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Analysis of Myosin II Minifilament Orientation at Epithelial Zonula Adherens
上皮粘着小带的微球蛋白II微丝排列分析   

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Abstract

Non-muscle myosin II (NMII) form bipolar filaments, which bind F-actin to exert cellular contractility during physiological processes (Vicente-Manzanares et al., 2009). Using a combinatorial approach to fluorescently label both N- and C-termini of the NMII heavy chain, recent works have demonstrated the ability to visualize NMII bipolar filaments at various subcellular localizations (Ebrahim et al., 2013; Beach et al., 2014). At the zonula adherens (ZA) of epithelia, NMII minifilaments bind the circumferential actin bundles in a pseudo-sarcomeric manner (Ebrahim et al., 2013), a conformation required to maintain junctional tension and tissue integrity (Ratheesh et al., 2012). By expressing green fluorescent protein (GFP)-NMIIA heavy chain and immunolabel it using a NMIIA C-terminus specific antibody, we were able to visualize the NMII minifilaments bound to F-actin bundles in Caco-2 cells (Michael et al., 2016), as previously reported (Ebrahim et al., 2013; Beach et al., 2014). In addition, we designed an FIJI/MATLAB analysis module to quantify the size, distance and alignment of these minifilaments with respect to junctional F-actin at the ZA. Measurements of the dispersion of minifilaments angles were proven to be a useful parameter that closely correlated to the extent of contractility at junctions (Michael et al., 2016).

Keywords: Myosin II minifilaments(肌球蛋白II微丝), Structured illumination microscopy(结构照明显微镜检查), Adherens junctions(黏着连接), Actin organization(肌动蛋白的组织)

Background

For decades, the assembly of NMII into bipolar filaments has been studied using electron microscopy (EM) techniques. These mainly involve the assembly of NMII minifilaments from purified proteins or the visualization of minifilaments in cells following extraction of the actin cytoskeleton (Pollard, 1982; Svitkina et al., 1989). Whilst these methods enabled measurements of NMII bipolar filament assemblies, they were technically challenging and did not accurately reflect the cellular distribution of these entities, notwithstanding the artifacts introduced due to the sample preparation. With the advent of super-resolution microscopy, we are now able to observe and measure these NMII minifilaments in various subcellular locations with high resolution, using a rapid process that is amenable for most laboratories equipped with a microscope that performs structured illumination microscopy (SIM, Yap et al., 2015). In this protocol, we describe a method that we have developed to assess NMII minifilaments properties at adherens junctions by measuring the lengths of the minifilaments as well as quantifying their angles with respect to the junctional F-actin and their distance from the junctions (Michael et al., 2016).

Materials and Reagents

  1. 1. 6-well plates (Corning, Costar®, catalog number: 3516 )
  2. Glass coverslips (13 mm, #1.5) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 1014355130NR15 )
  3. ShandonTM ColorFrostTM glass slides (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 6776214 )
  4. Caco-2 human colon adenocarcinoma cells (ATCC, catalog number: ATCCH®TB-37TM )
  5. Plasmid: GFP-NMIIA (Addgene, catalog number: 11347 )
  6. Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, GibcoTM, catalog number: 11875093 )
  7. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 26140079 )
  8. 100x penicillin/streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  9. 100x L-glutamine (200 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  10. Lipofectamine® 3000 (Thermo Fisher Scientific, InvitrogenTM, catalog number: L3000015 )
  11. Opti-MEM®, reduced serum media (Thermo Fisher Scientific, GibcoTM, catalog number: 31985070 )
  12. Alexa Fluor® 647 phalloidin (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: A22287 )
  13. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  14. Antibodies
    1. Anti-Myosin IIA rabbit polyclonal antibody (Biolegend, catalog number: PRB-440P )
      Note: This antibody targets the C-terminal portion of the NMIIA heavy chain.
    2. Goat anti-Rabbit IgG (H+L) secondary antibody, Alexa Fluor® 546 conjugate (Thermo Fisher Scientific, Invitrogen, catalog number: A11035 )
  15. TetraSpeckTM multi-speck 100 nm beads (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: T7279 )
  16. ProLong® Gold antifade mountant (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: P36934 )
  17. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: 158127 )
  18. Piperazine-N,N’-bis(2-ethanesulfonic acid) (PIPES) (Sigma-Aldrich, catalog number: P6757 )
  19. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  20. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  21. Ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
  22. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  23. Tris-Cl (Sigma-Aldrich, catalog number: T5941 )
  24. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
  25. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
  26. Phosphate buffered saline (PBS), without Ca2+ and Mg2+ (Thermo Fisher Scientific, GibcoTM, catalog number: 14190250 )
  27. 10x trypsin/EDTA (0.5%) (Thermo Fisher Scientific, GibcoTM, catalog number: 15400054 )
  28. 4% paraformaldehyde (PFA) (see Recipes)
  29. Tris buffered saline (TBS) (see Recipes)
  30. Blocking buffer (see Recipes)

