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Sep 2017
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An in vitro Microscopy-based Assay for Microtubule-binding and Microtubule-crosslinking by Budding Yeast Microtubule-associated Protein
利用芽殖酵母微管相关蛋白研究微管结合和微管交联的体外显微镜检测法   

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Abstract

In this protocol, we describe a simple microscopy-based method to assess the interaction of a microtubule-associated protein (MAP) with microtubules. The interaction between MAP and microtubules is typically assessed by a co-sedimentation assay, which measures the amount of MAP that co-pellets with microtubules by centrifugation, followed by SDS-PAGE analysis of the supernatant and pellet fractions. However, MAPs that form large oligomers tend to pellet on their own during the centrifugation step, making it difficult to assess co-sedimentation. Here we describe a microscopy-based assay that measures microtubule binding by direct visualization using fluorescently-labeled MAP, solving the limitations of the co-sedimentation assay. Additionally, we recently reported quantification of microtubule bundling by measuring the thickness of individual microtubule structures observed in the microscopy-based assay, making the protocol more advantageous than the traditional microtubule co-pelleting assay.

Keywords: Microtubule (微管), Yeast (酵母), Microtubule-associated protein (微管相关蛋白), Microtubule binding (微管结合), Microtubule crosslinking (微管交联), Microscopy-based assay (显微镜检测法), Imaging chamber (成像孵育池)

Background

Microtubules are dynamic polar filaments made up of polymerized tubulin subunits. Cells use microtubules to organize the cytosol and to build complex architectures necessary for cell growth and division. For instance, during interphase, microtubules act as molecular highways for motor proteins to manipulate the position of various cargoes in the cell. During mitosis, microtubules assemble the mitotic spindle to accomplish the critical task of chromosome segregation (Wadsworth and Khodjakov, 2004; Wadsworth et al., 2011). To date, we know that numerous regulators, including motor and non-motor microtubule-associated proteins (MAPs), interact with microtubules and participate in a variety of motile activities carried out by microtubules (Goshima and Scholey, 2010). In particular, many of these factors ensure that cells undergo proper mitotic progression, by helping to generate pulling and pushing forces on the microtubules for centrosome separation (Gonczy et al., 1999; Tanenbaum et al., 2008), nuclear envelope breakdown (Salina et al., 2002), mitotic spindle assembly (Rusan et al., 2002), chromosome capture and congression (Schmidt et al., 2005), spindle centering and positioning (Omer et al., 2018; Zulkipli et al., 2018), spindle checkpoint inactivation (Howell et al., 2001), and spindle elongation (Sharp et al., 1999; Khmelinskii et al., 2009).

We recently showed that She1, a MAP that regulates dynein motility along microtubule tracks (Markus et al., 2012), also crosslinks spindle microtubules to help maintain spindle integrity during spindle positioning in budding yeast (Zhu et al., 2017). In this bio-protocol, we describe a sensitive fluorescence-based assay that enables visualization and quantification of microtubule binding and crosslinking activities exhibited by a MAP. Because this assay does not need biochemical quantities of purified MAPs, we believe that it represents a significant advantage over the traditional microtubule co-sedimentation assay.

Materials and Reagents

  1. Avant 1.7 ml microcentrifuge tubes (MIDSCI, catalog number: AVSS1700)
  2. Pipette tips, 20 µl (Rainin Instrument, LLC, catalog number: 17005091)
  3. Pipette tips, 250 µl (Rainin Instrument, LLC, catalog number: 17005093)
  4. Razor blades (Fisher Scientific, catalog number: 12640)
  5. Microcentrifuge tube rack (Fisher Scientific, catalog number: 22-313630)
  6. Scotch double-sided tape, ¾ in. x 400 in. (3M)
  7. Microscope slides, 3 in. x 1 in. (Fisher Scientific, catalog number: 12518100A3)
  8. Cover glass, No. 1.5, 30 mm x 22 mm (Fisher Scientific, catalog number: 12544A)
  9. Filter forcep (EMD Millipore, catalog number: XX6200006P)
  10. Kimwipe (Fisher Scientific, catalog number: 06666A)
  11. Microscope lens paper, 4 in. x 6 in. sheets (Fisher Scientific, catalog number: 11996)
  12. Unlabeled porcine tubulin, > 99% pure, 1 mg (Cytoskeleton, Inc., catalog number: T240) (store at -80 °C)
  13. HiLyte Fluor 488 labeled porcine tubulin, 20 µg (Cytoskeleton, Inc., catalog number: TL488M) (store at -80 °C)
  14. Rhodamine labeled porcine tubulin, 20 µg (Cytoskeleton, Inc., catalog number: TL590M) (store at -80 °C)
  15. 100 mM GTP (Cytoskeleton, Inc., catalog number: BST06-010) (store at -20 °C)
  16. 10 mM Taxol (Cytoskeleton, Inc., catalog number: TXD01) (store at -20 °C)
  17. Precision Red Advanced Protein Assay Reagent (Cytoskeleton, Inc., catalog number: ADV02) (store at room temperature)
  18. PIPES (Sigma-Aldrich, catalog number: P1851) (store at room temperature)
  19. EGTA (Fisher Scientific, catalog number: O2783-100) (store at room temperature)
  20. Magnesium Chloride Hexahydrate (Fisher Scientific, catalog number: BP214-500)
  21. Ultrapure water from Milli-Q® Advantage A10 Water Purification System (Millipore Sigma)
  22. THETM alpha Tubulin Antibody, mAb, mouse (GenScript, catalog number: A01410) (store at -20 °C)
  23. Pluronic F-127, 100 g (Anatrace, catalog number: P305) (store at room temperature)
  24. Carl Zeiss ImmersolTM 518 F Immersion Oil (Fisher Scientific, catalog number: 1262466A) (store at room temperature)
  25. PEM Buffer (see Recipes)
  26. Blocking buffer (see Recipes)

