The endoplasmic reticulum contains a dynamic tubular domain which continuously interacts with the cytoskeleton undergoes constant motion and rearrangement. How the ER generates and maintains this organization is not yet fully understood. Here we present a convenient matter to form a bottom-up structural organelle model for the ER, which consists of a solid-supported protein-free membrane system that can be transferred to complex lipid nanotube networks.
Hydrophilics is an organic compound for energy. Protein purification and protein extraction are not required. The only essential components are the solid substrate and phospholipids providing the most basic ER model.
Transfer the dissolved lipid solutions into a 10 milliliter inverted pear shaped flask adding up to a total amount of 3, 000 micrograms of lipids and 300 microliters of chloroform. Then connect the flask to a rotary evaporator and position it with a tilt of 45 degrees. Rotate the flask at 24 RPM inside a water bath at 23 degrees Celsius for six hours with reduced air pressure to slowly and completely remove the chloroform.
Start reducing the pressure right after initiating the rotation by steps of 20 kilopascals every two minutes until the pressure reaches 20 kilopascals. After six hours, stop the rotation and increase the air pressure again gradually by steps of 20 kilopascals every two minutes until reaching 100 kilopascals. Remove the flask from the rotary evaporator and add three milliliters of PBS and 30 microliters of glycerol.
Gently swirl the flask to dissolve the glycerol. Use an air-tight glass stopper to seal the flask containing the lipids. Store the flask in the refrigerator at four degrees Celsius overnight for rehydration and swelling of the lipid films.
On the following day, sonicate the lipids with an ultrasonic water bath at room temperature and at 35 kilohertz frequency until achieving a uniform slightly turbid lipid suspension. Sonication can take around 10 to 30 seconds. Prolonged sonication produces heat and is detrimental to vesical formation.
These steps yield a suspension containing two types of vesicular structures. Multilamellar vesicles and giant unilamellar vesicles. For storage, divide the lipid suspension into 100 microliter aliquots using a total of 30 microcentrifuge tubes.
Store the aliquots in a freezer at minus 20 degrees Celsius. Flash freezing with liquid nitrogen is not necessary. Thaw the lipid suspension and transfer a four microliter droplet of suspension onto a clean glass microscope slide or cover slip.
After 20 minutes of desiccation, the droplet will collapse into a flat circular film of lipids which is visible to the eye. Rehydrate the lipid film with one milliliter of HEPES buffer for 3 minutes. Prepare the observation chamber with an open top to allow for buffer exchange by means of an automatic pipette, which is required to initiate the ER transformation as detailed in the text protocol.
After removing the cured PDMS slab with a spatula, use a scalpel to cut the frame into the dimensions and geometry appropriate for the available opening in the microscope stage. Dimensions of 1.5 by 1.5 by 0.5 centimeters are suitable for most setups. Bring the smooth side of the PDMS frame into contact with the active side of the surface where the aluminum oxide film resides and gently apply pressure to push the frame and surface against each other to make them adhere.
Immediately fill the observation chamber with calcium HEPES buffer. Do not fill the entire chamber volume to allow for addition of the rehydrated lipids in the subsequent step. Place the chamber onto the confocal microscope stage.
Transfer the rehydrated lipid material, now a suspension containing giant vesicles, into the chamber with a plastic pasture pipette. Wait 10 to 20 minutes to let the vesicles adhere onto the substrate and spread across the surface. The spreading starts immediately after deposition of lipids on the surface.
Before the lipid spreads the structure the buffer exchange must be done as gently as possible rapid removal or rapid addition of the buffer perturbs the lipid structures on the surface. After observing multiple lipid spreads, slowly remove the ambient buffer via automatic pipette. Such that only a thin buffer film remains on the bottom.
Take care to avoid rapid removal. Proceed to the ambient buffer exchange by slowly filling the observation chamber with chelator HEPES buffer using an automatic pipette. Avoid abrupt addition.
The final step yields dynamic nanotubular networks formed as a result of the chelator induced depinning and retraction of the DLBM to the multilamellar vesicles. Shown here are micrographs of the nanotube networks obtained in the protocol. In this image, the continuous bright red regions are the retracting fraction of the DLBM, which is marked with a blue dashed line.
Shown here, is a micrograph of a tubular network that has been inverted to increase contrast. Here, reduction of the tubular density is shown on a membrane region over the course of three hours and 20 minutes. The decrease of tubular density occurs due to the gradual depinning followed by retraction of the DLBM from the surface.
Locations of the calcium mediated pinning points can be established as the points where tubes have terminal or sharp turns. Sharp turns are referred to as v junctions or turning points because of the sudden shift in direction of the tubes alignment. The end point represents the terminus of the tube which prevents the tube from retracting.
If the spreading continues for prolonged periods, the membrane tension increases leading to ruptures. Since the lipids are fluorescently labeled, the rupturing can be directly observed. A key indicator of rupturing is the significant drop in fluorescence intensity in the ruptured region.
A complete dehydration of the sample or rapid exchange of buffers causes disturbance, rupturing, or deformation of the patches. The complexity of the model can be built up by adding ER associated components such as lipid proteins. And this model can also be implied to study self-assembly, nanofluidics, marangoni flow, and transport phenomena.
Chloroform is toxic and highly volatile it should always be handled under a fume hood and with associated personal protective equipment including polyvinyl acetate laboratory gloves, lab coat, and safety glasses.