Equipment

  1. NuncTM 75 cm2 cell culture flasks (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 156472 )
  2. Incubator
  3. Zeiss ELYRA superresolution microscope (ZEISS, model: ELYRA Superresolution Microscope )

Software

  1. Zen (black version; Zeiss)
  2. FIJI (http://imagej.net/Fiji)
  3. Prism, GraphPad (http://www.graphpad.com/scientific-software/prism/)
  4. Matlab, Mathworks (https://www.mathworks.com/index-c.html)
  5. FIJI and Matlab scripts for minifilament analysis (see Appendix I and II of this Bioprotocol)

Procedure

  1. Preparation and transfection of Caco-2 cells
    Note: This technique was used in our recent Developmental Cell article (Michael et al., 2016).
    1. Culture Caco-2 cells in RPMI media supplemented with 10% FBS, penicillin/streptomycin, L-glutamine and maintained at 37 °C in 5% CO2.
    2. For transfection, plate cells at 200,000 cells/well in a 6-well plate containing 4x coverslips evenly dispersed and lying flat within each well.
    3. Once cells reached ~50-80% confluence (~36-48 h post-plating, Figure 1), transfect cells with GFP-NMIIA plasmid using Lipofectamine 3000 reagent, according to manufacturer’s instructions.


      Figure 1. DIC image of Caco-2 cells after 48 h plating using a 20x objective. The image shows a field of view of 600 x 600 μm.

    4. For the transfection, 2 μg of DNA is diluted in 190 μl Opti-MEM containing 5 μl P3000 reagent. In a separate tube, 5 μl of Lipofectamine 3000 is diluted in 190 μl Opti-MEM. Both mixes are then combined and incubated at room temperature for 10 min and applied to cells containing 1 ml Opti-MEM per well.
    5. Return cells to the 37 °C incubator and after 6 h, replace the transfection media with growth media. Leave cells to grow within the incubator for a further 24 h before fixation. Expression of the GFP-tagged plasmid can be observed in 30-40% of the cells after 24 h transfection using a fluorescent microscope (Figure 2).


      Figure 2. Low magnification image of Caco-2 cells after 24 h transfection, taken with an epifluorescence microscope. Cells were fixed and stained using Alexa Fluor® 647 phalloidin.

  2. Immunofluorescence
    1. Fix cells on ice with 4% PFA for 20 min followed by permeabilization on ice with 0.25% Triton X-100 in TBS for 5 min.
    2. Block cells with blocking buffer (see Recipes) at room temperature for 1 h followed by incubation with anti-Myosin IIA antibody (1:1,000 dilution) for 1 h at room temperature. All antibodies used were diluted in blocking buffer.
    3. Wash 3 times with TBS. Then, incubate cells for 1 h with the goat anti-rabbit Alexa Fluor® 546 secondary antibody (1:500 dilution) and phalloidin Alexa Fluor® 647 (1:500 dilution).
    4. Wash cells again 3 times with TBS and then mount coverslips by inverting them onto glass slides containing 5 μl drops of ProLong® Gold mounting reagent.

  3. Imaging of Myosin IIA and F-actin at the zonula adherens
    1. Perform structured illumination microscopy (SIM) of the immunostained coverslips on a Zeiss Elyra superresolution microscope using a 60x objective, 1.4 NA Plan Apochromat oil immersion lens.
    2. Do 3-channel imaging, using 488, 561, 640 nm lasers, to detect GFP-NMIIA fluorescence, the NMIIA C-terminal antibody (Alexa Fluor® 546) and phalloidin (Alexa Fluor® 647) at the zonula adherens (ZA).
    3. Acquire images of a single z-slice through channel-specific gratings at 5 phases and 3 120° rotations of the grid pattern.
    4. Perform image reconstruction with the Zen software using a noise filtering level of -4.5 and an optical sectioning value of 100.
    5. Perform channel alignment on reconstructed images using pre-calibrated values that were previously obtained by measuring 4-colour bead alignment samples (multi-speck beads).
    6. The final images reveal a pseudo-sarcomeric organization of NMII decorating the phalloidin labeled F-actin filaments at the ZA (Figure 3A). Furthermore, due to the increased resolution of the SIM technique and the N-terminally tagged GFP-NMIIA and C-terminal NMIIA antibody labeling combination, the NMIIA bipolar filaments can be visualized in detail as a set of green-red-green puncta (Figures 3A and 3B).


      Figure 3. A magnified area of a final SIM image. A. SIM image of a cell-cell junction expressing GFP-NMIIA (green) and immunostained for the C-terminus of NMII (red). Phalloidin labeling (blue) served as a marker for junctional F-actin. B. Illustration of NMII bipolar filament organization and its visualization by SIM. Scale bar = 1 μm.