Equipment

  1. Ice bucket (Fisher Scientific, catalog number: 07210123)
  2. Milli-Q® Advantage A10 Water Purification System (Millipore Sigma)
  3. Sorvall centrifuge equipped with S120-AT2 rotor (Thermo Fisher Scientific, model: Discovery)
  4. 37 °C incubator (Fisher Scientific, catalog number: 11690525D)
  5. Microcentrifuge (Eppendorf, model: 5424)
  6. Inverted fluorescence microscope equipped with Perfect Focus System and a 100x/1.49 NA objective (Nikon, model: Ti-E)
  7. Laser launch system with 405/488/561/640 nm wavelengths at 15 mW per line (Nikon, model: LUN4)
  8. Electron multiplying CCD camera (Andor Technology, model: iXon 888)
  9. GFP filter cube set for imaging HiLyte Fluor 488 or Alexa Fluor 488 fluorescence (Chroma Technology Corp., catalog number: 49002)
  10. TRITC filter cube set for imaging rhodamine or TMR fluorescence (Chroma Technology Corp., catalog number: 49008)
  11. HP workstation with 3.5 GHz 6 core Xeon processor, 64 GB memory, 1 TB hard drive, and 64bit Windows 7 (Micro Video Instruments, Inc., catalog number: 99909)
  12. Pipet-Lite LTS pipette L-20XLS+ (Rainin Instrument, LLC, catalog number: 17014392)
  13. Pipet-Lite LTS pipette L-200XLS+ (Rainin Instrument, LLC, catalog number: 17014391)

Software

  1. NIS-Elements Advanced Research (Nikon) (https://www.nikoninstruments.com/Products/Software/NIS-Elements-Advanced-Research)
  2. ImageJ (NIH, https://imagej.nih.gov/ij/download.html)
  3. KaleidaGraph (http://www.synergy.com/wordpress_650164087/kaleidagraph/)

Procedure

  1. Prepare fluorescently-labeled microtubules
    1. Dissolve unlabeled porcine tubulin (Cytoskeleton, Inc.) on ice in PEM buffer (Recipe 1) to a final concentration of 5 mg/ml (e.g., add 200 µl of PEM buffer to 1 mg of unlabeled lyophilized tubulin).
    2. Dissolve HiLyte Fluor 488 or rhodamine-labeled tubulin (Cytoskeleton, Inc.) on ice in PEM buffer to a final concentration of 5 mg/ml (e.g., add 4 µl of PEM buffer to 20 µg of fluorescently-labeled lyophilized tubulin).
    3. Mix 50 µl of unlabeled tubulin with 4 µl of fluorescently-labeled tubulin in a clean 1.7 ml microfuge tube. This step and subsequent steps may be performed under normal room light. It is not necessary to pipet in the dark.
    4. Incubate the tubulin mixture on ice for 10 min without shaking.
    5. Centrifuge the tubulin mixture for 10 min at 298,000 x g and 4 °C (in Sorvall Discovery centrifuge with S120-AT2 rotor) to remove unwanted tubulin aggregates. A pellet is usually not visible after centrifugation in this step, since the lyophilized tubulin would normally dissolve completely in Steps A1 and A2 above.
    6. Transfer the supernatant containing the pre-cleared tubulin into a new 1.7 ml microfuge tube.
    7. Add 0.54 μl of 100 mM GTP (i.e., final GTP concentration at 1 mM) to polymerize the pre-cleared tubulin. Mix the solution well by pipetting up and down three times using the L-200XLS+ pipette. Do not over-pipet, as pipetting three times is sufficient to mix the solution and initiate the polymerization of tubulin subunits.
    8. Incubate the GTP/tubulin mixture in a 37 °C incubator for 20 min without shaking.
    9. Take the tube out of the 37 °C incubator and briefly spin it at 21,130 x g for 10 sec in an Eppendorf 5424 microcentrifuge to pellet any condensation that might have formed in the lid of the microfuge tube. Next, add 0.27 μl of 10 mM Taxol (i.e., final Taxol concentration at 50 μM) and mix well by pipetting up and down three times using the L-200XLS+ pipette.
    10. Incubate the tubulin/GTP/Taxol mixture at 37 °C for another 20 min without shaking.
    11. Centrifuge the tube containing the Taxol-stabilized microtubules at 20,156 x g for 10 min at room temperature in an Eppendorf 5424 microcentrifuge. This step will separate the polymerized microtubules from the free, unpolymerized tubulin.
    12. Discard the supernatant and resuspend the pellet in 50 μl of PEM buffer and 0.25 µl of 10 mM Taxol. To avoid shearing the polymerized microtubules, resuspend the pellet gently by pipetting up and down using the L-200XLS+ pipette and a pipet tip that has been shaved off with a razor blade.
    13. Once the pellet is completely dissolved, the concentration of the microtubules in the solution will be at ~50 μM. Alternatively, the concentration can be measured and determined by the Precision Red Advanced Protein Assay Reagent (Cytoskeleton, Inc.), following the manufacturer’s protocol.
    14. Store the microtubules at room temperature in the dark (e.g., in a drawer). This microtubule preparation is good for up to one month at the room temperature.

  2. Assemble the imaging chamber
    1. Cut two pieces of double-sided tape and stick them down on a clean microscope slide using filter forceps as shown in Part 1 of Video 1 and Figure 1A. The two pieces of tape should be placed in parallel and at 1.5 mm apart from each other in the middle of the microscope slide. No specific procedure to clean the surface of the microscope slide is required.

      Video 1. Preparing imaging chamber for microscopy. Part 1, a video clip showing Steps 1-3 of Procedure B. Part 2, a video clip showing how to pipet solution into the imaging chamber. Part 3, a video clip demonstrating how to replace the solution in the imaging chamber. For visualization purpose, PEM buffer spiked with bromophenol blue was used in the demonstration shown in the clips for Parts 2 and 3.

