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Apr 2019

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Optogenetic Tuning of Protein-protein Binding in Bilayers Using LOVTRAP
LOVTRAP对双层膜蛋白质结合的光遗传学调控   

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Abstract

Modern microscopy methods are powerful tools for studying live cell signaling and biochemical reactions, enabling us to observe when and where these reactions take place from the level of a cell down to single molecules. With microscopy, each cell or molecule can be observed both before and after a given perturbation, facilitating better inference of cause and effect than is possible with destructive modes of signaling quantitation. As many inputs to cell signaling and biochemical systems originate as protein-protein interactions near the cell membrane, an outstanding challenge lies in controlling the timing, location and the magnitude of protein-protein interactions in these unique environments. Here, we detail our procedure for manipulating such spatial and temporal protein-protein interactions in a closed microscopy system using a LOVTRAP-based light-responsive protein-protein interaction system on a supported lipid bilayer. The system responds in seconds and can pattern details down to the one micron level. We used this technique to unlock fundamental aspects of T cell signaling, and this approach is generalizable to many other cell signaling and biochemical contexts.

Keywords: Optogenetics (光遗传学), In-vitro reconstitution (体外重建), Cell signaling (细胞信号), LOV2 (LOV2), Microscopy (显微镜检查), Supported lipid bilayers (支撑脂质双层)

Background

Questions in cell signaling and cell biology commonly focus on how cells sense and respond to their environment. The signaling cascades that carry out these cellular decisions contain proteins that can move nanometers to microns in a matter of seconds to minutes. Some common methods, such as western blots, qPCR and deep sequencing, give us insight into the levels and activities of signaling proteins. However, because these approaches require destruction of the sample to make their measurements, they only give snapshots of the underlying biology. Light microscopy is an attractive complementary technique to fill in these gaps of knowledge by measuring how cells process and propagate signals in space and time.

Existing microscopy techniques can measure cell signaling on a variety of spatial and temporal scales. At the nanometer scale, fluorescence correlation spectroscopy can be used to monitor protein complex formation, and live cell FRET sensors can be used to measure protein conformation and protein-protein interactions. At around 10-100 nm, live cell super-resolution techniques like structured illumination (SIM) and stimulated emission depletion (STED) microscopy can map cellular sub-structures such as the cytoskeleton. Common wide-field and confocal techniques remain a standby to measure organelle-level localization of proteins and overall morphology of the cell. Images can be taken every millisecond to many minutes, depending on the technique and field of view.

Both conventional and super-resolution microscopy techniques are powerful because they capture the diverse temporal and spatial responses cells make. Although we can image cellular signals dynamically in space and time, our ability to manipulate these signals is much more limited. Stamps with micron-sized patterns can deposit molecules of interest onto a coverslip or other flat surface with near arbitrary shapes. Nanolithography techniques can take this further by carving complex 3-dimensional structures into a surface. While these techniques can create a variety of shapes, the shapes themselves cannot be readily manipulated in time and space to mimic the dynamics of actual stimuli. Micropipettes and/or microfluidics can deliver pulses of an extracellular ligand, but they are limited to controlling spatial distribution and are less suited to manipulate surface-presented ligands. For a better understanding of how cells sense and respond to their environments, we need new techniques to control stimuli in both space and time.

Cells commonly sense their environment through protein-protein interactions at the cell membrane. In this protocol, we mimic these cell-presented ligands using a light-gated protein-protein interaction system on a supported lipid bilayer (Figure 1). Light controllable protein-protein interactions are also powerful for studying biochemical reactions that occur close to a membrane, such as the phospho-inositol cycle or Ras signaling (Toettcher et al., 2011 and 2013).

Our approach for manipulating signals in bilayers is based on LOVTRAP, a light-induced protein dissociation technique (Wang et al., 2016) that consists of a naturally light-sensitive protein (the LOV2 domain of Avena sativa phototropin I) and an engineered binding partner, Zdk. In the ground state, Zdk binds to LOV2 with high affinity. When LOV2 is excited by blue light, it changes conformation, causing Zdk to dissociate. Active LOV2 slowly relaxes to the ground state, allowing Zdk to rebind and reset the system. By controlling when and where blue light is delivered, micron-sized patterns of a protein-protein interaction can be generated and altered in seconds. Because LOV2 is insensitive to red and infra-red light, this system is compatible with biosensors in these channels for microscopy-based quantification of the perturbation and cell response.


Figure 1. Diagram of the light-controlled protein-protein interaction system. The light-sensitive protein LOV2 is biochemically purified and attached to a supported lipid bilayer (SLB). Its binding partner, Zdk, binds to LOV2 in the dark and dissociates upon illumination with blue light. Zdk can be free in solution (as diagrammed here) or attached to a cell surface receptor to control cell signaling (as was done in Tischer and Weiner, 2019). In biochemical reconstitution, Zdk can be fused to a protein whose activity depends on membrane proximity, such as a lipid kinase or GTPase regulator.

We previously leveraged our optogenetic approach to stimulate the T cell signaling with precise temporal control, revealing that T cells measure the dynamics of ligand binding in their decision to activate (Tischer and Weiner, 2019). Ligands with longer binding half-lives signal disproportionately better than ligands with short binding half-lives, even when controlling for receptor occupancy. Such a conclusion was only possible because the optogenetic system allowed us to directly manipulate the variable of interest: protein-protein interaction half-life. This direct manipulation was not possible with existing experimental techniques. Because of the general nature of protein-protein interactions in initiating cell signaling, we detail our procedure here in the hopes that our approach will enable a powerful interrogation of other signaling systems.

Materials and Reagents

  1. Deep well 96-well block or alternative method for collecting fractions from an FPLC
  2. 0.22 μm filter
     For large volumes: Millipore, catalog number: SE1M179M6
    For small volumes: Millipore, catalog number: SLGV033RS
  3. Glass coverslips for flow chamber (Ibidi, catalog number: 10812 )
  4. Tin foil
  5. Aluminum foil
  6. 1.5 ml Eppendorf tubes
  7. PCR tubes (Sigma-Aldrich, catalog number: CLS3744 )
  8. Plastic Pasteur pipettes or plastic tubing (Celltreat Scientific, catalog number: 229276 )
  9. Plastic tubing
  10. Superdex 200 Increase 10/300 GL column (Sigma-Aldrich, catalog number: GE28-9909-44 )
  11. HiPrep 26/10 desalting column (GE Healthcare, catalog number: 45-000-266 )
  12. Glass vials (Thermo Fisher Scientific, catalog number: B7800-2 )
  13. Tongs or sturdy wire to transfer Coplin jars from a hot water bath.
  14. Pasteur pipettes (Thermo Fisher Scientific, catalog number: 13-678-20A )
  15. Plasmid for expressing LOV2 (pDT552; From Tischer and Wiener, 2019)
    This plasmid expresses the fusion protein 10xHis-TEV-AviTag-KCK-LOV2(V529N). Here, V529N denotes a mutation from the full length, wt phototropin 1 protein from Avena sativa. LOV2 refers to the second LOV domain of this protein. TEV denotes a cleavage site for the tobacco etch virus protease. AviTag is a 15 amino acid tag that can be biotinylated by the E. coli biotin ligase BirA. KCK is a tripeptide tag that makes the cysteine a stronger nucleophile; this helps to preferentially label this site over the LOV2 active site cysteine with maleimide-conjugated dyes. This construct is cloned into a modified pETM11-SUMO3 plasmid backbone.
  16. Plasmid for expressing Zdk, (pDT482; From Tischer and Wiener, 2019)
    This plasmid expresses the fusion protein 10xHis-SUMO3-KCK-SpyCatcher-Zdk1. SUMO3 is the human SUMO3 domain recognized by the SenP2 protease, which cleaves at the C-terminus of the domain. KCK is a tripeptide tag that makes the cysteine a stronger nucleophile; this makes the cysteine more reactive to maleimide-conjugated dyes. The SpyCatcher domain is not used for the current application but permits post-translational fusion to Spy-tag containing targets where desired. Zdk1 is the engineered binding partner to LOV2. This construct is cloned into a modified pETM11-SUMO3 plasmid backbone.
  17. Liquid nitrogen
  18. LB broth (Sigma-Aldrich, catalog number: L3522 )
  19. Kanamycin (Goldbio, catalog number: K-120-10 )
  20. Isopropyl-β-D-thiogalactoside (IPTG; Sigma-Aldrich, catalog number: I6758 )
  21. Biotin (Sigma-Aldrich, catalog number: B4639 )
  22. Flavin mononucleotide (FMN; Bio-Rad, catalog number: 161-0501 )
  23. Methanol (Sigma-Aldrich, catalog number: 179337 )
  24. Flow chamber sticky tops (Ibidi, catalog number: 80608 )
  25. POPC (Avanti Polar Lipids, catalog number: 850457C )
  26. PEG-PE (Avanti Polar Lipids, catalog number: 880230C )
  27. Biotinyl CAP PE (Avanti Polar Lipids, catalog number: 870277X )
  28. Alexa Fluor 488 C5 Maleimide (Thermo Fisher Scientific, catalog number: A10254 )
  29. Sulfo-Cyanine3 maleimide (Lumiprobe, catalog number: 11380 )
  30. Flavin mononucleotide (Bio-Rad, catalog number: 161-0501 )
  31. Anhydrous DMSO (Thermo Fisher Scientific, catalog number: D12345 )
  32. Chloroform (Electron Microscopy Sciences, catalog number: 12550 )
  33. DPBS (Thermo Fisher Scientific, catalog number: 14190144 )
  34. Hellmanex III (Sigma-Aldrich, catalog number: Z805939 )
  35. Acetone (Sigma-Aldrich, catalog number: 534064-4L )
  36. 190 proof ethanol (Koptec, catalog number: V1101 )
  37. KOH (Fisher, catalog number: P251 )
  38. Hydrogen peroxide (Fisher Scientific, catalog number: H325-500 )
  39. HCl (Acros, catalog number: 42379-5000 )
  40. Streptavidin (Rockland, catalog number: S000-01 )
  41. ProLong Live Antifade Reagent (Thermo Fisher Scientific, catalog number: P36975 )
  42. Roche cOmplete Mini, EDTA-free protease inhibitor tablet (Sigma-Aldrich, catalog number: 11836170001 )
  43. NaOH (Fisher, catalog number: 19232 )
  44. KH2PO4 (Sigma-Aldrich, catalog number: P5379 )
  45. K2HPO4
  46. NaCl (Sigma-Aldrich, catalog number: 793566 )
  47. Beta-mercaptoethanol (βME; Fisher, catalog number: 34461 )
  48. Imidazole (Fisher, catalog number: O3196-500 )
  49. N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES; Sigma-Aldrich, catalog number: H3375-250G )
  50. KCl (Fisher, catalog number: S77375-1 )
  51. Tris(2-carboxyethyl)phosphine (TCEP; Thermo, catalog number: 20491 )
  52. Tryptone (BD Biosciences, catalog number: 211705 )
  53. Yeast extract (BD Biosciences, catalog number: 288620 )
  54. Glycerol (Fisher, catalog number: BP229 )
  55. Beta-casein (Fisher, catalog number: 11461422 )
  56. Phenylmethylsulfonyl fluoride (PMSF; Pierce, catalog number: 36978 )
  57. 6xHis-TEV protease (Prepared recombinantly from E. coli in house. Commercial substitutes are easy to come by e.g., Sigma-Aldrich, catalog number: T4455-1KU )
  58. Protein concentrator (Vivaspin 20 MWCO 10,000; GE Life Sciences, catalog number: 28932360 )
  59. Dithiothreitol (DTT; Sigma-Aldrich, catalog number: D9779 )
  60. 6xHis-SenP2 protease (Prepared recombinantly from E. coli in house. Commercial substitutes are available e.g., Novus Biologicals, catalog number: E-710-050 )
  61. Compressed inert gas (Compressed nitrogen; Airgas, catalog number: NI 200 )
  62. Ethylenediamine tetraacetic acid (EDTA; Fisher Scientific, catalog number: S80007-1 )
  63. Cobalt(II) chloride hexahydrate (CoCl2·6H2O; Sigma-Aldrich, catalog number: 255599 )
  64. Clear nail polish
  65. IMAC binding buffer (see Recipes)
  66. IMAC elution buffer (see Recipes)
  67. HEPES buffered saline (HBS; see Recipes)
  68. Terrific Broth (TB; see Recipes)
  69. DPBS-BB (see Recipes)

