参见作者原研究论文

本实验方案简略版
May 2019
Advertisement

本文章节


 

Application of Mechanical Forces on Drosophila Embryos by Manipulation of Microinjected Magnetic Particles
机械力应用于果蝇胚胎的磁性粒子显微注射操作   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Cells generate mechanical forces to shape tissues during morphogenesis. These forces can activate several biochemical pathways and trigger diverse cellular responses by mechano-sensation, such as differentiation, division, migration and apoptosis. Assessing the mechano-responses of cells in living organisms requires tools to apply controlled local forces within biological tissues. For this, we have set up a method to generate controlled forces on a magnetic particle embedded within a chosen tissue of Drosophila embryos. We designed a protocol to inject an individual particle in early embryos and to position it, using a permanent magnet, within the tissue of our choice. Controlled forces in the range of pico to nanonewtons can be applied on the particle with the use of an electromagnet that has been previously calibrated. The bead displacement and the epithelial deformation upon force application can be followed with live imaging and further analyzed using simple analysis tools. This method has been successfully used to identify changes in mechanics in the blastoderm before gastrulation. This protocol provides the details, (i) for injecting a magnetic particle in Drosophila embryos, (ii) for calibrating an electromagnet and (iii) to apply controlled forces in living tissues.

Keywords: Drosophila embryos (果蝇胚胎), Tissue mechanics (力学), Force application (施力), Magnetic particle (磁性粒子), Tissue rheology (组织流变学), Morphogenesis (形态发生), Cellular forces (细胞受力), Cellularization (细胞化), Electromagnet (电磁铁)

Background

Drosophila melanogaster embryogenesis is a classical model for morphogenesis (Campos-Ortega and Hartenstein, 1985). While many tools have been developed to assess the role of specific proteins in morphogenesis, assessing cellular forces or mechanics still remains challenging. For the last fifteen years, laser dissection has been the most commonly used approach to assess cellular forces (Colombelli and Solon, 2013; Shivakumar and Lenne, 2016). However, laser dissection is invasive, it wounds tissues, and does not facilitate the application of ectopic forces. To overcome these limitations, a few methods have been developed to probe the mechanics of tissues by inducing deformations in droplets of magnetic fluid or through the optical trapping of cellular junctions (Bambardekar et al., 2015; Serwane et al., 2017). These methods have a limited range of force and require specific, complex instrumentation. Here, we present the protocol for an alternative, versatile and low-cost method for applying controlled forces within an epithelium of a living Drosophila embryo (D’Angelo et al., 2019). This method relies on the injection of a magnetic particle within a living embryo and on the application of a magnetic field with an electromagnet. Since the magnetic particle is coated with GBP nanobody, it is possible to target a specific intracellular attachment. Here, we injected the bead in a fly line expressing GFP at the plasma membrane (Resille GFP) to position the bead at the plasma membrane. Our methodology does not impair morphogenesis and repeated force application can be performed without cellular damage.

It is well established that tissue mechanics is an essential component to consider for the control of cellular behavior (differentiation, cell division, migration…) leading to morphogenesis during animal development and to the progression of diseases such as tumor formation and cancer progression (Lecuit et al., 2011; Heisenberg and Bellaiche, 2013; Engler et al., 2006; Frey et al., 2008; Godard and Heisenberg, 2019; Northcott et al., 2018). In these contexts, our method can be used to probe tissue mechanics and its changes or to apply local forces to investigate mechanosensation and mechanotransduction.

Materials and Reagents

  1. 1.5 ml Eppendorf tube
  2. Magic tape (Scotch)
  3. Glass capillaries 1 mm in diameter, 90 mm length (Narishige model G1)
  4. Cover slip 24 x 60 mm, thickness 1
  5. FrameSlides (Leica microsystem, catalog number: 11505151)
  6. Silicon grease (Kluber lubrication, catalog number: 0040260221)
  7. Neodymium magnet (RS, catalog number: 695-0169)
  8. Parafilm (VWR, catalog number: 291-1214)
  9. Mu-metal rod 100 x 4 mm (Sekels)
  10. Aluminium cylinder (45 mm x 200 mm)
  11. Copper wire 0.5 mm
  12. Drosophila melanogaster embryos expressing Resille GFP
    Note: This can be easily extended to other Drosophila line or organism.
  13. Dynabeads M-450 tosylactivated (Thermo Fisher, catalog number: 14013)
  14. GFP binding protein (GBP) custom made (D'Angelo et al., 2019)
    Note: The GBP can be substituted by commercial anti GFP or other types of antibodies.
  15. Boric Acid (Sigma-Aldrich, catalog number: B6768)
  16. PBS
  17. PDMS (Poly(dimethylsiloxane), hydroxy terminated 750 cSt (Sigma-Aldrich, catalog number: 481963)
  18. Heptane (Sigma-Aldrich, catalog number: H2198)
  19. Sodium Hypochlorite (Panreac, catalog number: 2119211211)
  20. Voltalef oil 10s (VWR, catalog number: 9002-83-9)
  21. BSA (Bovine Serum Albumin) (Sigma-Aldrich, catalog number: A2153)

Equipment

  1. Microinjection microscope (Leica, model: DMIL ) equipped with 20x objective and a micromanipulator (Narishige, model: MN-153 )
  2. Micro-puller (Sutter instruments, model: P30 ) equipped with a 3 mm wide Trough filament (Sutter Instrument, catalog number: FT00330B )
  3. Microinjector (WPI, model: PV 820 )
  4. Micro grinder (Narishige, model: EG-44 )
  5. Andor Revolution XD spinning disk confocal microscope equipped with 40x and 100x objectives and lasers emitting at 488 nm and 561 nm
  6. Three-axis micromanipulator (Narishige, model: UMM-3FC )
  7. Bioruptor (Diagenode, model: UCD-200TM-EX )
  8. Thermomixer (Eppendorf, model: 5350 )
  9. Hot plate (Stuart, model: US152 )
  10. Power supply 30 V, 5A (Blausonic, model: FA-350 )

