Myosin-I facilitates symmetry breaking and promotes the growth of actin 'comet tails'.

Abstract

Myosin-Is are single-headed, membrane associated members of the myosin superfamily that participate in crucial cellular processes related to membrane morphology and trafficking. Recent studies show that myosin-I isoforms frequently concentrate on membranes in areas of Arp2/3 complex-mediated actin polymerization that affect membrane shape and dynamics. To investigate how myosin-Is affect actin assembly, we performed a “comet tail assay” where branched actin networks were nucleated by Arp2/3 complex from a bead surface coated with a nucleation promoting factor (NPF). Actin filaments first formed a cloud around the bead, which transitioned into a polarized comet tail after symmetry breaking. We site-specifically coupled a range of densities of myosin-Is to the bead surface and assessed their effects on actin polymerization, network architecture, and symmetry breaking. We found that high myosin densities prevented comet tail formation. Instead, an extremely sparse actin cloud was created surrounding the bead. Decreasing the myosin density resulted in an actin network that broke symmetry more rapidly and formed a polarized comet tail. This actin comet tail was able to elongate from the bead at a faster rate and frequently showed smooth, rather than pulsatile motion. Myosin also changed the architecture of actin networks in comet tails. Compared with the coherent actin networks from non-myosin-coated beads, actin networks emerging from myosin-coated beads were sparser and more disordered, which might be due to the gliding and rotating effect of myosin. Strikingly, under low capping protein concentrations, where bead was embedded in a highly-dense actin shell and symmetry breaking was completely inhibited, myosin-coated beads were able to overcome this inhibition to break symmetry and form a comet tail. These studies show synergy between myosin activity and actin polymerization to power morphological changes at the cell membrane.