This protocol allows for the simple generation of large-scale lipid nanotube networks in which the individual nanotubes display phase separation behavior similar to that of parent vesicle. The key advantage of this approach is that we co-opt the work done by the molecular motors to self-assemble the lipid nanotubes in a highly parallel manner and without human intervention. To set up a gliding motility assay, first, affix three sets of double-sided tape strips onto a glass slide five millimeters apart.
Gently press a cover slip onto the tape and add 30 microliters of a one-micromolar kinesin solution into the prepared flow cell. After five minutes, add 30 microliters of a 10-microgram/milliliter microtubule solution into the cell, pressing a laboratory wipe gently against the opposite end of the flow channel to facilitate the solution exchange. After another five minute incubation, wash the cell one to three times with 50 microliters of room-temperature motility solution per wash before adding 30 microliters of 10-microgram/milliliter streptavidin solution to the flow cell.
After 10 minutes, add 30 microliters of a 10X GUV solution for a 30 minute incubation at room temperature. Then, add two microliters of a 100-millimolar AMP-PNP solution and close the chamber with sealant. Transfer the flow chamber to an inverted microscope for imaging and select the appropriate filter set.
Use a 100X oil objective to focus first the surface of the cover slip before acquiring an image of a network of interest. Then open the image in ImageJ and click Image, Color, and Composite to overlay the red and green channels to create a composite image. After imaging, open the image of interest and click Analyze and Set scale to calibrate the scale for the microscope.
Fill in the pixels to the micrometer conversion factor and click OK.Use the multi-point line tool to trace the nanotubes starting from the parent GUV and press and hold Control and M to measure the length. The image processing tool will save each new measurement in the results window. When all of the tubes have been measured, select Image, Adjust, and Threshold and click Apply to apply the threshold.
Then, draw a rectangle of known length over the desired tube. To measure the integrated density of the area, click Analyze and Measure To determine the lipid partitioning in the liquid nanotube nodes, use the line tool to draw a line over the node of interest and measure the node intensity in both the green and red channels. In this representative analysis, liquid nanotube networks were fabricated as demonstrated to generate liquid-disordered and ordered phases, as well as phase-separated vesicles.
For example, vesicles synthesized with 45%saturated lipid and 55%unsaturated lipid results in vesicles that separate into coexisting liquid-disordered and solid phases. If cholesterol is included, however, liquid-liquid coexistence can then be observed, creating GUVs that separate into coexisting liquid-ordered and liquid-disordered phases. Moreover, nodes can be observed within the phase-separated mixtures.
The most important thing to remember is to pick the appropriate exposure time and ND filter set to be able to visualize the lipid nanotubes without photo-bleaching the fluorophores. Tailoring the composition of phase behavior of the lipid nanotubes allows us to use a minimalistic model system to begin studying the biophysics of the tunneling nanotubes that connect cells.
