In Vitro Ricostituzione di Self-Organizing pattern proteico su lipidici supportati

This method can help answer key questions about cell's organization and membranes, for example, what role geometric cues play in the process. The main advantage of this technique is that it allows precise control over all aspects influencing pattern formation, such as protein concentrations. To begin, generate multilamellar vesicles as described in the text protocol.

Now, freeze-thaw the lipids for seven to 10 cycles, by first preparing a beaker with water at 70 to 99 degrees Celsius on a hot plate, as well as a container holding liquid nitrogen. Hold the vial in liquid nitrogen with large tweezers until the nitrogen stops boiling. Then transfer the vial to hot water until the solution is completely thawed.

Repeat these steps until the lipid mixture appears clear to the eye, depending on the mixture. Next, assemble a lipid extruder and pre-rinse the system with Min buffer. Extrude the lipid mixture between 35 and 41 times through a membrane of 50 nanometer pore size.

Make sure to end on an odd number of passes to avoid aggregates that never traversed the membrane. Distribute glass coverslips on an inverted glass Petri dish or other inert surface. With a glass pipette, add seven drops of concentrated sulfuric acid to the center of each coverslip.

Then add two drops of 50%hydrogen peroxide to the middle of the acid drops. Cover the reaction and incubate for at least 45 minutes. Now, pick up the coverslips individually using tweezers and rinse off the acid with ultrapure water.

Place the washed coverslips in nonstick holders or a similar transportation device. Now rinse each coverslip extensively with ultrapure water and dry the surface with pressurized gas. Mark the clean side of the coverslip with permanent marker.

To assemble the chamber, first cut off and discard the lid and the conical part of a 0.5 milliliter reaction tube with sharp scissors. Then apply UV glue to the upper rim of the tube and distribute the glue evenly by using a pipette tip. Glue the tube upside down to the clean side of the coverslip.

Finally, cure the UV glue by placing multiple chambers underneath a 360 nanometer lamp or LED for five to 15 minutes. For supported lipid bilayer formation, preheat a heat block to 37 degrees Celsius and incubate two milliliter reaction tubes with Min or SLB buffer at one tube per chamber. Blow nitrogen into the assembled and cured chambers to remove any dust or other particles that may have settled during the UV curing and assembly.

Then, place the chambers on the heat block. Dilute a 20 microliter aliquot of clear lipids with 130 microliters of Min buffer, building a working concentration of 0.53 milligrams per milliliter. Add 75 microliters of the lipid mixture to each chamber.

Now, set a timer to three minutes. During the incubation time, the vesicles burst on the hydrophilic glass surface, and fuse to form a coherent SLB. Once 60 seconds have passed, add 150 microliters of Min buffer to each chamber.

After the remaining 120 seconds, wash each chamber by adding 200 microliters of Min or SLB buffer, carefully pipetting up and down a few times, removing and adding another 200 microliters. Once each chamber has been washed once, proceed to wash the first chamber thoroughly until the two milliliters of buffer are used up. Washing of supported lipid bilayers needs some experience to perfect the extent of motions in the chamber, and find the correct washing intensity.

To produce PDMS microstructures from patterned silicon wafers, first use a plastic cup to weigh 10 grams of PDMS base and one gram of PDMS crosslinker. Use a mixing device to mix and degas the PDMS mixture. Now, use a pipette tip to drop a small amount of PDMS directly onto the structure on the silicon wafer.

Immediately place a 1 coverslip onto the PDMS drop and take the upper end of a clean pipette tip to gently press the coverslip onto the silicon wafer. The PDMS should be spreading thinly between the coverslip and the silicon wafer. Place the wafer with the coverslips into an oven and cure the PDMS for three to four hours, or overnight, at 75 degrees Celsius.

Next, remove the wafer from the oven and let it cool down to room temperature. With a razor blade, carefully remove the coverslip with the attached PDMS from the silicon wafer. Now, attach a plastic chamber to the PDMS as described before for glass.

Take the coverslips with attached chamber and place in plasma cleaner with oxygen as the process gas. Clean the coverslips with plasma. In this work, 30%power and 0.3 millibar oxygen pressure for one minute, was used.

Now, prepare an SLB in the chamber as described before on glass. To perform the self-organization assay, adjust the buffer volume in the chamber to 200 microliters of Min buffer, minus the amount of protein and ATP solution. Then add MinD, labeled MinD, MinE, and if desired, MinC, and gently mix the components by pipetting.

Now, add 2.5 millimolar ATP to start the self-organization of MinDE. Over the next 10 to 30 minutes, check for regular MinDE pattern formation and properly formed microstructures on a fluorescence microscope. When regular MinDE patterns have formed, gently pipette up and down twice to mix the components.

Remove the large bulk of buffer using a 100 microliter pipette, and then carefully remove the rest using a 10 microliter pipette. To allow for longer imaging times, plug a moistened piece of sponge inside the chamber. Make sure the sponge does not contact the surface of the coverslip.

Immediately close the chamber with a lid to avoid drying of the residual buffer in the microstructures. Before imaging of the microstructures, confirm that MinDE self-organization is halted on top of the PDMS microstructure while the proteins in the microcavities are oscillating. In this representative video, dual color imaging shows how MinD and MinE self-organize into traveling surface waves that conform spiral patterns on supported lipid bilayers.

Here, single color imaging shows MinD and MinE performing pole-to-pole oscillations in PDMS microstructures. The oscillations establish a time-averaged MinD concentration gradient with maximum concentration at the compartment poles and minimal concentration in the middle of the compartment. Following this procedure, other methods, like single particle tracking, can be performed in order to determine membrane binding kinetics and residence times.

Don't forget that working with piranha solution can be extremely hazardous, and precautions such as personal protective equipment should always be taken while performing this procedure.