Single-molecule live cell tracking and super-resolution microscopy to study stress related oligomerization and aggregation of the ALS-related protein TDP-43

Abstract

TAR DNA binding protein 43 (TDP-43) is a mostly nuclear nucleic acid binding protein and its mislocalization and aggregation was shown to be closely connected to the pathogenesis of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). ALS is a devastating neurodegeneration disease, leading to the progressive degeneration of motor neurons in the brain and spinal cord of affected individuals. TDP-43 is a highly aggregation prone protein and TDP-43 aggregates are found in brains of ALS patients. Despite its mainly nuclear function, TDP-43 shows translocation to cytoplasmic stress granules under stressed conditions. Stress granules are phase-separated compartments densely packed with RNAs and RNA-binding proteins, and are suspected to serve as a starting point for pathological protein aggregation. However, the exact role of stress granules in the formation of TDP-43 oligomers and aggregates is still under debate. In this context, the identification of possibly pathological TDP-43 species and the uncovering of TDP-43 aggregation pathways constitute highly relevant topic in the context of a better understanding of the pathogenesis of ALS and potential pharmacological interventions. In this work, TDP-43 mobility and cellular localization was analysed under different sodium arsenite stress and recovery conditions using live cell single-molecule tracking and super-resolution microscopy. Besides the expected reduced mobility within stress granules, single-molecule tracking showed a stress induced, strong decrease of TDP-43 mobility in the cytoplasm and in the nucleus. Stress removal lead to a recovery of TDP-43 mobility, whose extent strongly depended on the applied stress duration. Stimulated-emission depletion microscopy (STED) and tracking and localization microscopy (TALM) revealed TDP-43 substructure within stress granules, TDP-43 binding sites within the nucleus and more importantly cytoplasmic TDP-43 localization patches, exhibiting a reduced TDP-43 mobility throughout the cytoplasm. The here presented data provide new insight into stress-related TDP-43 mobility changes on a molecular level and show that TDP-43 exhibits region specific differences in its mobility. The data show a reduced mobility of TDP-43 within stress granules, most likely caused in part by a localization to defined binding regions seen by TALM. Most surprisingly, TDP-43 exhibits a strong mobility reduction within the cytoplasm and the nucleus, indicating that oligomerization happens within the cytoplasm distinct from stress granules. Visualization of the single-molecule tracking data showed patches of increased TDP-43 localization and reduced mobility throughout the cytoplasm, further supporting the idea of cytoplasmic, stress granule independent TDP-43 oligomerization. Recovery experiments showed that TDP-43 mobility can be regained after stress removal and that longer stress leads to the formation of insoluble TDP-43 species. Lastly, STED microscopy revealed TDP-43 substructures within stress granules. In summary, live cell single-molecule tracking and super-resolution microscopy serve as suitable tools to investigate TDP-43 mobility and localization on a molecular level and help to get new insights into the oligomerization process of TDP-43.