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

Class III myosins localize to inner ear hair cell stereocilia and are thought to be crucial for stereocilia length regulation. Mutations within the motor domain of MYO3A that disrupt its intrinsic motor properties have been associated with non-syndromic hearing loss, suggesting that the motor properties of MYO3A are critical for its function within stereocilia. In this study, we investigated the impact of a MYO3A hearing loss mutation, H442N, using both in vitro motor assays and cell biological studies. Our results demonstrate the mutation causes a dramatic increase in intrinsic motor properties, actin-activated ATPase and in vitro actin gliding velocity, as well as an increase in actin protrusion extension velocity. We propose that both "gain of function" and "loss of function" mutations in MYO3A can impair stereocilia length regulation, which is crucial for stereocilia formation during development and normal hearing. Furthermore, we generated chimeric MYO3A constructs that replace the MYO3A motor and neck domain with the motor and neck domain of other myosins. We found that duty ratio, fraction of ATPase cycle myosin is strongly bound to actin, is a critical motor property that dictates the ability to tip localize within filopodia. In addition, in vitro actin gliding velocities correlated extremely well with filopodial extension velocities over a wide range of gliding and extension velocities. Taken together, our data suggest a model in which tip-localized myosin motors exert force that slides the membrane tip-ward, which can combat membrane tension and enhance the actin polymerization rate that ultimately drives protrusion elongation.

Frequency and origin of the c.2090T>G p.(Leu697Trp) MYO3A variant associated with autosomal dominant hearing loss.

We recently described a novel missense variant [c.2090T>G:p.(Leu697Trp)] in the MYO3A gene, found in two Brazilian families with late-onset autosomal dominant nonsyndromic hearing loss (ADNSHL). Since then, with the objective of evaluating its contribution to ADNSHL in Brazil, the variant was screened in additional 101 pedigrees with probable ADNSHL without conclusive molecular diagnosis. The variant was found in three additional families, explaining 3/101 (~3%) of cases with ADNSHL in our Brazilian pedigree collection. In order to identify the origin of the variant, 21 individuals from the five families were genotyped with a high-density SNP array (~600 K SNPs- Axiom Human Origins; ThermoFisher). The identity by descent (IBD) approach revealed that many pairs of individuals from the different families have a kinship coefficient equivalent to that of second cousins, and all share a minimum haplotype of ~607 kb which includes the c.2090T>G variant suggesting it probably arose in a common ancestor. We inferred that the mutation occurred in a chromosomal segment of European ancestry and the time since the most common ancestor was estimated in 1100 years (CI = 775-1425). This variant was also reported in a Dutch family, which shares a 87,121 bp haplotype with the Brazilian samples, suggesting that Dutch colonists may have brought it to Northeastern Brazil in the 17th century. Therefore, the present study opens new avenues to investigate this variant not only in Brazilians but also in European families with ADNSHL.

Molecular insights into MYO3A kinase domain variants explain variability in both severity and progression of DFNB30 hearing impairment.

Hereditary hearing impairment (HI) is a common disease with the highest incidence among sensory defects. Several genes have been identified to affect stereocilia structure causing HI, including the unconventional myosin3A. Interestingly, we noticed that variants in MYO3A gene have been previously found to cause variable HI onset and severity. Using clinical exome sequencing, we identified a novel pathogenic variant p.(Lys50Arg) in the MYO3A kinase domain (MYO3A-KD). Previous in vitro studies supported its damaging effect as a 'kinase-dead' mutant. We further analyzed this variation through molecular dynamics which predicts that changes in flexibility of MYO3A structure would influence the protein-ATP binding properties. This Lys50Arg mutation segregated with congenital profound non-syndromic HI. To better investigate this variability, we collected previously identified MYO3A-KDs variants, p.(Tyr129Cys), p.(His142Gln) and p.(Pro189Thr), and built both wild type and mutant 3 D MYO3A-KD models to assess their impact on the protein structure and function. Our results suggest that KD mutations could either cause a congenital profound form of HI, when particularly affecting the kinase activity and preventing the auto-phosphorylation of the motor, or a late onset and progressive form, when partially or completely inactivating the MYO3A protein. In conclusion, we report a novel pathogenic variant affecting the ATP-binding site within the MYO3A-KD causing congenital profound HI. Through computational approaches we provide a deeper understanding on the correlation between the effects of MYO3A-KD mutations and the variable hearing phenotypes. To the best of our knowledge this is the first study to correlate mutations' genotypes with the variable phenotypes of DFNB30.Communicated by Ramaswamy H. Sarma.

