The Properties and Location of Anisosome-Forming RNA-binding deficient TAR DNA-binding Protein 43 inside the Nucleus
Open Access
- Author:
- Mertick Sykes, Eli
- Area of Honors:
- Biochemistry and Molecular Biology
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Xin Zhang, Thesis Supervisor
Song Tan, Thesis Honors Advisor - Keywords:
- Protein
Liquid-Liquid Phase Separation
TDP-43
Mammalian Cells
Immunofluorescence - Abstract:
- The accumulation of protein aggregates is a commonly displayed phenotype for patients at different stages of disease progression in neurodegenerative disorders. Much research has been done to combat these disorders, with many focusing on the ability of clearing protein aggregates or preventing their formation. The discovery that many protein aggregates initially form as misfolded or phase-separated oligomers has focused research to the prevention of these early-stage formations. TAR DNA-binding Protein 43 kDa (TDP-43) is a particular protein that has been implicated in patients with ALS; RNA-binding domain mutations that cause the protein to oligomerize and undergo liquid-liquid phase separation (LLPS) are a commonly displayed phenotype of patients with the disease. These phase-separated droplets form “anisosomes” when overexpressed in mammalian cells. This project seeks to characterize the phenotypes of multiple TDP-43 mutants as a model for the study of phase-separated oligomers. TDP-43 was expressed as a recombinant protein with an N-terminal SNAP-tag and incubated with a modified Green Fluorescent Protein that associates with the SNAP-tag. Fluorescence assays revealed a variety of anisosome phenotypes and quantified their propensities, size, and concentration. Structural analyses provided rationalizations for the formation of anisosomes but could not provide justifications for why certain mutants displayed differential phenotypes. Co-localization studies were performed using immunofluorescence of specific nuclear proteins to reveal potential protein-protein interactions. While some proteins exhibited signal overlap with TDP-43, many were statistically insignificant or were an artifact of improper procedural technique. Future localization assays should include enhanced microscopy techniques to support the existence of protein scaffolds or chaperones (if any) in the formation of anisosomes.