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Rapid IFM Dissection for Visualizing Fluorescently Tagged Sarcomeric Proteins
快速解剖间接飞行肌用于荧光标记肌节蛋白的观察   

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Abstract

Sarcomeres, the smallest contractile unit of muscles, are arguably the most impressive actomyosin structure. Yet a complete understanding of sarcomere formation and maintenance is missing. The Drosophila indirect flight muscle (IFM) has proven to be a very valuable model to study sarcomeres. Here, we present a protocol for the rapid dissection of IFM and analysis of sarcomeres using fluorescently tagged proteins.

Keywords: Dissection(解剖), Drosophila(果蝇), GFP(GFP), Indirect flight muscle(间接飞行肌肉), Sarcomere(肌节), Z-disc(Z盘)

Background

The cytoskeletal structures that enable contractility of striated muscle fibers are hundreds of cables called myofibrils. Myofibrils in turn are an array of serially arranged sarcomeres, all contracting simultaneously. The sarcomere is a perfectly symmetrical structure that contains all the elements required for contraction. At the center of the sarcomere lies the M-line, where myosin thick filaments are anchored. Flanking the sarcomere are the Z-discs, where actin thin filaments are anchored.

Muscular dystrophies are inherited disorders that cause progressive skeletal muscle weakness (Schröder and Schoser, 2009). There is no cure for muscular dystrophy, likely due to an incomplete understanding of the molecular mechanisms that underlie muscular dystrophies (Olive et al., 2013). Drosophila melanogaster is an effective genetic model organism to study muscle biology owing to its short life span, economical maintenance, and abundant available resources (Hales et al., 2015; Wangler et al., 2015).

Flight in Drosophila is powered by the synchronized action of the indirect flight muscles (IFM), the biggest muscles in flies, which are further subdivided into dorsal longitudinal muscles (DLM) and dorsal ventral muscles (DVM). The IFM share many fundamental similarities with human skeletal muscle: contraction mechanism, developmental steps, overall ultrastructure, and protein components (Vigoreaux, 2001). For example, the myopathy-related proteins ZASP and Filamin-C have fly homologs that when mutated develop muscle phenotypes (Liao et al., 2016; Gonzalez-Morales et al., 2017). Despite the advantages of using the IFM for muscle research, IFM dissection can be challenging and time-consuming. Here we present a protocol that combines fast and easy IFM dissection with high-quality imaging of the IFM using fluorescent proteins. We also provide a strategy for analyzing mutant phenotypes and quantifying sarcomeres by semi-automatic detection of sarcomere components.

Materials and Reagents

  1. Surgical blade (FEATHER Safety Razor, catalog number: No. 23 )
  2. 1.5 ml microcentrifuge tube
  3. Pipette tips
  4. Conventional needles PrecisionGlide 23 G 1 in. (Fisher Scientific, catalog number: 14-826-6B)
    Manufacturer: BD, catalog number: 305193 .
  5. BD disposable syringes (BD, catalog number: 309628 )
  6. Cover Glass No. 1 ½ 22 x 30 mm (e.g., Corning, catalog number: 2850-22 )
  7. Microscope slides (e.g., Fisher Scientific, catalog number: 12-552-3 )
  8. Flies expressing sarcomere fluorescent markers (e.g., Zasp52-GFP, Table 1)
  9. Custom-made 3.5% agar-filled 60 x 15 mm Petri dish plate (BioShop, catalog number: AGR003 )
  10. Phalloidin-Tetramethylrhodamine B isothiocyanate (TRITC) (Sigma-Aldrich, catalog number: P1951 )
  11. Phalloidin-Fluorescein Isothiocyanate (FITC) (Sigma-Aldrich, catalog number: P5282 )
  12. Mounting media ProLong Gold Antifade Mountant (Thermo Fisher Scientific, InvitrogenTM, catalog number: P36934 )
  13. Magnesium chloride (MgCl2)
  14. Ethylene glycol-bis-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
  15. Adenosine triphosphate (ATP) (BioShop, catalog number: ATP007 )
  16. 1,4-Dithiothreitol (DTT) (BioShop, catalog number: DTT001 )
  17. cOmpleteTM, EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics, catalog number: 11873580001 )
  18. Glycerol (BioShop, catalog number: GLY001 )
  19. Triton X-100 (BioShop, catalog number: TRX777 )
  20. 8% paraformaldehyde aqueous solution glass vial (Electron Microscopy Sciences, catalog number: 157-8 )
  21. Sodium chloride (NaCl)
  22. Potassium chloride (KCl)
  23. Sodium phosphate dibasic (Na2HPO4)
  24. Potassium phosphate dibasic (K2HPO4)
  25. Relaxing solution (see Recipes)
  26. Relaxing-Glycerol solution (see Recipes)
  27. 8% paraformaldehyde (see Recipes)
  28. 10x phosphate buffered saline (PBS) (see Recipes)
  29. 1x PBS, 0.1% Triton X-100 (PBST) (see Recipes)

Equipment

  1. CO2 Flypad, standard size (8.1 x 11.6 cm) (Genesee Scientific, Flystuff, catalog number: 59-114 )
  2. Blade handle for surgical blade (FEATHER Safety Razor, catalog number: No. 4 )
  3. Glass Petri dish; 60 x 15 mm (VWR, catalog number: 89000-770)
    Manufacturer: DWK Life Sciences, KIMBLE®, catalog number: 2306-10010 .
  4. Stereo microscope (Leica Microsystems, model: Leica MS5 )
  5. Dumont #5 forceps (Fine Science Tools, catalog number: 11251-30 )
  6. Incubator set to 25 °C
  7. P2, P20, P100, and P1000 Micro Pipettes (e.g., Gilson, catalog numbers: F144801 , F123600 , F123615 and F123602 )
  8. Platform mixers (e.g., Speci-Mix Test Tube Rocker) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: M71015Q )
  9. Medium-sized pointed brush
  10. Standard fly husbandry equipment

