Studying Distribution and Aggregation of TDP-43 in Mammalian Cells: A Comparison between Fluorescent Protein and Tetracysteine Labelling Strategies

Aggregation and deposition of TAR DNA-binding protein 43 (TDP-43) in motor neurons is a key pathological feature in nearly all amyotrophic lateral sclerosis (ALS) cases. However, little is known about the mechanism of TDP-43 aggregate formation, in particular how the protein structure changes over time and how this correlates with changes in cellular localisation. To date, a detailed analysis of the dynamic processes leading to TDP-43 aggregate deposits in live cells has not been reported. We have established neuronal cell lines that express the full length or C-terminal fragment (residue274-414) of TDP-43 tagged with a tetracysteine (TC) motif that binds specifically to biarsenical dyes (FlAsH, ReAsH), and we are comparing this to a cell model expressing TDP-43 fused to green fluorescent protein (GFP). Unlike GFP which is 27 kDa in size and thus, much larger than the C-terminal fragment, the TC-tag and biarsenical dyes are relatively small (∼1.1kDa); therefore, are less likely to interfere with the distribution and function of recombinant proteins. Using confocal microscopy and intermolecular FRET analysis, we demonstrate that biarsenical labelling technique enable the visualisation of TDP-43 localisation for the monomers and aggregate formed within live cells; in particular we can monitor the changes in distribution and aggregation as a function of time. Furthermore, using this approach we are able to compare, in a quantitative manner, the behaviour of ALS-associated variants of TDP-43 (A315T, M337V) that have been reported to possess enhanced aggregation as compared to the wild-type protein. Such models will prove useful towards investigating how changes in the structure of TDP-43 influences its localisation and aggregation under both physiological and cell stress conditions, and how these processes may relate to the pathogenicity and progression of ALS.