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Ex vivo Ooplasmic Extract from Developing Drosophila Oocytes for Quantitative TIRF Microscopy Analysis
从在发育的果蝇卵母细胞中离体提取卵磷脂用于定量TIRF显微镜分析   

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Abstract

Understanding the dynamic behavior and the continuously changing composition of macromolecular complexes, subcellular structures and organelles is one of areas of active research in both cell and developmental biology, as these changes directly relate to function and subsequently to the development and homeostasis of the organism. Here, we demonstrate the use of the developing Drosophila oocyte to study dynamics of messenger ribonucleoprotein complexes (mRNPs) with high spatiotemporal resolution. The combination of Drosophila genetics with total internal reflection (TIRF) microscopy, image processing and data analysis gives insight into mRNP motility and composition dynamics with unprecedented precision.

Keywords: Ooplasmic extract(卵磷脂提取), Intracellular motility(细胞内运动), TIRF microscopy(TIRF显微镜检查), Oocyte(卵母细胞), ex vivo assay(离体分析)

Background

Intracellular transport is one of the fundamental processes in living cells. Almost everything within the cell–ions, molecules, complexes, organelles–is transported actively such that the local entropy is reduced. Although in recent years we have gained considerable understanding of the mechanisms underlying these transport process, most of our knowledge comes from in vitro and cell culture studies. In these simplified systems, it is difficult to establish whether the full potential of the transport regulatory processes is utilized. Tissues, organs, organoids and organisms, on the other hand, are often too complex to be studied efficiently with spatiotemporal resolution sufficient to match the scale of these transport processes. To combine the advantages of the bottom-up and top-down approaches, techniques have been developed that, while preserving complexity, make these processes more accessible. One example is the preparation of mass cytoplasmic extract from ambiphian (e.g., Xenopus laevis) oocytes and embryos to study cell divisions (Lohka and Masui, 1983; Murray, 1991; Sawin and Mitchison, 1991). We have recently shown that in cytoplasmic drops–i.e., non-purified cytoplasm directly extracted from the cell–released from single Drosophila embryos mitotic activity of the contained nuclei continues, allowing the probing of spindle properties by simple physical and chemical perturbations (Telley et al., 2012 and 2013). Here, we describe a similar ex vivo preparation technique based on ooplasm of developing Drosophila egg-chambers. This method allows the study of intracellular transport processes (squash assay), such as the transport of localizing oskar mRNPs (Gaspar et al., 2017).

Materials and Reagents

  1. Coverslip stand for 5-10 coverslips (e.g., Wash-N-Dry, Diversified Biotech, catalog number: WSDR-1000 )
  2. Gloves
  3. A small plastic Petri dish or the cap of a 50 ml Falcon tube (~30 mm diameter)
  4. 15 x 20 cm black plastic plate
  5. High precision coverslips (e.g., Marienfeld Precision Cover Glass, 22 x 22 mm, Marienfeld-Superior, catalog number: 0107052 )
  6. X-ray film (e.g., Amersham HyperfilmTM ECL, GE Healthcare, catalog number: 28906835 )
  7. Double-sided adhesive tape (e.g., Tesa Doubleband Photostrip, Tesa, catalog number: 05338-00 )
  8. Cheesecloth
  9. A fly line or a cross that yields females of the appropriate genotype (see Note 1)
  10. Dry, granular baker’s yeast (e.g., Lesaffre Saf-Instant®)
  11. Halocarbon oil (e.g., Voltalef 10S, VWR, catalog number: 24627.188 )
  12. 100% ethanol (e.g., EMD Millipore, catalog number: 100983 )
  13. ~5% dichlorodimethylsilane (DCDMS) in heptane (e.g., Silanization Solution, Sigma-Aldrich, catalog number: 85126 )
  14. PIPES (e.g., Sigma-Aldrich, catalog number: P3768 )
  15. Magnesium chloride hexahydrate (MgCl2·6H2O) (e.g., EMD Millipore, catalog number: 105833 )
  16. EGTA (e.g., Sigma-Aldrich, catalog number: E3889 )
  17. HEPES (e.g., Sigma-Aldrich, catalog number: H3375 )
  18. Potassium chloride (KCl) (e.g., EMD Millipore, catalog number: 104936 )
  19. Dextran sulfate, MW ~10 kDa (e.g., Sigma-Aldrich, catalog number: D4911 )
  20. Agar (e.g., from Pro-BIO)
  21. Dry yeast (e.g., from Volk Klaus)
  22. Soya powder (e.g., from Ruckemann)
  23. Sirup (e.g., from Ruckemann)
  24. Malt extract (e.g., from Baeko Rhei)
  25. Corn powder (e.g., from Ruckemann)
  26. Propionic acid (e.g., VWR, catalog number: ACRO447231000)
    Manufacturer: Acros Organics, catalog number: 447231000 .
  27. Nipagin (e.g., Sigma-Aldrich, catalog number: H5501 )
  28. BRB80 (see Recipes)
  29. 1% injection buffer (1% IB) (see Recipes)
  30. Cornmeal agar (see Recipes)

Equipment

  1. Standard fly husbandry equipment (e.g., vials with cornmeal agar–see Recipes, 25 °C incubator with humidity controller, brushes, CO2 station, etc.)
  2. 3-9 well dissection plate (e.g., Corning, catalog number: 7220-85 )
  3. Vacuum desiccator (e.g., SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: F42025-0000 )
  4. Dumont #5 forceps (e.g., Fine Science Tools, catalog number: 11252-20 )
  5. Dumont #55 forceps (e.g., Fine Science Tools, catalog number: 11255-20 )
  6. Custom made coverslip holder for microscopy (Figure 1)


    Figure 1. Vacuum desiccator loaded with the coverslip holder and the silane reservoir. After replacing the lid of the device, apply vacuum by opening the vacuum valve and the water tap. Once the vacuum is formed–i.e., you can lift the entire device by the lid–close the vacuum valve and the water tap.

  7. Zooming stereomicroscope with 5-40x (or higher) magnification (e.g., Carl Zeiss, model: STEMI SV 11 )
  8. Sharp tungsten needles with handles (e.g., Fine Science Tools, catalog numbers: 10130-20 and 26018-17 )
  9. Total Internal Reflection Fluorescent (TIRF) microscope equipped with a high NA objective and a high sensitivity detector (Leica Microsystems, model: Leica AF7000 )
    Note: We use a Leica 7000 wide-field TIRF microscope with a 100x 1.46 NA oil immersion objective and a Photometrics Evolve Roper 512 EMCCD camera.

