Deafness mutation in the MYO3A motor domain impairs actin protrusion elongation mechanism

Abstract

Class III myosins are actin-based motors proposed to transport cargo to the distal tips of stereocilia in the inner ear hairs cells and/or to participate in stereocilia length regulation, which is especially important during development. Mutations in the MYO3A gene are associated with delayed onset deafness. A previous study demonstrated that L697W, a dominant deafness mutation, disrupts MYO3A ATPase and motor properties but does not impair its ability to localize to the tips of actin protrusions. In the current study, we characterized the transient kinetic mechanism of the L697W motor ATPase cycle. Our kinetic analysis demonstrates that the mutation slows the ADP release and ATP hydrolysis steps, which results in a slight reduction in the duty ratio and slows detachment kinetics. Fluorescence recovery after photobleaching (FRAP) of filopodia tip localized L697W and WT MYO3A in COS-7 cells revealed that the mutant does not alter turnover or average intensity at the actin protrusion tips. We demonstrate that the mutation slows filopodia extension velocity in COS-7 cells which correlates with its 2-fold slower in vitro actin gliding velocity. Overall, this work allowed us to propose a model for how the motor properties of MYO3A are crucial for facilitating actin protrusion length regulation.

The dynamics of actin protrusions can be controlled by tip-localized myosin motors

“…A MYO3B/3A fusion protein that replaces the MYO3A motor and neck with the MYO3B motor and neck (MYO3B.3Atail) was able to localize to the tips of filopodia in COS7 cells, though the amount of localized myosin, filopodia length, and filopodia density were all reduced ( 6 ). Similarly, we found that the loss-of-function deafness-associated mutation L697W reduced the motor function of MYO3A in vitro , as well as in COS7 cells ( 5 , 13 ). Kinetic studies demonstrated a reduction in actin-activated ATPase and in vitro motility velocity, while COS7 cells transfected with MYO3A L697W produced filopodia of shorter lengths that extended at a slower rate than WT.…”

“…We examined the biochemical and biophysical properties of the MYO3A motor by expressing and purifying a construct containing the motor domain and neck region but lacking the kinase domain, and containing a C-terminal GFP tag (MYO3A 2IQ; see structure and diagram in Fig. 1 , A and B ) ( 5 , 6 ). To investigate the cellular function of MYO3A we expressed a full-length construct lacking the kinase domain and containing an N-terminal GFP tag (MYO3A; see Fig.…”

“…The two vertebrate class III myosins, MYO3A and MYO3B, localize to the tips of stereocilia and have been implicated in stereocilia length regulation ( 4 , 5 , 6 ). The overall structure of MYO3A and MYO3B is similar, with both containing an N-terminal kinase domain followed by a canonical myosin motor and a neck region consisting of two calmodulin-binding IQ domains and an espin (ESPN-1/ESPN-L) binding domain in their tail region.…”

“…Both MYO3A and MYO3B have been proposed to be important for regulating stereocilia elongation during development, with mutations in MYO3A that alter this function ultimately leading to non-syndromic hearing loss (DFNB30) ( 5 , 8 , 9 , 10 , 11 , 12 ). Previous work from our lab has highlighted how changes to the MYO3A motor domain can alter actin protrusion dynamics in cell-based systems.…”

Thyroid Hormone T4 Mitigates Traumatic Brain Injury by Dynamically Remodeling Cell Type Specific Genes, Pathways, and Networks in Hippocampus and Frontal Cortex

“…Malat1 encodes a long non-coding RNA that is highly abundant in the nervous system and plays a role in neuronal cell injury; 39 this gene was increased by mTBI and decreased by T4 at 7-day but the direction was opposite at 24hr and 21-day post-injury. Myo3a is a cytoskeleton gene and variants are related to hearing and vision loss, 40,41 and this gene was primarily increased by mTBI but inhibited by T4 at 21-day post-injury. Ppia mediates inflammation and has shown both beneficial effects intracellularly 42 and detrimental function extracellularly.…”