Data analysis

  1. Quantification of NMIIA minifilament orientations at the ZA
    1. Overview
      1. Step 1 (FIJI software): Use an RGB image of the SIM data to draw (Figure 4) regions of interest (ROIs) corresponding to:
        1. The cell-cell junction according to the phalloidin (blue) staining as a guide, using the segmented line tool in FIJI.
        2. Each individual actomyosin minifilament (green-red-green dots), using the straight line tool in FIJI.


          Figure 4. A cropped area of the 3 color SIM image showing the details of ROIs used for analysis. Left, RGB image with ROI. Right, schematic of ROIs. The first ROI that needs to be drawn is the one that corresponds to the cell-cell junction (Blue) followed by the different ROIs that correspond to individual minifilaments.

      2. Step 2 (FIJI software): Save the ROIs as XY coordinates, using the FIJI script provided in this protocol (Appendix I), in a .txt format (Figure 5).


        Figure 5. Graphical representation of different ROIs in the XY plane. ROIs exported as .txt files using a FIJI script (Appendix I) are imported into MATLAB and shown as a figure (The figure is generated automatically after running the MATLAB script, Appendix II).

      3. Step 3 (MATLAB software): Measure the angle between the tangent of the junction (θ) and the orientation of the minifilament (γ). Angle = θ - γ (Figure 6).


        Figure 6. Schematic of how relative orientation of minifilaments is calculated based on the spatial information of the different ROIs. The red arrow represents a minifilament and γ indicates the angle that defines the local orientation of its vector. The blue arrow shows the local angle of the junction, indicated by θ, defined by the closest point of the junction to the corresponding minifilaments.

      4. Step 4 (MATLAB software): Measure the distance of the minifilament to the junction (Figure 7).


        Figure 7. Graphical representation of the calculation of the distance between minifilaments and the cell-cell junction

      5. Step 5 (MATLAB software): Measure the length of the ROIs (‘minifilament length’) corresponding to the different minifilaments (Figure 8).


        Figure 8. A magnified view of the SIM image with the position of the different ROIs

      6. Step 6 (PRISM or Excel software): Preparation of results
        Here the quantities that are obtained from this analysis are: (1) the standard deviation of the angle values, which is an index of the relative organization of minifilaments parallel to the junction; (2) the average distance of minifilaments to the junction, an index of how closely associated the minifilaments are to the cell-cell junctions and (3) the average length of minifilaments, which reflects the capacity of myosin (and mechanical tension) to organize actomyosin into sarcomeric structures.

    2. Procedure in practice
      1. Quantification of minifilaments alignment was performed using custom-made scripts for FIJI and MATLAB (see Appendix I and Appendix II).
      2. The 3 channel images were opened in FIJI. The F-actin at junctions is visualized as a bundle of linear F-actin filaments (Figure 3). Using the phalloidin channel as a reference, a freehand line is drawn in the middle of the F-actin bundle (corresponding to the junction) and this line is added to the ROI manager (refer to Figure 4, also step A1a.i).
      3. Using the merged 3-channel (RGB) image, each minifilament (defined by the green-red-green puncta) is marked using the line tool (step A1a.ii) and added to the ROI manager (refer to Figure 4). The ROI manager along with the RGB image is saved in a folder.
      4. Using the ‘FIJI script for minifilament analysis’, a custom-made macro for FIJI (Appendix I), the coordinates for the junctional F-actin and the minifilaments were extracted from the ROIs and these coordinates were saved as .txt that were used later in MATLAB to determine the minifilament angle, length and distance with respect to the junction. The macro can be easily created by selecting Plugins->New->Macro. A window appears where the script in Appendix I can be pasted. Then, from this window the macro can be run.
      5. Open MATLAB and select the directory that contains the RGB SIM images (where the .txt files with the XY coordinates of ROIs are stored).
      6. Open MATLAB and create a new script by copying and pasting the ‘MATLAB script for minifilament analysis’.
      7. Modify the first line of the code to include the correct number of ROIs to be analyzed.
      8. Run the script (‘command + alt + R’ in MacOS).
      9. After running the script 3 figures appear (Figure 9).
        These figures can be saved as EPS, JPEG or TIFF formats in MATLAB. We recommend saving these as EPS format, as these are fully editable, and amenable to subsequent figure preparation in Adobe Illustrator.


        Figure 9. Figure results from MATLAB using the script in Appendix II. A. Corresponds to the different minifilaments put on the same origin of coordinates. From here it is possible to appreciate the distribution of orientation of the minifilaments with respect to the cell-cell junction. B. Corresponds to the polar histogram of minifilament orientations shown in Figure 4. C. Shows the vectorial map of the different minifilaments alongside the position of the junction.