Software

  1. Microscope controller software (e.g., Leica LASAF)
  2. ImageJ/FIJI
  3. Custom-made particle detector and tracker plug-in for ImageJ (available to download under https://github.com/Xaft/xs/blob/master/_xs.jar)
  4. R (preferentially with RStudio), for data analysis

Procedure

  1. Fly husbandry
    1. Establish the cross(es) that yield female flies expressing the appropriate fluorescent marker(s) in the desired mutant genetic background (see Note 1). Depending on the complexity of the final genotype this might require multiple successive crosses.
    2. The day before the experiment, collect a few (maximum six) young (maximum one week old) female flies and place them together with 2-3 males in a vial with fresh cornmeal agar and 20-30 grains of dry baker’s yeast (see Note 2).

  2. Silanization of coverslips (see Note 3)
    1. On the day of the experiment, take 5-10 factory clean coverslips and load them into the coverslip holder (see Note 4).
      Note: Wear gloves.
    2. Place 200 µl of silanization solution (5% DCDMS) into the Petri dish/Falcon tube cap (silane reservoir).
    3. Place the coverslips next to the silane reservoir in the vacuum desiccator and apply vacuum (Figure 1).
    4. Incubate the coverslips for 90-120 min under vacuum at room temperature (RT). The vaporized DCDMS will coat the surface of the coverslips. No changes in color or in transparency of the glass should be observed.
    5. The silanized coverslips should be used on the same day and should not be stored.

  3. Prepare the TIRF microscope
    1. Before dissection, start the microscope, load the software and set up the imaging parameters.
    2. If controlled temperature is essential, start the incubation chamber around the microscope at least half an hour before starting the imaging.
    3. Make sure that the system is calibrated. The refraction index of the ooplasm is close to that of the halocarbon oil (η ~1.41), thus a coverslip with a drop of halocarbon oil could serve as reference.

  4. Dissection of the ovaries (see Video 1)

    Video 1. Dissection of the ovaries

    1. Load one well (~2 ml) of the dissection plate with 100% ethanol and two other wells with BRB80 (see Recipes). Use the black plastic plate as a workplace for the fly and ovary manipulations.
    2. Anesthetize a female fly and place it into the well with ethanol (see Note 5).
    3. After 4-5 sec incubation, transfer the fly into a well with BRB80 using a #55 forceps, and after another 4-5 sec transfer to the last well containing BRB80.
    4. Under a moderate magnification (10-15x) of the dissection microscope, with the #5 forceps in your non-dominant hand, crush the thorax of the fly to destroy it killing the animal and to maintain a firm grip. With the # 55 forceps, gently pierce the ventral-posterior side of the abdomen close to the midline.
    5. Open the abdomen by removing the posterior part by pulling. Take out the pair of ovaries and transfer it to a clean, non-silanized coverslip in a drop of BRB80.
    6. Place two drops of 1% IB (see Recipes) (~4-5 µl) on the same coverslip. Transfer the ovaries sequentially into each drop to remove BRB80 from around them.
    7. Transfer the ovaries into a ~2 µl drop of 1% IB in the center of a silanized coverslip.
      Note: Silanized coverslips needs to be used from this step onward.

  5. Extraction of the ooplasm (see Videos 2 and 3)
    1. Place a drop of halocarbon oil on the silanized coverslip such that it forms an interface with the drop containing the ovaries (see Video 2).

      Video 2. Separation and sorting of the ovarioles

    2. Wipe the tungsten needles with 100% ethanol and then rinse them in 1% IB.
    3. Increase the magnification to about 20-25x. Using the two needles, separate the ovarioles (strings of egg-chambers) from each other.
    4. Identify ovarioles that contain egg-chambers of the developmental stage of interest (stage 9 or older, Figure 2A). Carefully remove the older stages with the needles
      Note: By crossing them, using them as scissors.


      Figure 2. Drosophila egg-chambers under the microscope. A. Different stages of Drosophila oogenesis. The oocyte and its 15 sibling cells (nurse cells) form a syncytium in the germarium through a series of incomplete cell divisions. These 16 cells stay interconnected by cytoplasmic bridges and become encapsulated by a layer of somatic follicle cells forming the egg-chambers. The egg-chambers mature/go through 14 different stages of oogenesis to give rise to a mature egg (for more details, please refer to e.g., [Bastock and St Johnston, 2008]). To visualize the different cell types, wash-free in situ hybridization to oskar mRNA was performed using a mixture of three different FIT probes (Hovelmann et al., 2013). Scale bar is 50 µm. B-D. Different steps of ooplasm extraction from a stage 9 oocyte (stills taken from Video 3). Scale bar is 500 µm (B-D).

      Video 3. Ex vivo ooplasmic preparation

    5. Using your dominant hand, grab the trimmed ovariole on the side of the germarium and gently pull it under the oil.
    6. Repeat steps E4 and E5. Ideally, one should have 5-10 egg-chambers of interest under the oil before proceeding further.
    7. Zoom to about 40x magnification. Under the oil, first remove any egg-chambers younger than the stage of interest (e.g., stage 9), then start pricking the nurse cells (trophic sister cells of the oocyte) at the anterior tip of the egg-chamber (Figure 2B, see Video 3).
    8. When none or only a few nurse cells remain, gently grab the oocyte-containing follicle sack (‘oocyte sack’) at the posterior pole with the needle in your dominant hand and start pulling it away from the rubble of nurse cells (Figure 2C).
    9. The ooplasm should start flowing out. Adjust the speed of pulling to allow the ooplasm to touch the coverslip and adhere. You may help this adhesion with the other needle by creating tension anterior to the ooplasm (see Note 3).
    10. Depending on the speed of pulling, the size of the ooplasm and the hydrophobicity of the coverslip, one to several round droplets of ooplasm (containing numerous large 1-10 µm large yolk vesicles visible under 40x zoom) should form (Figure 2D).
    11. Troubleshooting: A too hydrophilic glass will result in an almost instantaneous, uncontrollable release of the ooplasm onto the surface, whereas a too hydrophobic glass usually repels the ‘oocyte sack’, causing it to float away from the surface. In either case, it is advised to restart the protocol from step B1, reducing or increasing the silanization time, respectively.
    12. Repeat steps E7-E9 to deposit more ooplasmic extracts on the same cover glass. Ideally, four-six of such preparations (i.e., replicates) can be placed on one 22 x 22 mm coverslip.

  6. TIRF microscopy
    1. Load the coverslip onto the stand of the microscope using a coverslip holder (Figure 3).


      Figure 3. Custom made coverslip holder. The basis of this coverslip holder is a plastic block with dimensions of a standard microscopy slide (76 x 25 x 2-3 mm) containing a 16-18 mm wide, 16-18 mm deep incision in one of its long sides. A hand cut piece of a (used) X-ray film (~40 x 25 mm, with a similar incision) is secured to the bottom of the plastic block by using two strips of double-sided adhesive tape. The two strips of adhesive tape should be placed such that the used coverslip (e.g., 22 x 22 mm) should fit firmly–in this example, these two strips are placed 22 mm apart. The silanized coverslips carrying the halocarbon oil with the ooplasmic squashes can be slid between the plastic block and the piece of X-ray film like a drawer. Scale unit is cm.

