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