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

Class III myosins are actin-based motors proposed to transport cargo to the distal tips of stereocilia in the inner ear hair 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 twofold 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.

Whole exome sequencing reveals pathogenic variants in MYO3A, MYO15A and COL9A3 and differential frequencies in ancestral alleles in hearing impairment genes among individuals from Cameroon.

There is scarcity of known gene variants of hearing impairment (HI) in African populations. This knowledge deficit is ultimately affecting the development of genetic diagnoses. We used whole exome sequencing to investigate gene variants, pathways of interactive genes and the fractions of ancestral overderived alleles for 159 HI genes among 18 Cameroonian patients with non-syndromic HI (NSHI) and 129 ethnically matched controls. Pathogenic and likely pathogenic (PLP) variants were found in MYO3A, MYO15A and COL9A3, with a resolution rate of 50% (9/18 patients). The study identified significant genetic differentiation in novel population-specific gene variants at FOXD4L2, DHRS2L6, RPL3L and VTN between HI patients and controls. These gene variants are found in functional/co-expressed interactive networks with other known HI-associated genes and in the same pathways with VTN being a hub protein, that is, focal adhesion pathway and regulation of the actin cytoskeleton (P-values <0.05). The results suggest that these novel population-specific gene variants are possible modifiers of the HI phenotypes. We found a high proportion of ancestral allele versus derived at low HI patients-specific minor allele frequency in the range of 0.0-0.1. The results showed a relatively low pickup rate of PLP variants in known genes in this group of Cameroonian patients with NSHI. In addition, findings may signal an evolutionary enrichment of some variants of HI genes in patients, as the result of polygenic adaptation, and suggest the possibility of multigenic influence on the phenotype of congenital HI, which deserves further investigations.

A novel missense variant in MYO3A is associated with autosomal dominant high-frequency hearing loss in a German family.

BACKGROUND: MYO3A, encoding the myosin IIIA protein, is associated with autosomal recessive and autosomal dominant nonsyndromic hearing loss. To date, only two missense variants located in the motor-head domain of MYO3A have been described in autosomal dominant families with progressive, mild-to-profound sensorineural hearing loss. These variants alter the ATPase activity of myosin IIIA. METHODS: Exome sequencing of a proband from a three-generation German family with prelingual, moderate-to-profound, high-frequency hearing loss was performed. Segregation analysis confirmed a dominant inheritance pattern. Regression analysis of mean hearing level thresholds per individual and ear was performed at high-, mid-, and low-frequencies. RESULTS: A novel heterozygous missense variant c.716T>C, p.(Leu239Pro) in the kinase domain of MYO3A was identified that is predicted in silico as disease causing. High-frequency, progressive hearing loss was identified. CONCLUSION: Correlation analysis of pure-tone hearing thresholds revealed progressive hearing loss, especially in the high-frequencies. In the present study, we report the first dominant likely pathogenic variant in MYO3A in a European family and further support MYO3A as an autosomal dominant hearing loss gene.

Mutations in the tail domain of MYH3 contributes to atrial septal defect.

