pH Modulation Assay on Supported Lipid Bilayers to Detect Protein-Phosphoinositide Interactions

Phosphatidylinositol 4,5-bisphosphate, or PIP2, is a minor membrane-bound phosphorylated phospholipid. Phospholipase C, or PLC, with specific PIP2-binding domains, binds to PIP2, generating secondary messengers essential for several cellular processes.

To detect PLC-PIP2 interactions in vitro, begin with a small unilamellar vesicle, SUV, acidic suspension, comprising PIP2 and phospholipid mixture, one of which is conjugated to a pH-sensitive fluorescent dye.

Pipette the solution into a pre-assembled fabricated microfluidic device's inlet and connect outlet tubing. Transfer and secure the device onto a fluorescence microscope stage. Through gravity, SUVs pass through the microchannel floor, made of a hydrophilic glass coverslip.

The SUVs adsorb on the glass surface, the solution's pH reducing electrostatic repulsion between the two. The SUVs interact with the surface, deforming and fusing to form large vesicles.

Following critical surface coverage, the vesicles rupture spontaneously, forming a supported lipid bilayer, SLB. Pass a buffer through the channel, removing unadsorbed vesicles.

Using a suitable fluorescence channel, image the microchannel to visualize and measure the pH-sensitive dye fluorescence — highly fluorescent at low pH — in the SLB.

Introduce a recombinant PLC solution into the microchannel. The solution's neutral pH, below the PLC's isoelectric point, makes the PLC positively charged.

The recombinant PLC, with its PIP2-binding domain, binds to the PIP2, recruiting negatively-charged hydroxide ions to the bilayer surface. This renders the bilayer surface more basic, shifting the protonation state of the dye and causing fluorescence quenching, indicating PLC-PIP2 interactions.

Transfer 100 microliters of the phosphatidyl inositol-4 5-bisphosphate containing small unilamellar vesicles into a 0.65-milliliter microcentrifuge tube. Adjust the pH of the solution to approximately 3.2 by adding 6.4 microliters of 0.2 Normal hydrochloric acid.

Pipette 10 microliters of the pH-adjusted small unilamellar vesicle solution into each channel through the inlet and apply pressure through the pipette until the solution reaches the outlet. Detach the tip from the pipette and leave it attached to the device. After repeating this step for each channel, incubate the device for 10 minutes at room temperature.

Injection of vesicles into microchannels should be performed immediately after the device assembly. Meanwhile, cut sets of inlet and outlet tubing. Using tweezers, connect the outlet tubing set to the device, and then, tape the device onto a microscope stage.

Submerge one end of the inlet tubing set in 25 milliliters of running buffer contained in a conical tube, and tape it to make sure that the tubing is secured. Using a lab jack, place the conical tube on a higher ground than the device in order to push the solution through the microchannels via gravity flow.

For each inlet tube, use a syringe to draw 1 milliliter of running buffer from the free end of the tubing. Remove the pipette tip from the inlet, and insert the free end of the inlet tubing into the device. Repeat this process to connect all the inlet tubing pieces to the device. Flowing running buffer through the channels helps to remove excess unruptured vesicles and equilibrate the bilayer to experimental conditions.

Next, open the microscope control software. On the left panel, click on the "Microscope" tab and choose the "10x" objective. Click on "Live," and then, the "Alexa 568" image icons on the toolbar. Using the Fine and Coarse adjustment knobs, focus on the microchannels. Scan through the device to check the quality of the SLBs and the channels.

Then, click the "FL Shutter Closed" image icon on the toolbar. Click on the "Acquisition" tab and, under "Basic Adjustments," select exposure time. Set the exposure time to 200 milliseconds. On the left panel, click on "Multidimensional Acquisition." Under the "Filters" menu, select the red channel. Then, click on the "Time-lapse" menu. Set the time interval to 5 minutes, duration to 30 minutes, and lapse menu.

Select the "Circle" tool under the "Measure" tab and draw a circle in any channel. Right-click while the circle is selected and choose "Properties." Under the "Profile" tab, check "all T" to view the fluorescence intensity as a function of time. Make sure this curve reaches a plateau, which indicates equilibrium, before proceeding to the next step.

Lower the buffer solution to an equal ground as the device to stop the flow. One at a time, detach each outlet tubing and apply 200 microliters of each protein dilution into the outlet channel using a pipette. Do not apply any pressure. Let gravity do the work. Detach the tip from the pipette and leave it attached to the microfluidic device. Repeat this process for each channel, and to make sure that air bubbles are not introduced into the channels during this process.

Next, lower the inlet tubing to a ground below the microfluidic device to start flowing the protein through the microchannels. Tape the free end of the tubing to a waste container. Flow the dilutions of the Pleckstrin homology domain for 30 minutes. On the left panel of the software, under the "Time-Lapse" tab, click on "Start" to begin imaging again.