Regulation of actin and microtubules by TDP-43 and profilin

TDP-43 is a classic DNA/RNA binding protein important for the normal function of numerous cellular activities that directly and indirectly impinge on the actin and microtubule cytoskeletons. When TDP-43 levels are reduced, neurons display phenotypes consistent with the loss of actin-microtubule crosstalk functions including aberrant morphology, fewer dendritic branches, and diminished area of growth cones. TDP-43 is direct binding partner of profilin (kD = 115.2 nM), which also regulates actin and microtubule dynamics, and variants of either protein are considered causative agents in several neurodegenerative diseases. First, we explored whether TDP-43 directly interacts with the actin or microtubules cytoskeletons and whether TDP-43 could mediate cytoskeletal crosstalk by forming phase-separated biomolecular condensates. We used TIRF microscopy assays to measure the impact of purified TDP-43 on actin and microtubule assembly. TDP-43 bound and sequestered both actin and tubulin (kD = 22.7 nM). inhibiting the assembly of either polymer. We also explored how interactions between profilin and TDP-43 modulated the dynamics of either cytoskeleton or phase separation. The addition of profilin increased TDP-43 condensate formation (number and size), further inhibited actin assembly, but stabilized microtubule assembly. To investigate this in a more physiological context, we imaged the cytoskeleton and TDP-43 in Neuroblastoma-2a cells. Cells lacking profilin displayed less robust actin and microtubule arrays compared to endogenous or rescue controls. In addition, TDP-43 is mislocalized to the cytoplasm, mirroring the disease state of amyotrophic lateral sclerosis (ALS). Rescue experiments expressing wild type profilin on plasmids restored actin and microtubule arrays and the nuclear localization of TDP-43, whereas profilin plasmids harboring ALS-related mutations did not. Thus, profilin and TDP-43 are important regulators of actin and microtubule dynamics in cells and in the context of ALS.