Myosin-driven Nucleation of Actin Filaments Drives Stereocilia Development Critical for Hearing

Abstract

SUMMARYThe assembly and maintenance of actin-based mechanosensitive stereocilia in the cochlea is critical for lifelong hearing. Myosin-15 (MYO15) is hypothesized to modulate stereocilia height by trafficking actin regulatory proteins to their tip compartments, where actin polymerization must be precisely controlled during development. We identified a mutation (p.D1647G) in the MYO15 motor-domain that initially maintained trafficking, but caused progressive hearing loss by stunting stereocilia growth, revealing an additional function for MYO15. Consistent with its maintenance of tip trafficking in vivo, purified p.D1647G MYO15 modestly reduced actin-stimulated ATPase activity in vitro. Using ensemble and single-filament fluorescence in vitro assays, we demonstrated that wild-type MYO15 directly accelerated actin filament polymerization by driving nucleation, whilst p.D1647G MYO15 blocked this activity. Collectively, our studies suggest direct actin nucleation by MYO15 at the stereocilia tip is necessary for elongation in vivo, and that this is a primary mechanism disrupted in DFNB3 hereditary human hearing loss.

Structural basis for tunable control of actin dynamics by myosin-15 in mechanosensory stereocilia

“…This defect occurring at the level of nuclei formation, rather than elongation, is consistent with our observation that F-actin elongation rates are indistinguishable in the presence of ADP-bound or rigor wild-type myosin-15 (Fig. 7, E and F), as well as the rigor jordan mutant (25), all of which slow actin polymerization relative to G-actin alone. Regardless, diminished affinity of the jordan mutant cannot be the sole explanation for its nucleation defect, as the presence of this myosin, which nevertheless binds F-actin with measurable affinity in an almost identical pose to the wild type, actually initially suppresses the rate of F-actin formation relative to G-actin alone (25).…”

“…We speculate that hair cells harness the differential efficiency of actin nucleation by myosin-15 in distinct nucleotide states as a regulatable mechanistic component of stereocilia height control. In our companion manuscript, we report that ATP markedly reduces the actin nucleation activity of myosin-15, to a much greater degree than we report here for ADP (25). It is thus feasible that the ADP-bound state, which substantially accelerates actin polymerization, albeit less than the rigor state, is sufficient for supporting myosin-15's direct contribution to actin nucleation in the tip compartment.…”

“…Although its actin-activated ATPase activity (wild type, k cat = 5.8 s −1 ; jordan, k cat = 0.87 s −1 ) and F-actin binding affinity (wild type, k ATPase = 29.1 M; jordan, k ATPase = 114.3 M) are somewhat compromised, purified jordan myosin-15 is nevertheless an active motor, and WHRN, EPS8, GPSM2 and GNAI3 localize to the tips of developing stereocilia in the jordan mouse, leading us to hypothesize that myosin-15 could control stereocilium height through additional mechanisms. Consistently, we found that a minimal motor domain truncation of myosin-15 (S1-like fragment) markedly stimulates actin polymerization in vitro, with the nucleotide-free "rigor" state specifically acting as a nucleation factor, while the jordan mutant instead suppresses actin assembly [figure 6 in (25)]. Early studies of the S1 fragment of skeletal muscle myosin demonstrated its capacity to nucleate actin polymerization in vitro (26)(27)(28), but a physiological role for this activity has not, to our knowledge, been reported in vivo.…”

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

“…In the stereocilia, we propose that MYO15A and MYO3A are both able to produce a protruding force on the membrane at stereocilia tips. In addition, MYO15A has recently been shown to nucleate actin filaments ( Moreland et al., 2021 ). However, since MYO3A is the slower motor, it may function as a “brake” to control the protrusion elongation rate, which in turn may prevent aberrant elongation and allow proper assembly of the components of the tip complex.…”

Promotion of row 1–specific tip complex condensates by Gpsm2-Gαi provides insights into row identity of the tallest stereocilia

“…Alternatively, Eps8-induced actin bundling may cause pronounced barbed-end elongation and thereby makes a longer bundle, as Espin (another actin-bundling protein) does in microvilli ( 35 ). It should be noted that the 5xTCD condensates might enhance actin polymerization/nucleation in addition to actin bundling, possibly by condensing higher amounts of Myo15 at row 1, especially when the motor domain of Myo15 was shown to directly accelerate actin filament polymerization by driving nucleation ( 36 ). Unfortunately, because of the technical challenges, we were unable to obtain qualified full-length Myo15 protein to prove this point in the current work.…”