    2. Gently put down a clean cover glass on top of the two pieces of double-sided tape. Press down on the cover glass along the double sided tape regions, creating a strip of chamber sandwiched between the cover glass and the microscope slide (i.e., the imaging chamber) as shown in Figure 1B. No specific treatment of the cover glass surface is required before it is pressed down on the microscope slide.
    3. Flip the cover glass/slide assembly over, so that the cover glass is now at the bottom relative to the microscope slide.
    4. Prepare the anti-tubulin antibody solution by diluting THETM alpha Tubulin Antibody (GenScript) from 500 µg/ml to 33.3 µg/ml using PEM buffer. Store unused antibody solution at 4 °C for further use.
    5. Pipet 7 µl of the anti-tubulin antibody into the imaging chamber. Fill the chamber with the antibody solution by injecting slowly at an angle from one side of the chamber using an L-20XLS+ pipette (see Figure 1C). Also see Part 2 of Video 1 for a demonstration of the technique. Consider rehearsing this step on a practice chamber with only PEM buffer because the anti-tubulin antibody solution might not flow into the chamber on the first try.
    6. Allow the anti-tubulin antibody to stick to the chamber by incubating at room temperature for 5 min.
    7. Replace the anti-tubulin antibody solution in the imaging chamber with blocking buffer (Recipe 2). To do so, cut a piece of Kimwipe into triangular shape and hold the pointed end against one side of the imaging chamber. While the antibody solution is being drawn out of the chamber by the Kimwipe, pipet 7 µl of blocking buffer solution into the other side of the chamber (see Figure 1D). Also see Part 3 of Video 1 for a demonstration of the technique to replace the solution in the imaging chamber.


      Figure 1. Chamber assembly. A. Adhere double-sided tapes to microscope slide. B. Apply cover glass onto the double-sided tapes. C. Turn the cover glass and slide over. Fill the chamber with anti-tubulin antibody solution. D. Flow-in blocking buffer by injecting on one side while drawing out with a Kimwipe on the other side.

    8. Allow the blocking buffer to incubate at room temperature for 5 min.
    9. Prepare the microtubule solution by diluting the microtubule stock (prepared as described in Procedure A above) from 50 µM to 0.125 µM using PEM buffer. To avoid shearing the microtubules, use a cut pipet tip when pipetting the microtubules, as described in Step 12 of Procedure A.
    10. Replace the blocking buffer in the imaging chamber with the diluted microtubule solution. Similar to Step B7 above, use a piece of Kimwipe to draw the blocking buffer out of the chamber from one side while pipetting the microtubule solution into the chamber on the other side.
    11. Allow the microtubules to attach to the anti-tubulin antibody by incubating at room temperature for 5 min.
    12. Bring the chamber to an inverted fluorescence microscope for imaging.

  3. Visualize microtubule binding and crosslinking
    The following steps require the experimenter to have in hand a small quantity of test microtubule-binding protein purified and labeled with a fluorescent probe that is different from that used for labeling the microtubules. We refer the experimenter to other studies (Markus et al., 2012; Zhu et al., 2017) for protocols involving purification and labeling of HALO-tagged fusion proteins with TMR or Alexa Fluor 488, which works well for the following steps.
    1. Mix 10 µl of 125 nM Taxol-stabilized microtubules (labeled with HiLyte Fluor 488 as described in Procedure A) with 1 μl of
      10 µM MAP (labeled with TMR) in PEM buffer. Add 9 µl of PEM buffer to bring the total reaction volume to 20 μl. Alternatively, the same volume and concentration of Taxol-stabilized microtubules labeled with rhodamine (prepared as described in Procedure A) can be mixed with MAP labeled with Alexa Fluor 488 in this step. Microtubules mixed with buffer alone should be performed separately as a control.
    2. Incubate the mixture at room temperature for 10 min.
    3. Flow the microtubules/MAP mixture into an imaging chamber that has been treated with anti-tubulin antibody and blocking buffer (Steps 1-8 in Procedure B). As described for Step 10 in Procedure B, use a piece of Kimwipe to draw the blocking buffer out of the chamber from one side while pipetting the microtubules/MAP solution into the chamber on the other side. To avoid shearing the microtubules, use a cut pipet tip when pipetting the microtubules into the imaging chamber.
    4. Allow the microtubules/MAP to attach to the anti-tubulin antibody by incubating at room temperature for 5 min.
    5. To wash away unattached microtubules/MAP, flow-in fresh blocking buffer once into the imaging chamber.
    6. Incubate at room temperature for 5 min. Bring the chamber to a microscope room for imaging.
    7. To visualize the microtubules/MAP and microtubules/buffer samples, use a Nikon Ti-E inverted microscope equipped with Perfect Focus System and a 100x/1.49 NA objective.
    8. Place a drop of immersion oil on the cover glass side of the imaging chamber and mount the slide on the microscope stage.
    9. Use a LUN4 laser launch system (Nikon) to illuminate the fluorophores and an iXon 888 electron multiplying CCD camera (Andor Technology) to image the fluorescently-labeled microtubules and MAP.
    10. Use a GFP filter cube set (Chroma Technology Corp.) to visualize Alexa Fluor 488 or HiLyte Fluor 488 fluorescence. Use a TRITC filter cube set (Chroma Technology Corp.) to visualize rhodamine or TMR fluorescence.
    11. Bring the microtubules into focus relative to the 100x/1.49 NA objective. Adjust focusing in the GFP channel for HiLyte Fluor 488-labeled microtubules. Alternatively, use the TRITC channel if Step C1 uses rhodamine-labeled microtubules. An example of an image of HiLyte Fluor 488-labeled microtubules is illustrated in Figure 2.