Software

  1. Fiji (Schindelin et al., 2012)
  2. Excel (or alternative plotting and fitting software)

Procedure

  1. Construction and calibration of the electro magnet
    To apply controlled forces, we designed an electromagnet similar to the one described in Kollmansberger and Fabry (Kollmannsberger and Fabry, 2007). The electromagnet is composed of three components: A Mu-metal rod (soft ferromagnetic alloy with very high magnetic permeability), a radiator in aluminium to evacuate the heat and a copper wire to connect to electric current (Figures 1A and 1B). The Mu-metal rod is sharpened to achieve a conical tip and then thermally annealed. (both operations can be done at the Sekels factory). A custom manufactured 45 mm long aluminium cylinder–with 4.5 mm inner diameter to host the Mu-metal rod was used to dissipate the heat generated by the magnet. The outer diameter of the cylinder was 200 mm. Close to one end, four, 1 mm wide and 40 mm deep troughs spaced by 2 mm were milled into the cylinder to increase the radiator surface. Another 220 mm wide trough of 100 mm diameter was milled 5 mm from the other end of the cylinder to host 100 coils of the copper wire. On the two extremities, three plastic screw were positioned at 120° interval and were used to hold the Mu-metal rod (Figures 1C and 1D).


    Figure 1. Electromagnet set up for force application. A. Photograph of the power supply (left) and electromagnet mounted on the micromanipulator. B. Photograph of the electromagnet mounted on the stage of the spinning disk microscope. C. Close up of the electromagnet, the left arrow indicates the Mu-metal rod conically shaped, the center arrow indicates the copper coil around the radiator and the right arrow indicates the aluminium radiator. D. Schematics showing the design of the aluminium radiator, a = 160 mm, b = 120 mm, c = 220 mm, d = 160 mm, e = 200 mm.

    Upon application of a direct current circulating within the cupper coils, a magnetic field emanates from the tip and decays spatially. Magnetic beads lying within the magnetic field are attracted towards the tip with a force proportional to the gradient of the field (Kollmannsberger and Fabry, 2007).
      To calibrate the force exerted on the bead as a function of the distance from the magnet, we developed an assay using beads embedded in PDMS. Since its viscosity is known, by measuring the velocity of the beads within the PDMS when the magnetic field is applied, we can calculate the force field as a function of the distance from the tip: Stoke’s law directly relates the force to the velocity (Figure 2 and Video 1).


    Figure 2. Construction of the calibration chamber. A and B. Sequential folding of a rectangular piece of Parafilm, dotted line represents the folding site. C. The dotted line represents the cut position to generate a window. D. The Parafilm is transferred onto a coverslip. E. Photograph of the calibration chamber mounted on the metal slide.

    Video 1. Calibration of the force applied to a 4.5 μm bead. (Left) Time lapse video of 4.5 μm magnetic beads embedded in PDMS moving toward the magnet during application of the magnetic field and respective tracking (Right).

    1. Place 5 μl of Dynabeads in 1.5 ml Eppendorf tube and put it at 37 °C with the lid open in the Thermomixer for a few hours in order to completely desiccate the bead.
    2. Add 1 ml of PDMS and stir it with a pipette tip in order to detach the pellet of beads from the bottom.
    3. Using the Bioruptor, sonicate at maximum power for 10 min. If the beads are not evenly resuspended repeat the sonication procedure.
    4. Cut a rectangular piece of Parafilm 10 cm x 3 cm and fold it as in Figure 2.
    5. Using a cutter, open a window of 5 x 10 mm in the center of the folded Parafilm.
    6. Transfer the Parafilm on top of a coverslip and heat it on top of a hot plate until the Parafilm attaches properly to the coverslip without melting.
    7. Transfer 50 μl of beads/PDMS (from Step A3) into the Parafilm window.
    8. Mount the coverslip onto a frame slide Leica using a drop of silicon grease.
    9. On the spinning disk microscope use a 40x lens and focus on the beads.
    10. Bring the electromagnet to one edge of the field of view (Video 1).
    11. Start the time-lapse using transmitted light and an exposure time of 20 ms.
    12. Start the force application.

  2. Injection procedure of individual 4.5 μm bead into Drosophila embryo
    A single magnetic bead is injected into the yolk of an embryo at stage 2 of Drosophila development (Figures 3-4). After injection, the coverslip supporting the embryos is left to develop over a neodymium magnet that will hold the bead in the embryo cortex. Thus, at cellularization time, the bead will be naturally encapsulated into a newly formed cell. By choosing the orientation of the embryo relative to the neodymium magnet, it is possible to embed the particle in a specific group of cells, which will later develop into specific tissues or organs.


    Figure 3. Immobilization of a single 4.5 μm bead at the tip of the injection needle. A. The injection needle is placed into a drop of 4.5 μm beads resuspended in 0.1% PBS/BSA. B. Negative pressure (P < 0) is applied to the needle in order to immobilize a single bead.


    Figure 4. Injection of a single 4.5 μm bead into Drosophila embryo. A and B. Negative pressure (P < 0) is applied to the needle in order to immobilize a single bead while approaching the embryo. C and D. Negative pressure is released and the bead remains inside the embryo. The inset shows a cartoon of the corresponding injection step where the needle is depicted in cyan, the bead in yellow and the embryo in light gray. The arrow represents the resulting force generated by the negative pressure inside the needle.

