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.

Myosins and Hearing.

Hearing loss is both genetically and clinically heterogeneous, and pathogenic variants of over a hundred different genes are associated with this common neurosensory disorder. A relatively large number of these "deafness genes" encode myosin super family members. The evidence that pathogenic variants of human MYO3A, MYO6, MYO7A, MYO15A, MYH14 and MYH9 are associated with deafness ranges from moderate to definitive. Additional evidence for the involvement of these six myosins for normal hearing also comes from animal models, usually mouse or zebra fish, where mutations of these genes cause hearing loss and from biochemical, physiological and cell biological studies of their roles in the inner ear. This chapter focuses on these six genes for which evidence of a causative role in deafness is substantial.

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.

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.

Functional Role of Class III Myosins in Hair Cells.

Cytoskeletal motors produce force and motion using the energy from ATP hydrolysis and function in a variety of mechanical roles in cells including muscle contraction, cargo transport, and cell division. Actin-based myosin motors have been shown to play crucial roles in the development and function of the stereocilia of auditory and vestibular inner ear hair cells. Hair cells can contain hundreds of stereocilia, which rely on myosin motors to elongate, organize, and stabilize their structure. Mutations in many stereocilia-associated myosins have been shown to cause hearing loss in both humans and animal models suggesting that each myosin isoform has a specific function in these unique parallel actin bundle-based protrusions. Here we review what is known about the classes of myosins that function in the stereocilia, with a special focus on class III myosins that harbor point mutations associated with delayed onset hearing loss. Much has been learned about the role of the two class III myosin isoforms, MYO3A and MYO3B, in maintaining the precise stereocilia lengths required for normal hearing. We propose a model for how class III myosins play a key role in regulating stereocilia lengths and demonstrate how their motor and regulatory properties are particularly well suited for this function. We conclude that ongoing studies on class III myosins and other stereocilia-associated myosins are extremely important and may lead to novel therapeutic strategies for the treatment of hearing loss due to stereocilia degeneration.

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.

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.

Macrophages promote matrix protrusive and invasive function of breast cancer cells via MIP-1ß dependent upregulation of MYO3A gene in breast cancer cells.

The potential of a tumor cell to metastasize profoundly depends on its microenvironment, or "niche" interactions with local components. Tumor-associated-macrophages (TAMs) are the most abundant subpopulation of tumor stroma and represent a key component of tumor microenvironment. The dynamic interaction of cancer cells with neighboring TAMs actively drive cancer progression and metastatic transformation through intercellular signaling networks that need better elucidation. Thus, current study was planned for discerning paracrine communication networks operational between TAMs, and breast cancer cells with special reference to cancer cell invasion and dissemination to distant sites. Here, we report role of MIP-1ß in enhancing invasive potential of metastatic breast cancer MDA-MB-231 and MDA-MB-468 cells. In addition, the poorly metastatic MCF-7 cells were also rendered invasive by MIP-1ß. The MIP-1ß-driven cancer cell invasion was dependent on upregulated expression levels of MYO3A gene, which encodes an unconventional myosin super-family protein harboring a kinase domain. Ex ovo study employing Chick-embryo-model and in vivo Syngenic 4T1/BALB/c mice-model further corroborated aforementioned in vitro findings, thereby substantiating their physiological relevance. Concordantly, human breast cancer specimen exhibited significant association between mRNA expression levels of MIP-1ß and MYO3A. Both, MIP-1ß and MYO3A exhibited positive correlation with MMP9, an established molecular determinant of cancer cell invasion. Higher expression of these genes correlated with poor survival of breast cancer patients. Collectively, these results point toward so far undisclosed MIP-1ß/MYO3A axis being operational during metastasis, wherein macrophage-derived MIP-1ß potentiated cancer cell invasion and metastasis via up regulation of MYO3A gene within cancer cells. Our study exposes opportunities for devising potential anti-metastatic strategies for efficient clinical management of breast cancer.