Atrial septal defect (ASD) is one of the most common congenital heart defects diagnosed in children. Sarcomeric genes has been attributed to ASD and knockdown of MYH3 functionally homologues gene in chick models indicated abnormal atrial septal development. Here, we report for the first time, a case-control study investigating the role of MYH3 among non-syndromic ASD patients in contributing to septal development. Four amplicons which will amplifies the 40 kb MYH3 were designed and amplified using long range-PCR. The amplicons were then sequenced using indexed paired-end libraries on the MiSeq platform. The STREGA guidelines were applied for planning and reporting. The non-synonymous c. 3574G>A (p.Ala1192Thr) [p = 0.001, OR = 2.30 (1.36-3.87)] located within the tail domain indicated a highly conserved protein region. The mutant model of c. 3574G>A (p.Ala1192Thr) showed high root mean square deviation (RMSD) values compared to the wild model. To our knowledge, this is the first study to provide compelling evidence on the pathogenesis of MYH3 variants towards ASD hence, suggesting the crucial role of non-synonymous variants in the tail domain of MYH3 towards atrial septal development. It is hoped that this gene can be used as panel for diagnosis of ASD in future.

Recessive marfanoid syndrome with herniation associated with a homozygous mutation in Fibulin-3.

We have previously reported on a consanguineous family where 2 siblings, a girl and a boy, presented with tall stature, long and triangular faces, prominent forehead, telecanthus, ptosis, everted lower eyelids, downslanting palpebral fissures, large ears, high arched palate, long arm span, arachnodactyly, advanced bone age, joint laxity, pectus excavatum, inguinal hernia, and myopia, suggestive of a new subtype of connective tissue disorder (Megarbane et al. AJMG, 2012; 158(A)5: 1185-1189). On clinical follow-up, both patients had multiple inguinal, crural, and abdominal herniae, intestinal occlusions, several huge diverticula throughout the gut and the bladder, and rectal prolapse. In addition, the girl had a mild hearing impairment, and the boy a left diaphragmatic hernia. Here we describe the molecular characterization of this disorder using Whole Exome Sequencing, revealing, in both siblings, a novel homozygous missense variant in the EFEMP1 gene, c.163T > C; p.(Cys55Arg) whose homozygous by descent, autosomal recessive transmission was confirmed through segregation analysis by Sanger sequencing. In addition, the girl exhibited a homozygous mutation in the MYO3A gene, c.1370_1371delGA; p.(Arg457Asnfs*25), associated with non-syndromic deafness. The siblings were also found to harbor a homozygous nonsense variant in the VCPKMT gene. We review the literature and discuss our updated clinical and molecular findings that suggest EFEMP1 to be the probable candidate gene implicated in this novel connective tissue disease.

Knock-In Mice with Myo3a Y137C Mutation Displayed Progressive Hearing Loss and Hair Cell Degeneration in the Inner Ear.

Myo3a is expressed in cochlear hair cells and retinal cells and is responsible for human recessive hereditary nonsyndromic deafness (DFNB30). To investigate the mechanism of DFNB30-type deafness, we established a mouse model of Myo3a kinase domain Y137C mutation by using CRISPR/Cas9 system. No difference in hearing between 2-month-old Myo3a mutant mice and wild-type mice was observed. The hearing threshold of the ≥6-month-old mutant mice was significantly elevated compared with that of the wild-type mice. We observed degeneration in the inner ear hair cells of 6-month-old Myo3a mutant mice, and the degeneration became more severe at the age of 12 months. We also found structural abnormality in the cochlear hair cell stereocilia. Our results showed that Myo3a is essential for normal hearing by maintaining the intact structure of hair cell stereocilia, and the kinase domain plays a critical role in the normal functions of Myo3a. This mouse line is an excellent model for studying DFNB30-type deafness in humans.

Characterization of a novel MYO3A missense mutation associated with a dominant form of late onset hearing loss.

Whole-exome sequencing of samples from affected members of two unrelated families with late-onset non-syndromic hearing loss revealed a novel mutation (c.2090 T > G; NM_017433) in MYO3A. The mutation was confirmed in 36 affected individuals, showing autosomal dominant inheritance. The mutation alters a single residue (L697W or p.Leu697Trp) in the motor domain of the stereocilia protein MYO3A, leading to a reduction in ATPase activity, motility, and an increase in actin affinity. MYO3A-L697W showed reduced filopodial actin protrusion initiation in COS7 cells, and a predominant tipward accumulation at filopodia and stereocilia when coexpressed with wild-type MYO3A and espin-1, an actin-regulatory MYO3A cargo. The combined higher actin affinity and duty ratio of the mutant myosin cause increased retention time at stereocilia tips, resulting in the displacement of the wild-type MYO3A protein, which may impact cargo transport, stereocilia length, and mechanotransduction. The dominant negative effect of the altered myosin function explains the dominant inheritance of deafness.

