A novel mode of capping protein-regulation by twinfilin.

Cellular actin assembly is controlled at the barbed ends of actin filaments, where capping protein (CP) limits polymerization. Twinfilin is a conserved in vivo binding partner of CP, yet the significance of this interaction has remained a mystery. Here, we discover that the C-terminal tail of Twinfilin harbors a CP-interacting (CPI) motif, identifying it as a novel CPI-motif protein. Twinfilin and the CPI-motif protein CARMIL have overlapping binding sites on CP. Further, Twinfilin binds competitively with CARMIL to CP, protecting CP from barbed-end displacement by CARMIL. Twinfilin also accelerates dissociation of the CP inhibitor V-1, restoring CP to an active capping state. Knockdowns of Twinfilin and CP each cause similar defects in cell morphology, and elevated Twinfilin expression rescues defects caused by CARMIL hyperactivity. Together, these observations define Twinfilin as the first 'pro-capping' ligand of CP and lead us to propose important revisions to our understanding of the CP regulatory cycle.

Species-Specific Functions of Twinfilin in Actin Filament Depolymerization.

Twinfilin is a highly conserved member of the actin depolymerization factor homology (ADF-H) protein superfamily, which also includes ADF/Cofilin, Abp1/Drebrin, GMF, and Coactosin. Twinfilin has a unique molecular architecture consisting of two ADF-H domains joined by a linker and followed by a C-terminal tail. Yeast Twinfilin, in conjunction with yeast cyclase-associated protein (Srv2/CAP), increases the rate of depolymerization at both the barbed and pointed ends of actin filaments. However, it has remained unclear whether these activities extend to Twinfilin homologs in other species. To address this, we purified the three mouse Twinfilin isoforms (mTwf1, mTwf2a, mTwf2b) and mouse CAP1, and used total internal reflection fluorescence microscopy assays to study their effects on filament disassembly. Our results show that all three mouse Twinfilin isoforms accelerate barbed end depolymerization similar to yeast Twinfilin, suggesting that this activity is evolutionarily conserved. In striking contrast, mouse Twinfilin isoforms and CAP1 failed to induce rapid pointed end depolymerization. Using chimeras, we show that the yeast-specific pointed end depolymerization activity is specified by the C-terminal ADF-H domain of yeast Twinfilin. In addition, Tropomyosin decoration of filaments failed to impede depolymerization by yeast and mouse Twinfilin and Srv2/CAP, but inhibited Cofilin severing. Together, our results indicate that Twinfilin has conserved functions in regulating barbed end dynamics, although its ability to drive rapid pointed end depolymerization appears to be species-specific. We discuss the implications of this work, including that pointed end depolymerization may be catalyzed by different ADF-H family members in different species.