      10. Numeric results
        1. In the workspace of MATLAB, the variable ‘AAResults’ will contain the numerical results for statistical analysis (Figure 10).
          This variable has 5 columns:
          1st Column: Minifilament length
          2nd Column: Distance of the minifilament from the junction
          3rd Column: Relative orientation (angle) of the minifilament with respect to the junction
          4th Column: X component of the minifilament vector
          5th Column: Y component of the minifilament vector


          Figure 10. A screen snapshot of the ‘AAResults’ table in MATLAB. This table contains the numerical results for the different analysis.

        2. Copy Columns 1 to 3 and paste into PRISM or Excel software to perform statistical analysis of the following parameters:
          Average the values of minifilament length (average of Column 1).
          Average the values of distance between minifilaments and the cell-cell junction (average of Column 2).
          Standard deviation of angles between minifilaments and the cell-cell junction (standard deviation of Column 3).
        3. These values then can be compared between different experimental conditions as in (Michael et al., 2016). 

Notes:

  1. If junctions to be analyzed are too vertical, modify the second line of the Matlab code to make it
    Rotate45 = 1.
    This would prevent a premature stop of the code while running.
  2. Analyze Column 4 of the AA results for the presence of negative values. The code and analysis are designed to run in such a way that values within this column should be positives. If some negative values are present for this column, the rows containing them should be excluded from analysis.

Recipes

  1. 4% paraformaldehyde (PFA, in cytoskeletal stabilization buffer)
    Dissolve PFA in 10 mM PIPES at pH 6.8
    100 mM KCl
    300 mM sucrose
    2 mM EGTA
    2 mM MgCl2
    Adjust pH to 7.0
    Store at -20 °C in 10 ml aliquots
  2. Tris buffered saline (TBS)
    50 mM Tris-Cl
    150 mM NaCl
    Dissolve in Milli-Q water
    Adjust pH to 7.5
  3. Blocking buffer
    Dissolve 5% (wt/vol) BSA into 1x TBS

Acknowledgments

This protocol is an adapted version of the one published in (Michael et al., 2016). This work was supported by grants from the National Health and Medical Research Council of Australia (1037320, 1067405), the Australian Research Council (DP120104667, 150101367) and the Kids Cancer Project of the Oncology Children’s Foundation. Optical imaging was performed at the ACRF/IMB Cancer Biology Imaging Facility, established with the generous support of the Australian Cancer Research Foundation, and the Queensland Brain Institute microscopy facility supported by ARC LIEF grant (LE130100078).

References

  1. Beach, J. R., Shao, L., Remmert, K., Li, D., Betzig, E. and Hammer, J. A., 3rd (2014). Nonmuscle myosin II isoforms coassemble in living cells. Curr Biol 24(10): 1160-1166.
  2. Ebrahim, S., Fujita, T., Millis, B. A., Kozin, E., Ma, X., Kawamoto, S., Baird, M. A., Davidson, M., Yonemura, S., Hisa, Y., Conti, M. A., Adelstein, R. S., Sakaguchi, H. and Kachar, B. (2013). NMII forms a contractile transcellular sarcomeric network to regulate apical cell junctions and tissue geometry. Curr Biol 23(8): 731-736.
  3. Michael, M., Meiring, J. C., Acharya, B. R., Matthews, D. R., Verma, S., Han, S. P., Hill, M. M., Parton, R. G., Gomez, G. A. and Yap, A. S. (2016). Coronin 1B reorganizes the architecture of F-actin networks for contractility at steady-state and apoptotic adherens junctions. Dev Cell 37(1): 58-71.
  4. Pollard, T. D. (1982). Structure and polymerization of Acanthamoeba myosin-II filaments. J Cell Biol 95(3): 816-825.
  5. Ratheesh, A., Gomez, G. A., Priya, R., Verma, S., Kovacs, E. M., Jiang, K., Brown, N. H., Akhmanova, A., Stehbens, S. J. and Yap, A. S. (2012). Centralspindlin and α-catenin regulate Rho signalling at the epithelial zonula adherens. Nat Cell Biol 14(8): 818-828.
  6. Svitkina, T. M., Surguchova, I. G., Verkhovsky, A. B., Gelfand, V. I., Moeremans, M. and De Mey, J. (1989). Direct visualization of bipolar myosin filaments in stress fibers of cultured fibroblasts. Cell Motil Cytoskeleton 12(3): 150-156.
  7. Vicente-Manzanares, M., Ma, X., Adelstein, R. S. and Horwitz, A. R. (2009). Non-muscle myosin II takes centre stage in cell adhesion and migration. Nat Rev Mol Cell Biol 10(11): 778-790.
  8. Yap, A. S., Michael, M. and Parton, R. G. (2015). Seeing and believing: recent advances in imaging cell-cell interactions. F1000Res 4(F1000 Faculty Rev): 273.