Impact of the Motor and Tail Domains of Class III Myosins on Regulating the Formation and Elongation of Actin Protrusions.

Class III myosins (MYO3A and MYO3B) are proposed to function as transporters as well as length and ultrastructure regulators within stable actin-based protrusions such as stereocilia and calycal processes. MYO3A differs from MYO3B in that it contains an extended tail domain with an additional actin-binding motif. We examined how the properties of the motor and tail domains of human class III myosins impact their ability to enhance the formation and elongation of actin protrusions. Direct examination of the motor and enzymatic properties of human MYO3A and MYO3B revealed that MYO3A is a 2-fold faster motor with enhanced ATPase activity and actin affinity. A chimera in which the MYO3A tail was fused to the MYO3B motor demonstrated that motor activity correlates with formation and elongation of actin protrusions. We demonstrate that removal of individual exons (30-34) in the MYO3A tail does not prevent filopodia tip localization but abolishes the ability to enhance actin protrusion formation and elongation in COS7 cells. Interestingly, our results demonstrate that MYO3A slows filopodia dynamics and enhances filopodia lifetime in COS7 cells. We also demonstrate that MYO3A is more efficient than MYO3B at increasing formation and elongation of stable microvilli on the surface of cultured epithelial cells. We propose that the unique features of MYO3A, enhanced motor activity, and an extended tail with tail actin-binding motif, allow it to play an important role in stable actin protrusion length and ultrastructure maintenance.

Identification of a novel homozygous mutation in MYO3A in a Chinese family with DFNB30 non-syndromic hearing impairment.

INTRODUCTION: Hearing loss is a common sensory impairment. Several genetic loci or genes responsible for non-syndrome hearing loss have been identified, including the well-known deafness genes GJB2, MT-RNR1 and SLC26A4. MYO3A belongs to the myosin superfamily. Previously only three mutations in this gene have been found in an Isreali family with DFNB30, in which patients demonstrated progressive hearing loss. METHODS: In this study, we characterized a consanguineous Kazakh family with congenital hearing loss. By targeted sequence capture and next-generation sequencing, we identified a homozygous mutation and did bioinformatics analysis to this mutation. RESULTS: A homozygous mutation, MYO3A:c.1841C>T (p.S614F), was identified to be responsible for the disease. Ser614 is located in the motor domain of MYO3A that is highly conserved among different species. Molecular modeling predicts that the conserved Ser614 may play an important role in maintaining the stability of ß-sheet and the interaction between neighboring ß-strand. CONCLUSIONS: This is the second report on MYO3A mutations in deafness and the first report in China. The finding help facilitate establishing a better relationship between MYO3A mutation and hearing phenotypes.

Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like.

Hair cells tightly control the dimensions of their stereocilia, which are actin-rich protrusions with graded heights that mediate mechanotransduction in the inner ear. Two members of the myosin-III family, MYO3A and MYO3B, are thought to regulate stereocilia length by transporting cargos that control actin polymerization at stereocilia tips. We show that eliminating espin-1 (ESPN-1), an isoform of ESPN and a myosin-III cargo, dramatically alters the slope of the stereocilia staircase in a subset of hair cells. Furthermore, we show that espin-like (ESPNL), primarily present in developing stereocilia, is also a myosin-III cargo and is essential for normal hearing. ESPN-1 and ESPNL each bind MYO3A and MYO3B, but differentially influence how the two motors function. Consequently, functional properties of different motor-cargo combinations differentially affect molecular transport and the length of actin protrusions. This mechanism is used by hair cells to establish the required range of stereocilia lengths within a single cell.

MYO3A Causes Human Dominant Deafness and Interacts with Protocadherin 15-CD2 Isoform.