Software

  1. Fiji (https://fiji.sc/)
  2. R Statistics package (https://www.r-project.org/)

Procedure

  1. Prepare flies to dissect
    1. Anesthetize flies using standard CO2 pads. Use 5-10 days old flies.
    2. Transfer flies to a custom-made agar plate using a fine brush. Concentrate all flies in the center of the plate.
    3. Add 100 µl of Relaxing-Glycerol solution (see Recipes) to the flies using a previously cut pipette tip.

  2. Cut half-thoraces
    The main goal of this step is to split the thorax into two symmetric halves (Figure 1 and Video 1). This step is relatively easy, however take note not to crush the muscles. If the blade is dull, replace it with a new blade.
    1. Use two fine forceps to remove the abdomen. Leave only the thorax and the head.
    2. Place and align the thorax with the dorsal side facing your eyes.
    3. Using a scalpel blade knife and a fast slicing movement cut the thorax in two symmetrical halves. Cut the head out if still attached to the thorax.
    4. At this point the IFM should be visible under the dissection microscope.
    5. Take the half-thoraxes with forceps and place them in a 1.5 ml microcentrifuge tube.
    6. Add 100 µl of Relaxing-Glycerol buffer.


      Figure 1. Dissection of the thorax into two halves. A-D. Snapshots from Video 1 showing critical dissection steps. A. Place the thorax with the dorsal side up; B. Gently touch the thorax to make sure the blade is positioned exactly at the middle and that the thorax will not move. C. Aim the tip of the blade at the head/thorax attachment site (arrowhead); D. With a sharp push cut the thorax; E. Representative thorax divided in two. Muscle fibers can be observed at this stage (red asterisks). Scale bars = 200 µm.

      Video 1. Dissection of the thorax into two halves. Dissection process recorded directly from the stereomicroscope. Snapshots of this video are presented in Figure 1.

  3. Remove mitochondria
    The mitochondria can be completely removed from the sample by an overnight incubation in Relaxing-Glycerol buffer at -20 °C without agitation (Figure 2). Removal of mitochondria makes imaging and staining easier. If mitochondria must be preserved, skip this section.


    Figure 2. Effect of glycerol extraction on mitochondria. A and B. Representative IFM expressing a mitochondrial:GFP protein and a mCherry-tagged Zasp52. A. In non-glycerinated IFM mitochondria can be detected; B. Mitochondria cannot be detected in glycerinated IFM. Scale bars = 10 µm.

  4. Paraformaldehyde fixation
    1. Add 150 µl of 8% paraformaldehyde (see Recipes) to the tube that contains the thoraces in relaxing buffer. The final paraformaldehyde concentration should be around 4%. A 4% formaldehyde fixation solution can also be used.
    2. Incubate at room temperature for 45 min with slow agitation (~50 rpm).
    3. Wash by replacing fixing solution with PBST (see Recipes). Incubate in PBST for 15 min under slow agitation (~50 rpm). Repeat this step three times. Thoraces quickly sink to the bottom of a microcentrifuge tube.

  5. Dissection of muscle fibers
    The main goal of this step is to separate the DLM fibers from the carcass without damaging them (Figure 3 and Video 2). Video 2 shows a detailed real-time dissection, see it before attempting to dissect the muscle fibers to get an idea of the overall process.
    1. Transfer the thoraces to a glass Petri dish using a previously cut pipette tip.
    2. Add enough PBST to just cover the tissue (excess liquid will make dissection challenging).
    3. Connect the needles to the syringes to make two dissection needles.
    4. Use two dissection needles to dissect the muscle fibers from the carcass (for details see Figure 4 and Video 2).
    5. Separate the fibers as much as possible without tearing the fibers apart.
    6. Transfer the fibers using a previously cut pipette tip back to a 1.5 ml microcentrifuge tube with PBST.


      Figure 3. General description of the IFM. A. Schematic view of a longitudinal section of a thorax, showing the position of the IFM. Two types of muscles compose the IFM, the dorsal longitudinal muscles (DLM, orange) and the dorsal ventral muscles (DVM, green). The goal of this protocol is to dissect the DLM. B. Representative isolated DLM, orange asterisks indicate individual DLM fibers (see also Video 2). Scale bar = 200 µm.


      Figure 4. Step-by-step guide to dissect DLM fibers. A-F. Snapshots from Video 2. A. Locate and hold the half-thorax using the left needle. Place the needle just below the DLM and above the legs. B. Make a small incision at the DLM posterior attachment site. C. Make a long cut above the DLM to completely separate the muscle fibers from the dorsal cuticle. D. Move the right needle to the ventral DLM (near the left needle). E. With the right needle push the DLM fibers out of the thorax. F. Isolated DLM can be further separated into individual fibers. G. Cartoon illustrating the dissection process. Dotted red lines mark the dissection needles. Note the small amount of liquid (~100 µl) in the dissection plate to prevent floating of half-thoraces. Scale bars = 200 µm.

      Video 2. Dissection of the DLM fibers. Stereomicroscope-recorded video showing the steps required for the correct dissection of the DLM fibers. Snapshots of this video are also presented in Figure 4.

