Cochlear hair cells transduce sound using stereocilia, and disruption to these delicate mechanosensors is a significant cause of hearing loss. Stereocilia architecture is dependent upon the nanomotor myosin 15. A short isoform (MYO15A-2) drives stereocilia development by delivering an elongation-promoting complex (EC) to stereocilia tips, and an alternatively spliced long isoform (MYO15A-1) tunes postnatal size in shorter stereocilia, which possess mechanosensitive ion channels. Disruption of these functions causes two distinct stereocilia pathologies, which underly human autosomal recessive non-syndromic hearing loss DFNB3. Here, we characterize a new isoform, MYO15A-3, that increases expression in postnatal hair cells as the developmental MYO15A-2 isoform wanes reciprocally. We show the critical EC complex is initially delivered by MYO15A-2, followed by a postnatal handover to MYO15A-3, which continues to deliver the EC. In a Myo15a-3 mutant mouse, stereocilia develop normally with correct EC targeting, but lack the EC postnatally and do not maintain their adult architecture, leading to progressive hearing loss. We conclude MYO15A-3 delivers the EC in postnatal hair cells and that the EC and MYO15A-3 are both required to maintain stereocilia integrity. Our results add to the spectrum of stereocilia pathology underlying DFNB3 hearing loss and reveal new molecular mechanisms necessary for resilient hearing during adult life.

Transmembrane protein 135 (TMEM135) is a highly conserved 52 kDa protein with five predicted transmembrane domains that colocalizes with mitochondria and peroxisomes. Previous studies have shown that TMEM135 is involved in mitochondrial dynamics, thermogenesis, and lipid metabolism across multiple tissues and species; however, its role in the inner ear and auditory system remains unknown. We investigated the function of TMEM135 in hearing using wild-type (WT) and Tmem135FUN025/FUN025 (FUN025) mutant mice on a CBA/CaJ background, a normal-hearing mouse strain. Although FUN025 mice displayed normal auditory brainstem response (ABR) thresholds at 1 month, we observed significantly elevated ABR thresholds at 8, 16, and 64 kHz by 3 months, which progressed to profound hearing loss by 12 months. Consistent with our auditory testing results, 13-month-old FUN025 mice exhibited a severe loss of outer hair cells and more modest changes in inner hair cell survival, spiral ganglion neuron density, and stria vascularis integrity in the cochlea. Our results using BaseScope RNA in situ hybridization indicate that TMEM135 is expressed in the inner hair cells, outer hair cells, supporting cells, and stria vascularis. Using Volocity software and Costes colocalization analysis, we found that TMEM135 closely colocalizes with mitochondria in hair cells. Together, these results demonstrate that the FUN025 mutation in Tmem135 causes progressive sensorineural hearing loss, and suggest that TMEM135 is crucial for maintaining key cochlear cell types and normal sensory function in the aging cochlea.

To understand the mechanics and kinetic properties of cytoskeletal molecular motors such as myosin, typically the motor of interest needs to be expressed and purified and then analyzed using a range of in vitro-based assays. In this chapter, we describe how to express and purify myosin using the insect cell system, how to characterize the purified protein by mass photometry and negative-stain EM to assess its quality, and how to perform in vitro assays in which fluorescently labeled myosin walks along actin tracks, including a brief description of adapting these assays for MINFLUX imaging.

Assembly of actin-based stereocilia is critical for cochlear hair cells to detect sound. To tune their mechanosensivity, stereocilia form bundles composed of graded rows of ascending height, necessitating the precise control of actin polymerization. Myosin 15 (MYO15A) drives hair bundle development by delivering critical proteins to growing stereocilia that regulate actin polymerization via an unknown mechanism. Here, we show that MYO15A is itself an actin nucleation-promoting factor. Moreover, a deafness-causing mutation in the MYO15A actin-binding interface inhibits nucleation activity but still preserves some movement on filaments in vitro and partial trafficking on stereocilia in vivo. Stereocilia fail to elongate correctly in this mutant mouse, providing evidence that MYO15A-driven actin nucleation contributes to hair bundle biogenesis. Our work shows that in addition to generating force and motility, the ATPase domain of MYO15A can directly regulate actin polymerization and that disrupting this activity can promote cytoskeletal disease, such as hearing loss.