Hereditary hearing loss (HL) is characterized by both allelic and locus genetic heterogeneity. Both recessive and dominant forms of HL may be caused by different mutations in the same deafness gene. In a family with post-lingual progressive non-syndromic deafness, whole-exome sequencing of genomic DNA from five hearing-impaired relatives revealed a single variant, p.Gly488Glu (rs145970949:G>A) in MYO3A, co-segregating with HL as an autosomal dominant trait. This amino acid change, predicted to be pathogenic, alters a highly conserved residue in the motor domain of MYO3A. The mutation severely alters the ATPase activity and motility of the protein in vitro, and the mutant protein fails to accumulate in the filopodia tips in COS7 cells. However, the mutant MYO3A was able to reach the tips of organotypic inner ear culture hair cell stereocilia, raising the possibility of a local effect on positioning of the mechanoelectrical transduction (MET) complex at the stereocilia tips. To address this hypothesis, we investigated the interaction of MYO3A with the cytosolic tail of the integral tip-link protein protocadherin 15 (PCDH15), a core component of MET complex. Interestingly, we uncovered a novel interaction between MYO3A and PCDH15 shedding new light on the function of myosin IIIA at stereocilia tips.

Class III myosins shape the auditory hair bundles by limiting microvilli and stereocilia growth.

The precise architecture of hair bundles, the arrays of mechanosensitive microvilli-like stereocilia crowning the auditory hair cells, is essential to hearing. Myosin IIIa, defective in the late-onset deafness form DFNB30, has been proposed to transport espin-1 to the tips of stereocilia, thereby promoting their elongation. We show that Myo3a(-/-)Myo3b(-/-) mice lacking myosin IIIa and myosin IIIb are profoundly deaf, whereas Myo3a-cKO Myo3b(-/-) mice lacking myosin IIIb and losing myosin IIIa postnatally have normal hearing. Myo3a(-/-)Myo3b(-/-) cochlear hair bundles display robust mechanoelectrical transduction currents with normal kinetics but show severe embryonic abnormalities whose features rapidly change. These include abnormally tall and numerous microvilli or stereocilia, ungraded stereocilia bundles, and bundle rounding and closure. Surprisingly, espin-1 is properly targeted to Myo3a(-/-)Myo3b(-/-) stereocilia tips. Our results uncover the critical role that class III myosins play redundantly in hair-bundle morphogenesis; they unexpectedly limit the elongation of stereocilia and of subsequently regressing microvilli, thus contributing to the early hair bundle shaping.

My oh my(osin): Insights into how auditory hair cells count, measure, and shape.

The mechanisms underlying mechanosensory hair bundle formation in auditory sensory cells are largely mysterious. In this issue, Lelli et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201509017) reveal that a pair of molecular motors, myosin IIIa and myosin IIIb, is involved in the hair bundle's morphology and hearing.

Myosin III-mediated cross-linking and stimulation of actin bundling activity of Espin.

Class III myosins (Myo3) and actin-bundling protein Espin play critical roles in regulating the development and maintenance of stereocilia in vertebrate hair cells, and their defects cause hereditary hearing impairments. Myo3 interacts with Espin1 through its tail homology I motif (THDI), however it is not clear how Myo3 specifically acts through Espin1 to regulate the actin bundle assembly and stabilization. Here we discover that Myo3 THDI contains a pair of repeat sequences capable of independently and strongly binding to the ankyrin repeats of Espin1, revealing an unexpected Myo3-mediated cross-linking mechanism of Espin1. The structures of Myo3 in complex with Espin1 not only elucidate the mechanism of the binding, but also reveal a Myo3-induced release of Espin1 auto-inhibition mechanism. We also provide evidence that Myo3-mediated cross-linking can further promote actin fiber bundling activity of Espin1.

Invertebrate and vertebrate class III myosins interact with MORN repeat-containing adaptor proteins.

