Protein Phase Separation Assay: An Optogenetic Method for Mutant RNA-Binding Protein Phase Separation in Spinal Motor Neurons of Zebrafish Larvae
Published: April 30, 2023
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<…
Disclosures
The authors have nothing to disclose.
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 |
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Cite This Article
Protein Phase Separation Assay: An Optogenetic Method for Mutant RNA-Binding Protein Phase Separation in Spinal Motor Neurons of Zebrafish Larvae. J. Vis. Exp. (Pending Publication), e21165, doi: (2023).