    1. Prepare 0.1 M boric acid solution at pH 9.5.
    2. Wash 3 times 50 μl of 4.5 μm Dynabeads with 1 ml of boric acid solution.
    3. Resuspend 10 μg of GBP in 400 μl of boric acid solution and incubate O/N at 37 °C.
    4. Wash 2 times the Dynabeads with 1 ml of boric acid solution.
    5. Resuspend in 1 ml boric acid solution 0.1% BSA (w/v).
    6. Prepare heptane glue by incubating O/N 10 ml of heptane with 120 cm of magic tape.
    7. Place a drop of 100 μl of heptane glue in the middle of a coverslip and allow the drop to flow down by gravity, creating a stripe of sticky surface in the middle of the coverslip, parallel to his long edge.
    8. Thirty to forty freshly laid embryos are chemically dechorionated using sodium hypochlorite for 90 s at RT, then rinsed under tap water for 90 s and then mounted on a coverslip coated with heptane glue.
      Note: All embryos should be mounted in a single row, regularly spaced and parallel to each other, to facilitate the injection.
    9. The embryos are dehydrated for 10-15 min in a 25 °C incubator, to reduce turgidity and avoid bursting upon injection, and then covered with a small drop of Voltalef oil. Please note that the dehydration time needs to be adjusted depending on the humidity in the incubator.
    10. A drop of 40 μl beads resuspended in PBS/0.1% BSA is deposited next to the embryos, aside of the Voltalef oil drop.
    11. Turn on the micro-puller and set the values of heat 1 at 860 and pull at 750. Then insert a glass capillary in the respective holder and generate an injection needle. Note that different pulling parameters can be used. The injection needle just need to be elongated enough to have a diameter allowing to hold the magnetic particle and to enter the embryo without damaging it (see Figures 3 and 4).
    12. The injection needle is precisely opened using a microgrinder to achieve an internal diameter of 3.5 μm (see Figure 3).
    13. The needle is connected to a Microinjector (WPI PV 820 ).
    14. The needle is positioned in the drop containing beads (in PBS/0.1% BSA) and, by applying negative pressure, a single bead is held at the tip of the needle. Figure 3 illustrates the process.
    15. Move the needle holding the bead close to the posterior pole of the embryo, set the pressure of the microinjector to neutral just before the injection. Then, the bead can be transferred into the center of the embryo (see Figure 4).
    16. Once all the embryos are injected, place the coverslip with the injected embryos on top of a permanent magnet for 2 h at 25 °C.
    Notes:
    1. In order to maximize the force applied by the neodymium magnet to the embryos, position the coverslip with the injected embryos directly on the neodymium magnet without the microscopy slide.
    2. To minimize the damage inferred to the embryos, the bead needs to be injected at early developmental stage (stage 2). In order to target specific tissues, the embryo needs to be mounted with the tissue of interest facing the coverslip. i.e., if we want to apply forces during the process of dorsal closure, the embryos have to be mounted with dorsal side facing the coverslip and the embryos must be left to develop for 20 h after injection at 18 °C.

  3. Imaging and force application
    1. The coverslip is mounted onto a frame slide Leica using a drop of silicon grease.
    2. Select a 40x lens at the spinning disk, focus on an embryo with the bead in right orientation and then position the tip of the electro-magnet at 150-200 μm from the chosen embryo. Care must be taken in choosing the bead-magnet distance in order to position the bead in a region of the force-distance curve with small steepness to allow for an accurate estimation of the force (see Figure 5B and Figure 6).
    3. Set up a two channel, x-y-z time lapse imaging protocol. One channel will use the 488 nm laser to acquire GFP signal and the other channel will use 561 nm laser will acquire the auto-fluorescence of the bead.
    4. Set up 5 z steps spaced by 1 μm and choose a time resolution of 5 s.
    5. Start the movie, after 1 min turn on the power supply and select 0.3 A to generate a magnetic field that will exert a force of 115pn on the bead. Keep the power supply on for 1 min, then, after switching off the power supply, allow the imaging to proceed for 1 more minute before stopping the movie (Figures 5A, 5C and Video 2).


      Figure 5. Force application on a single 4.5 μm bead to the Drosophila blastoderm. A. Time-lapse images showing the bead (magenta) in response to a force step (∼115 pN; onset at 0 s). The bead was embedded into an individual cell of a Resille-GFP embryo. White arrows indicate force application. White dashed lines mark the left side of the bead at time 0s. Scale bar = 10 μm. B. Snapshot in transmitted light of an embryo containing a 4.5 μm bead and the electromagnet placed at 190 μm. Scale bar = 20 μm. C. Bead displacement corresponding to the force application shown in A.


      Figure 6. Force-distance calibration plot. Example of force-distance plot for the calibration of the electromagnet. The force exerted on three single beads is plotted as function of the distance from the tip of the magnet. A power law fit on the force curve of bead number three is represented in red.

      Video 2. Force delivery to a single cell during Drosophila cellularization. Time-lapse video showing a force application on a 4.5 μm bead embedded into a Resille-GFP expressing embryo. The purple arrow indicates when a force of ~115 pN is applied on the bead. Scale bar = 10 μm.

Data analysis

  1. Force Calibration
    1. Open the time-lapse performed during the calibration with Fiji. Apply a median filter (2 pixels) to reduce the noise.
      Note: Other types of filter (like Gaussian blur filter for instance) can be used, the only criterion is to reduce sufficiently the background noise to correctly threshold the beads.
    2. Threshold to achieve a binary movie, and convert it to 8 bit.
    3. Remove the scale of the movie
    4. Use the MTrack2 plugins to extract the X and Y coordinates of the beads during the force application (Video 1). Define the following parameters in the window associated to the plugin:
      Minimum object size to 8 μm2 (corresponding to 20 pixels in our set up)
      Maximum object size to 120 μm2 (corresponding 300 pixels in our set up)
      Maximum velocity to 100
      Minimum track length (frames) to 20
      Tick Save Results File box
      Tick Display Path Lengths box
      Tick Show Labels box
      Tick Show Positions box
      Tick Show Paths box
    5. The MTrack2 plugin generates a trackresult file with X1 and Y1 coordinates of the tracked objects at each time point.
    6. Import the trackresult file in Excel or other data plotting software. If the detection is noisy, one can perform a time average of few consecutive data points into new X and Y columns to smooth the trajectories.
    7. From the X and Y coordinates, we calculate the distance D of the bead from the magnet at each time point t.



      where,
      pixel size is the size of a pixel in µm,
      xbead is the x value (in pixels) of the bead at each time point,
      ybead is the y value (in pixels) of the bead at each time point,
      xmagnet is the x value (in pixels) of the magnet,
      ymagnet is the y value (in pixels) of the magnet.

