Abstract: Actin is an essential protein required for force generation in key cellular processes including cytokinesis, cell migration, phagocytosis etc. Intracellular actin networks are thought to assemble by polymerization of filaments at their barbed ends and depolymerization at their pointed ends. This process, referred to as “treadmilling”, forms the central bedrock of our current understanding of actin dynamics. However, recent results from our lab suggest that this “dogma” might not be true. By using a combination of multicolor single-molecule and single filament experiments as well as mathematical modeling, we uncovered two distinct mechanisms which facilitate barbed end depolymerization and pointed ends polymerization; overturning the treadmilling “dogma”. Twinfilin, Cofilin, and Profilin, three key actin binding proteins have been shown to individually depolymerize barbed ends, however, how they collectively regulate barbed end depolymerization remains an open question. Therefore, we decided to investigate the multicomponent dynamics of twinfilin, cofilin, and profilin at the barbed end. We have discovered that twinfilin competes with profilin and promotes binding of cofilin to the sides. Interestingly, contrary to previous expectations, we found that profilin and cofilin can simultaneously bind the same barbed end resulting in its accelerated depolymerization. Additionally, we discovered the first ever pointed end polymerase VopF, which processively tracks pointed ends for several minutes and in the process accelerates their elongation. Using single-molecule force-spectroscopy, we found that VopF is mechanosensitive - its rate of polymerization increases under pN-range pulling forces. Taken together, our new findings challenge the basic tenets of our current understanding of intracellular actin dynamics and call for taking a fresh look.