0026,NR-15-IDEX-02 NeuroCoG,在“研究投资”计划的框架下,并获得了法国国家研究与发展部的资助。GIN是格勒诺布尔神经变性卓越中心(GREEN)的成员。格勒诺布尔研究所神经科学研究所的光子成像中心(格勒诺布尔阿尔卑斯大学– Inserm U1216)是ISdV核心设施的一部分,并获得了IBiSA标签的认证。


该协议源自Cuveillier等。(2020年)。


利益争夺


作者宣称没有利益冲突。


参考


Akhmanova,A.和Steinmetz,MO(2015)。控制微管的组织和动力学:众人瞩目的两个方面。Nat Rev Mol Cell Biol 16(12):711-726。
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Bechstedt,S.,Lu,K.和Brouhard,GJ (2014)。Doublecortin识别微管末端和晶格的纵向曲率。CURR生物化学24 (20):2366 - 2375 。
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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Cuveillier, C., Saoudi, Y., Arnal, I. and Delphin, C. (2021). Imaging Microtubules in vitro at High Resolution while Preserving their Structure. Bio-protocol 11(7): e3968. DOI: 10.21769/BioProtoc.3968.
  2. Cuveillier, C., Delaroche, J., Seggio, M., Gory-Fauré, S., Bosc, C., Denarier, E., Bacia, M., Schoehn, G., Mohrbach, H., Kulić, I., Andrieux, A., Arnal, I. and Delphin, C. (2020). MAP6 is an intraluminal protein that induces neuronal microtubules to coil. Sci Adv 6(14): eaaz4344.
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