The sarcomere is the fundamental contractile unit in skeletal muscle, and the regularity of its structure is critical for function. Emerging data demonstrates that nanoscale changes to the regularity of sarcomere structure can affect the overall function of the protein dense ~2μm sarcomere. Further, sarcomere structure is implicated in many clinical conditions of muscle weakness. However, our understanding of how sarcomere structure changes in disease, especially at the nanoscale, has been limited in part due to the inability to robustly detect and measure at sub-sarcomere resolution. We optimized several methodological steps and developed a robust pipeline to analyze sarcomere structure using structured illumination super-resolution microscopy in conjunction with commercially-available and fluorescently-conjugated Variable Heavy-Chain only fragment secondary antibodies (nanobodies), and achieved a significant increase in resolution of z-disc width (353nm vs. 62nm) compared to confocal microscopy. The combination of these methods provides a unique approach to probe sarcomere protein localization at the nanoscale and may prove advantageous for analysis of other cellular structures.

Our sense of hearing is mediated by cochlear hair cells, localized within the sensory epithelium called the organ of Corti. There are two types of hair cells in the cochlea, which are organized in one row of inner hair cells and three rows of outer hair cells. Each cochlea contains a few thousands of hair cells, and their survival is essential for our perception of sound because they are terminally differentiated and do not regenerate after insult. It is often desirable in hearing research to quantify the number of hair cells within cochlear samples, in both pathological conditions, and in response to treatment. However, the sheer number of cells along the cochlea makes manual quantification impractical. Machine learning can be used to overcome this challenge by automating the quantification process but requires a vast and diverse dataset for effective training. In this study, we present a large collection of annotated cochlear hair-cell datasets, labeled with commonly used hair-cell markers and imaged using various fluorescence microscopy techniques. The collection includes samples from mouse, human, pig and guinea pig cochlear tissue, from normal conditions and following in-vivo and in-vitro ototoxic drug application. The dataset includes over 90'000 hair cells, all of which have been manually identified and annotated as one of two cell types: inner hair cells and outer hair cells. This dataset is the result of a collaborative effort from multiple laboratories and has been carefully curated to represent a variety of imaging techniques. With suggested usage parameters and a well-described annotation procedure, this collection can facilitate the development of generalizable cochlear hair cell detection models or serve as a starting point for fine-tuning models for other analysis tasks. By providing this dataset, we aim to supply other groups within the hearing research community with the opportunity to develop their own tools with which to analyze cochlear imaging data more fully, accurately, and with greater ease.

In the auditory and vestibular systems, stereocilia are actin-based protrusions that convert mechanical stimuli into electrical signals. During development, stereocilia elongate and widen by adding filamentous actin (F-actin), attaining their mature shape necessary for mechanosensitive function. Myosin motors, including MYO3A/B and MYO15A, are required for normal stereocilia growth, but the regulation of actin and the impact of myosins on actin assembly remain unclear. We focused on stereocilia widening, which requires the addition of new filaments to the bundle of linear F-actin comprising the initial stereocilia core. Our findings revealed that newly expressed actin incorporates at the stereocilia tip first, then along the shaft to promote stereocilia widening. The newly incorporated F-actin surrounded the existing F-actin core, suggesting that the core is stable once formed, with additional actin adding only to the periphery. To better understand the nature of incorporating actin, we used several probes to detect globular (G-) actin, F-actin barbed ends, and F-actin pointed ends. While F-actin core filaments are parallel and thought to present only barbed ends at stereocilia tips, we also detected F-actin pointed ends, indicating a previously undetected population of short actin filaments. Overexpression of actin resulted in abundant F-actin pointed and barbed ends along the periphery of the stereocilia shaft, suggesting that short actin filaments contribute to stereocilia widening. Short actin filament levels correlated with the levels of MYO3A/B and MYO15A at stereocilia tips, suggesting these myosins generate or stabilize short actin filaments essential for stereocilia widening and elongation.

Inner ear hair cells assemble mechanosensitive hair bundles on their apical surface that transduce sounds and accelerations. Each hair bundle is comprised of ∼ 100 individual stereocilia that are arranged into rows of increasing height and width; their specific and precise architecture being necessary for mechanoelectrical transduction (MET). The actin cytoskeleton is fundamental to establishing this architecture, not only by forming the structural scaffold shaping each stereocilium, but also by composing rootlets and the cuticular plate that together provide a stable foundation supporting each stereocilium. In concert with the actin cytoskeleton, a large assortment of actin-binding proteins (ABPs) function to cross-link actin filaments into specific topologies, as well as control actin filament growth, severing, and capping. These processes are individually critical for sensory transduction and are all disrupted in hereditary forms of human hearing loss. In this review, we provide an overview of actin-based structures in the hair bundle and the molecules contributing to their assembly and functional properties. We also highlight recent advances in mechanisms driving stereocilia elongation and how these processes are tuned by MET.