Equipment

  1. 500 ml flask
  2. 2.8 L Baffled flask
  3. Heat block
  4. 1 L glass beaker
  5. Temperature controlled shaker, capable of 37 °C to 18 °C
  6. EmulsiFlex-C3 (or some other method of cell lysis)
  7. AKTA pure 25 (GE Healthcare, or similar FPLC system)
  8. 5 ml HiTrap Chelating column (GE Healthcare, catalog number: 17040801 )
  9. Milli-Q water purification system (or similar)
  10. Hamilton syringes (Hamilton Company, Gastight 1700 series, catalog numbers: 80265 and 81165 )
  11. Glass Coplin jar (Sigma-Aldrich, catalog number: BR472800 )
  12. Peristaltic pump and tubing
  13. Vacuum desiccator
  14. Dounce homogenizer
  15. Bath sonicator
  16. Tabletop ultracentrifuge 
  17. Floor ultracentrifuge (Beckman Coulter, model: Optima L-90K ) with Ti-45 rotor
  18. Swinging bucket floor centrifuge
  19. 470 nm LED (Lightspeed Technologies Inc., model: HPLS-36 )
  20. Inverted epifluorescence microscope, ideally capable of Total Internal Reflection (TIRF) microscopy (Nikon Eclipse Ti inverted microscope)

Software

  1. Microsoft Excel (Microsoft)
  2. Fiji (Schindelin et al., 2012)
  3. Micromanager (Edelstein et al., 2014)

Procedure

  1. LOV2 purification
    1. In a 500 ml flask, inoculate 200 ml of LB Kan (30 μg/ml) with E. coli BL21(DE3) transformed with a bicistronic plasmid expressing LOV2 and BirA (plasmid pDT552). Grow overnight (> 8 h) at 37 °C shaking at 225 rpm.
    2. Back-dilute 100 ml of the overnight culture into 1 L of TB Kan (30 μg/ml) in a 2.8 L baffled flask. Shake at 180 rpm at 37 °C until the OD600 (optical density at 600 nm) is between 0.5-0.6.
    3. Reduce the temperature to 18 °C, wait 30 min for the media to cool near to 18 °C, and induce expression with IPTG (250 μM, final concentration). From stock solutions, bring biotin to a final concentration of 50 μM and flavin mononucleotide (FMN) to 1 mM. Grow overnight.

    Note: Carry out all future steps on ice or at 4 °C.
    1. Pellet cells in a swinging bucket centrifuge (or similar) pre-chilled to 4 °C. Discard the supernatant. A typical spin is 800 RCF for approximately 10 min. The pellet usually appears noticeably yellow due to the high expression of LOV2 and the FMN cofactor.
    2. Use a scale and an empty centrifuge container to estimate the mass of the wet cell pellet. Add 2 ml of IMAC binding buffer for every 1 g wet cell pellet, and vigorously pipette up and down to crudely suspend the majority of the pellet.
    3. Transfer the cell suspension (some chunks are okay) to a dounce homogenizer that is surrounded by ice and add one Roche cOmplete Mini, EDTA-free protease inhibitor tablet. Thoroughly resuspend the cell pellet until it is completely homogenized (typically about around 10 plunges up and down).
    4. Immediately before lysing the cell on an EmulsiFlex-C3, add PMSF (phenylmethylsulfonyl fluoride) to a final concentration of 1 mM. PMSF has a half-life in aqueous environments of around 30 min, so it is important to add PMSF near the time of cell lysis. Make sure your PMSF stock is prepared in anhydrous methanol. If you think water might have gotten into your stock, make fresh PMSF. It is optional, but not required, to run the lysate through the EmulsiFlex a second time to reduce its viscosity. This can make the subsequent loading onto the IMAC column faster.
    5. Spin the lysate in an ultracentrifuge in a Ti-45 rotor at 40k rpm for one hour at 4 °C. Decant and retain the supernatant.
    6. Use a peristaltic pump to recirculate the supernatant over a 5 ml HiTrap Chelating column charged with Co2+ and equilibrated with the IMAC binding buffer. In the recirculation setup, the eluate from the column should gently drip back into the reservoir of supernatant that feeds the IMAC column. Make sure the inlet tube of the peristaltic pump is at the bottom of the reservoir so that all the supernatant will flow over the column. Estimate the flow rate by capturing some liquid in a 20-30 s interval. 1.5-2.0 ml/min is typical. Load the column for a duration that enables the entire supernatant to pass over the column twice. The column will turn visibly yellow from the top down as LOV2 binds the column.
    7. Transfer the loaded column to an AKTA and wash with IMAC binding buffer until the A280 of the effluent is less than 60 mAu. Elute the protein with a 10-column volume gradient (going from IMAC binding buffer to IMAC elution buffer) and collect in 1.5 ml fractions in a deep-well 96-well plate. After elution, equilibrate the column in IMAC binding buffer.
    8. Pool visibly yellow fractions in the 96-well block and then buffer exchange into IMAC binding buffer using a HiPrep 26/10 desalting column.
    9. Add purified 6xHis-TEV protease 1:10 w/w (weight to weight) TEV:LOV2 and incubate overnight at 4 °C covered in tin foil.
      Note: TEV protease was purified in house, but can be obtained commercially.
    10. Recirculate the digested LOV2 mixture over a HiTrap Chelating column (this can be the one used previously to elute LOV2 after equilibrating with IMAC binding buffer) with a peristaltic pump as before to remove the TEV protease, free His-tags, and any uncut LOV2. Recirculating at least twice over the column.
    11. Collect the flow-through and exchange into HEPES buffered saline (HBS) with the desalting column.
    12. Concentrate to approximately 4 mg/ml with a Vivaspin protein concentrator.
    13. Make a fresh 10 mM stock of Alexa Fluor 488 C5 Maleimide in anhydrous DMSO.
    14. On ice, add the dye to the protein at a final molar ratio of 2:1 dye:LOV2 and let the reaction proceed for 30 s.
    15. Quench with DTT at a final concentration of 10 mM. This procedure preferentially labels the N-terminal KCK tag (65) over the active site cysteine of LOV2. Restricting the final volume after quenching to under 500 μl is convenient, as you can proceed to gel filtration without concentrating the protein further.
    16. On an AKTA, run the quenched dye/LOV2 mixture over a Superdex 200 Increase 10/300 GL column equilibrated with HBS. Collect 1.5 ml fractions in a deep-well 96-well block.
    17. Pool fractions that have a high (> 100 mAu) A280 and are visibly yellow and concentrate to approximately 1.5 mg/ml using a Vivaspin protein concentrator.
    18. Add glycerol to a final volume of 10%. 
    19. Make 3 μl aliquots in PCR tubes and snap freeze in liquid nitrogen.
    20. Store at -80 °C. The aliquots have shown no signs of reduced activity after > 2 years stored at -80 °C.

  2. Zdk purification
    Purify and label Zdk in an identical manner to LOV2, except for the following changes.
    1. Do not supplement the bacterial growth media with biotin or flavin mononucleotide.
    2. Add the SUMO protease 6xHis-SenP2 to the IMAC eluted protein at 1:1,000 w/w instead of TEV protease (6xHis-SenP2 was purified in house, but can be obtained commercially).
    3. Label the protein with Sulfo-Cyanine3 maleimide instead of Alexa Fluor 488 C5 Maleimide.