    8. Calculate the velocity of the bead v(t)



      where,
      Δt is the time interval of the movie in seconds.

    9. The force F (t) applied on the bead can then be calculated at any time point using the following Stoke’s law.



      where,
      µ is the viscosity of the media in which the bead is embedded,
      R is the radius of the bead,
      v(t) is the velocity of the bead.

    10. From the obtained values of D(t) and F(t), generate a scatter plot of the force as a function of the respective distance in the plotting software of your choice and then fit the resulting scatter plot with a power law with the following shape:



      where A and α are fitting parameters (see Figure 6).
      Note: That each individual bead track can be fitted independently and the extracted fitting parameters will then be averaged or a single fit of all the tracks could be generated.

  2. Bead displacement analysis after force application in the blastoderm.
    1. Open the force application to blastoderm movie with Fiji and split the green (488 nm) and red (561 nm) channels (see Video 2).
    2. Sum project all the single z planes into single movie and then apply median filter (with a kernel of 2 pixels).
    3. Apply the steps 2 and 3 as in the Force Calibration section to the red channel.
    4. Folllow the step 4 as in the Force Calibration section but using the following parameters:
      Minimum object size to 32 µm2 (corresponding 200 pixels in our set up)
      Maximum object sixe to 400 µm2 (corresponding 2,500 pixels in our set up)
      Maximum velocity to 100
      Minimum track length (frames) to 20
      Tick Save Results File box
      Tick Display Path Lengths box
      Tick Show Labels box
      Tick Show Positions box
      Tick Show Paths box
    5. By applying equation 1 to the X and Y coordinates generated by the MTrack2 plugin, and replacing the coordinates of the magnet with the initial coordinates of the bead in the generated file, we can calculate the bead displacement during the force application at each time point t (Figure 6).
    6. To estimate the force applied to the bead we measure the distance D from the magnet to the initial position of the bead, again using the equation 1. Then, we calculate the force using the equation 4 with the parameters previously estimated during the calibration.
    Note: In order to position the magnet 190 μm from the bead, the reference bead-magnet snapshot has to be taken with a 40x lens (as in Figure 5B) but for better visualization the force application movie has to be taken with a 100x lens (as show in Figure 2A and Video 2).

Acknowledgments

We thank Peran Hayes for discussions and critical reading of the manuscript and Roger Uceda from the Fundació CIM for manufacturing the aluminium radiator. We are grateful to the ALMU team for providing help with the microscopy. The research leading to these results has received funding from the Spanish Ministry of Economy and Competitiveness, Plan Nacional, BFU2010-16546 and BFU2015-68754, “Centro de Excelencia Severo Ochoa” and to the EMBL partnership. We acknowledge the support of the CERCA Programme/Generalitat de Catalunya. This work was supported in part by the Fundaciòn Biofisika Bizkaia and the Basque Excellence Research Centre (BERC) program of the Basque Governement.

Competing interests

There are no conflicts of interest or competing interest.

References

  1. Bambardekar, K., Clement, R., Blanc, O., Chardes, C. and Lenne, P. F. (2015). Direct laser manipulation reveals the mechanics of cell contacts in vivo. Proc Natl Acad Sci U S A 112(5): 1416-1421.
  2. Campos-Ortega, J. A., and Hartenstein, V. (1985). The embryonic development of Drosophila melanogaster. Berlin, Springer-Verlag.
  3. Colombelli, J. and Solon, J. (2013). Force communication in multicellular tissues addressed by laser nanosurgery. Cell Tissue Res 352(1): 133-147.
  4. D'Angelo, A., Dierkes, K., Carolis, C., Salbreux, G. and Solon, J. (2019). In vivo force application reveals a fast tissue softening and external friction increase during early embryogenesis. Curr Biol 29(9): 1564-1571 e1566.
  5. Engler, A. J., Sen, S., Sweeney, H. L. and Discher, D. E. (2006). Matrix elasticity directs stem cell lineage specification. Cell 126(4): 677-689.
  6. Frey, B., Janko, C., Ebel, N., Meister, S., Schlucker, E., Meyer-Pittroff, R., Fietkau, R., Herrmann, M. and Gaipl, U. S. (2008). Cells under pressure - treatment of eukaryotic cells with high hydrostatic pressure, from physiologic aspects to pressure induced cell death. Curr Med Chem 15(23): 2329-2336.
  7. Godard, B. G. and Heisenberg, C. P. (2019). Cell division and tissue mechanics. Curr Opin Cell Biol 60: 114-120.
  8. Heisenberg, C. P. and Bellaiche, Y. (2013). Forces in tissue morphogenesis and patterning. Cell 153(5): 948-962.
  9. Kollmannsberger, P. and Fabry, B. (2007). High-force magnetic tweezers with force feedback for biological applications. Rev Sci Instrum 78(11): 114301.
  10. Lecuit, T., Lenne, P. F. and Munro, E. (2011). Force generation, transmission, and integration during cell and tissue morphogenesis. Annu Rev Cell Dev Biol 27: 157-184.
  11. Northcott, J. M., Dean, I. S., Mouw, J. K. and Weaver, V. M. (2018). Feeling stress: The mechanics of cancer progression and aggression. Front Cell Dev Biol 6:17.
  12. Serwane, F., Mongera, A., Rowghanian, P., Kealhofer, D. A., Lucio, A. A., Hockenbery, Z. M. and Campas, O. (2017). In vivo quantification of spatially varying mechanical properties in developing tissues. Nat Methods 14(2): 181-186.
  13. 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):676-82.
  14. Shivakumar, P. C. and Lenne, P. F. (2016). Laser ablation to probe the epithelial mechanics in Drosophila. Methods Mol Biol 1478: 241-251.