Supplementary Data from DNMT3A Harboring Leukemia-Associated Mutations Directs Sensitivity to DNA Damage at Replication Forks

The motor protein myosin-15 is necessary for the development and maintenance of mechanosensory stereocilia, and mutations in myosin-15 cause hereditary deafness. In addition to transporting actin regulatory machinery to stereocilia tips, myosin-15 directly nucleates actin filament (“F-actin”) assembly, which is disrupted by a progressive hearing loss mutation (p.D1647G, “ jordan ”). Here, we present cryo–electron microscopy structures of myosin-15 bound to F-actin, providing a framework for interpreting the impacts of deafness mutations on motor activity and actin nucleation. Rigor myosin-15 evokes conformational changes in F-actin yet maintains flexibility in actin’s D-loop, which mediates inter-subunit contacts, while the jordan mutant locks the D-loop in a single conformation. Adenosine diphosphate–bound myosin-15 also locks the D-loop, which correspondingly blunts actin-polymerization stimulation. We propose myosin-15 enhances polymerization by bridging actin protomers, regulating nucleation efficiency by modulating actin’s structural plasticity in a myosin nucleotide state–dependent manner. This tunable regulation of actin polymerization could be harnessed to precisely control stereocilium height.

Mechanosensory hair bundles are assembled from actin-based stereocilia that project from the apical surface of hair cells in the inner ear. Stereocilia architecture is critical for the transduction of sound and accelerations, and structural defects in these mechano-sensors are a clinical cause of hearing and balance disorders in humans. Unconventional myosin motors are central to the assembly and shaping of stereocilia architecture. A sub-group of myosin motors with MyTH4-FERM domains (MYO7A, MYO15A) are particularly important in these processes, and hypothesized to act as transporters delivering structural and actin-regulatory cargos, in addition to generating force and tension. In this review, we summarize existing evidence for how MYO7A and MYO15A operate and how their dysfunction leads to stereocilia pathology. We further highlight emerging properties of the MyTH4/FERM myosin family and speculate how these new functions might contribute towards the acquisition and maintenance of mechano-sensitivity.

Stereocilia on auditory sensory cells are actin-based protrusions that mechanotransduce sound into an electrical signal. These stereocilia are arranged into a bundle with three rows of increasing length to form a staircase-like morphology that is required for hearing. Stereocilia in the shorter rows, but not the tallest row, are mechanotransducing because they have force-sensitive channels localized at their tips. The onset of mechanotransduction during mouse postnatal development refines stereocilia length and width. However, it is unclear how actin is differentially regulated between stereocilia in the tallest row of the bundle and the shorter, mechanotransducing rows. Here, we show actin turnover is increased at the tips of mechanotransducing stereocilia during bundle maturation. Correspondingly, from birth to postnatal day 6, these stereocilia had increasing amounts of available actin barbed ends, where monomers can be added or lost readily, as compared with the non-mechanotransducing stereocilia in the tallest row. The increase in available barbed ends depended on both mechanotransduction and MYO15 or EPS8, which are required for the normal specification and elongation of the tallest row of stereocilia. We also found that loss of the F-actin-severing proteins ADF and cofilin-1 decreased barbed end availability at stereocilia tips. These proteins enriched at mechanotransducing stereocilia tips, and their localization was perturbed by the loss of mechanotransduction, MYO15, or EPS8. Finally, stereocilia lengths and widths were dysregulated in Adf and Cfl1 mutants. Together, these data show that actin is remodeled, likely by a severing mechanism, in response to mechanotransduction.

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 traffickingin vivo, purified p.D1647G MYO15 modestly reduced actin-stimulated ATPase activityin vitro. Using ensemble and single-filament fluorescencein vitroassays, 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 elongationin vivo, and that this is a primary mechanism disrupted in DFNB3 hereditary human hearing loss.