  3. Cobalt column charging procedure
    This charging protocol assumes the column begins stored in 20% ethanol. Throughout the procedure, be sure to capture any effluent that could contain cobalt(II) for proper waste disposal.
    1. Attach plastic tubing to the peristaltic pump and flush the tubing with ddH2O.
    2. Attach the plastic tubing to the IMAC column, taking care not to introduce any air bubbles.
    3. Remove the storage solution by flushing the column with 5-10 column volumes of ddH2O. Typical flow rates are between 1.5-2.0 ml/min.
    4. Pause the pump and switch the tubing intake to a 100 mM CoCl2 solution that has been passed through a 0.22 μm filter. Take care not to introduce bubbles into the line. Turn the pump back on.
    5. Flow the cobalt solution over the column until the column and the effluent are visibly pink. 
    6. Pause the pump and let the column incubate in the cobalt solution for 10 min.
    7. Switch the tubing intake back to ddH2O and turn on the pump. Wash the column with at least 10 column volumes of ddH2O. It is important to completely remove any traces of free cobalt(II), as cobalt(II) is easily reduced in buffers with reducing agents, causing it to precipitate and clog the column.
    8. Switch the tubing intake to the desired buffer (that has been passed through a 0.22 μm filter) and wash the column with 5 column volumes.
    9. The column is now ready to use.

  4. Column stripping, cleaning and storage
    Throughout the procedure, be sure to capture any effluent that could contain cobalt(II) for proper waste disposal.
    1. Attach the column to a peristaltic pump and tubing that has been flushed with ddH2O. Wash the column for 10 column volumes of ddH2O.
    2. Switch the tubing intake to a 1 M EDTA solution (that has been passed through a 0.22 μm filter) and flow over the column until it is visibly white again.
    3. Switch the tubing intake into ddH2O and wash for 5 column volumes.
    4. Switch the tubing intake into a 20% ethanol solution (that has been passed through a 0.22 μm filter) and wash for 5 column volumes.
    5. Cap both ends of the column and store at 4 °C.
    Note: For a more thorough cleaning of the column, it can optionally be treated with 1 M NaOH to hydrolyze any remaining protein. After Step D3 above, equilibrate the column with 1 M NaOH and let stand for 1 h. Wash with 5 column volumes of ddH2O and proceed to Step D4.

  5. Glassware cleaning
    SUV preparation and glassware cleaning protocols are modified from those previously published (Taylor et al., 2017).
    1. Add 8 glass vials (without the caps) and 8 Pasteur pipettes to a 1 L glass beaker and cover with 3 M NaOH. Place the beaker in a bath sonicator for 30 min.
    2. Decant the NaOH (it can be saved to future glass cleaning) and wash the glassware 5 times with ddH2O by filling the beaker and gently agitating.
    3. Cover the glassware with a 5% (volume/volume) Hellmanex III solution and incubate overnight (> 8 h).
    4. Decant the Hellmanex III solution and extensively wash the glassware with ddH2O. It is critical to remove all traces of the Hellmanex detergent from glassware. To thoroughly wash with ddH2O, attach a plastic pasteur pipette (or some small tubing that can fit inside the glass vials) to the ddH2O source with plastic tubing. Continually rinse the inside of the vial by holding it up-side-down and placing the plastic Pasteur pipette inside as far from the neck as possible. Rinse for approximately 30 s or until well after the water no longer makes bubbles. Also wash the outside of the vials extensively. Similarly, aggressively wash the inside and outside of each glass Pasteur pipette. The inside of the glass Pasteur pipettes can be effectively rinsed by threading the end of the glass Pasteur pipette just inside of the end of the plastic Pasteur pipette (you may have to trim the end of the plastic Pasteur pipette with scissors a bit) to ensure a continuous rinsing with water. For both the vials and the glass Pasteur pipettes, you may have to alternate between rinsing the inside and outside of the vial to ensure there is no cross contamination of the Hellmanex solution.
    5. Remove excess water by blow-drying with compressed nitrogen, air, or other compressed inert gas.
    6. Gently wrap the glassware in aluminum foil (to keep out dust) and set on an 80 °C heat block (or use a glass drying oven) for several hours until completely dry.
    7. Store glassware in a clean beaker covered with aluminum foil to protect from dust.
    8. To clean the plastic vial caps, sonicate in a beaker of ddH2O for 30 min, then dry and store in the same manner as the vials and glass Pasteur pipettes.

  6. Preparation of small unilamellar vesicles (SUVs)
    1. Rinse an NaOH-cleaned 4 ml glass vial with chloroform by adding approximately 500 μl chloroform with an NaOH-cleaned Pasteur pipette. Swirl several times to coat the inside of the vial and discard the chloroform. Repeat once more.
    2. Add approximately 500 μl of chloroform to the rinsed vial.
    3. Using Hamilton syringes, add 4 μmoles total lipids in a molar ratio of 97.5% POPC, 0.5% PEG-PE, and 2% biotinyl CAP PE to the vial. It is useful to dedicate a single Hamilton syringe to handle each lipid type, to reduce the change of cross contamination.
    4. Remove the chloroform by rotating the vial at an angle while slowly flowing nitrogen gas (or other inert gas) into the top. Once all visible solvent is evaporated (there should be white rings of residue on the inside of the vial), loosely cover the vial with a pre-washed cap and place in a vacuum desiccator overnight.
    5. The next day, rehydrate the lipids by adding 1.5 ml of 0.22 μm filtered DPBS, firmly screw on the cap, and gently vortex for 10 min. The vial can be secured to a benchtop vortexer by placing it vertically in the middle and running a piece of tape from under the vortex head, over the vial cap, and back down to the underside on the opposite side of the vortex head. Repeat with another piece of tape at 90 degrees to the first one. The vortexing speed is important. It should be no faster than is necessary to make the DPBS reach the neck of the vial so that all of the lipids can rehydrate. The vortexing is too fast if there are bubbles after 10 min of vortexing; this risks oxidizing the lipids.
    6. Once rehydrated, split the mixture equally between two 1.5 ml Eppendorf tubes and close under streaming nitrogen or other inert gas.
    7. Carry out 20 freeze-thaw cycles by moving the tubes between a dewar of liquid nitrogen and a 42 °C water bath or heat block. The mixture should turn from cloudy to noticeably clearer. If the solution doesn’t increase in clarity, it likely means the large lipid aggregates and multi-lamellar vesicles are not being broken up into SUVs. Since the PEG-PE helps the lipid aggregates to break up into SUVs, a cloudy solution could indicate either too much or too little PEG-PE was used or that the lipid has gone bad. Consider ordering new PEG-PE and/or double checking the correct molar amount of PEG-PE was used.
    8. Spin the lipid mixture at 60k RCF for 40 min at 4 °C in a tabletop ultracentrifuge. There will likely be a small, glassy pellet at the bottom of the tubes.
    9. Remove the supernatant to a 1.5 ml Eppendorf tube and store in liquid nitrogen until ready for use.

  7. RCA cleaning of microscopy coverslips
    1. Place one glass coverslip into each slot of a glass Coplin jar, cover with acetone and place in a bath sonicator for 10 min.
    2. Decant the acetone and repeat with 190 proof ethanol.
    3. Decant the ethanol and repeat with ddH2O. Fill and decant the Coplin jar 5 times with ddH2O before and after the ddH2O sonication step to remove excess organic solvents.
    4. Heat 1 L of ddH2O in a microwave (or hotplate) to approximately 70 °C. The warmed water will help prevent the Coplin jar from cracking from temperature stress during the upcoming washes.
    5. Separately, prepare a 70-80 °C water bath with enough water to submerge most of the Coplin jar without overrunning the lip.
    6. Dissolve 3.75 g KOH into 45 ml of the warmed ddH2O and add to the coverslips. Then add 15 ml 30% hydrogen peroxide.
    7. Use tongs or wire securely wrapped around the neck of the Coplin jar to transfer it to the water bath and let it react for 12 min: approximately 2 min for the solution to get up to temperature and 10 min for the reaction to proceed. The solution will constantly produce many small bubbles once it warms up.
    8. Remove the Coplin jar from the water bath. Carefully decant the base solution and wash 5 times with the warmed ddH2O.
    9. Add the following components in order to the Coplin jar: 38 ml warmed ddH2O, 9.5 ml 37% HCl and 12.6 ml 30% hydrogen peroxide.
    10. Incubate in the water bath for 12 min as before. The solution will bubble similarly to the base solution, but slightly less strongly.
    11. Remove the Coplin jar from the water bath, decant the acid solution, and wash 5 times with the warmed ddH2O. Cover the coverslips with warmed ddH2O, add the Coplin jar lid, and store at room temperature for up to one week.

  8. Forming SLBs
    While drying and preparing the glass coverslips, take care to only handle the edges and avoid touching any part near the center.
    1. Use forceps to remove a glass coverslip from the ddH2O in the Coplin jar. Hold it in one gloved hand by pinching between the two long sides and immediately blow dry with compressed nitrogen (or compressed air or other inert gas). The reflection off the coverslip should be spotless.
    2. Stack a sheet of microscopy lens paper on top of two paper towels and place the coverslip on top.
    3. Firmly press a six-well Ibidi sticky chamber onto the coverslip. Use very firm pressure to ensure contact, especially between the channels and between the ends of the channels and the sides of the coverslip.
    4. Seal the interface along the long edges of the coverslip with clear nail polish. The seal helps prevent any solution in the channels from wicking to the sides and evaporating during long microscopy time lapse experiments.
    5. Dilute a 30 μl aliquot of SUVs with 800 μl 0.22 μm filtered DPBS and evenly distribute between the six wells.
    6. Incubate at 37 °C for one hour. Once the SUVs are added, take care to never let the channel dry out during the subsequent washing and functionalization steps.
    7. Proceed the SLB functionalization

  9. Functionalizing SLBs
    1. To functionalize a well, flush out excess lipids with 500 μl of 0.22 μm filtered DPBS. It is useful to push the liquid in one end with a pipette and carefully aspirate from the other end (Figure 2).


      Figure 2. Exchanging solutions during SLB preparation and functionalization. Diagram of how to exchange solutions in the channels of the Ibidi chamber. In order to have an efficient exchange of solutions in the channels of the Ibidi chamber while keeping the SLB hydrated, it is useful to simultaneously push new solutions in one end and aspirate out the other. Keeping the tip of the aspirator high in the exit, above the top of the intervening channel, helps ensure liquid is only removed as a new solution is being pushed in by the pipette. Adding a small diameter pipette tip to the end of the aspirator can help moderate the suction to avoid over-aspirating the channel.

