TDP-43 phase transitions and co-demixing with stress and P-granule components
Haiyang Yu1, Shan Lu1, Fatima Gasset-Rosa1, Cong Chen1, Zevik Melamed1, Sandrine Da Cruz1, and Don W. Cleveland1,2,3*
1Ludwig Institute for Cancer Research; 2Department of Cellular and Molecular Medicine; 3Department of Neuroscience, University of California at San Diego, La Jolla, CA 92093, USA
Nuclear clearance and cytoplasmic aggregation of wild type TDP-43 is a pathological hallmark of over 90% of amyotrophic lateral sclerosis (ALS) patients. We have now identified de-mixing of wild type TDP-43 into membraneless, liquid-like granules in both the nucleus and the cytoplasm of cycling non-neuronal and neuron-like (Sh-Sy5Y) cells. The N-terminal ubiquitin-like and C-terminal low-complexity domains are necessary for stress-induced de-mixing of TDP-43 in the cytoplasm, while RNA-binding is not required. RNA-binding deficient TDP-43 mutant de-mixes in both the nucleus and the cytoplasm. Cytosolic TDP-43 co-de-mixes with stress granules, but a RNA-binding deficient mutant de-mixes into apparently membraneless granules that are independent of stress granules or stress granule marker proteins. Aging of these cytosolic TDP-43 granules leads to TDP-43 aggregation, cleavage, phosphorylation, and ubiquitination, a chain of events that produces aggregated TDP-43 that resembles the cytosolic TDP-43 aggregates observed in postmortem ALS. Co-demixing of TDP-43 with stress granules or components of stress granules enhances arsenite-induced cell death. By tagging TDP-43 with a 50-amino acid weakly self-associating domain (by folding into a beta-solenoid structure), TDP-43 readily de-mixes without added cellular stress. Using inducible TDP-43 de-mixing as a model, and APEX2-mediated proximity labeling technology, we have identified unique RNA binding proteins that favor de-mixed TDP-43, including stress granule and P-granule components. Cytosolic TDP-43 de-mixing causes dysregulation of P-granule components.
Funding: NIH 1-F32-AG059358-01