Characterization of drosophila myosin 7a mechanics

Abstract

Myosin 7a is an unconventional myosin which participates in the sensory cell functions of numerous organisms, including humans, zebra fish, and flies. In drosophila, myosin 7a (DmM7a) appears responsible for bristle morphology, including the antennae involved in auditory transduction. The composition of motifs within the molecule is as follows: a motor head, containing sub-domains broadly typical of the myosin super-family, which connects to 5 IQ's, followed by the tail region. Within the tail are a putative coiled-coil followed by two tandem MyTH4-FERM domains separated by an SH3 domain. Here, data obtained using the optical trap three bead assay - the practice of using photon force to manipulate micrometer-scale beads to observe single molecule events - are presented for DmM7a. A truncated DmM7a construct (DmM7aTD1), cropped after the tail SH3 domain, was observed to interact with an actin filament at low ionic strength (50 mM KCl). Under the same conditions no interactions were seen with the full length version (DmM7aFL), however, at high ionic strength (200 mM) DmM7aFL became active. These findings are in agreement with recent studies demonstrating that the tail performs an internal regulatory function which is electrostatic in nature. The actin detachment rates (Kdet), calculated from dwell times, were similar for DmM7aTD1 and DmM7aFL at 10 μM ATP, approximately 0.2 s-1. The Kdet for DmM7aFL was dependent on ATP concentration, and was increased at 1 mM ATP. These data support previous studies showing M7a to be a high duty motor with slow ATPase activity. Attempts to dimerise DmM7a on actin were unsuccessful based on the absence of “stepping” events which are a hallmark of processivity. This supports the case for DmM7a having a role in tension maintenance.