  6. Phalloidin staining and mounting
    1. Replace the PBST solution with detergent-free PBS. Be careful not to aspirate the fibers.
    2. Add FITC- or TRITC-phalloidin (1:1,250) in triton-free PBS.
    3. Incubate at room temperature for 1-2 h with slow agitation (~50 rpm).
    4. Wash three times with PBST under slow agitation (~50 rpm).
    5. Transfer the muscle fibers to a glass slide.
    6. Dry the samples using a Kimwipe.
    7. Add a drop of mounting media, compatible with fluorescent labelling. Cover with coverslip compatible with confocal microscopy.
    8. Store at 4 °C.

  7. Confocal microscopy
    1. Locate the muscle fibers under brightfield at 20x magnification.
    2. Change the objective to 63x/1.4 NA and adjust the focus using brightfield to protect the sample.
    3. Scan the muscle fiber using the confocal software, align the fiber and take a full resolution scanning image at 9x magnification 1,024 x 1,024 pixels.
    4. Take several 9x images at different locations within the muscle fiber to get an accurate representation of the phenotype, especially when dealing with tissue from a mutant.

Data analysis

  1. Image analysis
    1. Images obtained from this protocol can be used to identify sarcomere phenotypes. Direct comparison of images from mutant muscles and control muscles should be enough to detect even subtle sarcomeric phenotypes. As healthy IFM are very regular structures (see Figure 5 for control examples), the mutant phenotypes can be scored by directly counting the number of normal sarcomeres in control and mutant muscles.
    2. When counting phenotypes exclude all sarcomeres that appear out of focus, do not have well-defined actin staining, or have more than one Z-disc per sarcomere. These are indications of artifacts often caused by overlapping myofibrils. Actin staining should always be used as counterstain when counting phenotypes.


      Figure 5. Representative examples of IFM confocal images. A. Zasp52-mCherry stained with phalloidin to visualize actin thin filaments. B. GFP-Actinin (GFP-Actn) and Zasp52-mCherry show a clear Z-disc colocalization. C. Flightin-GFP (Fln-GFP) marks the part of the A-band where the myosin heads are present. Fln does not colocalize with Zasp52-mCherry. D. Obscurin-GFP localizes to the M-line. Scale bars = 10 µm.

  2. Semi-automatic Z-disc or M-line measurements
    The fluorescent signal from Z-disc, M-line, or A-band proteins can be used to automatically detect individual particles (Figure 6). This approach is useful when very precise measurements of a large number of sarcomeres are required (e.g., estimating Z-disc size). Video 3 shows a screen-recorded version of this protocol.
    1. Open the image with the Fiji package of ImageJ (Schindelin et al., 2012).
    2. Open the threshold menu, under the Image/Adjust menu. Select ‘Dark background’ and ‘Auto’.
    3. Under ‘Analyze’ menu, select ‘Set measurements’. Check the Area, Standard deviation, Bounding rectangle, Shape descriptors, Mean gray value, and Display label boxes.
    4. Under ‘Analyze’ menu, select ‘Analyze particles’
    5. Adjust the size value (0.3-1) and/or circularity to exclude undesirable objects (e.g., protein aggregates in mutant muscles).
    6. Select ‘outlines’ in the drop-down menu.
    7. The measurement and outline of the image should appear. If the outline contains undesirable objects, adjust the parameters in the ‘Analyze particles’ menu.
    8. Save the results.
    9. Analyze the results with any statistical software (e.g., R statistical programming language).


      Figure 6. Automatic measurement of individual Z-discs. A. Confocal image of Zasp52-mCherry; B. Thresholded image showing in black the pixels to be included. The thresholding step uses signal intensity as an inclusion criterion. C. Result from running the ‘analyze particles’ step. This step uses size and shape as inclusion criteria. For additional details see Video 3. Scale bars = 10 µm.

      Video 3. Automatic detection and measurement of individual Z-discs. Screen-recorded video showing the procedure in Fiji to obtain individual Z-discs and measure some properties of the isolated discs. First, a thresholded image is made using the automatic threshold tool. Then, individual Z-disc are obtained using the ‘Analyze particles’ tool. The particle size and the circularity options may be adjusted to optimize the procedure.

Notes

  1. Antibody stainings
    Antibody stainings work very well with IFM. However, they do not seem to work perfectly when this protocol is used. If antibodies should be used, the fixation and the dissection steps must be inverted. That is, after the overnight incubation instead of fixing the tissue first dissect the IFM fibers as shown in Video 2. Then wash the samples 3 times with Relaxing solution and then proceed to the fixation step. While this greatly enhances antibody stainings, unfixed DLM muscles are harder to dissect than pre-fixed fibers.

  2. Useful fluorescent markers
    This protocol works best when fluorescent markers are available. Many different stocks expressing GFP-tagged proteins are available from common stock centers. Table 1 shows a list of useful GFP stocks for IFM imaging. To complement the existing GFP stocks we made Zasp52MI02908-mCherry by integrating an mCherry cassette in frame with the Zasp52 sequence. This new Zasp52MI02908-mCherry allele recapitulates Zasp52 expression and localization (Figures 2, 5, and unpublished observations).

    Table 1. Useful fluorescent markers

    Note: * Not fully described, available upon request.

  3. Confocal images
    1. All images must be acquired at the same resolution, magnification, and orientation. This allows direct comparison of images and saves time when arranging figures. We recommend a 20 x 20 µm square image, scanned at 1,024 x 1,024 pixel resolution and averaged 3 times.
    2. Scan the muscle fiber as close to the surface as possible. Phalloidin has limited infiltration into the IFM fibers. Thus, actin staining will be sharp at the surface but fuzzy in the interior. More importantly, IFM phenotypes may slightly vary between the surface and the interior of the muscle fibers.