简介

非肌肉肌球蛋白II(NMII)形成双极细丝,其在生理过程期间结合F-肌动蛋白以施加细胞收缩(Vicente-Manzanares等人,2009)。使用组合方法荧光标记NMII重链的N末端和C末端,最近的工作已经证明了在各种亚细胞定位下可视化NMII双极细丝的能力(Ebrahim等人,2013; Beach 等。,2014)。在上皮细胞粘附分子(ZA)上,NMII小丝以假性肌节方式结合周围肌动蛋白束(Ebrahim等人,2013),这是维持连接张力和组织完整性所需的构象Ratheesh等人,2012)。通过表达绿色荧光蛋白(GFP)-NMIIA重链并使用NMIIA C-末端特异性抗体对其进行免疫标记,我们能够观察到在Caco-2细胞中与F-肌动蛋白束结合的NMII小丝(Michael等人。,2016),如以前报道的(Ebrahim等人,2013; Beach 等人,2014)。此外,我们设计了FIJI/MATLAB分析模块来量化这些小丝相对于在ZA处的连接F-肌动蛋白的大小,距离和比对。小纤维角度的分散性的测量被证明是与结合处收缩性程度密切相关的有用参数(Michael等人,2016)。
关键词:肌球蛋白II微丝,结构照明显微镜,Adherens连接,肌动蛋白组织

[背景]几十年来,NMII装配成双极细丝已经使用电子显微镜(EM)技术。这些主要涉及从肌动蛋白细胞骨架提取后,从纯化的蛋白质组装NMII小丝或细胞中小丝的可视化(Pollard,1982; Svitkina等人,1989)。虽然这些方法能够测量NMII双极灯丝组件,但是它们在技术上是有挑战性的并且不能准确地反映这些实体的细胞分布,尽管由于样品制备而引入了假象。随着超分辨率显微镜的出现,我们现在能够观察和测量这些NMII小丝在各种亚细胞位置具有高分辨率,使用快速过程是适合大多数实验室配备有显微镜执行结构化照明显微镜(SIM, Yap 。,2015)。在这个协议中,我们描述了一种方法,我们已经开发了评估NMII小纤维属性在粘附连接处通过测量小丝的长度以及量化他们的角度关于连接F-肌动蛋白和他们的距离连接(迈克尔等。,2016)。

关键字:肌球蛋白II微丝, 结构照明显微镜检查, 黏着连接, 肌动蛋白的组织

材料和试剂

  1. 1.6孔板(Corning,Costar ,目录号:3516)
  2. 玻璃盖玻片(13mm,#1.5)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:1014355130NR15)
  3. (Thermo Fisher Scientific,Thermo Scientific TM ,目录号:6776214),将所述玻璃载玻片置于玻璃载玻片上。
  4. Caco-2人结肠腺癌细胞(ATCC,目录号:ATCCH TB-37 TM )。
  5. 质粒:GFP-NMIIA(Addgene,目录号:11347)
  6. Roswell Park Memorial Institute(RPMI)1640培养基(Thermo Fisher Scientific,Gibco TM ,目录号:11875093)
  7. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM ,目录号:26140079)
  8. 100x青霉素/链霉素(10,000U/ml)(Thermo Fisher Scientific,Gibco TM ,目录号:15140122)
  9. 100x L-谷氨酰胺(200mM)(Thermo Fisher Scientific,Gibco TM ,目录号:25030081)
  10. Lipofectamine 3000(Thermo Fisher Scientific,Invitrogen TM ,目录号:L3000015)
  11. Opti-MEM ,血清培养基(Thermo Fisher Scientific,Gibco TM ,目录号:31985070)
  12. Alexa Fluor 647鬼笔环肽(Thermo Fisher Scientific,Moecular Probes TM ,目录号:A22287)
  13. Triton X-100(Sigma-Aldrich,目录号:X100)
  14. 抗体
    1. 抗肌球蛋白IIA兔多克隆抗体(Biolegend,目录号:PRB-440P)
      注意:该抗体靶向NMIIA重链的C末端部分。
    2. 山羊抗兔IgG(H + L)第二抗体,Alexa Fluor?546缀合物(Thermo Fisher Scientific,Invitrogen,目录号:A11035)
  15. TetraSpeck TM多多斑点100nm珠(Thermo Fisher Scientific,Molecular Probes TM ,目录号:T7279)
  16. ProLong Gold防褪色封固剂(Thermo Fisher Scientific,Molecular Probes TM ,目录号:P36934)
  17. 多聚甲醛(PFA)(Sigma-Aldrich,目录号:158127)
  18. 哌嗪-N,N'-双(2-乙磺酸)(PIPES)(Sigma-Aldrich,目录号:P6757)
  19. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  20. 蔗糖(Sigma-Aldrich,目录号:SO389)
  21. 乙二醇 - 双(β-氨基乙基醚)-N,N,N',N'-四乙酸(EGTA)(Sigma-Aldrich,目录号:E3889)
  22. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  23. Tris-Cl(Sigma-Aldrich,目录号:T5941)
  24. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014)
  25. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153)
  26. 没有Ca 2+和Mg 2+的磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco< sup>,目录号:14190250) br />
  27. 10x胰蛋白酶/EDTA(0.5%)(Thermo Fisher Scientific,Gibco TM ,目录号:15400054)
  28. 4%多聚甲醛(PFA)(参见配方)
  29. Tris缓冲盐水(TBS)(参见Recipes)
  30. 阻止缓冲区(参见配方)