    2. Using transmitted light, focus the specimen and locate one of the ooplasmic droplets. Droplets of ooplasm are recognized by the presence of yolk granules (Figures 4A and 4B), which are absent from the nurse cell cytoplasm (Figure 4C) (see Note 6). Sometimes, the membrane of the oocyte–reinforced by the forming vitelline layer–remains intact and therefore no ooplasm gets into contact with the glass surface. Such malformed oocytes can be recognized by the sharp, positively curved boundary with their surroundings (Figure 4B). Such ooplasms are inaccessible to TIRF microscopy (Figure 4D).


      Figure 4. Typical appearance of droplets after ex vivo preparation. A, B, D and E. Ooplasmic squash. A and B. Ooplasm from oocytes at mid-oogenetic stages onward can be recognized by the presence of large (1-10 µm) yolk granules. A and E. Sometimes, halocarbon oil droplets get mixed into the ooplasm, indicating that the plasma and the vitelline membranes surrounding the oocyte were removed. C. Droplet of nurse cell cytoplasm. Granularity is much finer than in the ooplasm A and B and very often the gigantic nurse cell nuclei remain intact. B and D. Ooplasm/oocyte with intact membranes (indicated by red arrows). D. In such preparations, the ooplasm cannot get into close contact with the glass surface and therefore no intraooplasmic complexes and organelles are detected by TIRF microscopy. E. In proper ooplasmic extracts, complexes and organelles contained within the ooplasm can be detected by TIRF microscopy (green–oskarMS2(10x)-MCP-EGFP mRNPs). Images in D and E were taken on the same day using the same microscopy setup.

    3. Optional: if the microscope is equipped with a 2D/3D motorized stage, it is recommended to localize and the store position of all droplets before switching to TIRF microscopy.
    4. Start TIRF imaging and adjust parameters, such as exposure time, laser intensity, and camera gain to obtain a good quality signal (Figure 4E).
    5. Temporal resolution is a critical parameter when studying the dynamics of moving objects. For motility analysis of oskar mRNPs, we exposed oskMS2-GFP particles for 20 msec, and simultaneously imaged EB1-mCherry (5 msec) to obtain information regarding the polarity of the underlying microtubule (MT) network. This resulted in a ~10-12 frame per second (FPS) acquisition, during which transported oskar mRNPs traveled ~50-150 nm (pixel size was 140 nm).

Data analysis

Note: The obtained images can be analyzed in any software/pipeline of the experimenter’s choice. Here, we describe the motility and co-localization/co-migration analysis of oskar mRNPs in ImageJ/FIJI using our custom-made particle recognition and tracking algorithms. A detailed manual of the plugins is available at https://github.com/Xaft/xs/blob/master/xs%20plug-ins.pdf.

  1. Motility analysis (see Videos 4 and 5)

    Video 4. Tutorial for the xsPT particle tracker plug-in of ImageJ, part one
    Note: Please watch the video in full screen for better clarity.

    Video 5. Tutorial for the xsPT particle tracker plug-in of ImageJ, part two
    Note: Please watch the video in full screen for better clarity.

    1. Load the acquired image sequence into ImageJ.
    2. Apply a Gaussian filter (0.8 pixel size) to all channels to reduce noise.
    3. Select the channel that contains information about the particles to be analysed (reference channel).
    4. Start the plugin xsPT.
    5. By using the pop-up window, enter the parameters for the particle detection and tracking.
      Note: See detailed description bundled with the plug-ins.
    6. After hitting ‘GO’, all frames of the reference channel are processed and initial tracks are created if selected (see Note 7).
    7. By clicking on the tracks shown after the detection, select those that are of interest.
      Note: In our example, these are tracks that appear long and linear.
    8. After selecting the tracks, press the ‘Selection mode’ button. This will switch to ‘Comment mode’.
    9. Change the channel to the EB1-mCherry signal. By playing the movie (e.g., ‘Play’ button, mouse scroll or dragging the scroll-bar), try to identify the MT along which a given particle run occurred.
      Note: In our example, we managed to identify 85-90% of the underlying MTs and their polarity.
    10. Click on the track and by using the 0-9 characters on the numerical keyboard, add a comment to represent the polarity of run (e.g., ‘1’ for plus tip directed, ‘2’ for minus end directed runs).
    11. Once finished, save the selected and commented tracks by pressing the ‘Save selected’ button. This will save a .csv file that contains the identifier of a track (no.), the frame number, calibrated x and y coordinates, the comment (step A9) and information (e.g., area, integrated density, background) about the sequence of recognized spots that compose the track.
    12. The motility information (e.g., speed, duration, displacement, pause and reversal frequencies) is extracted as described in (Gaspar et al., 2014).

  2. Co-migration analysis (see Videos 4 and 5)
    The second channel (target channel) contains the signal intensity of the protein of interest (e.g., Kinesin heavy chain or Tropomyosin1-I/C) instead of MT polarity information.
    1. Perform steps A1-A7 of section A.
    2. After selecting tracks of interest, save those selected.
      Note: This will save the integrated density of the detected spot in all channels.
    3. To detect if a protein-of-interest was present on the tracked particle at a given moment, compare the mean signal intensity (integrated density/area) of the appropriate channel (typically stored as ‘integral_2’) against a threshold.
    4. To determine the appropriate threshold, rerun the xsPT plugin by first selecting the target channel, i.e., making it the reference.
    5. Save data of all spots by pressing ‘Save all’.
    6. As a threshold, we used the lower 10th percentile of the mean signal intensity distribution determined in extracts of oocytes heterozygous for the fluorescently tagged protein-of-interest.
    7. Analyze the data as described in (Gaspar et al., 2017).

  3. Object based co-localization analysis (see Video 6)

    Video 6. Tutorial for the xsColoc colocalization analysis plug-in of ImageJ
    Note: Please watch the video in full screen for better clarity.