Cochlear hair cells possess an exquisite bundle of actin-based stereocilia that detect sound. Unconventional myosin 15 (MYO15A) traffics and delivers critical molecules required for stereocilia development and is essential for building the mechanosensory hair bundle. Mutations in the human MYO15A gene interfere with stereocilia trafficking and cause hereditary hearing loss, DFNB3. To understand the molecular mechanism of how MYO15A delivers proteins within stereocilia, we performed a kinetic study of the ATPase motor domain to characterize its mechano-chemical cycle. Using the baculovirus-Sf9 system, we purified a recombinant minimal motor domain (S1) by co-expressing the mouse MYO15 ATPase, essential and regulatory light chains that bind its IQ domains, and UNC45 and HSP90A chaperones required for correct folding of the ATPase. MYO15 purified with either UNC45A or UNC45B co-expression had similar ATPase activities ( k cat = ~ 6 s −1 at 20°C). Using stopped-flow and quenched-flow transient kinetic analyses, we measured the major rate constants describing the ATPase cycle, including ATP, ADP and actin binding, hydrolysis and phosphate release. Actin-attached ADP release was the slowest measured transition (~ 12 s −1 at 20°C), although this did not rate-limit the ATPase cycle. The kinetic analysis shows the MYO15 motor domain has a moderate duty ratio (~ 0.5) and weak thermodynamic coupling between ADP and actin binding. This is consistent with MYO15 being adapted for strain sensing as a monomer, or processive motility if oligomerized into ensembles. Our kinetic characterization enables future studies into how deafness-causing mutations affect MYO15 and ultimately disrupt stereocilia trafficking necessary for normal hearing.

Acute myeloid leukemia is characterized by recurrent somatic mutations in epigenetic modifier genes like DNA methyltransferase 3A (DNMT3A). Mutations in DNMT3A are commonly seen in older patients who are unfit for intensive chemotherapy and thus associated with unfavorable outcomes. Recent clinical trials have shown low-intensity regimens of nucleoside analogs (cytarabine and cladribine), to be effective in elderly AML patients, particularly those with DNMT3A mutations. Additionally, previous gene expression studies in DNMT3A cells uncovered signatures suggestive of replication stress and a resultant G2/M checkpoint adaptation. We hypothesized that this can be harnessed as a therapeutic vulnerability. We confirmed differential sensitivity to cytarabine in various model systems, with and without mutant DNMT3A, both in vitro and in vivo. We also observed that the cells with DNMT3A mutations accumulated more DNA damage, with the persistence of the intra S-phase checkpoint activation and elevated levels of activated p53. While DNMT3A-mutant cells remained proficient in engaging DNA double-strand break repair mechanisms, we observed impaired chromatin recruitment of proteins involved in nucleotide excision repair, after cytarabine treatment. Consistently, the DNMT3A-mutant cells displayed a higher rate of replication fork collapse after cytarabine wash-out. Despite persistent unrepaired DNA damage, the DNMT3A mutant cells were able to progress through mitosis, eventually leading to mitotic catastrophe. Consistent with these findings, transcriptomic analysis identified dysregulation of pathways related to cell cycle, DNA damage, and repair. Our studies aim to gain a better understanding of the role played by DNMT3A in modulating sensitivity to clinically relevant replication stalling drugs and thus making it a suitable approach to treat this poor prognosis subtype of AML. Acute myeloid leukemia is characterized by recurrent somatic mutations in epigenetic modifier genes like DNA methyltransferase 3A (DNMT3A). Mutations in DNMT3A are commonly seen in older patients who are unfit for intensive chemotherapy and thus associated with unfavorable outcomes. Recent clinical trials have shown low-intensity regimens of nucleoside analogs (cytarabine and cladribine), to be effective in elderly AML patients, particularly those with DNMT3A mutations. Additionally, previous gene expression studies in DNMT3A cells uncovered signatures suggestive of replication stress and a resultant G2/M checkpoint adaptation. We hypothesized that this can be harnessed as a therapeutic vulnerability. We confirmed differential sensitivity to cytarabine in various model systems, with and without mutant DNMT3A, both in vitro and in vivo. We also observed that the cells with DNMT3A mutations accumulated more DNA damage, with the persistence of the intra S-phase checkpoint activation and elevated levels of activated p53. While DNMT3A-mutant cells remained proficient in engaging DNA double-strand break repair mechanisms, we observed impaired chromatin recruitment of proteins involved in nucleotide excision repair, after cytarabine treatment. Consistently, the DNMT3A-mutant cells displayed a higher rate of replication fork collapse after cytarabine wash-out. Despite persistent unrepaired DNA damage, the DNMT3A mutant cells were able to progress through mitosis, eventually leading to mitotic catastrophe. Consistent with these findings, transcriptomic analysis identified dysregulation of pathways related to cell cycle, DNA damage, and repair. Our studies aim to gain a better understanding of the role played by DNMT3A in modulating sensitivity to clinically relevant replication stalling drugs and thus making it a suitable approach to treat this poor prognosis subtype of AML.

