Content mixing assay

1. Reagents setup
   

    i) lipids:

        POPC (10 mg/ml, Avanti Polar Lipids #850457)

        POPE (10 mg/ml, Avanti Polar Lipids #850757)

        DOPS (10 mg/ml, Avanti Polar Lipids #840035)

        PI-4,5-P₂ (1 mg/ml, Avanti Polar Lipids #840046)

        Cholesterol (Chol, 10 mM, Avanti Polar Lipids #700000)

     *All lipids were dissolved in chloroform except for PI-4,5-P2 in chloroform:methanol:water = 20:9:1

    ii) Chemicals:

        Tris(2-carboxyethyl)phosphine (TCEP, Sigma Aldrich #C4706), dissolved in deionized water at 0.5 M

        3-[(3-Cholamidopropyl)dimethylammonio]-1-propane sulfate (CHAPS, Amresco [now avantor VWR #VWRV0465]), dissolved in deionized water at 20% (w/v)

        Sulforhodamine B (SRB, Sigma Aldrich #230162), dissolved in lipid dissolving buffer (see below)

        Bio-Beads SM2 (Bio-Rad #1523920)

        poly-D-lysine hydrobromide (mol wt 30,000-70,000, Sigma Aldrich #P7886), dissolved in deionized water at 200 ug/ml

    iii) Buffers:

        Lipid dissolving buffer I: 25mM HEPES-K pH 7.40, 150 mM KCl, 10% (v/v) Glycerol, 0.2 mM TCEP, 1% (w/v) CHAPS

        Lipid dissolving buffer II: 25mM HEPES-K pH 7.40, 150 mM KCl, 10% (v/v) Glycerol, 0.2 mM TCEP, 50 mM SRB, 1% (w/v) CHAPS

        Dialysis buffer I: 5mM HEPES-K pH 7.40, 150 mM KCl, 10% (v/v) Glycerol, 0.1 mM TCEP

        Dialysis buffer II: 5mM HEPES-K pH 7.40, 150 mM KCl, 10% (v/v) Glycerol, 0.1 mM TCEP, 40 mM SRB

        Reaction buffer: 5mM HEPES-K pH 7.40, 150 mM KCl, 10% (v/v) Glycerol, 0.1 mM TCEP

        CaCl₂ solution: 0.1 M, dissolved in deionized water

        EDTA-2Na⁺ solution: 50 mM, dissolved in deionized water

  2. Instruments and consumables

        QM40 spectrofluorimeter (Photo Technology International, now part of Horiba) 

        Vortex-Genie 2 (Scientific industries)

        Magstir Genie (Scientific industries)

        Oil pump (Oerlikon Leybold Vacuum)

        Vacuum chamber (Shanghai Everone)

        Flasks 250ml, 1L (Fisher)

        Glass test tube Φ8mm (Customization)

        Slide-A-Lyzer™ Dialysis Cassette (3.5K MWCO, 0.5 mL, Thermo Scientific #66335)

  3. Experiments

    i) Proteoliposome preparation

        Lipids formula (for prepare 200 ul, 5mM total lipids):

            Target liposome (no SRB loading)                    Donor liposome (SRB loading)

            Lipids        mol%    volume                                    Lipids    mol%    volume

            POPC        59%        45.8ul                                     POPC    55%        42.7ul

            POPE        20%        14.0ul                                     POPE     20%        14.0ul

            DOPS        20%        16.0ul                                    DOPS    15%        12.0ul

            PI-4,5-P₂   1%          11.0ul                                    Chol      10%        10.0ul

        Lipids were mixed in glass test tubes, dry under nitrogen flow with gentle rotation.

        * Important: For lipid mixtures containing PI-4,5-P₂, one should use 45°C water bath when drying under nitrogen flow. This is because of the higher transition temperature (~40°C) of currently used PI-4,5-P₂.

        After drying under nitrogen flow (until no visible liquids, one could easily see lipid films stick on the glass tube), the glass test tubes were transferred to a vacuum chamber equipped with an oil pump, keep the lipid films under vacuum for at least 3 hours (room temperature).

        Then dissolve the lipid films using the formula as follow:

            Target liposome (no SRB loading)                        Donor liposome (SRB loading)

            Lipid dissolving buffer I            160ul                    Lipid dissolving buffer II (containing SRB)       165ul 

            Syx1/SN25 complex(125 uM)    40ul                    Syb2(200 uM)                                                      25ul

                                                                                            Syt1(100 uM)                                                      10ul

        The protein-to-lipid (p/l) ratio is 1:200 for Syx1/SN25 complex and Syb2, 1:1,000 for Syt1. Vortex the mixtures on ice for 5 minutes until the lipid film fully dissolved, then incubate the mixture at room temperature for 30 minutes.

         * It is recommended to incubate the dissolved lipid films at room temperature for better performance (transmembrane protein incorporation).

        Transfer the mixtures to Slide-A-Lyzer™ Dialysis Cassette, for SRB-loaded donor liposome, firstly dialyze in 250 ml Dialysis buffer II supplied with 1.25 g Bio-Beads SM2 (5g/L) at 4°C in the dark for 6 hours.

        * Important: this step aims to keep constant 40 mM SRB loaded into donor proteoliposome. 

        For the following dialysis, target liposomes and SRB-loaded donor liposomes are the same: using 1L Dialysis buffer I (no SRB supplied) supplied with 5g Bio-Beads SM2 (5g/l). Dialyze 3 times at 4°C in the dark, 6 hours for each time.

        * Prepared proteoliposomes could be stored at 4°C in the dark no more than 3 days for better performance.

    ii) Content mixing assay

        Reaction setup: (60ul cuvette)

            Donor liposome (5 mM)                  1.2 ul  (100 uM)

            Target liposome (5 mM)                  0.6 ul  (50 uM)

            EDTA-2Na⁺(50 mM)                        0.24 ul (0.2 mM)

            Complexin-1 (200 uM)                     6.0 ul  (20 uM)

            poly-D-lysine (200 ug/ul)                0.6 ul  (2 ug/ml)

            Reaction buffer                                51.4 ul

        Instrument setup:

            Excitation wave-length                    565 nm

            Emission wave-length                      580 nm

            Slit width (excitation/emission)        1.25 mm/1.25 mm

            Temperature                                    25 °C

            Sampling rate                                   1 Hz

        During the sampling at 1,500 s, supply 0.6 ul CaCl₂ solution (the final concentration is 1 mM) to trigger fast liposome fusion.

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