Formins are actin-binding proteins that construct nanoscale machinery with the growing barbed end of actin filaments and serve as key regulators of actin polymerization and depolymerization. To maintain the regulation of actin dynamics, formins have been proposed to processively move at every association or dissociation of a single actin molecule toward newly formed barbed ends. However, the current models for the motile mechanisms were established without direct observation of the elementary processes of this movement. Here, using optical tweezers, we demonstrate that formin mDia1 moves stepwise, observed at a nanometer spatial resolution. The movement was composed of forward and backward steps with unitary step sizes of 2.8 and -2.4 nm, respectively, which nearly equaled the actin subunit length (∼2.7 nm), consistent with the generally accepted models. However, in addition to steps equivalent to the length of a single actin subunit, those equivalent to the length of two or three subunits were frequently observed. Our findings suggest that the coupling between mDia1 stepping and actin polymerization is not tight but loose, which may be achieved by the multiple binding states of mDia1, providing insights into the synergistic functions of biomolecules for the efficient construction and regulation of nanofilaments.
Keywords: Formin; actin; cytoskeleton; individual steps; optical tweezers; single molecule.