简介

[ 摘要] 电池产生的机械力塑造组织形态发生过程。这些力量可以激活一些生化途径和触发多元化Ç Ellular回应机械性-感,如分化,分裂,迁移和凋亡。评估的机械性-响应细胞在活生物体需要在生物组织内施加受控局部力的工具,为此,我们建立了一种方法来对嵌入果蝇胚胎所选组织内的磁性粒子产生受控力,并设计了将单个粒子注入早期胚胎的方案。并使用永久磁铁将其定位在我们选择的组织内。可以使用事先已校准的电磁体,将微微至纳牛顿范围内的控制力施加到微粒上。施加力后上皮变形可通过实时成像进行跟踪,并使用简单分析进行进一步分析 该工具已成功用于识别胃胚形成之前胚盘中的力学变化。该协议提供了以下细节:(i)在果蝇胚胎中注入磁性粒子,(ii)校准电磁体,以及(iii)在活体组织中施加控制力。

[ 背景] 果蝇胚胎发育是一个经典模型对于形态(坎波斯,奥尔特加和哈滕斯坦,1985年)。尽管许多工具已经开发评估具体蛋白质形态的作用,评估细胞部队或力学仍然具有挑战性。在过去的十五年来,激光解剖是评估细胞力最常用的方法(Colombelli和Solon,2013; Shivakumar和Lenne,2016)。然而,激光解剖是侵入性的,会伤及组织,不利于异位力的施加。。为了克服这些限制,一些方法已经发展到探头的磁流体或通过光阱蜂窝结组织通过诱导变形以液滴的力学(Bambardekar 等人,2015年; Serwane 。等,2017) 。方法有这些有限的力范围,并要求具体,复杂的仪器。在这里,我们目前的协议,用于替代方案,通用和大号 一种在果蝇活体胚胎的上皮内施加控制力的低成本方法(D'Angelo et al。,2019)。该方法依赖于在活体胚胎内注入磁性颗粒并施加磁场电磁体:由于磁性颗粒被GBP纳米抗体包裹,因此有可能靶向特定的细胞内附着。在此,我们将磁珠注入到质膜上表达GFP的蝇线中(Resille GFP),以将磁珠定位在血浆中我们的方法不会损害形态发生,并且可以在不损伤细胞的情况下重复施加力。

它是公认的组织力学是一个重要组成部分考虑为控制细胞行为中(分化,细胞分裂,迁移...),导致形态发生动物开发与要的进展性疾病,如肿瘤形成和癌症进展(Lecuit的Et Al。,2011; Heisenberg和Bellaiche,2013; Engler 等,2006; Frey 等,2008; Godard和Heisenberg,2019 ; Northcott 等,2018)。在这些情况下,我们的方法可用于探测组织力学及其变化或施加局部力来研究机械感觉和机械转导。

关键字:果蝇胚胎, 力学, 施力, 磁性粒子, 组织流变学, 形态发生, 细胞受力, 细胞化, 电磁铁

材料和试剂


 


1.5 ml Eppendorf管
魔术胶带(苏格兰威士忌)
玻璃毛细管直径1毫米,长度90毫米(Narishige型号G1)
盖玻片24 x 60毫米,厚度1
FrameSlides(Leica microsystem,货号:11505151)
硅脂(克鲁勃润滑,目录号:0040260221)
钕磁铁(RS,目录号:695-0169)
封口膜(V WR ,目录号:291-1214)
Mu金属棒100 x 4 mm(塞克尔)
铝制圆筒(45毫米x 200毫米)
铜线0.5毫米
果蝇表达Resille GFP的果蝇。
注意:这可以很容易地扩展到其他果蝇系或有机体。


Dynabeads M-450甲苯磺酰活化(Thermo F 垫圈,货号:14013)
制备了GFP结合蛋白(GBP)(D'Angelo et al。,2019)
注:牛逼他GBP可以替代商业防GFP或其他类型的抗体。


硼酸(Sigma - Aldrich ,目录号:B6768)
PBS
PDMS(聚(二甲基硅氧烷),羟基封端的750 cSt (Sigma - Aldrich ,目录号:481963)
庚烷(Sigma - Aldrich,目录号:H2198)
次氯酸钠(Panreac,目录号:2119211211)
Voltalef油10s(V WR ,目录号:9002-83-9)
BSA(牛血清白蛋白)(Sigma - Aldrich,目录号:A2153)
 


配套设备


 


显微镜显微注射机(Leica,型号:DMIL)配备有20 X 物镜和微操作(NARISHIGE,型号:MN-153)
拔轮器,微型(萨特仪器,型号:P30)配备了一块3 毫米宽的槽丝(萨特我Nstrument,目录号:FT00330B)
微型注射器(WPI,型号:PV 820)
微型研磨机(成茂,型号:EG-44)
革命XD纺纱安道尔磁盘共聚焦显微镜配备了40 X 100 X 目标和落色卢比发光在488 nm和561牛米
三轴微操纵器(成茂,型号:UMM-3FC)
生物破坏者(Diagenode,型号:UCD-200TM-EX)
温控器(Eppendorf,型号:5350)
热板(Stuart,型号:US152)
电源30 V,5A (Blausonic,型号:FA-350)
 


软体类


 


斐济(Schindelin 等人,2012)
Excel(或其他绘图和拟合软件)
 


程序


 