      Figure 2. Example of HiLyte Fluor 488-labeled microtubules. Microtubules bound to anti-tubulin antibody in the imaging chamber were visualized in a Nikon Ti-E inverted microscope in this picture. Scale bar = 7 µm.

    12. Switch to the TRITC channel to visualize the bound MAP on the microtubules. Alternatively, switch to GFP channel if Step C1 uses Alexa Fluor 488-labeled MAP.
    13. To acquire two-color pictures, use a PC workstation with NIS-Elements software (Nikon), which controls channel switching, laser intensity, and camera settings. An example of two-color images of HiLyte Fluor 488-labeled microtubules and TMR-labeled MAP is illustrated in Figure 3.


      Figure 3. Example of two-color images of fluorescently-labeled microtubules and MAP. HiLyte Fluor 488-labeled microtubules mixed with a TMR-labeled recombinant MAP, She1-C-TMR, purified as described in Zhu et al. (2017), were visualized in the imaging chamber in this picture. Scale bar = 3.6 µm.

    14. To collect enough data for subsequent analysis, capture at least 20 different fields of two-color microtubules/MAP images per chamber. Repeat the experiment with at least three independent preparations of MAP.
    15. Use identical camera settings to compare the amount of MAP bound to the microtubules from chamber to chamber or when different MAP concentrations are used (if needed).
    16. Some MAPs might crosslink or bundle microtubules. Image microtubules in the presence and absence of MAP to determine the frequency of observing microtubule bundles. Bundles can be distinguished from individual microtubules by measuring the microtubule cross-sectional intensity (as described in Data Analysis).

Data analysis

  1. To quantify the relative amount of bound MAP on microtubules, use the segmented line tool in ImageJ software (NIH). Trace a line along individual microtubules observed in the two-color images (as illustrated in Figure 4, left panel). Measure the mean fluorescence intensity of MAP per unit length for at least 50 individual microtubules (see Figure 4). Subtract the background of each measurement by moving the line tool to an adjacent area without microtubules (as illustrated in Figure 4, right panel). Calculate the average value of the corrected mean intensity per unit length.


    Figure 4. Example of quantifying bound MAP on microtubules. Left, sample lines drawn along individual microtubules (indicated by 1 and 3). Right, mean intensity of She1-C-TMR per unit length of the lines, as measured by ImageJ (under Analyze tab). Background intensity is measured by moving the line to an adjacent area without microtubules (indicated by 2 and 4).

  2. Compare the values measured in the presence of MAP with that in the buffer control. Plot the distribution of the values for the experimental versus the control conditions using KaleidaGraph software. Perform a two-tailed unpaired Student's t-test to determine the statistical significance between the two distributions.
  3. To determine whether microtubules are crosslinked by MAP, use the segmented line tool in ImageJ to draw a line across individual microtubule structures observed in the two-color images (as illustrated in Figure 5, left panel). Use the plot profile tool in ImageJ to measure the cross-sectional fluorescence intensities along the line (Figure 5, middle panel). Perform these measurements for at least 50 different microtubule structures. Plot the fluorescence intensities as a function of the line using KaleidaGraph software (see Figure 3C in Zhu et al., 2017 for sample plots). Calculate the average peak intensity of the fluorescence profiles.
  4. Compare the peak intensities measured for microtubule structures observed in the presence of MAP with that in the buffer only control (see Figure 3C in Zhu et al., 2017 for examples). Perform a two-tailed unpaired Student's t-test to determine the statistical significance between the two groups of peak intensities.


    Figure 5. Example of measuring cross-sectional fluorescence intensities. Left, line across a microtubule structure. Middle, fluorescence profile along the line. Right, plot values of the profile.

Notes

  1. Because the imaging chamber is not sealed, the liquid in the chamber will slowly evaporate, altering sample concentration and compromising image quality. Thus, each imaging chamber should be used only for approximately 15 min of imaging at room temperature.
  2. Because microtubules are attached to the glass surface by anti-tubulin antibodies, capturing single focal plane images should suffice to encompass the entire thickness of the microtubule.

Recipes

  1. PEM Buffer (100 ml)
    2.42 g of PIPES
    20.33 mg of MgCl2 hexahydrate
    38.04 mg of EGTA
    1 ml of 100 mM GTP
    Ultrapure water
    Adjust the pH to 6.9 with 0.1 M NaOH
  2. Blocking buffer (100 ml)
    1 g of Pluronic F-127
    100 ml of PEM buffer

Acknowledgments

This work was supported by an NIH/NIGMS grant (GM076094) to W.-L. L..

Competing interests

The authors declare no competing financial interests.