Recipes

  1. Relaxing solution
    1. Dissolve in 40 ml of ddH2O:
      1,000 µl (final concentration is 20 mM) of sodium phosphate buffer stock solution 1 M pH 7.0
      500 µl (final concentration is 5 mM) of 0.5 M MgCl2
      2.5 ml (final concentration is 5 mM) of 0.1 M EGTA
      2.5 ml (final concentration is 5 mM) of 0.1 M ATP stock solution
      250 µl (final concentration is 5 mM) of 1 M DTT
    2. Add 1 tablet of protease inhibitors (cOmpleteTM, EDTA-free Protease Inhibitor Cocktail, Roche)
    3. Adjust volume to 50 ml with ddH2O
    4. Store at -20 °C
  2. Relaxing-Glycerol solution
    Note: Prepare Relaxing-Glycerol solution based on York Modified Glycerol solution (Peckham, 1990).
    1. Mix the relaxing solution with glycerol 1:1 (v/v)
    2. Add 0.5% Triton X-100
    3. Store in 10 ml aliquots at -20 °C
  3. 8% paraformaldehyde
    1. Open the 8% paraformaldehyde aqueous solution glass vial (Electron Microscopy Sciences)
    2. Divide the vial content into 400 µl aliquots and store them at -20 °C. Aliquots can then be stored for more than 6 months
  4. 10x phosphate buffered saline (PBS)
    1. Dissolve in 800 ml ddH2O
      80 g of NaCl
      2.0 g of KCl
      14.4 g of Na2HPO4
      2.4 g of K2HPO4
    2. Adjust pH to 7.4.
    3. Adjust volume to 1 L with ddH2O
    4. Dilute to 1x PBS with ddH2O
  5. 1x PBS, 0.1% Triton X-100 (PBST)
    Add 500 µl of Triton X-100 to 500 ml of 1x PBS

Acknowledgments

This protocol is adapted from the work of others. The original IFM dissection technique was described and distributed by Belinda Bullard and John Sparrow. We thank Anja Katzemich for technical advice, and the CIAN imaging facility for help with confocal microscopy. This work was supported by operating grant MOP-142475 from the Canadian Institutes of Health Research. We have no conflict of interest to declare.

References

  1. Gonzalez-Morales, N., Holenka, T. K. and Schöck, F. (2017). Filamin actin-binding and titin-binding fulfill distinct functions in Z-disc cohesion. PLoS Genet 13(7): e1006880.
  2. Hales, K. G., Korey, C. A., Larracuente, A. M. and Roberts, D. M. (2015). Genetics on the Fly: A primer on the Drosophila model system. Genetics 201(3): 815-842.
  3. Katzemich, A., Kreisköther, N., Alexandrovich, A., Elliott, C., Schöck, F., Leonard, K., Sparrow, J. and Bullard, B. (2012). The function of the M-line protein obscurin in controlling the symmetry of the sarcomere in the flight muscle of Drosophila. J Cell Sci 125(Pt 14): 3367-3379.
  4. Katzemich, A., Liao, K. A., Czerniecki, S. and Schöck, F. (2013). Alp/Enigma family proteins cooperate in Z-disc formation and myofibril assembly. PLoS Genet 9(3): e1003342.
  5. Liao, K. A., Gonzalez-Morales, N. and Schöck, F. (2016). Zasp52, a core Z-disc protein in Drosophila indirect flight muscles, interacts with α-Actinin via an extended PDZ domain. PLoS Genet 12(10): e1006400.
  6. Olive, M., Kley, R. A. and Goldfarb, L. G. (2013). Myofibrillar myopathies: new developments. Curr Opin Neurol 26(5): 527-535.
  7. Orfanos, Z., Leonard, K., Elliott, C., Katzemich, A., Bullard, B. and Sparrow, J. (2015). Sallimus and the dynamics of sarcomere assembly in Drosophila flight muscles. J Mol Biol 427(12): 2151-2158.
  8. Peckham, M., Molloy, J. E., Sparrow, J. C. and White, D. C. (1990). Physiological properties of the dorsal longitudinal flight muscle and the tergal depressor of the trochanter muscle of Drosophila melanogaster. J Muscle Res Cell Motil 11(3): 203-15.
  9. Sarov, M., Barz, C., Jambor, H., Hein, M. Y., Schmied, C., Suchold, D., Stender, B., Janosch, S., K, J. V., Krishnan, R. T., Krishnamoorthy, A., Ferreira, I. R., Ejsmont, R. K., Finkl, K., Hasse, S., Kampfer, P., Plewka, N., Vinis, E., Schloissnig, S., Knust, E., Hartenstein, V., Mann, M., Ramaswami, M., VijayRaghavan, K., Tomancak, P. and Schnorrer, F. (2016). A genome-wide resource for the analysis of protein localisation in Drosophila. Elife 5: e12068.
  10. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J. Y., White, D. J., Hartenstein, V., Eliceiri, K., Tomancak, P. and Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7): 676-682.
  11. Schröder, R. and Schoser, B. (2009). Myofibrillar myopathies: a clinical and myopathological guide. Brain Pathol 19(3): 483-492.
  12. Stronach, B. (2014). Extensive nonmuscle expression and epithelial apicobasal localization of the Drosophila ALP/Enigma family protein, Zasp52. Gene Expr Patterns 15(2): 67-79.
  13. Vakaloglou, K. M., Chountala, M. and Zervas, C. G. (2012). Functional analysis of parvin and different modes of IPP-complex assembly at integrin sites during Drosophila development. J Cell Sci 125(Pt 13): 3221-3232.
  14. Vigoreaux, J. O. (2001). Genetics of the Drosophila flight muscle myofibril: a window into the biology of complex systems. Bioessays 23(11): 1047-1063.
  15. Wangler, M. F., Yamamoto, S. and Bellen, H. J. (2015). Fruit flies in biomedical research. Genetics 199(3): 639-653.