    2. Flush the well with 200 μl of streptavidin diluted into DPBS-BB (2 μg/ml final) and incubate at room temperature for 5 min.
    3. Flush the well with 500 μl DPBS-BB.
    4. Flush the well with 200 μl of LOV2 diluted into DPBS-BB to the desired concentration (typically between 20-200 nM) and incubate at room temp for 5 min.
    5. Finally, flush the channel with 500 μl of DPBS-BB. The SLB is now functionalized with LOV2.

    Regardless of the intended use of the LOV2-functionalized SLBs, it is important that the solution the experiment takes place in has been pretreated with an oxygen scavenger. This helps prevent blue light from bleaching the LOV2 chromophore and dramatically increases the possible rounds of recruitment and release of Zdk to LOV2. One method that works well is to dilute ProLong Live Antifade Reagent 1:100 into the solution in which microscopy will be performed and let it incubate at room temp for at least 90 min for the dissolved oxygen to be depleted. It is useful to begin the oxygen scavenger treatment before functionalizing the SLBs so that the solution is ready by the time the SLBs are fully functionalized.

  10. Microscope
    A microscope capable of TIRF (total internal reflection fluorescence) microscopy is necessary for quantifying Zdk recruitment and release from a LOV2-functionalized bilayer and is a natural choice for imaging a resulting change in cell signaling or of a biochemical reaction.
      We imaged using an Eclipse Ti inverted microscope (Nikon) with two tiers of dichroic turrets to allow simultaneous fluorescence imaging and optogenetic stimulation. The microscope was also equipped with a motorized laser TIRF illumination unit, a 60x Apochromat TIRF 1.49 NA objective (Nikon), an iXon Ultra EMCCD camera, and a laser merge module (LMM5; Spectral Applied Research) equipped with 405-, 440-, 488-, 514-, and 561-nm laser lines. The microscope and associated hardware were controlled with MicroManager (Edelstein et al., 2014) in combination with custom-built Arduino controllers (Advanced Research Consulting Corporation). Blue light for optogenetic stimulation was from a 470 nm LED independently controlled with MATLAB.

  11. Checking bilayer fluidity
    Before performing experiments, it is important to confirm that your bilayers are functionalized with LOV2 and fluid. This is easily done with a fluorescence recovery after photobleaching (FRAP) experiment. One can use almost any fluorescent imaging modality (e.g., TIRF, confocal, wide field) to check that the bilayer is functionalized with LOV2.
    1. Load an SLB functionalized with a saturating amount of fluorescently labeled LOV2 onto the microscope and use a 60x or 100x objective to focus at the glass-water interface. It may be useful to dope in a small concentration of fluorescent beads to make locating this interface easier.
    2. Close down the episcopic field diaphragm so that only part of the field of view is illuminated. There should be an obvious increase in fluorescence inside the illuminated region if the bilayer is functionalized with fluorescently labeled LOV2.
    3. A qualitative FRAP measurement is usually all that is needed to assess bilayer fluidity. While it is useful to have a proper FRAP system or high power TIRF laser, it is sometimes possible to use a lower-powered light source (such as a wide field epifluorescence light source) if neither is available. In such a case, it is useful to label the LOV2 with a fluorophore like FITC (fluorescein isothiocyanate) that is easy to bleach. Not treating the imaging buffer with an oxygen scavenger also makes fluorophores easier to bleach.
    4. Bleach a small region of the field of view with the method of your choice. If you are not using a proper FRAP system (e.g., using a TIRF laser or wide field epifluorescence light source instead), close down the episcopic field diaphragm so only a small portion of the field of view is illuminated. If using a TIRF laser, point it straight up. For non-FRAP systems, the power will likely need to be increased to its maximum. The time it takes to bleach the area of interest will vary depending on the intensity of the light source, the small molecule fluorophore attached to LOV2, and the imaging media, among other factors. Bleaching times longer than 30 seconds are generally not effective, as diffusion in the bilayer (if it is fluid) will equilibrate LOV2 between the illuminated and non-illuminated regions.
    5. Image the entire field of view every 5-10 s for the next 2-3 min. (Open the episcopic field diaphragm if it was closed down.) A bleached hole with a diameter of ~5-10 µm should largely recover in ~30-60 s if the bilayer is fluid (Figure 3 and Video 1). No border from the original bleached hole should remain visible.


      Figure 3. Assessing bilayer fluidity with FRAP. A small hole was bleached into a bilayer functionalized with fluorescent LOV2 using a TIRF laser. Because the bilayer is fluid, new molecules of LOV2 diffused into the bleached area, largely returning to its initial state in 60 s. Non-fluid bilayers fail to recover over this time course. Scale bar indicates 1 μm.

      Video 1. Time lapse of a FRAP experiment showing the SLB is fluid and functionalized with LOV2. A small hole was photobleached into an SLB functionalized with fluorescent LOV2. Over the course of about 60 s, the fluorescence signal recovers, owing to new molecules of LOV2 diffusing in from outside the bleached region. A bilayer that was not functionalized with LOV2 would not show a "hole" in the beginning. A bilayer that was functionalized but not fluid would not recover in about 60 s. The exact recovery rate depends on the diameter of the bleached region. Scale bar indicates 1 μm.

  12. Testing LOV2 functionality
    The most direct way to test LOV2 functionality on the bilayer is to use TIRF to measure the recruitment and release of fluorescently labeled Zdk. This test should be performed after you have confirmed the bilayers are functionalized with LOV2 and fluid.
    1. Functionalize an SLB with saturating amounts of LOV2 and flow in imaging media containing 250 nM Zdk that has been treated with the oxygen scavenger. It is best to use the same media you intend to perform your experiments in, but DPBS can serve as a good starting point. Make sure the Zdk is labeled with a dye that is excited by light with a wavelength longer than 560 nm so that you can continuously image it without activating LOV2.
    2. Set up a 10 min time course where Zdk is imaged with TIRF every 5 s. Every 2 min, expose the LOV2 to blue light of around 470 nm for 1 s. Any sort of GFP imaging setting usually works well. Depending on the intensity of blue light, Zdk should be rapidly released from the bilayer in ~1-10 s and then slowly recover over the following 2 min. Similar cycles of release and recruitment should be observed over the next 10 min (Figure 4). If more than a 50% reduction in Zdk recruitment occurs over 10 min, the most likely reason is that the oxygen scavenger treatment was not effective.


      Figure 4. Time course of Zdk recruitment and release from lipid bilayers. Fluorescently labeled Zdk was recruited and released from a LOV2 functionalized bilayer for five cycles. LOV2 quickly releases Zdk after being excited with blue light (470 nm light from an LED) and slowly rebinds after the blue light is removed. (Vertical blue bars indicate the 470 nm LED is on.) Even if the microscope is only capable of periodic blue light illumination (instead of continuous illumination as ours is), the recruitment and release kinetics of Zdk should be very similar.

    For protocols using Zdk on a cell surface receptor to initiate signaling, we refer the interested reader to our previous publication (Tischer and Weiner, 2019). For our previous work with T cell signaling, we were able to make low-dozens of single cell measurements from a single field of view. With three biological replicates, we recorded on the order of ~50-100 single cell measurements per condition, which provided sufficient numbers for statistical significance in our experiments.

Recipes

Media and buffers:

  1. IMAC binding buffer
    50 mM KH2PO4
    400 mM NaCl
    0.5 mM βME
    pH 7.5
  2. IMAC elution buffer
    50 mM KH2PO4
    400mM NaCl
    500 mM imidazole
    0.5 mM βME
    pH 7.5
  3. HBS
    20 mM HEPES
    100 mM KCl
    0.5 mM TCEP
    pH 7.5
  4. Terrific Broth (TB)
    Nutrient Base
    12 g tryptone
    24 g yeast extract
    4 ml glycerol
    900 ml ddH2O

    10x TB Salts
    170 mM KH2PO4
    720 mM K2HPO4
    1. Autoclave the nutrient base and 10x TB salts separately
    2. Then add 100 ml of 10x TB salts to 900 ml of the nutrient base to make 1 L of TB

Buffers for SLB functionalization and imaging:
  1. DPBS-BB
    DPBS (Thermo Fisher Scientific)
    1 mg/ml beta-casein
    0.5 mM βME
    Filter with a 0.22 μm filter immediately prior to use

Acknowledgments

This work was supported by a Genentech Fellowship (D.T.), NIH grant GM118167 and the Novo Nordisk Foundation (O.D.W.) and the Center for Cellular Construction (DBI-1548297), an NSF Science and Technology Center. This protocol was adapted from Tischer and Weiner (2019).

Competing interests

The authors declare no financial or non-financial competing interests.

References

  1. Edelstein, A. D., Tsuchida, M. A., Amodaj, N., Pinkard, H., Vale, R. D. and Stuurman, N. (2014). Advanced methods of microscope control using μManager software. J Biol Methods 1(2): e11.
  2. 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.
  3. Taylor, M. J., Husain, K., Gartner, Z. J., Mayor, S. and Vale, R. D. (2017). A DNA-based T cell receptor reveals a role for receptor clustering in ligand discrimination. Cell 169(1): 108-119 e120.
  4. Tischer, D. K. and Weiner, O. D. (2019). Light-based tuning of ligand half-life supports kinetic proofreading model of T cell signaling. Elife 8: 42498.
  5. Toettcher, J. E., Gong, D., Lim, W. A. and Weiner, O. D. (2011). Light-based feedback for controlling intracellular signaling dynamics. Nat Methods 8(10): 837-839.
  6. Toettcher, J. E., Weiner, O. D. and Lim, W. A. (2013). Using optogenetics to interrogate the dynamic control of signal transmission by the Ras/Erk module. Cell 155(6): 1422-1434.
  7. Wang, H., Vilela, M., Winkler, A., Tarnawski, M., Schlichting, I., Yumerefendi, H., Kuhlman, B., Liu, R., Danuser, G. and Hahn, K. M. (2016). LOVTRAP: an optogenetic system for photoinduced protein dissociation. Nat Methods 13(9): 755-758.