References

  1. Gonczy, P., Pichler, S., Kirkham, M. and Hyman, A. A. (1999). Cytoplasmic dynein is required for distinct aspects of MTOC positioning, including centrosome separation, in the one cell stage Caenorhabditis elegans embryo. J Cell Biol 147(1): 135-150. 
  2. Goshima, G. and Scholey, J. M. (2010). Control of mitotic spindle length. Annu Rev Cell Dev Biol 26: 21-57.
  3. Howell, B. J., McEwen, B. F., Canman, J. C., Hoffman, D. B., Farrar, E. M., Rieder, C. L. and Salmon, E. D. (2001). Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation. J Cell Biol 155(7): 1159-1172.
  4. Khmelinskii, A., Roostalu, J., Roque, H., Antony, C. and Schiebel, E. (2009). Phosphorylation-dependent protein interactions at the spindle midzone mediate cell cycle regulation of spindle elongation. Dev Cell 17(2): 244-256.
  5. Markus, S. M., Kalutkiewicz, K. A. and Lee, W. L. (2012). She1-mediated inhibition of dynein motility along astral microtubules promotes polarized spindle movements. Curr Biol 22(23): 2221-2230.
  6. Omer, S., Greenberg, S. R. and Lee, W. L. (2018). Cortical dynein pulling mechanism is regulated by differentially targeted attachment molecule Num1. Elife 7: e 36745.
  7. Rusan, N. M., Tulu, U. S., Fagerstrom, C. and Wadsworth, P. (2002). Reorganization of the microtubule array in prophase/prometaphase requires cytoplasmic dynein-dependent microtubule transport. J Cell Biol 158(6): 997-1003.
  8. Salina, D., Bodoor, K., Eckley, D. M., Schroer, T. A., Rattner, J. B. and Burke, B. (2002). Cytoplasmic dynein as a facilitator of nuclear envelope breakdown. Cell 108(1): 97-107.
  9. Schmidt, D. J., Rose, D. J., Saxton, W. M. and Strome, S. (2005). Functional analysis of cytoplasmic dynein heavy chain in Caenorhabditis elegans with fast-acting temperature-sensitive mutations. Mol Biol Cell 16(3): 1200-1212.
  10. Sharp, D. J., McDonald, K. L., Brown, H. M., Matthies, H. J., Walczak, C., Vale, R. D., Mitchison, T. J. and Scholey, J. M. (1999). The bipolar kinesin, KLP61F, cross-links microtubules within interpolar microtubule bundles of Drosophila embryonic mitotic spindles. J Cell Biol 144(1): 125-138.
  11. Tanenbaum, M. E., Macurek, L., Galjart, N. and Medema, R. H. (2008). Dynein, Lis1 and CLIP-170 counteract Eg5-dependent centrosome separation during bipolar spindle assembly. EMBO J 27(24): 3235-3245.
  12. Wadsworth, P. and Khodjakov, A. (2004). E pluribus unum: towards a universal mechanism for spindle assembly. Trends Cell Biol 14(8): 413-419. 
  13. Wadsworth, P., Lee, W. L., Murata, T. and Baskin, T. I. (2011). Variations on theme: spindle assembly in diverse cells. Protoplasma 248(3): 439-446. 
  14. Zhu, Y., An, X., Tomaszewski, A., Hepler, P. K. and Lee, W. L. (2017). Microtubule cross-linking activity of She1 ensures spindle stability for spindle positioning. J Cell Biol 216: 2759-2775.
  15. Zulkipli, I., Clark, J., Hart, M., Shrestha, R. L., Gul, P., Dang, D., Kasichiwin, T., Kujawiak, I., Sastry, N. and Draviam, V. M. (2018). Spindle rotation in human cells is reliant on a MARK2-mediated equatorial spindle-centering mechanism. J Cell Biol 217(9): 3057-3070.

简介

在该协议中,我们描述了一种简单的基于显微镜的方法来评估微管相关蛋白(MAP)与微管的相互作用。 MAP和微管之间的相互作用通常通过共沉降测定来评估,所述共沉淀测定通过离心测量与微管共沉淀的MAP的量,然后对上清液和沉淀级分进行SDS-PAGE分析。然而,形成大的低聚物的MAP在离心步骤期间倾向于自身沉淀,使得难以评估共沉淀。在这里,我们描述了一种基于显微镜的分析,通过使用荧光标记的MAP直接观察来测量微管结合,解决了共沉淀分析的局限性。另外,我们最近报道了通过测量在基于显微镜的测定中观察到的单个微管结构的厚度来定量微管束,使得该方案比传统的微管共制粒测定更有利。

【背景】微管是由聚合的微管蛋白亚基组成的动态极性细丝。细胞使用微管来组织细胞质并构建细胞生长和分裂所必需的复杂结构。例如,在间期期间,微管充当运动蛋白的分子通路,以操纵细胞中各种货物的位置。在有丝分裂期间,微管组装有丝分裂纺锤体以完成染色体分离的关键任务(Wadsworth和Khodjakov,2004; Wadsworth 等人,>,2011)。迄今为止,我们知道许多调节因子,包括运动和非运动微管相关蛋白(MAPs),与微管相互作用并参与由微管进行的各种运动活动(Goshima和Scholey,2010)。特别是,这些因子中的许多因素通过帮助在微管上产生拉动和推力以进行中心体分离来确保细胞经历适当的有丝分裂进展(Gonczy et al。>,1999; Tanenbaum et al 。>,2008),核膜破裂(Salina et al。>,2002),有丝分裂纺锤体组装(Rusan et al。>,2002),染色体捕获和板集合(施密特等人>,2005年),主轴中心和定位(奥马尔等人>,2018; Zulkipli 等人>,2018) ,纺锤体检查点失活(Howell et al。>,2001)和纺锤体伸长率(Sharp et al。>,1999; Khmelinskii et al。>, 2009)。

我们最近发现,She1,沿着微管轨道调节动力蛋白活力的MAP(马库斯的等>的,2012),还交联纺锤体微管,以帮助维持主轴定位在纺锤体的完整性在芽殖酵母(朱等人>,2017)。在该生物方案中,我们描述了一种灵敏的基于荧光的分析,该分析能够实现MAP显示的微管结合和交联活性的可视化和定量。因为该测定不需要生化量的纯化MAP,我们认为它代表了比传统微管共沉降测定的显着优势。