    For cases in which no dynamic information is available, or if the signals of the two channels are not expected to overlap completely–e.g., the objects in the two channels are large, we developed a modified version of our particle tracker that determines the co-localization of objects (as opposed to raw signals) in single snapshot frames. This plugin estimates the likelihood of random (expected) co-localization by running a series of Monte Carlo simulations using the particle information of the image.
    1. Load one frame of the acquired image into ImageJ.
    2. Run the xsColoc plugin.
    3. By selecting the channels from the drop-down menu of the pop-up window, adjust the parameters of particle detection for each channel (a detailed description is bundled with the plugins).
    4. After hitting ‘GO’, select the reference and the target channels and the maximal distance allowed (in nm) between the centers of two neighboring objects (see Note 8).
    5. Optional: if parts of the image do not contain any information about the specimen (usually blank regions), it is advised to restrict the analysis to a region of interest (drawn by the standard ROI tools of ImageJ). This will ensure accuracy of the simulated (expected) co-localization.
    6. Run the analysis by hitting the ‘Test colocalization’ button.
    7. A ‘Save’ dialog will open. Here, three files are saved: (i) the actual image (.tif), (ii) a descriptor file containing the parameters of particle detection and the coordinates of the ROI (.mcc), and (iii) a file (.csv) containing the features of all objects in the reference channels, the features of a co-localizing object (if any, otherwise -1) from the target channel, and 100 distance entries (in nm, for the 100 Monte-Carlo simulation runs) between the reference object and a simulated particle of the target channel.
    8. Analyze the data as described in (Gaspar et al., 2017).

Notes

  1. To label oskar mRNPs we used transgenic oskMS2(10x) combined with monomeric MCP-EGFP (Zimyanin et al., 2008). To polarity mark MTs, we expressed UASp-EB1-mCherry (gift of Damian Brunner) in the female germline using the oskar-Gal4 driver (Telley et al., 2004). For co-migration analyses, we endogenously tagged Kinesin heavy chain and Tropomyosin1-I/C with mKate2 and mCherry, respectively (Gaspar et al., 2017).
  2. Protein-poor diet and other sources of stress (overcrowding, lack of mates) were shown to negatively influence oskar mRNP motility and result in rapid redistribution of oskar mRNA into sponge/processing bodies (Snee and Macdonald, 2009; Shimada et al., 2011).
  3. Proper hydrophobicity is key for successful ooplasmic extraction. Glass that is too hydrophilic results in aspecific binding of proteins–including components of oskar mRNPs–to the surface, rendering the particles immotile, whereas a surface that is too hydrophobic prevents adherence of the ooplasm.
  4. In our experience, washes with 1 N HCl, 2 N H2SO4 or 1 N NaOH result in the appearance of blinking, red fluorescent spots on the glass that are often indistinguishable or stronger than the red fluorescence of the tagged protein molecules. Therefore, we recommend against cleaning the coverslips with acidic or alkaline washes before silanization.
  5. 100% ethanol quickly dissolves the hydrophobic wax layer that covers the fly cuticle and thus allows flies to sink to the bottom of the wells. Moreover, it terminally anesthetizes the fly.
  6. The droplets derived from a single egg-chamber are often connected by thin strings of liquid that can be traced under the halocarbonoil, facilitating the sometimes tedious search for the droplets using the 1,000x magnification on the TIRF microscope.
  7. The xsPT plugin allows online switching between automated and manual tracking. Moreover, tracks selected from automated tracking are transferred to the manual tracking mode, allowing the manual fixing of tracking errors.
  8. If there is a third channel available, it may be used as a mask channel, i.e., only those objects of the reference and target channel that are within a certain distance (‘mask distance’ in nm) to an object of the mask channel are taken into consideration.

Recipes

  1. BRB80
    80 mM PIPES, pH 6.9
    2 mM MgCl2
    1 mM EGTA
  2. 1% injection buffer (1% IB)
    10 mM HEPES, pH 7.7
    120 mM KCl
    1 mM MgCl2
    1% (w/v) dextran sulfate (MW ~10 kDa)
  3. Cornmeal agar
    10 L H2O
    120 g agar (e.g., from Pro-BIO)
    180 g dry yeast (e.g., from Volk Klaus)
    100 g soya powder (e.g., from Ruckemann)
    220 g sirup (e.g., from Ruckemann)
    800 g malt extract (e.g., from Baeko Rhei)
    800 g corn powder (e.g., from Ruckemann)
    62.5 ml propionic acid (e.g., VWR)
    24 g nipagin (e.g., Sigma-Aldrich)
    1. Boil agar (90 °C) in half of the water in a kettle until agar has dissolved completely. Dissolve everything except the propionic acid and nipagin in the rest of water and add to the agar-water mixture in the kettle
    2. Cook for 2 h, stirring every 10 min
    3. Reduce heat (80 °C)
    4. Add nipagin and propionic acid
    5. Pour vials, bottles and cover them immediately with cheesecloth
    6. Let bottles dry for several hours; vials should be dried overnight before stoppering
    7. Store fly food at 18 °C or 4 °C

Acknowledgments

This work was funded by the EMBL. This protocol is adapted from Gaspar et al., 2017.

References

  1. Bastock, R. and St Johnston, D. (2008). Drosophila oogenesis. Curr Biol 18: R1082-1087.
  2. Gaspar, I., Sysoev, V., Komissarov, A. and Ephrussi, A. (2017). An RNA-binding atypical tropomyosin recruits kinesin-1 dynamically to oskar mRNPs. EMBO J 36(3): 319-333.
  3. Gaspar, I., Yu, Y. V., Cotton, S. L., Kim, D. H., Ephrussi, A. and Welte, M. A. (2014). Klar ensures thermal robustness of oskar localization by restraining RNP motility. J Cell Biol 206(2): 199-215.
  4. Hovelmann, F., Gaspar, I., Ephrussi, A. and Seitz, O. (2013). Brightness enhanced DNA FIT-probes for wash-free RNA imaging in tissue. J Am Chem Soc 135: 19025-19032.
  5. Lohka, M. J. and Masui, Y. (1983). Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science 220(4598): 719-721.
  6. Murray, A. W. (1991). Cell cycle extracts. Methods Cell Biol 36: 581-605.
  7. Sawin, K. E. and Mitchison, T. J. (1991). Mitotic spindle assembly by two different pathways in vitro. J Cell Biol 112(5): 925-940.
  8. Shimada, Y., Burn, K. M., Niwa, R. and Cooley, L. (2011). Reversible response of protein localization and microtubule organization to nutrient stress during Drosophila early oogenesis. Dev Biol 355(2): 250-262.
  9. Snee, M. J. and Macdonald, P. M. (2009). Dynamic organization and plasticity of sponge bodies. Dev Dyn 238(4): 918-930.
  10. Telley, I. A., Gaspar, I., Ephrussi, A. and Surrey, T. (2012). Aster migration determines the length scale of nuclear separation in the Drosophila syncytial embryo. J Cell Biol 197(7): 887-895.
  11. Telley, I. A., Gaspar, I., Ephrussi, A. and Surrey, T. (2013). A single Drosophila embryo extract for the study of mitosis ex vivo. Nat Protoc 8(2): 310-324.
  12. Zimyanin, V. L., Belaya, K., Pecreaux, J., Gilchrist, M. J., Clark, A., Davis, I. and St Johnston, D. (2008). In vivo imaging of oskar mRNA transport reveals the mechanism of posterior localization. Cell 134(5): 843-853.