简介

肌肉最小的收缩单位肌肉,可以说是最令人印象深刻的肌动球蛋白结构。 然而,缺少对肌节形成和维护的完整理解。 果蝇间接飞行肌肉(IFM)已被证明是研究肌节的一个非常有价值的模型。 在这里,我们提出了快速解剖IFM和使用荧光标记的蛋白质分析肌节的协议。

【背景】能够使横纹肌纤维收缩的细胞骨架结构是数以百计的称为肌原纤维的电缆。肌原纤维又是一系列连续排列的肌节,全部同时收缩。肌节是完美对称的结构,包含收缩所需的所有元素。在肌节的中心位置是M-线,其中肌球蛋白粗丝被锚定。在肌小节的侧面是Z-盘,其中肌动蛋白细丝被锚定。

肌营养不良是导致进行性骨骼肌无力的遗传性疾病(Schröder和Schoser,2009)。对于肌营养不良症,目前尚无法治愈,可能是由于对肌营养不良症基础的分子机制的不完全理解(Olive et al。,2013)。黑腹果蝇是一种有效的遗传模式生物,由于其生命周期短,经济的维护和丰富的可利用资源而被用于研究肌肉生物学(Hales等人,2015; Wangler等,等人,2015年)。

在果蝇中的飞行由间接飞行肌肉(IFM)(苍蝇中最大的肌肉)的同步动作驱动,后者被进一步细分为背纵肌(DLM)和背腹肌(DVM)。 IFM与人类骨骼肌具有许多基本相似之处:收缩机制,发育步骤,总体超微结构和蛋白质成分(Vigoreaux,2001)。例如,肌病相关蛋白ZASP和Filamin-C具有飞行同源物,当突变发生肌肉表型时(Liao等人,2016; Gonzalez-Morales等人 ,2017)。尽管使用IFM进行肌肉研究的优势,但是IFM解剖可能是具有挑战性和耗时的。在这里我们提出了一个议定书,结合快速和容易的IFM解剖与IFM使用荧光蛋白质的高品质成像。我们还提供了一种分析突变表型和通过半自动检测肌节成分来量化肌节的策略。

关键字:解剖, 果蝇, GFP, 间接飞行肌肉, 肌节, Z盘

材料和试剂

  1. 外科刀片(FEATHER安全剃刀,目录编号:23号)
  2. 1.5 ml微量离心管
  3. 移液器提示
  4. 传统针PrecisionGlide 23 G 1英寸(Fisher Scientific,目录号:14-826-6B)
    制造商:BD,目录号:305193。
  5. BD一次性注射器(BD,目录号:309628)
  6. 1号盖玻片22×30毫米(例如,康宁,目录号:2850-22)
  7. 显微镜载玻片(如,Fisher Scientific,目录号:12-552-3)
  8. 苍蝇表达肌节荧光标记(例如,Zasp52-GFP,表1)
  9. 定制的3.5%琼脂填充的60×15mm培养皿平板(BioShop,目录号:AGR003)
  10. 鬼笔环肽 - 四甲基若丹明B异硫氰酸酯(TRITC)(Sigma-Aldrich,目录号:P1951)
  11. 鬼笔环肽 - 荧光素异硫氰酸酯(FITC)(Sigma-Aldrich,目录号:P5282)
  12. 安装介质ProLong Gold Antifade Mountant(Thermo Fisher Scientific,Invitrogen TM,产品目录号:P36934)
  13. 氯化镁(MgCl 2)
  14. 乙二醇 - 双 - 四乙酸(EGTA)(Sigma-Aldrich,目录号:E3889)
  15. 三磷酸腺苷(ATP)(BioShop,目录号:ATP007)
  16. 1,4-二硫苏糖醇(DTT)(BioShop,目录号:DTT001)
  17. 无EDTA蛋白酶抑制剂混合物(Roche Diagnostics,目录号:11873580001)
  18. 甘油(BioShop,目录号:GLY001)
  19. Triton X-100(BioShop,目录号:TRX777)
  20. 8%多聚甲醛水溶液玻璃瓶(电子显微镜科学,目录号:157-8)
  21. 氯化钠(NaCl)
  22. 氯化钾(KCl)
  23. 磷酸氢二钠(Na 2 HPO 4)
  24. 磷酸二氢钾(K 2 HPO 4)
  25. 放松的解决方案(见食谱)
  26. 放松甘油溶液(见食谱)
  27. 8%多聚甲醛(见食谱)
  28. 10倍磷酸盐缓冲盐水(PBS)(见食谱)
  29. 1x PBS,0.1%Triton X-100(PBST)(见食谱)

