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