设备

  1. 细胞培养瓶(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:156472)的微量滴定板
  2. 孵化器
  3. Zeiss ELYRA超分辨率显微镜(ZEISS,型号:ELYRA超分辨率显微镜)

软件

  1. 禅(黑版;蔡司)
  2. FIJI( http://imagej.net/Fiji
  3. Prism,GraphPad( http://www.graphpad.com/scientific - 软件/棱镜/
  4. Matlab,Mathworks( https://www.mathworks.com/index- c.html
  5. 用于minifilament分析的FIJI和Matlab脚本(请参阅附录I II  

程序

  1. 制备和转染Caco-2细胞
    注意:这种技术用于我们最近的Developmental Cell文章(Michael等人,2016)。
    1. 在补充有10%FBS,青霉素/链霉素,L-谷氨酰胺并在37℃,5%CO 2中维持的RPMI培养基中培养Caco-2细胞。
    2. 对于转染,在含有4x盖玻片的6孔板中以200,000细胞/孔平板细胞均匀分散并平放在每个孔内。
    3. 一旦细胞达到〜50-80%汇合(铺板后约36-48小时,图1),使用Lipofectamine 3000试剂根据制造商的说明用GFP-NMIIA质粒转染细胞。


      图1.使用20x物镜48小时电镀后Caco-2细胞的DIC图像。图像显示600 x 600μm的视野。

    4. 对于转染,将2μgDNA在含有5μlP3000试剂的190μlOpti-MEM中稀释。在单独的试管中,将5μlLipofectamine 3000在190μlOpti-MEM中稀释。然后将两种混合物合并,并在室温下温育10分钟,并施加到每孔含有1ml Opti-MEM的细胞中。
    5. 将细胞返回37℃培养箱,6小时后,用生长培养基替换转染培养基。离开细胞在孵化器内生长另外24小时,然后固定。使用荧光显微镜在24小时转染后可以在30-40%的细胞中观察到GFP标记的质粒的表达(图2)。


      图2.用epifluorescence显微镜取得的24小时转染后Caco-2细胞的低放大倍数图像。将细胞固定并使用Alexa Fluor 647鬼笔环肽染色。

  2. 免疫荧光
    1. 固定细胞在冰上用4%PFA 20分钟,然后在冰上用0.25%Triton X-100在TBS中透化5分钟。
    2. 在室温下用封闭缓冲液(参见Recipes)封闭细胞1小时,随后与抗肌球蛋白IIA抗体(1:1000稀释)在室温下温育1小时。使用的所有抗体在封闭缓冲液中稀释
    3. 用TBS洗涤3次。然后,用山羊抗兔Alexa Fluor 546二抗(1:500稀释)和鬼笔环肽Alexa Fluor 647(1:500稀释)孵育细胞1小时)。
    4. 用TBS再次洗涤细胞3次,然后通过将盖玻片倒置到含有5μlProLong Gold安装试剂滴的载玻片上来安装盖玻片。

  3. 肌球蛋白IIA和F-肌动蛋白在子宫粘膜上的成像
    1. 在Zeiss Elyra超分辨率显微镜上使用60倍物镜,1.4 NA Plan Apochromat油浸镜头对免疫染色的盖玻片进行结构化照明显微镜(SIM)。
    2. 使用488,561,640nm激光器进行3通道成像以检测GFP-NMIIA荧光,NMIIA C-末端抗体(Alexa Fluor 546)和鬼笔环肽(Alexa Fluor 647)在zonula adherens(ZA)。
    3. 通过通道特定的光栅在网格图案的5个相位和3 120°旋转获取单个z切片的图像。
    4. 使用噪声过滤级别为-4.5和光学截面值为100的Zen软件执行图像重建。
    5. 使用先前通过测量4色珠粒对齐样品(多斑点珠粒)获得的预校准值对重建的图像进行通道对齐。
    6. 最终的图像显示NMII的假性肌节组织在ZA装饰鬼笔环肽标记的F-肌动蛋白丝(图3A)。此外,由于SIM技术和N末端标记的GFP-NMIIA和C末端NMIIA抗体标记组合的增加的分辨率,NMIIA双极细丝可以被详细地可视化为一组绿色 - 红色 - 绿色斑点(图3A和3B)。