设备

  1. CO 2 2 Flypad,标准尺寸(8.1×11.6厘米)(Genesee Scientific,Flystuff,目录号:59-114)
  2. 刀片刀柄(FEATHER安全剃刀,目录号:4)
  3. 玻璃培养皿; 60×15毫米(VWR,目录号:89000-770)
    制造商:DWK生命科学公司,KIMBLE ®,产品目录号:2306-10010。
  4. 立体显微镜(徕卡显微系统,型号:徕卡MS5)
  5. 杜蒙#5镊子(精细科学工具,目录号:11251-30)
  6. 孵化器设置为25°C
  7. P2,P20,P100和P1000微量移液器(例如,Gilson,目录号:F144801,F123600,F123615和F123602)
  8. 平台混合器(例如,Speci-Mix试管摇杆)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:M71015Q)
  9. 中型尖头刷子
  10. 标准的飞行饲养设备

软件

  1. 斐济( https://fiji.sc/
  2. R统计软件包( https://www.r-project.org/

程序

  1. 准备苍蝇解剖
    1. 麻醉苍蝇使用标准的CO 2垫。使用5-10天的苍蝇。
    2. 使用细刷将苍蝇转移到定制的琼脂平板上。将所有苍蝇集中在盘子的中心。
    3. 添加100μL的松弛甘油溶液(见食谱)苍蝇使用以前削减枪头。

  2. 切半个胸部
    这一步的主要目标是将胸部分成两个对称的一半(图1和视频1)。这一步是比较容易的,但要注意不要粉碎肌肉。如果刀片变钝,请用新的刀片替换。
    1. 使用两个罚款镊子去除腹部。只留下胸部和头部。

    2. 放置并对齐胸部和背侧朝向你的眼睛。
    3. 使用手术刀刀片和快速切片运动将胸部切成两半对称。
      如果仍然附着在胸部,请将头部剪掉
    4. 在这一点上,IFM应该在解剖显微镜下可见。

    5. 镊子半胸部,并放置在1.5毫升微量离心管
    6. 加入100μl的放松甘油缓冲液。


      图1.胸部分为两半。 A-D。视频1中的快照显示了重要的解剖步骤。 A.放置胸部,背侧向上; B.轻轻触摸胸部,确保刀片正好位于中间位置,胸部不会移动。 C.将刀片的尖端对准头部/胸部固定部位(箭头)。 D.用尖锐的推开胸部; E.代表性的胸部分为两部分。在这个阶段可以观察到肌肉纤维(红色星号)。比例尺= 200微米。

      视频1

  3. 去除线粒体
    在不加搅拌的情况下,在-20℃的放松 - 甘油缓冲液中孵育过夜,可以将样品中的线粒体完全去除(图2)。去除线粒体使成像和染色更容易。如果线粒体必须保存,请跳过这一节。


    图2.甘油提取对线粒体的影响。 A和B.代表性的IFM表达线粒体:GFP蛋白和mCherry标记的Zasp52。 A.在非甘油化IFM中可以检测到线粒体; B.在甘油IFM中不能检测到线粒体。比例尺= 10微米。

  4. 多聚甲醛固定
    1. 加入150μL的8%多聚甲醛(见食谱),在缓冲缓冲液中含有胸腔的管中。最终的多聚甲醛浓度应该在4%左右。也可以使用4%的甲醛固定溶液。

    2. 在慢速搅拌(〜50 rpm)下室温孵育45分钟。
    3. 用PBST代替固定溶液清洗(见食谱)。在慢速搅拌(〜50rpm)下,在PBST中孵育15分钟。重复这个步骤三次。胸腔迅速沉入微量离心管的底部。

  5. 解剖肌肉纤维
    这个步骤的主要目标是将DLM纤维与屠体分离而不损坏它们(图3和视频2)。视频2显示了详细的实时解剖,在尝试解剖肌肉纤维以了解整个过程之前,先看看它。

    1. 使用先前切割的移液器尖将转移到玻璃培养皿
    2. 添加足够的PBST来覆盖组织(多余的液体将使解剖具有挑战性)。
    3. 将针连接到注射器,以制造两个解剖针。
    4. 使用两个解剖针解剖尸体肌纤维(细节见图4和视频2)。
    5. 将纤维尽可能分开,不要撕裂纤维。

    6. 使用先前切割的移液管尖端将纤维转移至1.5 ml带有PBST的微量离心管中。


      图3. IFM的一般描述A.一个胸部纵断面的示意图,显示了IFM的位置。两种类型的肌肉构成IFM,背侧纵肌(DLM,橙色)和背侧腹肌(DVM,绿色)。这个协议的目标是剖析DLM。 B.具有代表性的孤立DLM,橙色星号表示单独的DLM光纤(另见视频2)。比例尺= 200微米。


      图4.分解DLM光纤的分步指南 A-F。视频2中的快照。A.使用左针找到并保持半胸部。将针放在DLM正下方和腿上方。 B.在DLM后部附着部位做一个小切口。 C.在DLM上方做一个长切口,将肌纤维从背侧角质层完全分开。 D.将右侧针移到腹侧DLM(靠近左侧针)。 E.用右针将DLM纤维推出胸腔。 F.分离的DLM可以进一步分离成单独的纤维。 G.说明解剖过程的动画片。虚线的红线标记解剖针。注意解剖板中的少量液体(〜100μl)以防止半胸的浮动。比例尺= 200微米。

      视频2

  6. 鬼笔环肽染色和安装
    1. 用无洗涤剂的PBS代替PBST溶液。小心不要吸入纤维。
    2. 在无Triton的PBS中加入FITC-或TRITC-鬼笔环肽(1:1,250)。

    3. 在慢速搅拌(〜50 rpm)下室温孵育1-2小时

    4. 在慢速搅拌(〜50 rpm)下,用PBST清洗三次
    5. 将肌纤维转移到载玻片上。
    6. 使用Kimwipe干燥样品。
    7. 添加一滴安装介质,与荧光标签兼容。盖上与共焦显微镜兼容的盖玻片。
    8. 在4°C储存。