关键字:微管, 酵母, 微管相关蛋白, 微管结合, 微管交联, 显微镜检测法, 成像孵育池

材料和试剂

  1. Avant 1.7 ml微量离心管(MIDSCI,目录号:AVSS1700)
  2. 移液器吸头,20μl(Rainin Instrument,LLC,目录号:17005091)
  3. 移液器吸头,250μl(Rainin Instrument,LLC,目录号:17005093)
  4. 剃刀刀片(Fisher Scientific,目录号:12640)
  5. 微量离心管架(Fisher Scientific,目录号:22-313630)
  6. Scotch双面胶带,¾英寸x 400英寸(3M)
  7. 显微镜载玻片,3英寸x 1英寸(Fisher Scientific,目录号:12518100A3)
  8. 盖玻片,编号1.5,30 mm x 22 mm(Fisher Scientific,目录号:12544A)
  9. Filter forcep(默克密理博中国,目录号:XX6200006P)
  10. Kimwipe(Fisher Scientific,目录号:06666A)
  11. 显微镜镜头纸,4英寸x 6英寸(Fisher Scientific,目录号:11996)
  12. <未标记的猪微管蛋白> 99%纯度,1毫克(Cytoskeleton,Inc。,目录号:T240)(储存在-80°C)
  13. HiLyte Fluor 488标记猪微管蛋白,20μg(Cytoskeleton,Inc。,目录号:TL488M)(在-80°C保存)
  14. 罗丹明标记的猪微管蛋白,20μg的(细胞骨架,公司,目录号:TL590M)(储存在-80℃)
  15. 100 mM GTP(Cytoskeleton,Inc。,目录号:BST06-010)( - 20°C保存)
  16. 10 mM Taxol(Cytoskeleton,Inc。,目录号:TXD01)( - 20°C保存)
  17. Precision Red Advanced Protein Assay Reagent(Cytoskeleton,Inc。,目录号:ADV02)(在室温下保存)
  18. PIPES(Sigma-Aldrich公司,目录号:P1851)(在室温下储存)
  19. EGTA(Fisher Scientific,目录号:O2783-100)(室温下保存)
  20. 氯化镁六水合物(Fisher Scientific,目录号:BP214-500)
  21. 来自Milli-Q ® Advantage A10净水系统(Millipore Sigma)的超纯水
  22. TM alpha Tubulin抗体,单克隆抗体,因子(GenScript,目录号:A01410)( - 20°C以下保存)
  23. Pluronic F-127,100克(Anatrace,目录号:P305)(室温下储存)
  24. Carl Zeiss Immersol TM 518 F Immersion Oil(Fisher Scientific,目录号:1262466A)(室温下储存)
  25. PEM缓冲液(见食谱)
  26. 阻塞缓冲区(见食谱)

设备

  1. 冰桶(Fisher Scientific,目录号:07210123)
  2. Milli-Q ® Advantage A10净水系统(Millipore Sigma)
  3. Sorvall离心机配备S120-AT2转子(Thermo Fisher Scientific,型号:Discovery)
  4. 37°C培养箱(Fisher Scientific,目录号:11690525D)
  5. 微量离心机仪(Eppendorf,型号:5424)
  6. 倒置荧光显微镜配备完美聚焦系统和100x / 1.49 NA物镜(尼康,型号:Ti-E)
  7. 激光发射系统,每线15 mW,波长为405/488/561/640 nm(尼康,型号:LUN4)
  8. 电子倍增CCD相机(Andor Technology,型号:iXon 888)
  9. 用于成像HiLyte Fluor 488或Alexa Fluor 488荧光的GFP滤光片立方体(Chroma Technology Corp.,目录号:49002)
  10. 用于成像罗丹明或TMR荧光的TRITC滤光片立方体(Chroma Technology Corp.,目录号:49008)
  11. HP工作站,配备3.5 GHz 6核Xeon处理器,64 GB内存,1 TB硬盘和64位Windows 7(Micro Video Instruments,Inc。,目录号:99909)
  12. Pipet-Lite LTS移液器L-20XLS +(Rainin Instrument,LLC,目录号:17014392)
  13. Pipet-Lite LTS移液器L-200XLS +(Rainin Instrument,LLC,目录号:17014391)

软件

  1. NIS-Elements高级研究(尼康)( https://www.nikoninstruments。 COM /产品/软件/ NIS元素 - 高级研究)
  2. ImageJ(NIH, https://imagej.nih.gov/ij/download.html )
  3. KaleidaGraph( http://www.synergy.com/wordpress_650164087/kaleidagraph/ )

程序

  1. 准备荧光标记的微管
    1. 将未标记的猪微管蛋白(Cytoskeleton,Inc。)溶解在PEM缓冲液(配方1)中的冰上至终浓度为5 mg / ml(例如>,将200μlPEM缓冲液加入1 mg未标记的冻干液中微管蛋白)。
    2. 将HiLyte Fluor 488或罗丹明标记的微管蛋白(Cytoskeleton,Inc。)溶解在PEM缓冲液中的冰上至终浓度为5 mg / ml(例如>,加入4μlPEM缓冲液至20μg荧光 - 标记的冻干微管蛋白)。
    3. 将50μl未标记的微管蛋白与4μl荧光标记的微管蛋白混合在干净的1.7 ml微量离心管中。该步骤和后续步骤可以在正常室内光下进行。没有必要在黑暗中移液。
    4. 将微管蛋白混合物在冰上孵育10分钟而不摇动。
    5. 将微管蛋白混合物在298,000 x g >和4°C(在具有S120-AT2转子的Sorvall Discovery离心机中)离心10分钟以除去不需要的微管蛋白聚集体。在该步骤中离心后通常不能看到沉淀,因为冻干的微管蛋白通常在上述步骤A1和A2中完全溶解。
    6. 将含有预先清除的微管蛋白的上清液转移到新的1.7ml微量离心管中。
    7. 加入0.54μl的100mM GTP(,即>,最终GTP浓度为1mM)以聚合预先澄清的微管蛋白。使用L-200XLS +移液器上下移液三次,充分混合溶液。不要过度移液,因为移液三次足以混合溶液并引发微管蛋白亚基的聚合。
    8. 将GTP /微管蛋白混合物在37°C培养箱中孵育20分钟,不要摇动。
    9. 将管从37°C培养箱中取出并在Eppendorf 5424微量离心机中以21,130 x g >短暂旋转10秒,以沉淀微量离心管盖子中可能形成的任何冷凝物。接下来,加入0.27μl10mM紫杉醇(,>,最终紫杉醇浓度为50μM)并使用L-200XLS +移液管通过上下吸移三次充分混合。
    10. 将微管蛋白/ GTP /紫杉醇混合物在37℃下孵育另外20分钟而不摇动。
    11. 在室温下,在Eppendorf 5424微量离心机中将含有紫杉醇稳定的微管的管在20,156 x g >离心10分钟。该步骤将聚合的微管与游离的未聚合的微管蛋白分开。
    12. 弃去上清液,将沉淀重悬于50μlPEM缓冲液和0.25μl10mM紫杉醇中。为避免剪切聚合的微管,使用L-200XLS +移液管和用剃刀刀片剃掉的移液管尖端上下移液轻轻重悬沉淀。
    13. 一旦沉淀完全溶解,溶液中微管的浓度将为~50μM。或者,可以按照制造商的方案,通过Precision Red Advanced Protein Assay Reagent(Cytoskeleton,Inc。)测量和测定浓度。
    14. 将微管在室温下避光保存(例如>,在抽屉中)。这种微管制剂在室温下可长达一个月。