简介

了解大分子复合物,亚细胞结构和细胞器的动态行为和不断变化的组成是细胞和发育生物学中积极研究的领域之一,因为这些变化与功能和随后的生物体的发育和稳态直接相关。 在这里,我们演示了使用发展中的果蝇卵母细胞来研究具有高时空分辨率的信使核糖核蛋白复合物(mRNPs)的动力学。 果蝇的遗传学与全内反射(TIRF)显微镜,图像处理和数据分析结合,以前所未有的精确度了解了mRNP的运动性和组成动力学。
【背景】细胞内运输是活细胞的基本过程之一。几乎细胞内的所有细胞 - 离子,分子,复合物,细胞器 - 被积极地传输,使局部熵减少。尽管近年来,我们对这些运输过程的机理有了很大的了解,但我们的大部分知识来自于体外和细胞培养研究。在这些简化的系统中,很难确定是否利用运输监管流程的全部潜力。另一方面,组织,器官,组织和生物体通常太复杂,无法有效地研究时空分辨率,足以匹配这些运输过程的规模。为了结合自下而上和自上而下的方法的优点,已经开发了在保持复杂性的同时,使这些过程更易于访问的技术。一个例子是从ambiphian(例如,非洲爪蟾)卵母细胞和胚胎中制备大量细胞质提取物来研究细胞分裂(Lohka和Masui,1983; Murray,1991; Sawin和Mitchison,1991)。我们最近显示,在细胞质液滴 - 即中,从包含细胞核的单一果蝇胚胎有丝分裂活性释放的细胞中直接提取的非纯化细胞质继续,允许通过简单的物理和化学扰动探测主轴性能(Telley等人,2012年和2013年)。在这里,我们描述了基于开发果蝇蛋壳的卵的类似的离体制备技术。该方法允许研究细胞内转运过程(南瓜测定),例如迁移本地化的oskar mRNPs(Gaspar等人,2017)。

关键字:卵磷脂提取, 细胞内运动, TIRF显微镜检查, 卵母细胞, 离体分析

材料和试剂

  1. 盖玻片放置5-10个盖玻片(例如,Wash-N-Dry,Diversified Biotech,目录号:WSDR-1000)
  2. 手套
  3. 小塑料培养皿或50ml Falcon管(直径约30 mm)的帽子
  4. 15 x 20厘米黑色塑料板
  5. 高精度盖玻片(例如,Marienfeld Precision Cover Glass,22 x 22 mm,Marienfeld-Superior,目录号:0107052)
  6. X光胶片(例如,Amersham HyperfilmTM ECL,GE Healthcare,目录号:28906835)
  7. 双面胶带(例如,,Tesa Doubleband Photostrip,Tesa,目录号:05338-00)
  8. 粗棉布
  9. 产生适合基因型女性的飞线或十字架(见注1)
  10. 干燥的粒状面包酵母(例如,Lesaffre Saf-Instant < / sup>)
  11. 卤代烃油(例如,,Voltalef 10S,VWR,目录号:24627.188)
  12. 100%乙醇(例如,EMD Millipore,目录号:100983)
  13. 在庚烷中的约5%二氯二甲基硅烷(DCDMS)(例如,硅烷化溶液,Sigma-Aldrich,目录号:85126)
  14. PIPES(例如,,Sigma-Aldrich,目录号:P3768)
  15. 氯化镁六水合物(MgCl 2·6H 2 O)(例如,EMD Millipore,目录号:105833)
  16. EGTA(例如,Sigma-Aldrich,目录号:E3889)
  17. HEPES(例如,,Sigma-Aldrich,目录号:H3375)
  18. 氯化钾(KCl)(例如,EMD Millipore,目录号:104936)
  19. 硫酸葡聚糖,MW〜10kDa(例如,Sigma-Aldrich,目录号:D4911)
  20. 来自Pro-BIO的Agar(例如,)
  21. 来自Volk Klaus的干酵母(例如,)
  22. 来自Ruckemann的大豆粉末(例如)
  23. Sirup(例如,,来自Ruckemann)
  24. 来自Baeko Rhei的麦芽提取物(例如,)
  25. 来自Ruckemann的玉米粉(例如,)
  26. 丙酸(例如,VWR,目录号:ACRO447231000)
    制造:Acros Organics,目录号:447231000。
  27. 尼泊金(例如,,Sigma-Aldrich,目录号:H5501)
  28. BRB80(见配方)
  29. 1%注射缓冲液(1%IB)(见配方)
  30. 玉米粉琼脂(见食谱)

设备

  1. 标准的饲养设备(例如,具有玉米面琼脂的小瓶 - 食谱,带有湿度控制器的25℃培养箱,刷子,CO 2站,等等。 em>)
  2. 3-9孔解剖板(例如,,康宁,目录号:7220-85)
  3. 真空干燥器(例如,SP Scienceware - Bel-Art Products-H-B Instrument,目录号:F42025-0000)
  4. Dumont#5镊子(例如,,Fine Science Tools,目录号:11252-20)
  5. Dumont#55钳子(例如,,Fine Science Tools,目录号:11255-20)
  6. 用于显微镜的定制盖玻片支架(图1)


    图1.装有盖玻片支架和硅烷储存器的真空干燥器。更换设备盖后,打开真空阀和水龙头进行真空。一旦形成真空,即,您可以通过盖子提起整个设备 - 关闭真空阀和水龙头。

  7. 具有5-40x(或更高)放大倍率的放大立体显微镜(例如,Carl Zeiss,型号:STEMI SV 11)
  8. 具有手柄的锋利钨针(例如,,Fine Science Tools,目录号:10130-20和26018-17)
  9. 具有高NA物镜和高灵敏度检测器的全内反射荧光(TIRF)显微镜(Leica Microsystems,型号:Leica AF7000)
    注意:我们使用带有100x 1.46 NA油浸物镜和Photometrics Evolve Roper 512 EMCCD相机的Leica 7000宽幅TIRF显微镜。

软件

  1. 显微镜控制器软件(例如,,Leica LASAF)
  2. ImageJ / FIJI
  3. 用于ImageJ的定制粒子检测器和跟踪器插件(可从 https://github.com/Xaft/xs/blob/master/_xs.jar
  4. R(优先使用RStudio),用于数据分析

