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

Abstract

Source: Asakawa, K. et al. Optogenetic Phase Transition of TDP-43 in Spinal Motor Neurons of Zebrafish Larvae. J. Vis. Exp. (2022)

In this video, we describe a phase separation assay, wherein intracellular proteins comprising intrinsically disordered regions fused to a photosensitive oligomerization domain are induced via blue-light exposure to self-associate into membrane-less liquid-like condensates.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board 1. Preparation of LED for blue light illumination Turn on an LED panel by using the associated application installed on a tablet/phone. Put the probe of a spectrometer into an empty well of a 6-well dish and adjust the LED light to the wavelength peaking at ~456 nm through the application. Place the optical sensor of an opt…

Representative Results

Figure 1: Ratiometric comparisons of opTDP-43h and EGFP-TDP-43z before and after light stimulation. (A) ROIs covering the somas of four single mnr2b-positive cells at 48 and 72 hpf were drawn based on the EGFP-TDP-43z signal and are shown in magenta. The rectangular ROIs (bg) were used to subtract the background signal (background ROI). Figures are adapted from Asakawa et al<…

Materials

Confocal microscope	Olympus	FV1200
Epifluorescence microscope	ZEISS	Axioimager Z1
Fluorescence stereomicroscope	Leica	MZ16FA
Glass base dish	IWAKI	3910-035
Incubator	MEE	CN-25C
LED panel	Nanoleaf Limited	Nanoleaf AURORA smarter kit
Objective lens	Olympus	XLUMPlanFL N 20×/1.00
Objective lens	ZEISS	Plan-Neofluar 5x/0.15
Optical power meter	HIOKI	3664
Optical sensor	HIOKI	9742-10
Six-well dish	FALCON	353046
Spectrometer probe BLUE-Wave	StellerNet Inc.	VIS-50
Syringe needle	TERUMO	NN-2725R
Tricane	Sigma-Aldrich	A5040
NuSieve GTG Agarose	LONZA	50181