电磁体的构造和校准
为了施加受控力,我们设计了一种类似于Kollmansberger和Fabry (Kollmannsberger and Fabry,2007)中描述的电磁体。电磁体由三部分组成:Mu- 金属棒(具有很高磁导率的软铁磁合金),用铝制成的散热器散发热量,用铜线连接电流(图1A和1B)。将Mu金属棒磨成圆锥形尖端,然后进行扩散退火(这两种操作都可以在Sekels进行)厂),一个定制生产的45毫米长的铝合金缸体–内径为4.5 mm 的Mu- 金属棒用于散发磁铁产生的热量。圆柱体的外径为200 mm。靠近一端,四个,宽1 mm ,深40 mm。在圆柱体上隔开2mm的距离,以增加散热器的表面。从圆柱体的另一端到另外的5mm处铣削另一个220mm宽的直径100mm的槽,容纳100个铜线线圈。在两个末端,三个螺杆在120定位塑料° 间隔和使用了-保持Mu金属。杆(FIGUR Ë 小号1C和1 d)。


D:\重新格式化\ 2020-3-2 \ 1902737--1344JérômeSolon 793986 \图jpg \图1.jpg


1图。电磁铁设置为强制的应用。阿。照片的电源(大号EFT)和电磁铁安装在微操作。乙。照片的电磁铁安装在台旋转盘显微镜中E. C. 关闭了电磁体,左箭头表示锥形的Mu金属棒,中心箭头表示散热器周围的铜线圈,右箭头表示铝散热器D.表示铝散热器设计的示意图,a = 160 mm ,b =120mm ,c = 220mm ,d =160mm,e = 200mm 。


 


在铜线圈中施加直流电后,磁场从尖端散发出来并在空间上衰减,位于磁场中的磁珠以与磁场梯度成比例的力被吸向尖端(Kollmannsberger和Fabry, 2007)。


为了校准施加在磁珠上的力(与距磁铁的距离的函数),我们开发了一种使用嵌入PDMS的磁珠的测定法,由于其粘度是已知的,因此可以通过在施加磁场时测量PDMS中磁珠的速度来进行测定。 ,我们可以根据距尖端的距离来计算力场:斯托克定律直接将力与速度相关联(图2和视频1)。






D:\重新格式化\ 2020-3-2 \ 1902737--1344JérômeSolon 793986 \图jpg \图2.jpg


图2 ,建设的校准室。a和b 。顺序折叠一张长方形的封口膜,虚线表示折叠遗址。Ç 。虚线表示切割位置生成一个窗口。d 。封口膜转移到盖玻片A.ê 。照片的校准室安装在金属滑轨。


 






1视频。校准力施加在4.5 Myuemu珠。(左)视频经过时间4.5 Myuemu中磁珠嵌入在PDMS移向磁铁在磁场和各自的跟踪(右)应用。


 


将5μlDynabeadads放入1.5 ml Eppendorf管中,并将其置于37°C,在Thermomixer中打开盖子几个小时,以完全干燥珠子。
加入1 ml PDMS ,并用移液器吸头搅拌,以从底部分离出沉淀的珠子。
该Bioruptor使用,小号Onicate最大功率10分钟。如果珠不是均匀重悬浮重复超声镨Ocedure。
切成一片10 厘米x 3厘米的矩形胶片,然后如图2所示折叠。
使用切割器在折叠的Parafilm中心打开一个5 x 10 mm的窗口。
将Parafilm转移到盖玻片的顶部,然后在加热板上加热,直到Parafilm正确附着在盖玻片上而不会熔化。
将50μl的珠子/ PDMS(来自步骤A3 )转移到Parafilm窗口中。
使用一滴硅脂将盖玻片安装到镜架Leica上。
在旋转圆盘显微镜上,使用40倍透镜并聚焦在微珠上。
将电磁体移到视场的一侧(视频1)。
使用透射光和20 ms的曝光时间开始延时拍摄。
启动强制应用程序。
 


将4.5μm小珠注入果蝇胚胎的步骤
单磁珠被注入蛋黄的胚胎在第2阶段果蝇发展(图小号3 - 4)注射后,盖玻片支持胚胎留给开发超过钕磁铁,将持有的珠在胚胎皮质。因此,在细胞化时,珠子会自然封装到一个新成立的细胞。通过选择胚胎相对于取向的钕磁铁,可以嵌入粒子在一个特定的细胞群,这将在后面发育成特定的组织或器官。


 


D:\重新格式化\ 2020-3-2 \ 1902737--1344JérômeSolon 793986 \图jpg \图3.jpg


3图。固定化的单个4.5 Myuemu珠在尖端注射针头。中A.注射针放入一滴4.5 Myuemu珠重悬浮于0.1 Pasento PBS / BSA。B.负压(P < 0)是为了固定单个小珠,将其涂在针上。


 


D:\重新格式化\ 2020-3-2 \ 1902737--1344JérômeSolon 793986 \图jpg \图4.jpg


4图。注射的单个4.5 Myuemu珠子进入果蝇胚胎。A和B负压(P < 0)被施加到针为了固定单个珠子在接近的萌芽。c和d 。负压解除插图显示了相应注射步骤的卡通插图,其中,针头用青色表示,针头用黄色表示,胚胎用浅灰色表示。箭头表示由针头内部的负压产生的合力。针。


 


准备pH 9.5的0.1 M硼酸溶液。
用1毫升硼酸溶液洗涤50升4.5微米Dynabeads 3次。
将10μgGBP重悬于400μl硼酸溶液中,并在37 °C 下孵育O / N。
用1毫升硼酸溶液洗涤2次Dynabeads。
重悬于1毫升0.1%BSA(w / v)的硼酸溶液中。
通过将O / N 10 ml庚烷与120 cm魔术胶带温育来准备庚烷胶。
在盖玻片的中间放置一滴100μl庚烷胶,并使其在重力作用下流下,在盖玻片的中部形成一条平行于其长边的粘性表面条纹。
使用次氯酸钠在室温下将30至40个刚产下的胚胎化学去绒毛化90 s,然后在自来水下冲洗90 s,然后将其安装在涂有庚烷胶的盖玻片上。
注意:所有胚胎均应以规则的间隔且彼此平行的方式排成一排,以利于注射。


