BPS2026 – TetrathymosinBeta (TTH-1) inhibits actin assembly through the coordinated function of 4 tandem WH2 domains

Spatiotemporal actin assembly and disassembly is precisely regulated by the coordinated action of various actin binding proteins (ABPs) with diverse functions. The C. elegans ABP TetrathymosinBeta (TTH-1) is known to be critical during development. TTH-1 is hypothesized to function both alone and in combination with other ABPs to help maintain a pool of unassembled actin for rapid F-actin network assembly, but the mechanism(s) underlying its function(s) are poorly understood. TTH-1 consists of four repeated WH2 domains. The best-characterized WH2 domain is found in human thymosinBeta4 sequence, which is known to sequester G-actin, preventing both nucleation and elongation. However, in other proteins, the WH2 domain can exhibit multiple or opposite functions. To learn the function(s) of the WH2 domains in TTH-1, here we compare the ability of TTH-1 and thymosinBeta4 to bind and inhibit actin assembly. We combined “bulk” pyrene assays with single molecule TIRF microscopy to measure how TTH-1 and its domains bind actin and influence assembly. We observe that TTH-1 significantly decreases both the nucleation of F-actin and their subsequent elongation to a greater extent than thymosinBeta4, indicating that TTH-1 is a G-actin sequestering protein. As such, knockdown of TTH-1 significantly increases F-actin network assembly in the C. elegans zygote. Fits of kinetic models to our pyrene data support the hypothesis that TTH-1 can bind multiple actin monomers simultaneously. Additionally, we have purified individual and combinations of TTH-1 WH2 domains. Preliminary data on these constructs suggest that the domains are much less active when in isolation as full-length TTH-1. Together, our results reveal previously unknown functions of full-length TTH-1 and its individual WH2 domains that help explain its role in maintaining G-actin for the rapid assembly of functionally diverse F-actin networks during C. elegans development.