Tuning the Mechanical Output of Nonmuscle Myosin-2 Filaments

Abstract

Three nonmuscle myosin-2 (NM2) paralogs participate in many mammalian cellular phenomena. Each NM2 paralog forms 310nm bipolar filaments containing either 30 (NM2A/NM2B) or 16 myosins (NM2C). The three paralogs are all slow enzymatically and mechanically compared to other myosins, but have distinct kinetic signatures with NM2B having the highest duty ratio. NM2B bipolar filaments show robust processive movements on single actin filament in vitro with a run length of about 2 µm and a velocity of 43 nm/s. Rings of branched actin filaments formed in the presence Arp2/3 are contracted by addition of NM2B filaments. In addition, NM2B filaments bundle long parallel actin filaments that emanate from the rings. To determine the number of NM2B motor domains that are necessary for processive movement we co-assembled NM2B molecules with headless Halo-tagged-myosin rods allowing us to vary the number of motor domains in a bipolar filament. More than five NM2B motors/half filament are required for processive movements. This number agrees well with calculations based on the duty ratio of single headed NM2B S1. As the number of myosin motor domains are reduced in the co-filaments the run-length, but not the velocity decreases. In contrast, under the same buffer conditions, NM2A filaments do not move processively. This is not surprising given the lower duty ratio of NM2A. Processive movements with NM2A can be achieved by including methylcellulose in the assay to mimic the viscosity of a cell or by forming co-polymeric filaments with NM2B. The co-filaments exhibit mechanical properties that are intermediate between those of the individual myosins. This demonstrates that cells can fine tune the mechanical output of a myosin filament by controlling the isoforms present in the filament.