TDP-43 (TAR-DNA-binding protein-43) is a nuclear protein that regulates gene expression, RNA splicing, and transcript stabilization. It is essential for neuronal development and forms biomolecular condensates (liquid phase separated droplets). TDP-43 condensates can become pathogenic when exported from the nucleus to the cytoplasm, but it is unclear whether all cytoplasmic condensates are toxic (or if this feature is limited to fibrils). Pathogenic TDP-43 disrupts the integrity of cells by interfering with essential cytoskeleton-controlled processes to cause disease.
We became interested in TDP-43 for several reasons: 1) familial ALS-causing mutations in TDP-43 are more prevalent than those in profilin-1; 2) although the classic cytoskeletal regulator profilin does not form condensates, it is a direct binding partner of TDP-43; and, 3) curiosity regarding whether condensates could link actin and microtubule dynamics; and 4) in profilin-deficient cells, TDP-43 is localized almost exclusively to the cytoplasm, phenocopying an ALS-related disease state. Using our single-molecule TIRF system, we visualized labeled actin alone and in the presence of TDP-43. Strikingly, all reactions that contained TDP-43 (even unlabeled TDP-43 reactions) display fluorescent condensates and also appear to have reduced actin assembly (see above).
We are investigating the following questions:
1) Do profilin and actin (alone or combined) affect the size, structure, or assembly dynamics of TDP-43 condensates?
2) Do TDP-43 condensates facilitate actin-microtubule crosstalk?
3) Which residues in profilin, actin, tubulin, or TDP-43 mediate or affect these interactions?
Condensates, the cytoskeleton & ALS (lou gehrig's disease)
Profilin-1 KO cells
actin filaments, TDP-43, & nucleus
in vitro TIRF of purified actin & TDP-43-GFP
merge: actin & TDP-43-GFP condensates
1) Define the mechanisms that underlie how TDP-43 regulates actin and microtubules.
2) Determine the cellular consequences of cytoplasmic TDP-43 on the cytoskeleton.