      图3.最终SIM图像的放大区域 A.表达GFP-NMIIA(绿色)和NMII(红色)C末端免疫染色的细胞 - 细胞连接的SIM图像。鬼笔环肽标记(蓝色)用作连接F-肌动蛋白的标记。 B. NMII双极细丝组织及其通过SIM的可视化的图示。比例尺= 1μm。

数据分析

  1. 在ZA的NMIIA微丝取向的定量
    1. 概述
      1. 步骤1(FIJI软件):使用SIM数据的RGB图像绘制(图4)感兴趣区域(ROI),其对应于:
        1. 根据鬼笔环肽(蓝色)染色的细胞 - 细胞连接作为指导,使用FIJI中的分段线工具。
        2. 每个单独的肌动球蛋白微丝(绿 - 红 - 绿点),使用FIJI中的直线工具。


          图4.三色SIM图片的裁剪区域,显示用于分析的ROI详细信息。左图,具有ROI的RGB图像。右,ROI的示意图。需要绘制的第一个ROI是对应于细胞 - 细胞连接(蓝色),然后是对应于单个小丝的不同ROI的那个。

      2. 步骤2(FIJI软件):使用此协议中提供的FIJI脚本将感兴趣区域保存为XY坐标(附录I ),以.txt格式(图5)。


        图5. XY平面中不同ROI的图形表示。使用FIJI脚本导出为.txt文件的ROI(附录I)被导入到MATLAB中并显示为一个图形(该图是自动生成的运行MATLAB脚本,附录II )。

      3. 步骤3(MATLAB软件):测量结点(θ)的切线和小丝(γ)的取向之间的角度。角度=θ-γ(图6)。


        图6.基于不同ROI的空间信息计算小纤维的相对取向的示意图。红色箭头表示微丝,γ表示定义其向量的局部取向的角度。蓝色箭头表示接合处的局部角度,由θ表示,由与相应小丝的接合点的最近点定义。

      4. 步骤4(MATLAB软件):测量小丝到接合处的距离(图7)。


        图7.小细丝和细胞 - 细胞连接之间的距离的计算的图形表示

      5. 步骤5(MATLAB软件):测量对应于不同小丝的ROI("小丝长度")的长度(图8)。


        图8.具有不同ROI的位置的SIM图像的放大视图

      6. 步骤6(PRISM或Excel软件):结果的准备
        这里,从该分析获得的量是:(1)角度值的标准偏差,其是平行于接合处的小丝的相对组织的指数; (2)小丝对结合部的平均距离,小纤维与细胞 - 细胞接合部紧密相关的指数,以及(3)小纤维的平均长度,其反映肌球蛋白的能力(和机械张力)肌动球蛋白进入肌节结构
    2. 实践程序
      1. 使用针对FIJI和MATLAB的定制脚本进行小丝比对的定量(参见附录一附录二)。
      2. 在FIJI中打开3通道图像。在连接处的F-肌动蛋白被可视化为一束线性F-肌动蛋白丝(图3)。使用鬼笔环肽通道作为参考,在F-肌动蛋白束(对应于结)的中间绘制手绘线,并且将该线添加到ROI管理器(参见图4,也是步骤A1a.i)。
      3. 使用合并的3通道(RGB)图像,使用线工具(步骤A1a.ii)标记每个小丝(由绿 - 红 - 绿斑点定义)并添加到ROI管理器(参见图4)。 ROI管理器与RGB图像一起保存在文件夹中。
      4. 使用'FIJI脚本进行minifilament分析',为FIJI定制的宏( Appendix I ),从ROI中提取连接F-肌动蛋白和小丝的坐标,并将这些坐标保存为.txt,稍后在MATLAB中使用以确定小丝角度,长度和距离。通过选择插件 - >新建 - >宏可以轻松创建宏。将出现一个窗口,其中的脚本位于附录I 可以粘贴。然后,从该窗口可以运行宏。
      5. 打开MATLAB并选择包含RGB SIM图像的目录(其中存储具有ROI的XY坐标的.txt文件)。
      6. 打开MATLAB并通过复制和粘贴'MATLAB脚本进行minifilament分析'创建一个新脚本
      7. 修改代码的第一行以包括要分析的正确ROI数。
      8. 运行脚本(MacOS中的"command + alt + R")。
      9. 运行脚本后出现3个数字(图9)。
        这些数字可以保存为EPS,JPEG或TIFF格式在MATLAB中。我们建议将这些格式保存为EPS格式,因为这些格式是完全可编辑的,并且适合Adobe Illustrator中的后续图形准备。


        图9.使用附录II中的脚本从MATLAB中得到的结果。 A.对应于放在同一坐标原点上的不同小光盘。从这里可以了解小丝相对于细胞 - 细胞连接的取向分布。 B.对应于图4所示的微丝取向的极坐标直方图。C.显示不同微丝沿着连接位置的矢量图。

