Protein Phase Separation Assay: An Optogenetic Method for Mutant RNA-Binding Protein Phase Separation in Spinal Motor Neurons of Zebrafish Larvae

Nuclear RNA-binding proteins, RBPs, are multifunctional proteins composed of ordered regions with well-defined, three-dimensional structure and less stable, intrinsically disordered regions, IDRs.

In several neurodegenerative diseases, IDR-specific mutations cause RBPs to mislocalize into the cytoplasm, and aberrantly engage in weak interactions, forming dynamic, membrane-less inclusions - a phenomenon called phase separation. Eventually, these inclusions mature into toxic, insoluble aggregates, resulting in neuronal dysfunction and degeneration.

To investigate RBP phase separation in vivo using optogenetics, begin with an anesthetized, dechorionated transgenic zebrafish larva, expressing an IDR-mutated RBP fused to a fluorescent reporter and a blue light-sensitive photoreceptor domain, in suitable buffer.

Next, mount the larva laterally onto a molten agarose drop for spinal cord imaging. Allow the agarose to solidify, immobilizing the larva. Using a confocal microscope, acquire spinal cord images to visualize the fluorescent RBPs, localized within the neuronal nuclei.

Now, remove the larva from the agarose and transfer into a buffer-containing multi-well plate. Position the plate onto an optimized blue light-emitting diode panel and illuminate the larva.

Upon exposure to blue light, the mutant RBPs mislocalize to the cytoplasm, where the photoreceptor domains oligomerize and form clusters, bringing the IDRs of the RBPs in close proximity and facilitating their self-association into membrane-less inclusions.

Post-illumination, embed the larva in agarose, and image its spinal cord. Fluorescent RBPs appear as discrete compartments dispersed within the neuronal cytoplasm.

For imaging of the zebrafish larvae expressing optogenetic TDP-43, dechorionate the double-transgenic fish, and anesthetize them in E3 buffer containing 250 micrograms per microliter of Tricane.

Next, pre-heat 1% low-melting temperature agarose containing 250 micrograms per microliter of ethyl 3-aminobenzoate methanesulfonate at 42 degrees Celsius, then, put a drop of the agarose on the glass base dish. The diameter of the dome-shaped agarose drop on the glass dish should be 8 to 10 millimeters.

Next, using a Pasteur pipette, add the anesthetized fish to the agarose on the glass base dish. Minimize the amount of E3 buffer added to the agarose along with the fish. Then, mix by pipetting a few times.

During solidification of the agarose, maintain the fish on its side using a syringe needle, such that the spinal cord is in an appropriate horizontal position. After the agarose has solidified, add a couple drops of E3 buffer onto the dome-shaped agarose-mounted fish.

Using a confocal microscope equipped with a 20x water immersion objective lens, acquire serial confocal z-sections of the spinal cord. Include the cloaca on the ventral side of the fish in the regions of interest as a reference to identify and compare the spinal segments across the time points.

As soon as the imaging is complete, use a syringe needle to carefully crack the agarose and remove the fish from the agarose. Keep the amount of time the fish is embedded in the agarose as short as possible, although agarose embedding for less than 30 minutes does not affect the viability of the fish.

Add 7.5 milliliters of E3 buffer to one well of a 6-well dish, and place the imaged double-transgenic fish into that well. Then, place the dish on the LED panel, keeping the dish, and LED panel 5 millimeters apart with a spacer, and turn on the blue LED light.

Keep some double-transgenic fish in a separate 6-welled dish covered with aluminum foil for unilluminated control fish. After the desired illumination time, image the spinal cord of the illuminated fish as demonstrated earlier.