简介

[摘要] 现代显微镜方法是研究活细胞信号转导和生化反应的强大工具,使我们能够观察这些反应的时间和位置,从细胞水平到单个分子。利用显微镜,可以在给定的扰动之前和之后观察每个细胞或分子,比起破坏性的信号定量方法,可以更好地推断因果关系。由于细胞信号传导和生化系统的许多输入源于细胞膜附近的蛋白质-蛋白质相互作用,因此一个巨大的挑战在于控制时间,位置 以及这些独特环境中蛋白质与蛋白质相互作用的程度。在这里,我们详细介绍了在封闭的显微镜系统中使用这种基于时空的蛋白质-蛋白质相互作用系统,在支持的脂质双分子层上使用​​基于LOVTRAP的光反应性蛋白质-蛋白质相互作用系统的程序。系统可以在几秒钟内做出响应,并且可以将细节图案化到1微米级别。我们使用了该技术来解锁T细胞信号传导的基本方面,并且该方法可推广到许多其他细胞信号传导和生化环境。

背景技术细胞信号传导和细胞生物学中的问题通常集中在细胞如何感知和响应其环境上。进行这些细胞决定的信号级联反应包含的蛋白质可以在几秒钟到几分钟的时间内将纳米级移动到微米级。某些常用方法,例如蛋白质印迹,qPCR 和深度测序,使我们可以深入了解信号蛋白的水平和活性。但是,由于这些方法需要销毁样本才能进行测量,因此它们只能提供基础生物学的热点信息。光学显微镜是一种有吸引力的补充技术,可以通过测量细胞如何在空间和时间上处理和传播信号来填补这些知识空白。

现有的显微镜技术可以在各种空间和时间尺度上测量细胞信号传导。在纳米级,荧光相关光谱可用于监测蛋白质复合物的形成,而活细胞FRET传感器可用于测量蛋白质构象和蛋白质-蛋白质相互作用。在大约10-100 nm处,活细胞超分辨率技术(例如结构化照明(SIM)和受激发射损耗(STED)显微镜)可以绘制细胞亚结构,例如细胞骨架。常用的宽视野和共聚焦技术仍然可以用来测量蛋白质的细胞器水平定位和细胞的整体形态。根据技术和视野的不同,可以每毫秒到数分钟拍摄一次图像。

常规和超分辨率显微镜技术都很强大,因为它们可以捕获细胞做出的各种时间和空间响应。尽管我们可以在空间和时间上动态地对细胞信号进行成像,但是我们处理这些信号的能力受到很大限制。具有微米尺寸图案的印模可以将感兴趣的分子沉积到具有几乎任意形状的盖玻片或其他平坦表面上。纳米光刻技术可以通过将复杂的3维结构雕刻到一个表面中来进一步实现这一目标。尽管这些技术可以创建各种形状,但是形状本身在时间和空间上不易操纵以模仿实际刺激的动力学。微量移液器和/或微流控剂可以传递细胞外配体的脉冲,但是它们仅限于控制空间分布并且不适合操纵表面呈现的配体。为了更好地了解细胞如何感知和响应其环境,我们需要新的技术来控制时空刺激。

细胞通常通过细胞膜上的蛋白质相互作用来感知环境。在此协议中,我们在支持的脂质双层上使用光门控蛋白质-蛋白质相互作用系统模拟这些细胞呈递的配体(图1 )。光可控的蛋白质-蛋白质相互作用对于研究膜附近发生的生化反应(例如磷酸肌醇循环或Ras信号传导)也很有效(Toettcher 等人,2011和2013)。

我们在双层中操纵信号的方法基于LOVTRAP,这是一种光诱导的蛋白质解离技术(Wang 等人,2016),它由天然感光蛋白(Avena sativa phototropin I 的LOV2结构域)和工程结合合作伙伴Zdk 。在基态下,Zdk以高亲和力与LOV2 结合。当LOV2被蓝光激发时,它会改变构象,导致Zdk 解离。活动的LOV2缓慢放松到基态,从而允许Zdk 重新绑定并重置系统。通过控制何时何地发出蓝光,可以在几秒钟内生成并改变微米级蛋白质-蛋白质相互作用的模式。由于LOV2对红色和红外光不敏感,因此该系统与这些通道中的生物传感器兼容,可用于基于显微镜的扰动和细胞反应定量分析。



D:\ Reformatting \ 2020-7-1 \ 1902649--1485 Orion Weiner 784707 \ Figs jpg \ fig 1.jpg

图1.光控蛋白-蛋白相互作用系统图。光敏蛋白LOV2经过生物化学纯化,并附着在支持的脂质双层(SLB)上。它的结合伴侣Zdk在黑暗中与LOV2 结合,并在蓝光照射下解离。Zdk 可以在溶液中游离(如此处所示),也可以附着在细胞表面受体上以控制细胞信号传导(如 Tischer和Weiner,2019年)。在生化重建中,Zdk 可以与活性取决于膜邻近性的蛋白质融合,例如脂质激酶或GTPase调节剂。



我们之前利用光遗传学方法通过精确的时间控制来刺激T细胞信号传导,揭示了T细胞在决定激活配体时测量了配体结合的动力学(Tischer and Weiner,2019)。结合半衰期较长的配体比结合半衰期较短的配体更好地发出信号,即使控制受体的占有率也是如此。这样的结论是唯一可能的,因为光遗传学系统使我们能够直接操纵目标变量:蛋白质-蛋白质相互作用的半衰期。使用现有的实验技术无法进行这种直接操作。由于在启动细胞信号转导过程中蛋白质与蛋白质相互作用的一般性质,我们在此详细介绍我们的程序,希望我们的方法能够对其他信号转导系统进行有力的询问。

关键字:光遗传学, 体外重建, 细胞信号, LOV2, 显微镜检查, 支撑脂质双层

材料和试剂


 


深孔96孔模块或用于从FPLC收集馏分的替代方法
0.22 微米过滤器
˚F Ø [R大容量:Millipore公司,目录号:SE1M179M6


小批量:密理博,目录号:SLGV033RS


流动室用玻璃盖玻片(Ibidi ,目录号:10812)
锡纸
铝箔
1.5 ml Eppendorf管
PCR试管(Sigma-Aldrich,目录号:CLS3744)
巴斯德塑料移液管或塑料管(Celltreat Scientific,目录号:229276)
塑料管
Superdex 200增加10/300 GL色谱柱(Sigma - Aldrich,目录号:GE28-9909-44)
HiPrep 26/10脱盐柱(GE Healthcare,目录号:45-000-266)
玻璃瓶(Thermo Fisher Scientific,目录号:B7800-2)
用钳子或坚固的电线从热水浴中转移Coplin 罐。
巴斯德移液器(Thermo Fisher Scientific,目录号:13-678-20A)
用于表达LOV2的质粒(pDT552; F rom Tischer and Wiener,2019)
该质粒表达融合蛋白10xHis-TEV-AviTag-KCK-LOV2(V529N)。在此,V529N表示来自Avena的全长wt phototropin 1蛋白的突变 苜蓿。LOV2是指此蛋白质的第二个LOV域。TEV表示烟草蚀刻病毒蛋白酶的切割位点。AviTag 是一种15个氨基酸的标签,可以通过大肠杆菌生物素连接酶BirA 进行生物素化。KCK是使半胱氨酸成为更强亲核试剂的三肽标签;这有助于用马来酰亚胺偶联的染料在LOV2活性位点半胱氨酸上优先标记该位点。将该构建体克隆到修饰的pETM11-SUMO3质粒主链中。


用于表达Zdk的质粒(pDT482;来自Tischer and Wiener,2019)
该质粒表达融合蛋白10xHis-SUMO3-KCK-SpyCatcher-Zdk1。SUMO3是被SenP2蛋白酶识别的人SUMO3结构域,该酶在该结构域的C末端切割。KCK是使半胱氨酸成为更强亲核试剂的三肽标签;这使半胱氨酸对马来酰亚胺偶联的染料更具反应性。该抓间谍者域不用于当前应用程序,但允许翻译后融合到所需含目标间谍标签。Zdk1是LOV2的工程绑定伙伴。将该构建体克隆到修饰的pETM11-SUMO3质粒主链中。