将胚胎在25°C的培养箱中脱水10-15分钟,以减少僵硬度并避免注射后破裂,然后用一小滴Voltalef油覆盖。请注意,需要根据湿度调整脱水时间在孵化器中。
除了Voltalef油滴,一滴重悬浮于PBS / 0.1%BSA中的40μl珠子还沉积在胚胎附近。
转动微拉器,将热量1的值设置为860,将热值设置为750,然后将玻璃毛细管插入相应的支架中并生成注射针,请注意可以使用不同的牵引参数。拉长到足以有Diamet 尔允许持有磁粉并打入胚胎无坝老化(参见图小号3和4)。
使用微粉碎机将注射针精确打开,以达到3.5μm的内径(见图3)。
针头连接到微型注射器(WPI PV 820)。
针被定位在在下拉含珠(在PBS / 0.1 Pasento BSA),并且通过施加负压,单个珠子被保持在尖端处的针。图3型插画中速率小号的流程。
针支撑移动珠关闭后极胚胎,设定的压力微量注射器至中性只是在注射前,然后,珠可以转移到该中心的胚胎(小号的Ee图4) 。
注射完所有胚胎后,将盖有注射胚胎的盖玻片放在永久磁铁顶部,在25°C下放置2 h。
注意:


为了最大程度地将钕磁铁施加到胚胎的力,将盖玻片和注入的胚胎直接放置在钕磁铁上,而无需显微镜载玻片。
为了最大程度地减少对胚胎造成的损害,需要在发育的早期阶段(第2阶段)注射小珠。为了靶向特定组织,需要将感兴趣的组织面向盖玻片进行固定。要在背侧闭合过程中施加力,必须将胚胎的背侧朝向盖玻片安装,并且在18 ° C 注射后必须使胚胎发育20 h 。
 


成像和施力
用一滴硅脂将盖玻片安装到徕卡镜框滑架上。
在旋转盘上选择40倍透镜,将珠子对准正确的位置聚焦在胚胎上,然后将电磁铁的尖端定位在距所选胚胎150-200μm的位置。选择珠子-磁体距离时必须格外小心为了将胎圈定位在具有较小陡度的力-距离曲线的区域中,以允许对力进行精确估计(请参见图5B和图6)。
设置两个通道的xyz延时成像协议,一个通道将使用488 nm激光获取GFP信号,另一个通道将使用561 nm激光获取珠子的自发荧光。
设置5个z 步距,间隔为1μm,并选择5 s的时间分辨率。
开始播放影片,在打开电源1分钟后,选择0.3 A产生磁场,该磁场会在磁珠上施加115 pn的力。打开电源1分钟,然后在关闭电源后,映,以允许进行1更微小停止电影(图之前小号5A,5 ℃和视频2) 。
 






5.强制应用图上的单个4.5 Myuemu 焊道到果蝇胚盘。阿。延时图像显示在胎圈(品红)在响应于力工序(〜115 PN;开始于0 S)珠粒嵌入到单个细胞的Resille-GFP胚胎。白色箭头指示部队的应用。白色虚线的标志左侧的中珠在时间0s。比例尺= 10 Myuemu; B 。快照在透射光的胚胎含有4.5 Myuemu 珠电磁铁放置并在190 Myuemu。比例尺= 20 Myuemu 。C.珠位移对应于力的应用显示在A.


 


D:\重新格式化\ 2020-3-2 \ 1902737--1344JérômeSolon 793986 \图jpg \图6.jpg


图6. 力距标定图。用于电磁铁标定的力距标图示例。施加在三个单个磁珠上的力随距磁铁尖端距离的函数而弯曲。功率定律适用于该力第三珠的曲线用红色表示。


 






2.兵力投送视频单个单元格在果蝇细胞化。延时录像显示力应用在4.5 Myuemu珠嵌入Resille-GFP表达胚胎。紫色箭头指示当力〜115 PN是应用撒谎比例尺= 10μm 。


 


数据一Nalysis


                           


力标定
使用Fiji打开在校准过程中执行的延时。应用中值滤波器(2像素)以减少噪声。             
注:Ø 疗法类型的滤波器(像高斯模糊滤镜为例),可以使用,唯一的标准是噪音充分降低的背景,正确门槛珠。


阈值以实现二进制电影,并将其转换为8位。
删除影片的缩放比例。
在强制应用(视频1)期间,使用MTrack2插件提取小珠的X和Y坐标。在与插件关联的窗口中定义以下参数:
对象大小T最低O 8 Myuemu 2 (对应于20像素,我们设立)


对象大小为最大120 Myuemu 2 (对应300像素,我们设立)             


最高速度到100             


最小轨道长度(帧)为20


勾选保存结果文件框


刻度线显示路径长度框


勾选显示标签框


勾选显示位置框


勾选显示路径框


MTrack2插件会在每个时间点生成带有跟踪对象的X1和Y1坐标的trackresult文件。
将跟踪结果文件导入Excel或其他数据绘图软件中。如果检测到噪声,则可以将几个连续数据点的时间平均值输入到新的X和Y列中以平滑轨迹。
根据X和Y坐标,我们计算出每个时间点t处磁珠与磁铁的距离D。
 






(等式1)


 


w ^ 这里,


像素大小是像素的大小,单位为µm ,


x bead 是每个时间点上珠子的x值(以像素为单位),


y 珠是每个时间点的y值(以像素为单位),


x magnet 是磁铁的x值(以像素为单位),


y 磁铁是磁铁的y 值(以像素为单位)。


 


计算磁珠的速度v(t)
 


 






(式2)