  2. 组装成像室
    1. 切割两片双面胶带,并使用过滤钳将其粘在干净的显微镜载玻片上,如视频1和图1A的第1部分所示。两片胶带应平行放置,并在显微镜载玻片中间相互间隔1.5毫米。无需特定程序清洁显微镜载玻片表面。


      视频1.准备用于显微镜的成像室。 第1部分>,显示程序B的步骤1-3的视频剪辑。第2部分>,视频夹子显示如何将溶液吸移到成像室。 第3部分>,一个视频片段,演示如何更换成像室中的溶液。为了实现可视化目的,在第2部分和第3部分的夹子中显示的示例中使用了掺有溴酚蓝的PEM缓冲液。

    2. 轻轻地在两片双面胶带上面放一块干净的盖玻片。沿着双面胶带区域向下按压盖玻片,形成夹在盖玻片和显微镜载玻片(即>,成像室)之间的腔室条带,如图1B所示。在将显微镜载玻片压下之前,不需要对盖玻片表面进行特殊处理。
    3. 翻转盖玻片/滑动组件,使盖玻片相对于显微镜载玻片位于底部。
    4. 通过使用PEM缓冲液将 TM α微管蛋白抗体(GenScript)从500μg/ ml稀释至33.3μg/ ml来制备抗微管蛋白抗体溶液。将未使用的抗体溶液储存在4°C以备后用。
    5. 将7μl抗微管蛋白抗体吸移到成像室中。通过使用L-20XLS +移液管从腔室的一侧以一定角度缓慢注射来向腔室填充抗体溶液(参见图1C)。另请参阅视频1的第2部分以了解该技术。考虑在仅使用PEM缓冲液的练习室上排练该步骤,因为抗微管蛋白抗体溶液可能在第一次尝试时不会流入腔室。
    6. 通过在室温下孵育5分钟使抗微管蛋白抗体粘附到腔室。
    7. 用阻断缓冲液替换成像室中的抗微管蛋白抗体溶液(配方2)。为此,将一片Kimwipe切成三角形,并将尖端固定在成像室的一侧。当Kimwipe将抗体溶液从室中抽出时,将7μl封闭缓冲液吸移到室的另一侧(参见图1D)。另请参阅视频1的第3部分,了解更换成像室中溶液的技术。


      图1.腔室组装。 A.将双面胶带粘贴到显微镜载玻片上。 B.将盖玻片盖在双面胶带上。 C.转动盖玻片并滑过。用抗微管蛋白抗体溶液填充腔室。 D.通过在另一侧用Kimwipe抽出而一边注入流入阻塞缓冲区。

    8. 使封闭缓冲液在室温下孵育5分钟。
    9. 通过使用PEM缓冲液将微管原液(如上文步骤A中所述制备)从50μM稀释至0.125μM来制备微管溶液。为避免剪切微管,在移液微管时使用切割的移液管尖端,如过程A的步骤12所述。
    10. 用稀释的微管溶液替换成像室中的阻断缓冲液。与上述步骤B7类似,使用一片Kimwipe从一侧将阻塞缓冲液从腔室中抽出,同时将微管溶液吸移到另一侧的腔室中。
    11. 通过在室温下孵育5分钟使微管附着于抗微管蛋白抗体。
    12. 将腔室置于倒置荧光显微镜下进行成像。

  3. 可视化微管结合和交联
    以下步骤要求实验者掌握少量用荧光探针纯化和标记的测试微管结合蛋白,该荧光探针不同于用于标记微管的荧光探针。我们将实验者转介到其他研究(Markus et al。>,2012; Zhu et al。>,2017),用于涉及用TMR纯化和标记HALO标记的融合蛋白的方案。或Alexa Fluor 488,适用于以下步骤。
    1. 将10μl125nM紫杉醇稳定的微管(如方法A中所述用HiLyte Fluor 488标记)与1μl10μMMAP(用TMR标记)在PEM缓冲液中混合。加入9μlPEM缓冲液使总反应体积达到20μl。或者,在该步骤中,可以将用罗丹明标记的相同体积和浓度的紫杉醇稳定的微管(如方法A中所述制备)与用Alexa Fluor 488标记的MAP混合。与缓冲液单独混合的微管应作为对照单独进行。
    2. 将混合物在室温下孵育10分钟。
    3. 将微管/ MAP混合物流入已经用抗微管蛋白抗体和封闭缓冲液处理的成像室中(步骤B中的步骤1-8)。如过程B中步骤10所述,使用一片Kimwipe从一侧将阻塞缓冲液从腔室中抽出,同时将微管/ MAP溶液吸移到另一侧的腔室中。为避免剪切微管,在将微管移液到成像室时使用切割的移液管尖端。
    4. 通过在室温下孵育5分钟使微管/ MAP附着于抗微管蛋白抗体。
    5. 为了洗掉未附着的微管/ MAP,将新鲜的封闭缓冲液一次流入成像室。
    6. 在室温下孵育5分钟。将腔室带到显微镜室进行成像。
    7. 要观察微管/ MAP和微管/缓冲液样品,请使用配备Perfect Focus System和100x / 1.49 NA物镜的Nikon Ti-E倒置显微镜。
    8. 将一滴浸油放在成像室的盖玻片侧,并将载玻片安装在显微镜载物台上。
    9. 使用LUN4激光发射系统(尼康)照亮荧光团和iXon 888电子倍增CCD相机(安道尔技术),对荧光标记的微管和MAP进行成像。
    10. 使用GFP滤光片立方体组(Chroma Technology Corp.)可视化Alexa Fluor 488或HiLyte Fluor 488荧光。使用TRITC滤光片立方体组(Chroma Technology Corp.)可视化罗丹明或TMR荧光。
    11. 使微管相对于100x / 1.49 NA物镜聚焦。调整聚焦在GFP通道中,用于HiLyte Fluor 488标记的微管。或者,如果步骤C1使用罗丹明标记的微管,则使用TRITC通道。 HiLyte Fluor 488标记的微管图像的一个例子如图2所示。


