Cellular membranes are highly crowded due to the presence of embedded proteins and sugars. We demonstrate single molecule imaging on synthetic crowded supported lipid bilayers. This platform is useful for investigating the effect of crowding on membrane biomolecular reactions, at the molecular level.
The protocol explains the analysis of binding, diffusion, and assembly of membrane biomolecules. Success in the experiment depends on rigorous cleaning of the substrate, avoiding damage to the bilayer, and maintaining a high signal to noise ratio in single molecule imaging. Demonstrating the procedure with me is Aditya Upasani, and Vishwesh Haricharan Rai, graduate students from the lab.
Begin by placing the plasma treated acrylic slide on clean tissue paper. Peel the double sided laser cut tape and stick it to the slide. Using a pipette tip, flatten the tape to prevent leakage from one channel to the other.
Close the chamber by placing the plasma treated cover slip on the taped slide. Using a pipette tip, gently press the cover slip on the taped regions to make the channel water tight. If using glass slides seal off the edges using epoxy resin.
Store the prepared microfluidic imaging chambers for one to two weeks under desiccated conditions. Take a glass vial pre-cleaned using Piranha solution, and add chloroform solution of POPC and DOPE-PEG(2000)at the desired mole fraction of PEG 2000 lipids to get the final lipid concentration of three millimolar, after adding the buffer. Similarly other membrane compositions of lipids can be prepared.
Next, dry out the chloroform from the vial using a gentle stream of nitrogen with a small swirl so that the desired lipids are uniformly coated on the surface of the vial. Remove residual chloroform by keeping the vial in a vacuum desiccator for one hour. Then add one milliliter of PBS to the vial and incubate overnight at 37 degrees Celsius.
After overnight incubation, gently vortex the vial for one to two minutes until the solution turns turbid and milky. Now transfer 100 microliters of this solution into a small 500 microliter centrifuge tube. To generate small unilamellar vesicles of 50 to 100 nanometer diameter sonicate the solution for about one hour in a bath sonicator.
At the end of the sonication the solution will become clear, if the solution is still turbid then sonicate for an additional 30 minutes. Then add calcium chloride to the sonicated vesicles to a final concentration of 30 millimolar in the lipid solution. Cut a micro pipette tip to inject the sample solution so it fits in the hole tightly.
Mix the lipid solution and inject it through one of the holes in the imaging chamber. Wipe the excess solution that comes out of the chamber from the outlet with clean tissue to prevent the contamination of adjacent channels. Prepare a humidifying chamber by placing wet tissue at the end of a 50 milliliter centrifuge tube.
Place the slide in the tube and close the tube cap. Lay the tube sideways and leave this assembly in a humidified chamber for 90 minutes, preferably in an incubator at 37 degrees Celsius. Wash the chamber thoroughly with a copious amount of PBS buffer, preventing air bubbles from entering the chamber while washing, as this can generate defects in the membrane.
Before conducting any experiment on mobility and assembly, align the TIRF microscope by preparing a fluorescent bead sample and adding them into the microfluidic channel at low concentrations such that fluorescent spots from single beads do not overlap in the images. First, visualize the beads in epifluorescence mode. While illumination is in epifluorescence mode, move the M5 mirror sitting on a translation stage such that the beam coming out of the objective bends until total internal reflection configuration is achieved.
To verify whether TIR illumination has been achieved, check that only the beads on the surface are visible when the TIR evanescent field is illuminating the bead sample and free floating beads away from the surface are not observed. At the start of the experiment, set the laser power at the objective back focal plane to five to 10 milliwatts to prevent photo destruction of the fluorophores. Keep the slide on the microscope stage and focus on the bare membrane first, small traces of impurities in lipid membranes are sufficient to visualize the membrane.
Inject the sample using a micro pipette into the PEG SLB coated imaging chamber. For measuring single molecule binding kinetics, use a syringe pump to flow the labeled biomolecules through the inlet holes into the microfluidic chamber at a flow rate of 50 to 500 microliters per minute. To record 5, 000 frames at 25 to 50 frames per second, set the required movie acquisition parameters.
Start movie acquisition and acquire more than 5, 000 frames under continuous flow from the syringe pump until there is no further increase in the appearance of new spots on the membrane surface, and analyze the binding kinetics as described in the manuscript. For single particle tracking add the labeled biomolecules at low concentrations to the channel using a micro pipette. To ensure the high fidelity of tracks recovered from the data sets, optimize the concentration to a density of less than 0.1 particles per square micrometer, so that individual particles rarely cross paths.
If required, incubate the chamber in a humidifying environment and wash with the buffer before imaging. For single particle trajectory analysis, acquire 200 to 500 frames at 10 to 100 frames per second and maintain the objective lens focused on the bilayer plane with minimal stage drift during the image acquisition. For measuring subunits, add the relevant concentration of labeled biomolecules to the microfluidic imaging chamber, and incubate the slide in a humidifying chamber at the desired temperature for the required time.
Before imaging, wash the imaging chamber with a buffer. Place the slide on a microscope and adjust the focus to visualize the labeled molecules. Set the laser power to a level at which the bleaching of the fluorophore occurs gradually.
Ideally for each assembled complex control the laser power to keep the photo bleaching step rate between 10 to 20 frames apart as described in the manuscript, and acquire the images until all the molecules are completely photo bleached. Perform a time dependent assembly measurement by starting movie acquisition as soon as the sample is introduced into the chamber and acquiring new photo bleaching movies at fixed time intervals after membrane binding from different segments of the PEG SLB. Binding of Cytolysin A to lipid membranes with five mole percent DOPE-PEG(2000)displayed increased particle density and attained saturation.
An exponential decay function fit to the bound particles gives the time constant for Cytolysin A membrane binding. Without any PEG polymer in the membrane, most tracers displayed restricted diffusion on SLBs. Small levels of PEG 2000 in the membrane bilayer lifted away from the underlying surface, allowing the tracer molecules to diffuse without surface constraints.
However, extreme confinement is observed at a high concentration of PEG in the bilayer membrane. ISD2 distribution for the diffusion of lipophilic DNA tracer in two different PEG 2000 lipid membranes showed decreased mobility at high concentrations of PEG. After incubating on the crowded lipid bilayer membrane Cytolysin A displayed many distinct photo bleaching steps, suggesting the formation of various assembly intermediates.
After correcting for the labeling efficiency, the final distribution of oligomers showed the dodecameric Cytolysin A species as the dominant structure. Membrane protein binding assembly and other reactions should be conducted in appropriate membrane crowding environments. Attention should be paid to cleaning the imaging chamber, avoiding disrupting the bilayer, and setting up the TIRF microscope.