 


w ^ 这里,


 是电影的时间间隔(以秒为单位)。


然后,可以使用以下斯托克定律在任何时间点计算施加在胎圈上的力F(t)。
 


 






(式3)


 


w ^ 这里,


µ 是嵌入磁珠的介质的粘度,


R 是磁珠的半径,


v(t)是磁珠的速度。


 


根据所获得的和的值,在您选择的绘图软件中生成力的散点图,将其作为相应距离的函数,然后将所产生的散点图与幂律拟合为以下形状:
 


 






(式4)


w ^ 这里并Arufa 为拟合参数(参见图6)。


注意:每个单独的磁珠轨道都可以独立地拟合,然后对提取的拟合参数进行平均,或者可以生成所有轨道的单个拟合。


 


在胚盘中施加力后的胎圈位移分析。
打开施力要胚盘电影斐济与分立的绿色(488纳米)和红色(561 Nm)的通道(小号的Ee视频2)。
求和将所有单个z平面投影到单个影片中,然后应用中值滤镜(内核为2个像素)。
将步骤2和步骤3如“力校准”部分中所述应用到红色通道。
按照“力校准”部分中的步骤进行步骤4,但使用以下参数:
最小物体尺寸为32 µm 2 (在我们的设置中相当于200像素)


物体最大六分之一为400 µm 2 (在我们的设置中相当于2500像素)             


最高速度到100             


最小轨道长度(帧)为20


勾选保存结果文件框


刻度线显示路径长度框


勾选显示标签框


勾选显示位置框


勾选显示路径框


通过将方程式1应用于MTrack2插件生成的X和Y坐标,并将磁体的坐标替换为生成文件中磁珠的初始坐标,我们可以计算在每个时间点施加力时的磁珠位移t(图6)。
为了估算施加到磁珠上的力,我们再次使用等式1测量了从磁体到磁珠初始位置的距离D。然后,使用等式4和先前在校准期间估算的参数,使用了等式4计算了力。
注意:为了将磁体从磁珠上定位190μm,必须使用40倍透镜拍摄参考磁珠-磁体快照(如图5B所示),但是为了更好地显示,必须使用一个100倍镜头(如图2 A和视频2所示)。


 


Acknowled 摹发言:


 


我们感谢Peran Hayes对手稿的讨论和批判性阅读,也感谢FundacióCIM的Roger Uceda的铝制散热器的制造。我们感谢ALMU团队为显微镜提供了帮助,导致这些结果的研究已获得了资金的支持。西班牙经济与竞争力部计划,国家计划BFU2010-16546和BFU2015-68754,“ Centro de Excelencia Severo Ochoa”和EMBL合作伙伴关系,我们感谢CERCA计划/加泰罗尼亚大将军的支持。这项工作得到了由Bioafisika Bizkaia基金会和巴斯克政府巴斯克卓越研究中心(BERC)计划的一部分。


 


竞争利益


 


没有利益冲突或利益冲突。


 


参考文献


 


。Bambardekar,K.,克莱门特,R.,布兰克,O.,Chardes,C。和莱内,PF(2015)直接激光操纵揭示细胞接触的力学体内。国家科学院院刊USA 112(5):1416 -1421。
Campos-Ortega,J . A.和Hartenstein,V.(1985)。Drosophila melanogaster的胚胎发育,柏林,Springer-Verlag。
Colombelli,J.和Solon,J.(2013)。激光纳米外科技术解决的多细胞组织中的力传递。细胞组织研究352(1):133-147。
D'Angelo,A.,Dierkes,K.,Carolis,C.,Salbreux,G.和Solon,J.(2019)。体内力的施加显示了在早期胚胎发生过程中组织快速软化和外摩擦增加 .Curr Biol 29 (9):1564-1571 e1566。
恩格勒,AJ,森,S.,理发师,HL和Discher,DE(2006)矩阵弹性。指导干细胞谱系规范。细胞126(4):677-689。
弗雷,B.,扬科,C.,玉宝,N.,梅斯特,S.,Schlucker,E.,迈耶-Pittroff,R.,Fietkau,R.,赫尔曼,M。和Gaipl,US(2008)。将细胞从生理学方面到压力诱导的细胞死亡,对具有高静水压的真核细胞进行加压处理。Curr Med Chem 15(23):2329-2336。
Godard,BG和Heisenberg,CP(2019)。细胞分裂和组织力学。Curr Opin Cell Biol 60:114-120。
海森堡,Cp和Bellaiche,Y.(2013)。部队在组织形态发生和图案化。细胞153(5):948-962。
Kollmannsberger,P.和Fabry,B.(2007)。带有力反馈的高力磁力镊子在生物应用中的应用。Sci Instrum 78(11):114301。
Lecuit,T.,Lenne,PF和Munro,E.(2011)。细胞和组织形态发生过程中的力产生,传递和整合。Annu Rev Cell Dev Biol 27:157-184。
Northcott,JM,Dean,IS,Mouw,JK和Weaver,VM(2018)。压力感:癌症发展和侵略的机理。Front Cell Dev Biol 6:17。
Serwane,F.,Mongera,A.,Rowghanian,P.,Kealhofer,DA,Lucio,AA,Hockenbery,ZM和Campas,O.(2017)。体内定量分析发育中组织的空间变化力学特性。自然方法14 (2):181-186。
Schindelin,J.,Arganda-Carreras,I.,Frise,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. and Cardona,A.(2012)。斐济:一种用于生物图像分析的开源平台。Nat Methods 9( 7):676-82。
Shivakumar宣读,PC和莱内,PF(2016)。激光消融探头上皮力学d Rosophila 。方法分子生物学1478:241-251。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用:D’Angelo, A. and Solon, J. (2020). Application of Mechanical Forces on Drosophila Embryos by Manipulation of Microinjected Magnetic Particles. Bio-protocol 10(9): e3608. DOI: 10.21769/BioProtoc.3608.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。