  7. 共聚焦显微镜

    1. 在放大20倍的明视野下找到肌纤维。
    2. 将目标更改为63x / 1.4 NA,并使用明场调整焦点以保护样品。
    3. 使用共聚焦软件扫描肌纤维,对准光纤,并以9x放大倍数1,024 x 1,024像素拍摄全分辨率扫描图像。
    4. 在肌肉纤维内的不同位置拍摄几张9x图像,以获得表型的准确表示,特别是在处理来自突变体的组织时。

数据分析

  1. 图像分析
    1. 从这个协议获得的图像可以用来识别肌节表型。直接比较来自突变肌肉和对照肌肉的图像应该足以检测甚至微妙的肌节表型。由于健康的IFM是非常规则的结构(对照例见图5),突变表型可以通过直接计数对照和突变肌肉中正常肌节的数量来评分。
    2. 当计数表型排除出现在焦点外的所有肌节,不具有定义明确的肌动蛋白染色或每个肌节有多于一个Z-盘。这些通常是由重叠的肌原纤维引起的伪迹的迹象。计数表型时,应始终使用肌动蛋白染色作为复染色。


      图5. IFM共聚焦图像的代表性实例A. Zasp52-mCherry用鬼笔环肽染色以显现肌动蛋白细丝。 B.GFP-肌动蛋白(GFP-Actn)和Zasp52-mCherry显示清晰的Z-盘共定位。 C. Flightin-GFP(Fln-GFP)标志着肌球蛋白头部存在的A带的部分。 Fln不与Zasp52-mCherry共同定位。 D. Obscurin-GFP定位到M线。比例尺= 10微米。

  2. 半自动Z盘或M线测量
    来自Z-disc,M-line或A-band蛋白的荧光信号可用于自动检测单个颗粒(图6)。当需要对大量肌节进行非常精确的测量时(例如,估计Z-盘大小),这种方法是有用的。视频3显示了该协议的屏幕录制版本。
    1. 使用斐济的ImageJ软件包打开图像(Schindelin ,2012年)。
    2. 打开图像/调整菜单下的阈值菜单。选择“黑暗的背景”和“自动”。
    3. 在“分析”菜单下,选择“设置测量”。检查面积,标准偏差,边界矩形,形状描述符,平均灰度值和显示标签框。
    4. 在“分析”菜单下,选择“分析粒子”
    5. 调整尺寸值(0.3-1)和/或圆度以排除不希望的对象(例如,突变肌肉中的蛋白质聚集体)。
    6. 在下拉菜单中选择“轮廓”。
    7. 应显示图像的测量和轮廓。如果轮廓包含不想要的对象,请调整“分析粒子”菜单中的参数。
    8. 保存结果。
    9. 用任何统计软件(例如,R统计编程语言)分析结果。


      图6.自动测量单个Z盘。 :一种。 Zasp52-mCherry的共焦图像; B.以黑色显示要包含的像素的阈值图像。阈值步骤使用信号强度作为包含标准。 C.运行“分析粒子”步骤的结果。这一步使用大小和形状作为纳入标准。有关更多详细信息,请参阅视频3.比例尺= 10μm。

      视频3

笔记

  1. 抗体染色
    抗体染色与IFM非常吻合。但是,使用这个协议时,它们似乎并不完美。如果应该使用抗体,那么固定和解剖步骤必须颠倒。也就是说,过夜培养后,不要固定组织,首先解剖IFM纤维,如视频2所示。然后用放松的溶液清洗样品3次,然后进行固定步骤。虽然这极大地增强了抗体染色,但未固定的DLM肌肉比预先固定的纤维更难于解剖。

  2. 有用的荧光标记
    当荧光标记可用时,该协议效果最好。从普通股中心可以获得许多不同的表达GFP标签蛋白的原料。表1显示了用于IFM成像的有用GFP储备的列表。为了补充现有的GFP库存,我们通过将mCherry盒与Zasp52序列整合在一起,制成了ZmiP 52MI02908-mCherry 这个新的 Zamsp 52MI02908-mCherry 等位基因概括了Zasp52的表达和定位(图2,5和未发表的观察结果)。

    表1.有用的荧光标记

    注:*没有完整描述,可根据要求提供。

  3. 共焦图像
    1. 所有图像都必须以相同的分辨率,放大倍率和方向进行采集。这可以直接比较图像,并节省时间安排数字。我们建议使用20 x 20μm的正方形图像,以1,024 x 1,024像素分辨率扫描并平均3次。
    2. 将肌纤维尽可能靠近表面扫描。鬼笔环肽对IFM纤维的渗透有限。因此,肌动蛋白染色将在表面锐利但在内部模糊。更重要的是,IFM表型在肌纤维的表面和内部之间可能略有不同。

食谱

  1. 放松的解决方案

    1. 溶于40ml ddH 2 O中 1000μl(终浓度为20mM)磷酸钠缓冲液储备液1M pH 7.0
      500μl(终浓度为5mM)0.5M MgCl 2 2 2.5 ml(最终浓度为5 mM)0.1 M EGTA
      2.5毫升(终浓度为5毫米)的0.1 M ATP储备液 250μl(终浓度为5 mM)1 M DTT
    2. 添加1片蛋白酶抑制剂(cOmplete TM,不含EDTA的蛋白酶抑制剂混合物,Roche)。
    3. 用ddH 2 O调节体积至50ml
    4. 在-20°C储存
  2. 放松甘油溶液
    注意:准备基于约克改性甘油溶液(Peckham,1990)的放松 - 甘油溶液。
    1. 混合松弛的解决方案与甘油1:1(V / V)
    2. 加0.5%Triton X-100