      图2.HiLyte Fluor 488标记的微管的实例在该图中,在Nikon Ti-E倒置显微镜中观察成像室中与抗微管蛋白抗体结合的微管。比例尺=7μm。

    12. 切换到TRITC通道以显示微管上的结合MAP。或者,如果步骤C1使用Alexa Fluor 488标记的MAP,则切换到GFP通道。
    13. 要获取双色图片,请使用带有NIS-Elements软件(尼康)的PC工作站,该软件控制通道切换,激光强度和相机设置。图3显示了HiLyte Fluor 488标记的微管和TMR标记的MAP的双色图像示例。


      图3.荧光标记的微管和MAP的双色图像示例。 HiLyte Fluor 488标记的微管与TMR标记的重组MAP,She1-C-TMR混合,如Zhu et al。>(2017)所述纯化,在成像室中可视化在这幅图片中。比例尺=3.6μm。

    14. 为了收集足够的数据用于后续分析,每个室捕获至少20个不同的双色微管/ MAP图像区域。用至少三种独立的MAP制剂重复实验。
    15. 使用相同的相机设置来比较从腔室到腔室的微管结合的MAP量,或者当使用不同的MAP浓度时(如果需要)。
    16. 一些MAP可能交联或捆绑微管。在存在和不存在MAP的情况下的图像微管以确定观察微管束的频率。通过测量微管横截面强度可以将束与单个微管区分开(如数据分析中所述)。

数据分析

  1. 要量化微管上结合MAP的相对量,请使用ImageJ软件(NIH)中的分段线工具。沿着双色图像中观察到的单个微管描绘一条线(如图4,左图所示)。测量至少50个单独微管的每单位长度MAP的平均荧光强度(参见图4)。通过将线条工具移动到没有微管的相邻区域来减去每个测量的背景(如图4,右图所示)。计算每单位长度校正平均强度的平均值。


    图4.定量微管上结合MAP的实例。 左>,沿着单个微管绘制的样本线(由1和3表示)。 右>,通过ImageJ(在Analyze选项卡下)测量的每单位线长度的She1-C-TMR的平均强度。通过将线移动到没有微管的相邻区域(由2和4表示)来测量背景强度。

  2. 将存在MAP时测量的值与缓冲液控制中的值进行比较。使用KaleidaGraph软件绘制实验值与对照条件的值分布。执行双尾未配对Student's t > -test以确定两个分布之间的统计显着性。
  3. 为了确定微管是否通过MAP交联,使用ImageJ中的分段线工具在双色图像中观察到的各个微管结构上画一条线(如图5,左图所示)。使用ImageJ中的绘图轮廓工具测量沿线的横截面荧光强度(图5,中间面板)。对至少50种不同的微管结构进行这些测量。使用KaleidaGraph软件绘制荧光强度作为线的函数(参见Zhu 等人的图3C,>,2017用于样品图)。计算荧光分布的平均峰强度。
  4. 比较在MAP存在下观察到的微管结构的峰强度与仅缓冲液对照中的峰值强度(参见Zhu 等人的图3C,<2017;实施例中的图3C)。执行双尾未配对学生的 t > - 测试,以确定两组峰值强度之间的统计显着性。


    图5.测量横截面荧光强度的示例。 左>,穿过微管结构。 中间>,沿线的荧光剖面。 正确>,绘制个人资料的值。

笔记

  1. 由于成像室未密封,腔室中的液体将缓慢蒸发,从而改变样品浓度并影响图像质量。因此,每个成像室应仅在室温下成像约15分钟。
  2. 由于微管通过抗微管蛋白抗体附着在玻璃表面,因此捕获单焦平面图像应足以涵盖微管的整个厚度。

食谱

  1. PEM缓冲液(100毫升)
    2.42克PIPES
    20.33mg MgCl 2 六水合物
    38.04毫克EGTA
    1毫升100毫摩尔GTP
    超纯水
    用0.1M NaOH调节pH至6.9
  2. 封闭缓冲液(100毫升)
    1克Pluronic F-127
    100毫升PEM缓冲液

致谢

这项工作得到了W.H的NIH / NIGMS拨款(GM076094)的支持。 L.

利益争夺

作者声明没有竞争性的经济利益。

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Copyright: © 2018 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. Zhu, Y., Tan, W. and Lee, W. (2018). An in vitro Microscopy-based Assay for Microtubule-binding and Microtubule-crosslinking by Budding Yeast Microtubule-associated Protein. Bio-protocol 8(23): e3110. DOI: 10.21769/BioProtoc.3110.
  2. Zhu, Y., An, X., Tomaszewski, A., Hepler, P. K. and Lee, W. L. (2017). Microtubule cross-linking activity of She1 ensures spindle stability for spindle positioning. J Cell Biol 216: 2759-2775.
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