液氮
LB肉汤(Sigma - Aldrich,目录号:L3522)
卡那霉素(Goldbio ,目录号:K-120-10)
异丙基-β-D- 硫代半乳糖苷(IPTG; Sigma - Aldrich,目录号:I6758)
生物素(Sigma - Aldrich,目录号:B4639)
黄素单核苷酸(FMN; Bio- R ad,目录号:161-0501)
甲醇(Sigma - Aldrich,目录号:179337)
流动室粘顶布(伊比迪(Ibidi),目录号:80608)
POPC(Avanti极性脂质,目录号:850457C)
PEG-PE(Avanti极性脂质,目录号:880230C)
生物素CAP PE(Avanti极性脂质,目录号:870277X)
Alexa Fluor 488 C5马来酰亚胺(Thermo Fisher Scientific,目录号:A10254)
磺基花青3 马来酰亚胺(Lumiprobe ,目录号:11380)
黄素单核苷酸(B io -R ad ,目录号:161-0501)
无水DMSO(Thermo Fisher Scientific,目录号:D12345)
氯仿(电子显微镜科学,目录号:12550)
D PBS(Thermo Fisher Scientific,目录号:14190144)
Hellmanex III(Sigma-Aldrich,目录号:Z805939)
丙酮(Sigma-Aldrich,目录号534064-4L)
190标准乙醇(Koptec ,目录号:V1101)
KOH(Fisher,货号:P251)
过氧化氢(Fisher Scientific,目录号:H325-500)
HCl(Acros ,目录号:42379-5000)
链霉亲和素(Rockland,目录号:S000-01)
ProLong Live Antifade试剂(Thermo Fisher Scientific,目录号:P36975)
Roche cOmplete Mini,不含EDTA的蛋白酶抑制剂片剂(Sigma - Aldrich,目录号:11836170001)
NaOH(Fis 她,货号:19232)
KH 2 PO 4 (Sigma - Aldrich,目录号:P5379)
K 2 HPO 4
NaCl(Sigma - Aldri ch,目录号:793566)
的β- 巯基乙醇(βME ;费舍尔,目录号:34461)
I 咪唑(Fisher,目录号:O3196-500)
N-(2- 羟乙基)哌嗪-N'-(2-乙磺酸)(HEP ES; Sigma-Aldrich,目录号:H3375-250G)
KCl (Fisher,目录号:S77375-1)
三(2- 羧乙基)膦(TCEP; Thermo,目录号:20491)
胰蛋白((BD B iosciences,目录号:211705)
酵母提取物(BD B iosciences,目录号:288620)
甘油(Fisher,目录号:BP229)
β-酪蛋白(Fisher,目录号:11461422)
苯甲基磺酰氟(PMSF;皮尔斯(Pierce),目录号:36978)
的6xHis-TEV蛋白酶(来自重组制备的大肠杆菌。在房子商业替代品容易得到例如,西格玛- Aldrich公司,目录号:T4455-1KU)
蛋白质浓缩器(Vivaspin 20 MWCO 10,000; GE Life Sciences,目录号:28932360)
二硫苏糖醇(DTT; Sigma - Aldrich,目录号:D9779)
的6xHis-SENP2蛋白酶(来自重组制备的大肠杆菌。在房子的Comme rcial替代是可用例如,Novus公司Biologicals公司,目录号:E-710-050)
压缩惰性气体(压缩氮气;空气,目录号:NI 200)
乙二胺四乙酸(EDTA; Fisher Scientific,目录号:S80007-1)
钴(II)六水合物(氯化钴2· 6H 2 O;Sigma - Aldrich,目录号:255599)
透明指甲油
IMAC绑定缓冲区(请参阅食谱)
IMAC洗脱缓冲液(请参阅配方)
HEPES缓冲盐水(H BS;请参见食谱)
很棒的汤(TB ;请参阅食谱)
DPBS-BB(请参阅食谱)
 


设备


 


500毫升烧瓶
2.8升带挡板的烧瓶
热块
1 L玻璃烧杯
温控振荡器,温度范围为37°C至18°C
EmulsiFlex-C3(或其他细胞裂解方法)
AKTA纯25 (GE医疗集团,或类似的FPLC系统)
5 ml HiTrap 螯合柱(GE Healthcare,目录号:17040801)
Milli-Q净水系统(或类似设备)
汉密尔顿注射器(Hamilton Company,Gastight 1700系列,目录号:80265和81165)
玻璃Coplin广口瓶(Sigma-Aldrich,目录号:BR472800)
蠕动泵和管道
真空干燥器
杜恩斯均质机
浴超声仪
台式超速离心机
带有Ti-45转子的地面超速离心机(Beckman Coulter ,型号:Optima L-90 K )
摆动斗式地板离心机
470 nm LED(Lightspeed Technologies Inc.,型号:HPLS-36)
倒置落射荧光显微镜,非常适合全内反射(TIRF)显微镜(Nikon Eclipse Ti 倒置显微镜)
 


软件


 


Microsoft Excel(Microsoft)
斐济(Schindelin 等人,2012年)
微观经理(Edelstein et al。,2014)
 


程序


 


LOV2纯化
在500 ml烧瓶中,向200 ml LB Kan(30μg / ml)中接种用表达LOV2和BirA (质粒pDT552)的双顺反子质粒转化的大肠杆菌BL21(DE3 )。在225 rpm摇动下于37°C过夜生长(> 8 h)。
背稀将100ml过夜培养物到1L TB阚(30 μ 克/毫升)在一个2.8升挡板烧瓶。在37°C下以180 rpm的速度摇动,直到OD 600 (600 nm处的光密度)在0.5-0.6之间。
将温度降低至18°C,等待30分钟,使介质冷却至接近18°C,并诱导IPTG(250 μM ,终浓度)。从储备溶液中,使生物素的终浓度为50μM ,黄素单核苷酸(FMN)的终浓度为1 mM。过夜生长。
 


注意:在冰上或在4°C下执行所有以后的步骤。


在摆动式离心机(或类似装置)中将沉淀细胞预冷至4°C。丢弃上清液。典型的旋转是800 RCF,持续约10分钟。由于LOV2和FMN辅助因子的高表达,沉淀通常看起来明显为黄色。
使用磅秤和空的离心容器估计湿细胞沉淀的质量。每1 g湿细胞沉淀添加2 ml IMAC结合缓冲液,并剧烈上下移液以粗略悬浮大部分沉淀。
细胞悬液转移(一些块都还可以),以一个杜恩斯均化器是由冰包围并添加一个罗氏完全迷你,无EDTA蛋白酶抑制剂片剂。彻底重悬细胞沉淀,直至其完全匀浆(通常上下约10次下降)。
在EmulsiFlex-C3上裂解细胞之前,立即添加PMSF(苯甲基磺酰氟)至终浓度1 mM。PMSF在水性环境中的半衰期约为30分钟,因此在细胞裂解时添加PMSF非常重要。确保在无水甲醇中制备PMSF库存。如果您认为水可能已经进入您的库存,请制作新鲜的PMSF。可以选择(但不是必需)第二次将裂解液通过EmulsiFlex,以降低其粘度。这样可以使后续加载到IMAC列的速度更快。
在Ti-45转子中以40k rpm在超速离心机中将裂解物在4°C下旋转一小时。倒出并保留上清液。
使用蠕动泵使上清液在装有Co 2+ 并与IMAC结合缓冲液平衡的5 ml HiTrap 螯合柱上再循环。在再循环设置中,来自色谱柱的洗出液应轻轻滴回注入IMAC色谱柱的上清液容器中。确保蠕动泵的入口管在储液罐的底部,以使所有上清液均流过色谱柱。通过在20-30 s的间隔内捕获一些液体来估计流速。典型的是1.5-2.0 ml / min。加载色谱柱的持续时间应可使整个上清液两次通过色谱柱。当LOV2绑定该列时,该列将从上到下明显变为黄色。
将上样的色谱柱转移至AKTA,并用IMAC结合缓冲液洗涤,直到流出液的A 280 小于60 mAu 。以10列的体积梯度洗脱蛋白质(从IMAC结合缓冲液到IMAC洗脱缓冲液),并在96孔深孔板中收集1.5 ml馏分。洗脱后,在IMAC结合缓冲液中平衡色谱柱。
在96孔模块中合并可见的黄色馏分,然后使用HiPrep 26/10脱盐柱将缓冲液交换到IMAC结合缓冲液中。
加入纯化的6xHis-TEV蛋白酶1:10 w / w(重量对重量)TEV:LOV 2,并在锡箔覆盖的4°C下孵育过夜。
注意:TEV蛋白酶在纯化房子,B UT 可以商购获得。


像以前一样,用蠕动泵在HiTrap 螯合柱(可以是用IMAC结合缓冲液平衡后用于洗脱LOV2的柱)上循环消化的LOV2混合物,以除去TEV蛋白酶,游离的His标签和任何未切割的LOV2。在色谱柱上至少循环两次。
收集流出液,并用脱盐柱交换到HEPES缓冲盐水(HBS)中。
用Vivaspin 蛋白浓缩仪浓缩至约4 mg / ml 。
在无水DMSO中制作新鲜的10 mM Alexa Fluor 488 C5马来酰亚胺库存。
在冰上,以2:1 染料:LOV 2 的最终摩尔比将染料添加到蛋白质中,并使反应进行30 s。
用DTT淬灭,终浓度为10 mM。该程序优先在LOV2的活性位点半胱氨酸上标记N端KCK标签(65)。淬火至低于500限制后的最终体积微升是方便,因为可以继续凝胶过滤而不蛋白质进一步浓缩。
在AKTA上,将淬灭的染料/ LOV2混合物在用HBS平衡的Superdex 200增加10/300 GL色谱柱上运行。在一个深孔96孔块中收集1.5 ml馏分。
池级分具有较高(> 100 mAu )A 280 ,明显呈黄色,并使用Vivaspin 蛋白浓缩器浓缩至约1.5 mg / ml 。
加入甘油至最终体积为10%。
使3分微升在PCR管中的等分试样并卡冷冻在液氮中。
储存在-80°C。等分试样在-80°C下保存> 2年后,未显示活性降低的迹象。
 


ZDK 净化
除以下更改外,以与LOV2相同的方式纯化和标记Zdk 。


不要在细菌生长培养基中添加生物素或黄素单核苷酸。
添加SUMO蛋白酶的6xHis-SENP2到IMAC在1洗脱的蛋白质:1 ,000瓦特/ w的代替TEV蛋白酶(的6xHis-SENP2在纯化的房子,但可以商购获得)。
用磺化花青3马来酰亚胺标记蛋白质,而不用Alexa Fluor 488 C5马来酰亚胺标记。
 


钴柱充电程序
该充电方案假定色谱柱开始存储在20%的乙醇中。在整个过程中,请确保捕获可能含有钴(II)的所有废水,以进行适当的废物处理。


将塑料管连接到蠕动泵上,并用ddH 2 O 冲洗该管。
将塑料管连接到IMAC色谱柱,注意不要引入任何气泡。
用5-10倍柱体积的ddH 2 O 冲洗色谱柱,以除去存储溶液。典型的流速是1.5-2.0ml / min。
暂停泵,将管路入口切换为已通过0.22 的100 mM CoCl 2 溶液 μ米过滤器。注意不要在管路中引入气泡。重新打开泵。
使钴溶液流过色谱柱,直到色谱柱和流出物明显呈粉红色为止。
暂停泵,将色谱柱在钴溶液中孵育10分钟。
将管路入口切换回ddH 2 O并打开泵。用至少10倍柱体积的ddH 2 O 洗涤色谱柱。重要的是要完全除去任何痕量的游离钴(II),因为在还原剂缓冲液中钴(II)易于还原,从而导致沉淀和阻塞。柱。
切换管道进气到所需的缓冲液(已经通过0.22传递μ 米过滤器),并用5个柱体积洗涤柱。
该列现在可以使用了。
色谱柱汽提,清洁和储存
在整个过程中,请确保捕获可能含有钴(II)的所有废水,以进行适当的废物处理。