程序

  1. 飞养
    1. 建立产生在所需突变体遗传背景中表达适当荧光标记的雌性苍蝇的交叉(见注1)。根据最终基因型的复杂性,这可能需要多个连续的交叉
    2. 在实验前一天,收集少数(最多六只)年轻(最多一周龄)的雌性蝇,并将它们与2-3个男性一起放在一个小瓶中,新鲜的玉米面琼脂和20-30粒干燥的面包酵母(见注2)
  2. 盖玻片的硅化(见注3)
    1. 在实验当天,将5-10个工厂清洁的盖玻片装入盖玻片夹(见附注4)。
      注意:戴手套。
    2. 将200μl硅烷化溶液(5%DCDMS)放入培养皿/ Falcon管帽(硅烷储存器)中。
    3. 将盖玻片放在真空干燥器中的硅烷储存器旁边并施加真空(图1)
    4. 在室温(RT)下在真空下孵育盖玻片90-120分钟。蒸发的DCDMS将覆盖盖玻片的表面。不应该观察到玻璃的颜色或透明度的变化
    5. 硅藻盖玻片应在同一天使用,不应该储存。

  3. 准备TIRF显微镜
    1. 在解剖之前,启动显微镜,加载软件并设置成像参数。
    2. 如果控制温度至关重要,请在开始成像前至少半小时启动显微镜周围的孵育室。
    3. 确保系统已校准。 质量的折射率接近于卤烃油的折射率(η〜1.41),因此可以作为参考使用含有一滴卤代烃油的盖玻片。

  4. 解剖卵巢(见视频1)

    Video 1. Dissection of the ovaries

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player


    1. 用100%乙醇和另外两个具有BRB80的孔装载一个孔(约2ml)的解剖板(参见食谱)。使用黑色塑料板作为飞行和卵巢操作的工作场所。
    2. 麻醉一只雌性蝇,并用乙醇将其放入井中(见注5)
    3. 孵化4-5秒后,使用#55镊子,用BRB80将蝇转运到一个井中,另外4-5秒转移到含有BRB80的最后一个井。
    4. 在解剖显微镜的中等放大倍率(10-15倍)下,用非支配手的#5镊子,粉碎飞行的胸部以摧毁它,杀死动物并保持牢固的握力。用#55钳子,轻轻地刺穿腹部的腹侧后侧靠近中线。
    5. 通过拉拔去除后部部分来打开腹部。取出一对卵巢,并在一滴BRB80中将其转移到干净,非硅烷化的盖玻片上。
    6. 将两滴1%IB(见食谱)(约4-5μl)放在同一盖玻片上。将卵巢顺序转移到每个滴液中以从其周围移除BRB80。
    7. 将卵巢转移到硅藻土盖玻片中心的〜2微升1%IB中。
      注意:需要从此步骤使用硅化盖玻片。

  5. 卵的提取(见视频2和3)
    1. 将一滴卤代烃油放在硅胶盖玻片上,使其与含有卵巢的液滴形成界面(见视频2)。

      Video 2. Separation and sorting of the ovarioles

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


    2. 用100%乙醇擦拭钨针,然后在1%IB中冲洗。
    3. 将放大倍数提高到约20-25倍。使用两根针,将卵子(鸡蛋串)彼此分开。
    4. 鉴定包含感兴趣发育阶段(第9阶段或更早的图2A)的卵室的卵巢。用针
      小心地取出较旧的舞台 注意:穿过它们,使用它们作为剪刀。


      果蝇 发育。卵母细胞及其15个同胞细胞(护士细胞)通过一系列不完全的细胞分裂在胚胎中形成合胞体。这16个细胞通过细胞质桥连接并被形成蛋室的体细胞卵泡细胞层包围。卵巢成熟/经过14个不同阶段的卵子发生,以产生一个成熟的卵子(更多细节请参考,[Bastock和St Johnston,2008])。为了可视化不同的细胞类型,使用三种不同的FIT探针(Hovelmann等人)的混合物进行与oskar mRNA的原位杂交的无洗涤 em>,2013)。刻度棒为50μm。 B-D。从9期卵母细胞提取的不同步骤(静止图像来自视频3)。刻度棒为500μm(B-D)
      Video 3. Ex vivo ooplasmic preparation

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


    5. 使用你的优势手,抓住镶嵌在金刚石边上的修剪后的圆顶,轻轻地将其拉到油底下。
    6. 重复步骤E4和E5。理想情况下,在进一步进一步处理之前,应该有5-10个石油兴趣小屋。
    7. 缩放到大约40倍的放大倍数。在油下,首先清除比感兴趣的阶段更小的蛋室(例如,第9阶段),然后开始在护士细胞(卵母细胞的营养性姐妹细胞)的前端刺穿蛋室(图2B,见视频3)。
    8. 当没有一个或只有几个护士细胞残留时,轻轻地用针在你的优势手中的后极上抓取含卵母细胞的卵泡袋(“卵母细胞袋”),并开始将其从护士细胞的瓦砾中拉出(图2C) 。
    9. 卵子应该开始流出。调整拉力的速度,使质地接触盖玻片并粘附。您可以通过在卵巢前方产生张力来帮助其他针头粘连(见注3)。
    10. 根据拉动速度,卵形体的大小和盖玻片的疏水性,应形成(图2D),一至数个圆形的卵浆液滴(含有大量1-10μm大的蛋黄泡泡囊泡囊,可见于40x变焦)。 br />
    11. 故障排除:太亲水的玻璃会导致几乎瞬间的,无法控制地将表面释放出来,而太疏水的玻璃通常会排斥“卵母细胞袋”,使其从表面浮起。在任一情况下,建议从步骤B1重新启动协议,分别减少或增加硅烷化时间。
    12. 重复步骤E7-E9将更多的卵磷脂提取物沉积在同一个玻璃上。理想情况下,这样的准备工作中有四十六份(即复制)可放在一张22 x 22毫米的盖玻片上。

  6. TIRF显微镜
    1. 使用盖玻片支架将盖玻片加载到显微镜的支架上(图3)

      图3.定制的盖玻片支架。此盖玻片支架的基础是塑料块,尺寸为标准显微镜(76 x 25 x 2-3 mm),包含16-18 mm宽,其长边16-18毫米深切口。通过使用两条双面胶带将(使用)X射线胶片(约40×25mm,具有相似切口)的手工切割片固定到塑料块的底部。应该将两条胶带放置成使得所使用的盖玻片(例如,22×22mm)应该牢固地配合 - 在这个例子中,这两个条分开放置22毫米。带有卤素油的硅烷化盖玻片可以像抽屉一样在塑料块和X射线胶片之间滑动。刻度单位为cm。

    2. 使用透射光,聚焦样品并定位其中一个液体液滴。通过卵巢颗粒(图4A和4B)的存在可以认识到卵磷脂,其在护士细胞质中不存在(图4C)(见附注6)。有时,通过形成卵黄层增强的卵母细胞膜保持完整,因此不会与玻璃表面接触。这种畸形的卵母细胞可以被其周围的锐利,正弯曲的边界识别(图4B)。 TIRF显微镜无法获得这种肿瘤(图4D)