      10. 数值结果
        1. 在MATLAB的工作区中,变量"AAResults"将包含用于统计分析的数值结果(图10)。
          此变量有5列:
          第一列:细丝长度
          第二列:小丝距离连接点的距离
          第三列:小丝相对于连接点的相对取向(角度)
          第4列:微丝矢量的X分量
          第5列:微丝矢量的Y分量


          图10. MATLAB中"AAResults"表的屏幕快照。此表包含不同分析的数值结果。

        2. 复制列1到3并粘贴到PRISM或Excel软件中,以执行以下参数的统计分析:
          平均细丝长度(第1列的平均值)。
          平均小丝和细胞 - 细胞接合处之间的距离值(第2列的平均值)。
          微丝与细胞 - 细胞连接处的角度的标准偏差(第3列的标准偏差)
        3. 然后可以在不同的实验条件之间比较这些值,如在(Michael等人。,2016)中。

注意:

  1. 如果要分析的连接点太垂直,请修改Matlab代码的第二行以使其
    Rotate45 = 1.
    这将防止代码在运行时过早停止。
  2. 分析AA结果的第4列的负值的存在。代码和分析设计为以此列中的值应为正数的方式运行。如果此列存在某些负值,则应从分析中排除包含它们的行。

食谱

  1. 4%多聚甲醛(PFA,在细胞骨架稳定缓冲液中) 将PFA溶解在pH 6.8的10mM PIPES中
    100 mM KCl
    300mM蔗糖 2 mM EGTA
    2mM MgCl 2/
    将pH调节至7.0
    在-20℃下以10ml等分试样存储
  2. Tris缓冲盐水(TBS)
    50mM Tris-Cl 150mM NaCl 溶解在Milli-Q水中
    将pH调节至7.5
  3. 阻塞缓冲区
    将5%(wt/vol)BSA溶解于1×TBS中

致谢

该协议是在(Michael等人。,2016)中发布的协议的改编版本。这项工作得到澳大利亚国家卫生和医学研究委员会(1037320,1067405),澳大利亚研究委员会(DP120104667,150101367)和儿童癌症项目的肿瘤学儿童基金会的资助。光学成像在由澳大利亚癌症研究基金会的慷ous支持建立的ACRF/IMB癌症生物学成像设施和由ARC LIEF授权(LE130100078)支持的昆士兰大学研究所显微镜设备建立。

参考文献

  1. 在这种情况下,我们可以使用一个简单的例子来描述这个例子,这个例子中,我们可以看到,/inf> 24(10):1160-368(2004)。在这些实施方案中,本发明提供了一种用于治疗和预防非肌肉肌球蛋白II同种型的方法,
  2. Ebrahim,S.,Fujita,T.,Millis,BA,Kozin,E.,Ma,X.,Kawamoto,S.,Baird,MA,Davidson,M.,Yonemura,S.,Hisa,Y.,Conti, MA,Adelstein,RS,Sakaguchi,H。和Kachar,B。(2013)。  NMII形成收缩性跨细胞肌节网,以调节顶端细胞连接和组织几何形状。 Curr Biol 23(8):731-736。
  3. Michael,M.,Meiring,JC,Acharya,BR,Matthews,DR,Verma,S.,Han,SP,Hill,MM,Parton,RG,Gomez,GA和Yap,AS(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27046832"target ="_ blank"> Corone 1B在稳态下重组F-肌动蛋白网络的收缩性结构,凋亡粘附连接。 Dev Cell 37(1):58-71。
  4. Pollard,TD(1982)。  棘阿米巴的结构和聚合肌球蛋白II丝。细胞生物 95(3):816-825。
  5. 本研究结果表明,该方法可以有效地提高生产力,提高生产效率,降低生产成本,提高生产效率。 Centralspindlin和α-catenin调节上皮粘膜粘附的Rho信号。 Nat Cell Biol 14(8):818-828
  6. Svitkina,TM,Surguchova,IG,Verkhovsky,AB,Gelfand,VI,Moeremans,M.and De Mey,J。(1989)。  直接可视化培养的成纤维细胞的应激纤维中的双极肌球蛋白细丝。细胞Motil细胞骨架12(3): 150-156。
  7. Vicente-Manzanares,M.,Ma,X.,Adelstein,RS和Horwitz,AR(2009)。  非肌肉肌球蛋白II在细胞粘附和迁移中占据中心阶段。 Nat Rev Mol Cell Biol 10(11):778-790。
  8. Yap,AS,Michael,M. and Parton,RG(2015)。  见到和相信:成像细胞 - 细胞相互作用的最新进展。 F1000Res 4(F1000 Faculty Rev):273。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Michael, M., Liang, X. and Gomez, G. A. (2016). Analysis of Myosin II Minifilament Orientation at Epithelial Zonula Adherens. Bio-protocol 6(23): e2054. DOI: 10.21769/BioProtoc.2054.
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