将色谱柱连接到已用ddH 2 O冲洗过的蠕动泵和管道中。用10倍柱体积的ddH 2 O 清洗色谱柱。
管道进气切换到1种摩尔EDTA溶液(已经通过0.22传递μ 米过滤器),并流过该柱,直到它是明显白试。
将进样口切换为ddH 2 O,并洗涤5 倍柱体积。
切换管道吸入到20%乙醇溶液(已经通过0.22传递μ 米过滤器)和洗5个柱体积。
盖上色谱柱的两端并存放在4 ℃。
注意:为了更彻底地清洁色谱柱,可以选择将其用1 M NaOH 处理,以水解任何残留的蛋白质。后小号TEP d 3 的上方,与平衡1列1M的NaOH,静置1个小时。洗涤用的DDH的5倍柱体积2 O和前进到小号TEP d 4。


 


玻璃器皿清洗
SUV的制备和玻璃器皿清洁方案是根据先前发布的内容进行修改的(Taylor 等,2017)。


将8个玻璃小瓶(无瓶盖)和8个巴斯德移液器添加到1 L的玻璃烧杯中,并盖上3 M NaOH。将烧杯放在超声波浴中30分钟。
倒出NaOH(可将其保存以备将来清洗玻璃时使用),并通过填充烧杯并轻轻搅拌,用ddH 2 O 将玻璃器皿洗涤5次。
用5%(体积/体积)的Hellmanex III溶液覆盖玻璃器皿,并孵育过夜(> 8小时)。
倒出Hellmanex III溶液,并用ddH 2 O 彻底清洗玻璃器皿。从玻璃器皿中除去所有痕量的Hellmanex 洗涤剂至关重要。要用ddH 2 O 彻底清洗,请用塑料管将塑料巴氏移液器(或一些可以装在玻璃小瓶内的小管)连接到ddH 2 O源。颠倒并连续冲洗小瓶的内部,然后将塑料的巴斯德移液器放置在尽可能远离颈部的位置。冲洗大约30 s或直到水不再产生气泡为止。还要彻底清洗小瓶的外部。同样,用力清洗每个巴斯德移液器的内部和外部。可以通过将巴斯德玻璃移液器的末端穿入塑料巴斯德移液器末端的内部,来有效地冲洗巴斯德玻璃移液器的内部(您可能需要用剪刀稍微修剪一下塑料巴斯德移液器的末端),以确保用水连续冲洗。对于小瓶和巴斯德玻璃移液器,您可能必须在冲洗小瓶的内部和外部之间进行交替操作,以确保不会交叉污染Hellmanex 溶液。
用压缩氮气,空气或其他压缩惰性气体吹干以除去多余的水。
将玻璃器皿轻轻包裹在铝箔中(以防止灰尘),然后将其放在80°C的加热块上(或使用玻璃干燥箱)放置数小时,直到完全干燥。
将玻璃器皿存放在干净的装有铝箔的烧杯中,以防止灰尘进入。
要清洁塑料瓶盖,请在ddH 2 O 烧杯中超声处理30分钟,然后干燥并以与小瓶和巴斯德玻璃移液管相同的方式存储。
 


制备单层小囊泡(SUVs)
通过将大约500冲洗用氯仿一个的NaOH清洁4 ml玻璃管制瓶内微升氯仿用的NaOH清洗巴斯德吸管。旋转几次以覆盖小瓶的内部,并丢弃氯仿。再重复一次。
加入约500 微升Ò ˚F氯仿到冲洗小瓶中。
使用汉密尔顿注射器,以77.5%POPC,0.5%PEG-PE和2%生物素CAP PE 的摩尔比添加4μ 摩尔总脂质。专门使用一个汉密尔顿注射器处理每种脂质类型,以减少交叉污染的变化是有用的。
缓慢旋转氮气(或其他惰性气体)到顶部,同时旋转小瓶以除去氯仿。一旦所有可见溶剂蒸发掉(小瓶内部应有白色残留环),用预先清洗的盖子松开小瓶盖,然后放置在真空干燥器中过夜。
第二天,通过加入1.5毫升0.22再水合脂质微米过滤d PBS,牢固地拧上盖子,轻轻涡旋10分钟。可以将小瓶垂直放置在台式涡流器中,方法是将小瓶垂直放置在中间,然后从涡流头下方通过一条胶带穿过小瓶盖,然后再向下旋至涡流头另一侧的下侧。与第一条磁带成90度角重复另一条磁带。该涡旋速度是很重要的。它应该不会比使D PBS到达小瓶的颈部所需的速度快,这样所有脂质都可以重新水化。如果涡流10分钟后有气泡,涡流太快; 这有氧化脂质的风险。
一旦再水化,分割2支1.5毫升的微量管之间,靠近下在流动的氮气或其他同样的混合物ERT 气体。
进行20次冻融循环通过移动之间的管杜瓦瓶的液氮和42℃的水浴或加热块。混合物应从浑浊变成明显澄清。如果解决方案的清晰度没有提高,则可能意味着大型脂质聚集体和多层囊泡不会分解为SUV。由于PEG-PE可以帮助脂质聚集体分解为SUV,因此浑浊的溶液可能表明使用了太多或太少的PEG-PE或脂质已经变质。考虑订购新的PEG-PE和/或仔细检查使用的PEG-PE的正确摩尔量。
在台式超速离心机中,将脂质混合物在60°RCF下于4°C于40°C下旋转40分钟。管的底部可能会有小的玻璃状颗粒。
将上清液移至1.5 ml Eppendorf管中,并储存在液氮中直至准备使用。
RCA清洁显微镜盖玻片
将一个玻璃盖玻片放入玻璃Coplin广口瓶的每个插槽中,盖上丙酮,然后放入超声波浴中10分钟。
倒出丙酮,并用190标准乙醇重复。
倒出乙醇,并用ddH 2 O 重复。在ddH 2 O超声处理步骤之前和之后,用ddH 2 O 填充并倒出Coplin 罐5次,以去除多余的有机溶剂。
在微波炉(或加热板)中将1 L ddH 2 O加热至大约70°C。热水将有助于防止Coplin广口瓶在即将到来的洗涤过程中因温度应力而破裂。
另外,准备70-80°C的水浴,并用足够的水将大部分Coplin广口瓶浸没,而不会使唇部溢出。
将3.75 g KOH溶于45 ml加热的ddH 2 O中,并加到盖玻片上。然后加入15毫升30%过氧化氢。
用钳子或金属丝牢固地缠绕在Coplin广口瓶的脖子上,将其转移到水浴中,使其反应12分钟:溶液大约需要2分钟才能升至温度,反应需要10分钟。一旦加热,该溶液将不断产生许多小气泡。
从水浴中取出Coplin 罐。小心倾析基础溶液,并用温热的ddH 2 O 洗涤5次。
添加下列组分,以在科普林罐:38毫升温热的DDH 2 O,9.5毫升37%HC 升和12.6毫升30%过氧化氢。
如前所述,在水浴中孵育12分钟。溶液将像基本溶液一样起泡,但起泡的强度稍弱。
卸下科普林从水浴罐子,滗出溶液,并用温热的DDH洗5次2 O.盖上温热的DDH盖玻片2 O,所述添加科普林罐盖,并储存在室温下长达一周。
 


形成SLB
在干燥和准备玻璃盖玻片时,请注意仅握住边缘,并避免接触中央附近的任何部位。


使用镊子从Coplin广口瓶中的ddH 2 O中取出玻璃盖玻片。用一只戴手套的手捏住两个长边,立即用压缩氮气(或压缩空气或其他惰性气体)吹干。盖玻片上的反射应该是一尘不染的。
在两张纸巾上叠放一张显微镜镜头纸,然后将盖玻片放在上面。
将六孔的Ibidi 粘性腔室牢固地按到盖玻片上。使用非常牢固的压力以确保接触,尤其是通道之间以及通道末端与盖玻片侧面之间的接触。
用透明指甲油沿盖玻片的长边密封界面。密封件有助于防止长时间的显微镜时间间隔实验中通道中的任何溶液芯吸到侧面和蒸发。
稀释30 微升的SUV的等分试样用800 微升0.22 微米过滤d PBS和六个井之间均匀地分布。
在37°C下孵育1小时。一旦添加了SUV,请注意不要在随后的清洗和功能化步骤中使通道干燥。
进行SLB功能化
 


功能化SLB
官能井,用500排出多余的脂质微升的0.22 微米过滤d PBS。用移液器将液体一端推入,然后从另一端小心吸出(图2 ),这很有用。
 


D:\ Reformatting \ 2020-7-1 \ 1902649--1485 Orion Weiner 784707 \ Figs jpg \ fig 2.jpg


图2.在SLB准备和功能化期间交换解决方案。在伊比迪会议厅通道中如何交换溶液的示意图。为了在Ibidi 腔室的通道中进行有效的溶液交换,同时保持SLB处于水合状态,同时将新溶液推入一端并从另一端吸出是很有用的。将抽吸器的尖端保持在居间通道顶部上方的出口较高位置,有助于确保仅在移液器将新溶液推入时才除去液体。在吸气器的末端增加一个小直径的移液器吸头,可以帮助减轻吸力,避免过度吸出通道。


 


用200冲洗井微升的链霉稀释到d PBS-BB(2 微克/毫升终浓度)并在室温下孵育erature 5分钟。
用500冲洗井微升d PBS-BB。
用200冲洗井微升LOV2的稀释到d PBS-BB至所需浓度(典型地在20-200 纳米)和孵化在室温下5分钟。
最后,冲洗用500通道微升的d PBS-BB。现在,SLB已通过LOV2进行了功能化。
 