      图4. 离开 离子 制备之后的液滴的典型外观。A,B,D和E。卵泡南瓜。 A和B.卵母细胞在中期生育阶段的卵质可以通过存在大(1-10μm)的卵黄颗粒来识别。 A和E.有时,卤代烃油滴混入卵质,表明卵母细胞周围的血浆和卵黄膜被去除。 C.护士细胞浆液滴。颗粒度比卵母细胞A和B更精细,并且通常巨大的护士细胞核保持完整。 B和D.具有完整膜的卵母细胞/卵母细胞(由红色箭头指示)。 D.在这种制剂中,卵磷脂不能与玻璃表面紧密接触,因此不能通过TIRF显微镜检测到内质网络合物和细胞器。 E.在适当的卵泡提取物中,通过TIRF显微镜(绿色 - oskarMS2(10x) - MCP-EGFP mRNPs)可以检测到在卵质内含有的复合物和细胞器。在同一天使用相同的显微镜设置拍摄D和E中的图像。

    3. 可选:如果显微镜配备了2D / 3D电动舞台,建议在切换到TIRF显微镜之前将所有液滴定位和存储位置。
    4. 启动TIRF成像并调整参数,如曝光时间,激光强度和摄像机增益,以获得良好的质量信号(图4E)。
    5. 时间分辨率是研究运动物体动力学的关键参数。对于oskar mRNP的运动性分析,我们暴露了oskMS2 -GFP粒子20毫秒,同时成像EB1-mCherry(5毫秒),以获得关于底层的极性的信息微管(MT)网络。这导致每秒约10-12帧(FPS)采集,其间运送的oskar mRNP行进〜50-150nm(像素大小为140nm)。

数据分析

注意:可以在实验者选择的任何软件/管道中分析获得的图像。在这里,我们使用我们定制的粒子识别和跟踪算法描述了ImageJ / FIJI中的oskar mRNP的运动和共定位/共同迁移分析。 https://github.com/Xaft/xs/blob/master/xs%20plug-ins.pdf

  1. 运动分析(见视频4和5)

    Video 4. Tutorial for the xsPT particle tracker plug-in of ImageJ, part one

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player

    Note: Please watch the video in full screen for better clarity.

    Video 5. Tutorial for the xsPT particle tracker plug-in of ImageJ, part two

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player

    注意:请观看全屏视频以获得更好的清晰度。

    1. 将获取的图像序列加载到ImageJ中。
    2. 对所有通道应用高斯滤波器(0.8像素),以减少噪声
    3. 选择包含要分析的粒子信息的通道(参考通道)。
    4. 启动插件xsPT。
    5. 通过使用弹出窗口,输入粒子检测和跟踪的参数。
      注意:请参阅插件附带的详细说明。
    6. 在“GO”之后,参考通道的所有帧都被处理,如果选择了创建初始轨迹(见注7)。
    7. 通过点击检测后显示的曲目,选择感兴趣的曲目。
      注意:在我们的示例中,这些轨迹看起来很长和线性。
    8. 选择轨道后,按“选择模式”按钮。这将切换到“注释模式”。
    9. 将通道更改为EB1-mCherry信号。通过播放电影(例如,“播放”按钮,鼠标滚动或拖动滚动条),尝试识别发生给定粒子运行的MT。
      注意:在我们的例子中,我们设法确定了85-90%的底层MT及其极性。
    10. 点击轨道,使用数字键盘上的0-9个字符,添加一个注释来表示运行的极性( eg ,加号指针'1',负端的'2'定向运行)。
    11. 完成后,按“保存所选”按钮保存所选和已注释的曲目。这将保存.csv文件,该文件包含轨道(号)的标识符,帧号,校准的x和y坐标,注释(步骤A9)和信息(例如,区域,集成密度,背景)关于构成轨道的识别点的顺序。
    12. 如(Gaspar等人,2014年)所述提取运动信息(例如,速度,持续时间,位移,暂停和反转频率)。

  2. 共同迁移分析(见视频4和5)
    第二通道(靶通道)包含感兴趣的蛋白质(例如,驱动蛋白重链或原肌球蛋白1-I / C)的信号强度,而不是MT极性信息。
    1. 执行A部分的A1-A7步骤
    2. 选择感兴趣的曲目后,保存所选的曲目。
      注意:这将保存所有通道中检测到的斑点的集成密度。
    3. 为了检测在给定时刻跟踪的粒子上是否存在感兴趣的蛋白质,将相应信道的平均信号强度(积分密度/面积)(通常以“integral_2”存储)与阈值进行比较。
    4. 要确定适当的阈值,首先选择目标通道,即重新运行xsPT插件,使其成为参考。
    5. 按“全部保存”保存所有点的数据。
    6. 作为阈值,我们使用在荧光标记的兴趣蛋白质杂合的卵母细胞提取物中确定的平均信号强度分布的下10个百分位数。
    7. 按照(Gaspar等人,2017)所述分析数据。

  3. 基于对象的协同定位分析(见视频6)

    Video 6. Tutorial for the xsColoc colocalization analysis plug-in of ImageJ

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    注意:请观看全屏视频以获得更好的清晰度。

    对于没有动态信息可用的情况,或者如果两个信道的信号预计不会完全重叠 - 例如,则两个通道中的对象很大,我们开发了一个修改版本的粒子跟踪器确定单个快照帧中对象(与原始信号相反)的共定位。该插件通过使用图像的粒子信息运行一系列蒙特卡罗模拟来估计随机(预期)共定位的可能性。
    1. 将获取的图像的一帧加载到ImageJ中。
    2. 运行xsColoc插件。
    3. 通过从弹出窗口的下拉菜单中选择通道,调整每个通道的粒子检测参数(详细说明与插件捆绑在一起)。
    4. 点击“GO”后,选择两个相邻对象的参考和目标通道和允许的最大距离(以nm为单位)(见注8)。
    5. 可选:如果图像的部分不包含关于样本(通常为空白区域)的任何信息,建议将分析限于感兴趣区域(由ImageJ的标准ROI工具绘制)。这将确保模拟(预期)共定位的准确性。
    6. 按“测试colocalization”按钮运行分析。
    7. 将打开“保存”对话框。这里保存三个文件:(i)实际图像(.tif),(ii)包含粒子检测参数和ROI(.mcc)坐标的描述符文件,(iii)文件(.csv )包含参考通道中所有对象的特征,来自目标通道的共定位对象(如果有的话,否则为-1)的特征,以及100个距离条目(以nm为单位,用于100个蒙特卡罗模拟运行)在参考对象和目标通道的模拟粒子之间。
    8. 按照(Gaspar等人,2017)所述分析数据。

