A Hearing Loss-Associated myo1c Mutation (R156W) Decreases the Myosin Duty Ratio and Force Sensitivity

Abstract

Myo1c is a member of the myosin superfamily that has been proposed to function as the adaptation motor in vestibular and auditory hair cells. A recent study identified a myo1c point mutation (R156W) in a person with bilateral sensorineural hearing loss. This mutated residue is located at the start of the highly conserved switch-1 region, which is a crucial element for the binding of nucleotide. We characterized the key steps on the ATPase pathway at 37 °C using recombinant wild-type (myo1c 3IQ ) and mutant myo1c (R156W-myo1c 3IQ ) constructs that consist of the motor domain and three IQ motifs. The R156W mutation only moderately affects the rates of ATP binding, ATP-induced actomyosin dissociation, and ADP release. The actin-activated ATPase rate of the mutant is inhibited > 4-fold, which is likely due to a decrease in the rate of phosphate release. The rate of actin gliding, as measured by the in vitro motility assay, is unaffected by the mutation at high myosin surface densities, but actin gliding is substantially reduced at low surface densities of R156W-myo1c 3IQ . We used a frictional-loading assay to measure the affect of resisting forces on the rate of actin gliding and found that R156W-myo1c 3IQ is less force sensitive than myo1c 3IQ . Taken together, these results indicate that myo1c with the R156W mutation has a lower duty ratio than the wild-type protein and motile properties that are less sensitive to resisting forces.Myo1c is the single-headed member of the myosin superfamily that plays roles in trafficking of GLUT4-containing vesicles to the plasma membrane in response to insulin stimulation (1,2) and in compensatory endocytosis following regulated exocytosis (3). Myo1c has also been proposed to play a key role in the process of adaptation in specialized sensory cells, where it is thought to dynamically adjust tension on mechanosensitive ion channels via its interaction with actin (4,5).Cell biological studies have shown that myo1c localizes and fractionates with cell membranes, and biochemical experiments have shown myosin-I isoforms bind directly to phosphoinositides (6,7). Additionally, mechanical experiments have shown that a related myosin isoform (myo1b) acts as a tension sensor by responding to small resisting loads by increasing its actin-attachment lifetime, allowing it to generate and sustain tension for

Myosin IC generates power over a range of loads via a new tension-sensing mechanism

“…Myo1b is suited to function as a tension-sensitive anchor, transforming from a low duty-ratio motor to a high duty ratio-motor with forces resisting its working stroke >0.5 pN. Moreover, the rate of actin translocation in the in vitro motility assay is similar for both myo1b (21) and myo1c (16) (when corrected for the differences in length of the light chain-binding domain). Given the similarity of their unloaded biochemical properties, one may have expected myo1c to share the force-sensing properties of myo1b (22,23).…”

“…The rate constants that define the myo1c actin-activated ATPase pathway in solution are very similar to myo1b (10,12,16,19), a closely related myosin-I isoform that has actin-detachment kinetics that are exquisitely sensitive to tension (20). Myo1b is suited to function as a tension-sensitive anchor, transforming from a low duty-ratio motor to a high duty ratio-motor with forces resisting its working stroke >0.5 pN.…”

“…Previous biochemical analyses have shown that myo1c is a lowduty ratio motor (i.e., it spends most of its biochemical cycle detached from actin) (10,12,16), albeit with an actin-attachment lifetime that is approximately 500-fold longer than fast skeletal muscle myosin-II (17) and approximately 10-fold longer than the processive motor, myosin-V (18). The rate constants that define the myo1c actin-activated ATPase pathway in solution are very similar to myo1b (10,12,16,19), a closely related myosin-I isoform that has actin-detachment kinetics that are exquisitely sensitive to tension (20).…”

Structural Analysis of the Myo1c and Neph1 Complex Provides Insight into the Intracellular Movement of Neph1

“…Expression and purification of His-ZO1-PDZ1 protein have also been described previously (14,39). Recombinant proteins Flag-Myo1c-FL and GFP-HisMyo1c-tail were produced in a baculovirus expression system as described previously (40,41). Two peptides, ERTPYEAYDPIGKYATATRF and YE KFNSHPFPGAAGYPTYRL, derived from the K761 region of Neph1 and the region of Neph1 not involved in Myo1c binding, respectively, were synthesized chemically using standard 9-fluorenylmethoxy carbonyl (Fmoc) solid-phase chemistry, and 2-chloro trityl resin was used as a solid support (PTI peptide synthesizer).…”

Myosin-I molecular motors at a glance

“…Additionally, in vitro and cellular studies have shown that cargo docking by Myo1c is regulated by the presence of non-muscle tropomyosin, which might spatially regulate the location of cargo docking to tropomyosin-free filaments just beneath the plasma membrane (Kee et al, 2015;McIntosh et al, 2015). Myo1c has also been implicated in the mechanical adaptation of signaling in inner ear hair cells (Batters et al, 2004;Gillespie et al, 1993;Holt et al, 2002;Lin et al, 2011), as well as in the regulation of a Na + channel after antidiuretic hormone stimulation in the kidney collection ducts (Wagner et al, 2005). Myo1b has been proposed to tether amino acid transporters to the apical plasma membrane of kidney cells, thereby facilitating neutral amino acid transport across the membrane; however, more investigation is needed to differentiate this hypothesis from other potential roles, such as facilitating membrane fusion of vesicles containing amino acid transporters, and/or mediating the transport and/or sorting of these cargo to the apical plasma membrane (Komaba and Coluccio, 2015).…”