无论LOV2官能化SLB的预期用途是什么,重要的是实验进行时所用的溶液已用除氧剂进行了预处理。这有助于防止蓝光漂白LOV2发色团,并显着增加Zdk 募集和释放到LOV2的可能次数。一种有效的方法是将ProLong Live Antifade Reagent 1:100 稀释到将要进行显微镜操作的溶液中,并使其在室温下孵育至少90分钟,以消除溶解氧。在将SLB功能化之前开始除氧剂处理非常有用,这样在SLB完全功能化之前就可以准备溶液。


 


显微镜
具备TIRF(全内反射荧光)显微镜的显微镜对于定量Zdk 募集和从LOV2功能化的双层中释放是必不可少的,并且是对细胞信号或生化反应的结果变化进行成像的自然选择。


我们使用带有两层二向色性转塔的Eclipse Ti 倒置显微镜(Nikon)进行成像,以允许同时进行荧光成像和光遗传学刺激。显微镜还配备了电动激光TIRF照明装置,60倍复消色差TIRF 1.49 NA物镜(Nikon),iXon Ultra EMCCD相机以及配备405-,440-, 488、514和561 nm激光线。显微镜和相关硬件由MicroManager (Edelstein 等人,2014)结合定制的Arduino控制器(Advanced Research Consulting Corporation)进行控制。用于光遗传学刺激的蓝光来自由MATLAB单独控制的470 nm LED。


 


检查双层流动性
在进行实验之前,重要的是要确认双层已经被LOV2和液体功能化。通过光漂白(FRAP)实验后的荧光恢复可以轻松完成此操作。可以使用几乎任何荧光成像方式(例如TIRF,共焦,宽视场)来检查双层是否已被LOV2功能化。


将用饱和量的荧光标记LOV2功能化的SLB装入显微镜,并使用60倍或100倍物镜聚焦在玻璃水界面上。掺杂少量浓度的荧光珠可能会很有用,以使该界面的定位更容易。
关闭向下的落射场光阑所以视场的,只有一部分被照射。如果双层被荧光标记的LOV2功能化,则照明区域内的荧光应明显增加。
通常需要定性FRAP测量来评估双层流动性。尽管拥有合适的FRAP系统或高功率TIRF激光器很有用,但有时可能无法使用功率较低的光源(例如宽场落射荧光光源)。在这种情况下,用易于漂白的荧光团(如FITC(异硫氰酸荧光素))标记LOV2是有用的。不使用除氧剂处理成像缓冲液也会使荧光团更容易漂白。
用您选择的方法漂白视场的一小部分。如果未使用适当的FRAP系统(例如,使用激光TIRF或宽视场落射荧光光源代替),靠近下的落射场光阑所以只有视场的一小部分被照亮。如果使用TIRF激光,请将其指向正上方。对于非FRAP系统,可能需要将功率增加到最大。漂白目标区域所需的时间将取决于光源的强度,连接到LOV2的小分子荧光团和成像介质等因素。漂白时间长于30秒通常是无效的,因为双层中的扩散(如果是流体)会平衡照明区域和非照明区域之间的LOV2。
接下来的2-3分钟,每5-10秒钟对整个视场成像一次。(打开落射视场光阑,如果它是关闭)。A漂白孔的直径为〜5-10微米如果双层是流体应当基本上恢复在〜30-60秒(图3 和视频1 )。原始漂白孔的边界应保持可见。
 






图3.用FRAP评估双层流动性。使用TIRF激光将一个小孔漂白到用荧光LOV2功能化的双层中。因为双层是流体,所以新的LOV2分子扩散到漂白区,在60 s内大体上恢复了其初始状态。非流体双层无法在这段时间内恢复。比例尺指示1μm 。


 


D:\ Reformatting \ 2020-7-1 \ 1902649--1485 Orion Weiner 784707 \ video1.jpg


视频1. FRAP实验的时间流逝,显示SLB处于流动状态并通过LOV2进行了功能化。将一个小孔光漂白到用荧光LOV2功能化的SLB中。在大约60 s的过程中,由于新的LOV2分子从漂白区域外部扩散而来,荧光信号得以恢复。未使用LOV2功能化的双层在开始时不会显示“空洞”。被功能化但不能流动的双层将在大约60 s内恢复。确切的回收率取决于漂白区域的直径。比例尺为1 μ 米。


 


测试LOV2功能
测试双层LOV2功能的最直接方法是使用TIRF来测量荧光标记Zdk 的募集和释放。在确认双层已被LOV2和液体官能化后,应执行此测试。


用饱和量的LOV2使SLB功能化,并在包含250nM Zdk的成像介质中流动,该成像介质已用除氧剂处理。最好使用打算用于实验的媒体,但D PBS可以作为一个很好的起点。确保Zdk 标记有被波长大于560 nm的光激发的染料,以便您可以连续成像而不激活LOV2。
设置一个10分钟的时间过程,其中每5秒钟用TIRF对Zdk 进行一次成像。每2分钟,将LOV2暴露在约470 nm的蓝光下1秒钟。任何种类的GFP成像设置通常都可以正常工作。根据蓝光的强度,Zdk 应在约1-10秒钟内从双层中快速释放出来,然后在接下来的2分钟内缓慢恢复。在接下来的10分钟内,应观察到类似的释放和募集周期(图4 )。如果在10分钟内Zdk 募集减少了50%以上,则最可能的原因是除氧剂治疗无效。
 


D:\ Reformatting \ 2020-7-1 \ 1902649--1485 Orion Weiner 784707 \ Figs jpg \ fig 4.jpg


图4. Zdk 募集和从脂质双层释放的时间过程。招募荧光标记的Zdk ,并从LOV2功能化的双层中释放五个周期。LOV2 在被蓝光(LED发出的470 nm光)激发后迅速释放Zdk ,并在去除蓝光后缓慢重新结合。(垂直的蓝色条表示470 nm LED处于打开状态。)即使显微镜只能进行周期性的蓝光照明(而不是像我们这样的连续照明),Zdk 的募集和释放动力学也应该非常相似。


 


                对于在细胞表面受体上使用Zdk 启动信号的方案,我们将感兴趣的读者介绍给我们之前的公开出版物(Tischer and Weiner,2019)。对于我们以前与T细胞信号转导有关的工作,我们能够从一个单一的视野进行数十个单细胞测量。通过三个生物学重复,我们在每种条件下记录了约50-100个单细胞测量值,为我们的实验中的统计意义提供了足够的数量。


 


菜谱


 


媒体和缓冲区:


IMAC绑定缓冲区
50毫米KH 2 PO 4


400毫米氯化钠


0.5毫米βME


pH值7.5


IMAC洗脱缓冲液
50毫米KH 2 PO 4


400mM氯化钠


500 mM咪唑


0.5毫米βME


pH值7.5


哈佛商学院
20毫米HEPES


100毫米氯化钾


0.5毫米TCEP


pH值7.5


很棒的汤(TB)
营养基础


12克try


24克酵母提取物


4毫升甘油


900毫升ddH 2 O


 


10 TB盐


170毫米KH 2 PO 4


720毫米K 2 HPO 4


分别高压灭菌营养盐和10x TB盐
然后将100 ml的10x TB盐添加到900 ml的营养基质中,制成1 L TB
 


用于SLB功能化和成像的缓冲液:


D PBS-BB
D PBS(Thermo Fisher Scientific)


1 mg / mlβ-酪蛋白


0.5毫米βME


使用0.22滤波器微米过滤器之前立即使用


 


Acknowledg 发言:


 


这项工作得到了Genentech奖学金(DT),NIH赠款GM118167和Novo Nordisk基金会(ODW)以及NSF 科学技术中心细胞建设中心(DBI-1548297)的支持。该协议改编自Tischer 和Weiner (2019)。


 


利益争夺


 


作者声明没有任何金融或非金融竞争利益。


 


参考文献


 


Edelstein,AD,Tsuchida ,MA,Amodaj ,N.,Pinkard,H.,Vale,RD和Stuurman,N.(2014)。使用μManager 软件进行显微镜控制的高级方法。 J生物学方法1(2):e11。
Schindelin ,J.,阿尔甘-Carreras,I.,雄犬,E.,Kaynig ,V.,Longair ,M.,Pietzsch ,T.,Preibisch ,S.,Rueden酒店,C.,萨尔费尔德,S.,施密德,B ,Tinevez ,JY,White,DJ,Hartenstein,V.,Eliceiri ,K.,Tomancak ,P。和Cardona,A。(2012)。斐济:一个用于生物图像分析的开源平台。Nat Methods 9(7):676-682。
泰勒,MJ,侯赛因,K.,加特纳,ZJ,市长,S.和谷,RD(2017)。基于DNA的T细胞受体揭示了受体簇在配体识别中的作用。单元格169(1):108-119 e120。
Tischer ,DK和Weiner,OD(2019)。基于光的配体半衰期调节支持T细胞信号转导的动力学校对模型。网上生活8:42498。
Toettcher ,JE,Gong,D.,Lim,WA和Weiner,OD(2011)。基于光的反馈,用于控制细胞内信号传导动力学。Nat Methods 8(10):837-839。
Toettcher ,JE,Weiner,OD和Lim,WA(2013)。使用光遗传学来询问Ras / Erk 模块对信号传输的动态控制。细胞155(6):1422-1434。
Wang,H.,Vilela ,M.,Winkler,A.,Tarnawski ,M.,Schlichting,I.,Yumerefendi ,H.,Kuhlman,B.,Liu,R.,Danuser ,G.和Hahn,KM(2016 )。LOVTRAP:用于光诱导蛋白解离的光遗传系统。Nat Methods 13(9):755-758。              
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Copyright Tischer and Weiner. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Tischer, D. and Weiner, O. D. (2020). Optogenetic Tuning of Protein-protein Binding in Bilayers Using LOVTRAP. Bio-protocol 10(17): e3745. DOI: 10.21769/BioProtoc.3745.
  2. Tischer, D. K. and Weiner, O. D. (2019). Light-based tuning of ligand half-life supports kinetic proofreading model of T cell signaling. Elife 8: 42498.
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