笔记

  1. 为了标记oskar mRNPs,我们使用转基因oskMS2(10x)与单体MCP-EGFP(Zimyanin等人,2008)组合。对于极性标记MT,我们使用 oskar -Gal4驱动程序(Telley等人,2004年)在女性种系中表达了UASp-EB1-mCherry(Damian Brunner的礼物) 。对于共同迁移分析,我们分别用mKate2和mCherry分别标记了驱动蛋白重链和原肌球蛋白1-I / C(Gaspar等人,2017)。
  2. 蛋白质不足的饮食和其他压力来源(过度拥挤,缺乏配偶)显示出负面影响oskar的mRNP运动性,并导致将海马mRNA快速重新分配到海绵/加工中身体(Snee和Macdonald,2009; Shimada等人,2011)。
  3. 适当的疏水性是成功的卵磷脂提取的关键。太亲水的玻璃导致蛋白质(包括oskar mRNPs的组分)与表面的非特异性结合,使得颗粒不稳定,而疏水的表面防止了卵石的粘附。
  4. 根据我们的经验,用1N HCl,2NH 2 SO 4或1N NaOH洗涤导致玻璃上闪烁的红色荧光斑点的出现,这些斑点通常是不可区分的或比标记的蛋白质分子的红色荧光强。因此,我们建议在硅烷化之前用酸性或碱性洗涤剂清洁盖玻片。
  5. 100%乙醇快速溶解覆盖飞角角质层的疏水性蜡层,从而允许苍蝇沉入井底。此外,它终于麻醉了苍蝇。
  6. 从单个蛋室衍生的液滴通常由可以追踪在卤代烃下面的薄的液体串连接,有助于在TIRF显微镜上使用1,000倍放大率,有时繁琐地搜索液滴。
  7. xsPT插件允许在自动和手动跟踪之间进行在线切换。此外,从自动跟踪中选择的轨迹将转移到手动跟踪模式,允许手动修复跟踪错误。
  8. 如果有第三个通道可用,则可以将其用作掩模通道,即仅将参考和目标通道中的那些物体在一定距离内(“nm距离”)考虑到掩模通道的对象。

食谱

  1. BRB80
    80 mM PIPES,pH 6.9
    2mM MgCl 2
    1 mM EGTA
  2. 1%注射缓冲液(1%IB)
    10 mM HEPES,pH 7.7
    120 mM KCl
    1mM MgCl 2
    1%(w / v)硫酸葡聚糖(MW〜10kDa)
  3. 玉米棒琼脂
    10 L H 2 O O
    来自Pro-BIO的120g琼脂(例如,)
    来自Volk Klaus的180g干酵母(例如,)
    来自Ruckemann的100g大豆粉(例如)
    220克sirup(例如,,来自Ruckemann)
    来自Baeko Rhei的800g麦芽提取物(例如,)
    来自Ruckemann的800克玉米粉(例如,)
    62.5ml丙酸(例如,VWR)
    24g nipagin(例如,Sigma-Aldrich)
    1. 将一半水中的一半水煮沸琼脂(90℃),直到琼脂完全溶解。将除了丙酸和nipagin之外的所有物质溶解在其余的水中,并加入水壶中的琼脂 - 水混合物中
    2. 煮2小时,每10分钟搅拌一次
    3. 减少热量(80°C)
    4. 添加nipagin和丙酸
    5. 倒入瓶子,瓶子,并立即用干酪包布覆盖
    6. 让瓶子干几个小时;瓶子应该在堵塞之前干燥一整夜
    7. 在18°C或4°C下存放飞行食物

致谢

这项工作由EMBL资助。该协议是从2017年的Gaspar等人改编的。

参考

  1. Bastock,R.和St Johnston D.(2008)。 果蝇 oogenesis。 Curr Biol 18:R1082-1087。
  2. Gaspar,I. Sysoev,V.,Komissarov,A.和Ephrussi,A.(2017)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov / pubmed / 28028052“target =”_ blank“>一种RNA结合非典型原肌球蛋白可以动态地将kinesin-1招募到oskar mRNPs。 EMBO J 36(3) :319-333。
  3. Gaspar,I.,Yu,YV,Cotton,SL,Kim,DH,Ephrussi,A.and Welte,MA(2014)。< a class =“ke-insertfile”href =“http://www.ncbi .nlm.nih.gov / pubmed / 25049271“target =”_ blank“> Klar通过抑制RNP运动来确保 定位的热稳定性。 206(2):199-215。
  4. Hovelmann,F.,Gaspar,I.,Ephrussi,A.和Seitz,O。(2013)。  亮度增强的DNA FIT探针用于组织中的无洗涤RNA成像。 J Am Chem Soc 135:19025-19032。 >
  5. Lohka,MJ和Masui,Y.(1983)。  在两栖类卵泡成分诱导的精子原核和有丝分裂染色体的体外形成。 220(4598):719-721。
  6. Murray,AW(1991)。细胞周期提取物。< / a>方法细胞周期36:581-605。
  7. Sawin,KE和Mitchison,TJ(1991)。&nbsp; 有丝分裂通过体外两种不同途径的纺锤体组装。 .J Cell Biol.112(5):925-940。
  8. Shimada,Y.,Burn,KM,Niwa,R。和Cooley,L。(2011)。早期卵子发生的蛋白质定位和微管组织对营养胁迫的可逆反应 355(2) :250-262。
  9. Snee,MJ和Macdonald,PM(2009)。动态组织和可塑性的海绵体。 Dev Dyn 238(4):918-930。
  10. Telley,IA,Gaspar,I.,Ephrussi,A.and Surrey,T。(2012)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/ peter / 22711698“target =”_ blank“> Aster迁移决定了果蝇合胞体胚中核分离的长度尺度。细胞生物学197(7) :887-895。
  11. Telley,IA,Gaspar,I.,Ephrussi,A.and Surrey,T。(2013)。&nbsp; 用于研究有丝分裂离子的单一的果蝇胚胎提取物。 Nat Protoc 8(2):310-324。
  12. Zimyanin,VL,Belaya,K.,Pecreaux,J.,Gilchrist,MJ,Clark,A.,Davis,I. and St Johnston,D。(2008)。 oskar的体内成像 > mRNA转运揭示了后定位的机制。细胞 134(5):843-853。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Gáspár, I. and Ephrussi, A. (2017). Ex vivo Ooplasmic Extract from Developing Drosophila Oocytes for Quantitative TIRF Microscopy Analysis. Bio-protocol 7(13): e2380. DOI: 10.21769/BioProtoc.2380.
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