In Drosophila photoreceptors, the NINAC-encoded myosin III is found in a complex with a small, MORN-repeat containing, protein Retinophilin (RTP). Expression of these two proteins in other cell types showed NINAC myosin III behavior is altered by RTP. NINAC deletion constructs were used to map the RTP binding site within the proximal tail domain of NINAC. In vertebrates, the RTP ortholog is MORN4. Co-precipitation experiments demonstrated that human MORN4 binds to human myosin IIIA (MYO3A). In COS7 cells, MORN4 and MYO3A, but not MORN4 and MYO3B, co-localize to actin rich filopodia extensions. Deletion analysis mapped the MORN4 binding to the proximal region of the MYO3A tail domain. MYO3A dependent MORN4 tip localization suggests that MYO3A functions as a motor that transports MORN4 to the filopodia tips and MORN4 may enhance MYO3A tip localization by tethering it to the plasma membrane at the protrusion tips. These results establish conserved features of the RTP/MORN4 family: they bind within the tail domain of myosin IIIs to control their behavior.

Phosphorylation of the kinase domain regulates autophosphorylation of myosin IIIA and its translocation in microvilli.

Motor activity of myosin III is regulated by autophosphorylation. To investigate the role of the kinase activity on the transporter function of myosin IIIA (Myo3A), we identified the phosphorylation sites of kinase domain (KD), which is responsible for the regulation of kinase activity and thus motor function. Using mass spectrometry, we identified six phosphorylation sites in the KD, which are highly conserved among class III myosins and Ste20-related misshapen (Msn) kinases. Two predominant sites, Thr¹84 and Thr¹88, in KD are important for phosphorylation of the KD as well as the motor domain, which regulates the affinity for actin. In the Caco2 cells, the full-length human Myo3A (hMyo3AFull) markedly enlarged the microvilli, although it did not show discrete localization within the microvilli. On the other hand, hMyo3AFull(T184A) and hMyo3AFull(T188A) both showed clear localization at the microvilli tips. Our results suggest that Myo3A induces large actin bundle formation to form microvilli, and phosphorylation of KD at Thr¹84 and Thr¹88 is critical for the kinase activity of Myo3A, and regulation of Myo3A translocation to the tip of microvilli. Retinal extracts potently dephosphorylate both KD and motor domain without IQ motifs (MDIQo), which was inhibited by okadaic acid (OA) with nanomolar range and by tautomycetin (TMC) with micromolar range. The results suggest that Myo3A phosphatase is protein phosphatase type 2A (PP2A). Supporting this result, recombinant PP2Ac potently dephosphorylates both KD and MDIQo. We propose that the phosphorylation-dephosphorylation mechanism plays an essential role in mediating the transport and actin bundle formation and stability functions of hMyo3A.

Myosin 3A kinase activity is regulated by phosphorylation of the kinase domain activation loop.

Class III myosins are unique members of the myosin superfamily in that they contain both a motor and kinase domain. We have found that motor activity is decreased by autophosphorylation, although little is known about the regulation of the kinase domain. We demonstrate by mass spectrometry that Thr-178 and Thr-184 in the kinase domain activation loop and two threonines in the loop 2 region of the motor domain are autophosphorylated (Thr-908 and Thr-919). The kinase activity of MYO3A 2IQ with the phosphomimic (T184E) or phosphoblock (T184A) mutations demonstrates that kinase activity is reduced 30-fold as a result of the T184A mutation, although the Thr-178 site only had a minor impact on kinase activity. Interestingly, the actin-activated ATPase activity of MYO3A 2IQ is slightly reduced as a result of the T178A and T184A mutations suggesting coupling between motor and kinase domains. Full-length GFP-tagged T184A and T184E MYO3A constructs transfected into COS7 cells do not disrupt the ability of MYO3A to localize to filopodia structures. In addition, we demonstrate that T184E MYO3A reduces filopodia elongation in the presence of espin-1, whereas T184A enhances filopodia elongation in a similar fashion to kinase-dead MYO3A. Our results suggest that as MYO3A accumulates at the tips of actin protrusions, autophosphorylation of Thr-184 enhances kinase activity resulting in phosphorylation of the MYO3A motor and reducing motor activity. The differential regulation of the kinase and motor activities allows for MYO3A to precisely self-regulate its concentration in the actin bundle-based structures of cells.