    3. 储存在10毫升等分试样中
  3. 8%多聚甲醛
    1. 打开8%多聚甲醛水溶液玻璃瓶(电子显微镜科学)
    2. 将小瓶内容物分成400μl等分试样并储存在-20℃。等分试样可以储存6个月以上
  4. 10x磷酸盐缓冲盐水(PBS)
    1. 溶于800ml ddH 2 O
      80克的NaCl
      2.0克KCl
      14.4克Na 2 HPO 4 4 2.4克的K 2 HPO 4 4
    2. 调整pH值到7.4。
    3. 用ddH <2> O调整音量到1L
    4. 用ddH 2 O稀释至1x PBS
  5. 1x PBS,0.1%Triton X-100(PBST)
    加500μl的Triton X-100到500ml的1xPBS

致谢

这个协议是改编自其他人的工作。最初的IFM解剖技术由Belinda Bullard和John Sparrow描述和分发。我们感谢Anja Katzemich提供的技术建议,以及CIAN成像设备,帮助共聚焦显微镜。这项工作得到了加拿大卫生研究院的运营资金MOP-142475的支持。我们没有利益冲突要申报。

参考

  1. Gonzalez-Morales,N.,Holenka,T.K。和Schöck,F。(2017)。 Filamin肌动蛋白结合和titin结合在Z-disc凝聚力中完成不同的功能。 PLoS Genet 13(7):e1006880。
  2. Hales,K. G.,Korey,C. A.,Larracuente,A. M.和Roberts,D. M.(2015)。 动态遗传学:果蝇模型系统的入门< / a> Genetics 201(3):815-842。
  3. Katzemich,A.,Kreisköther,N.,Alexandrovich,A.,Elliott,C.,Schöck,F.,Leonard,K.,Sparrow,J.和Bullard,B。(2012)。 M线蛋白obscurin在控制飞行肌肉中肌小节的对称性中的功能果蝇。 J Cell Sci 125(Pt 14):3367-3379。
  4. Katzemich,A.,Liao,K.A。,Czerniecki,S。和Schöck,F。(2013)。 Alp / Enigma家族蛋白在Z形盘形成和肌原纤维组装中合作。 > PLoS Genet 9(3):e1003342。
  5. Liao,K. A.,Gonzalez-Morales,N.和Schöck,F。(2016)。 Zasp52是果蝇间接飞行肌肉中的Z型核心蛋白,通过扩展的PDZ结构域与α-肌动蛋白相互作用。 PLoS Genet 12(10):e1006400。
  6. Olive,M.,Kley,R.A。和Goldfarb,L.G。(2013)。 肌原纤维肌病:新进展 Curr Opin Neurol 26 (5):527-535。
  7. Orfanos,Z.,Leonard,K.,Elliott,C.,Katzemich,A.,Bullard,B。和Sparrow,J。(2015)。 Sallimus和果蝇组织中肌节组装的动力学 J Mol Biol 427(12):2151-2158。
  8. Peckham,M。,Molloy,J.E。,Sparrow,J.C。和White,D.C。(1990)。 果蝇背部纵行飞行肌肉和转子肌肉抑制物的生理特性melanogaster 。 J肌肉Res Cell Motil 11(3):203-15。
  9. Sarov,M.,Barz,C.,Jambor,H.,Hein,MY,Schmied,C.,Suchold,D.,Stender,B.,Janosch,S.,K,JV,Krishnan,RT,Krishnamoorthy,A Ferreira,IR,Ejsmont,RK,Finkl,K.,Hasse,S.,Kampfer,P.,Plewka,N.,Vinis,E.,Schloissnig,S.,Knust,E.,Hartenstein,V., Mann,M.,Ramaswami,M.,VijayRaghavan,K.,Tomancak,P。和Schnorrer,F。(2016)。 在果蝇中分析蛋白质定位的全基因组资源 。 Elife 5:e12068。
  10. Schindelin,J.,Arganda-Carreras,I.,Frize,E.,Kaynig,V.,Longair,M.,Pietzsch,T.,Preibisch,S.,Rueden,C.,Saalfeld,S.,Schmid,B Tinevez,JY,White,DJ,Hartenstein,V.,Eliceiri,K.,Tomancak,P.和Cardona,A。(2012)。 斐济:生物图像分析的开源平台。 Nat方法 9(7):676-682。
  11. Schröder,R.和Schoser,B。(2009)。 肌原纤维肌病:临床和肌病指南 脑病 19(3):483-492。
  12. Stronach,B。(2014)。 果蝇的广泛非肌肉表达和上皮apicobasal本地化 ALP / Enigma家族蛋白质,Zasp52。 Gene Expr Patterns 15(2):67-79。
  13. Vakaloglou,K. M.,Chountala,M.和Zervas,C. G.(2012)。 在果蝇期间整合素位点的parvin功能分析和IPP复合体组装的不同模式 / em>发展。 J Cell Sci 125(Pt 13):3221-3232。
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  15. Wangler,M.F。,Yamamoto,S。和Bellen,H.J。(2015)。 果蝇生物医学研究 遗传学 199(3) ):639-653。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Xiao, Y., Schöck, F. and González-Morales, N. (2017). Rapid IFM Dissection for Visualizing Fluorescently Tagged Sarcomeric Proteins. Bio-protocol 7(22): e2606. DOI: 10.21769/BioProtoc.2606.
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