Actin-rich structures, like stereocilia and microvilli, are assembled with precise control of length, diameter, and relative spacing. By quantifying actin-core dimensions of stereocilia from phalloidin-labeled mouse cochleas, we demonstrated that inner hair cell stereocilia developed in specific stages, where a widening phase is sandwiched between two lengthening phases. Moreover, widening of the second-tallest stereocilia rank (row 2) occurred simultaneously with the appearance of mechanotransduction. Correspondingly, Tmc1(KO/KO); Tmc2(KO)(/)(KO) or Tmie(KO/KO) hair cells, which lack trans- duction, have significantly altered stereocilia lengths and diameters, including a narrowed row 2. EPS8 and the short splice isoform of MYO15A, identity markers for mature row 1 (the tallest row), lost their row exclusivity in transduction mutants. GNAI3, another member of the mature row 1 complex, accumulated at mutant row 1 tips at considerably lower levels than in wild-type bundles. Alterations in stereocilia dimensions and in EPS8 distribution seen in transduction mutants were mimicked by block of transduction channels of cochlear explants in culture. In addition, proteins normally concentrated at mature row 2 tips were also distributed differently in transduction mutants; the heterodimeric capping protein subunit CAPZB and its partner TWF2 never concentrated at row 2 tips like they do in wild-type bundles. The altered distribution of marker proteins in transduction mutants was accompanied by increased variability in stereocilia length. Transduction channels thus specify and maintain row identity, control addition of new actin filaments to increase stereocilia diameter, and coordinate stereocilia height within rows.

Mutations in the DNA methyltransferase 3A (DNMT3A) gene are recurrent in de novoacute myeloid leukemia (AML) and are associated with poor prognosis. Although studies demonstrated survival benefit of induction chemotherapy dose intensification, outcomes remain unsatisfactory in most patients due to advanced age, comorbidities, and hence inability to tolerate treatment. Clinical trials of low-intensity regimens combining cytarabine and cladribine, nucleoside analog chain terminators that stall DNA replication, appear to be safe and effective, and tend to particularly benefit patients with DNMT3Amutations. Consistently, we observe increased sensitivity to cytarabine, fludarabine, and cladribine in multiple cellular systems harboring mutant DNMT3Ain vitro (Figure 1A, B). Differential sensitivity to cytarabine was confirmed in normal and leukemic primary bone marrow cells derived from mice with and without Dnmt3a mutations ex vivo (Figure 1C). Dynamic chromatin organization plays a pivotal role in DNA-associated cellular processes including DNA replication and damage repair. We previously found altered chromatin remodeling in cells expressing mutant DNMT3A after genotoxic stress. Gene expression studies by us and others demonstrated negative enrichment of cell cycle related signatures including G2/M checkpoint adaptation, in cells with DNMT3A mutations. These signatures are implicated in DNA damage response and replication fork integrity and suggest sensitivity to replication stress. To investigate the mechanism of differential sensitivity to cytarabine-induced DNA damage, we overexpressed wildtype (WT) or R882 mutant (MUT) forms of DNMT3A in U2OS cells, a well-established model for DNA damage studies. Analysis of the DNA damage signaling proficiency in response to cytarabine revealed persistent intra-S phase checkpoint activation (phospho-CHK1), accompanied by accumulation of DNA damage visualized by γH2A.X foci and by Comet assay in the DNMT3A(mut) overexpression cells (Figure 1D). This damage was only partially resolved after drug had been removed and cells were allowed to repair the DNA (Figure 1E), and was carried through mitosis, resulting in increased rate of micronucleation.At the same time, DNMT3A mutant cells remained proficient in initiating homology-directed repair (HDR) and non-homologous end joining (NHEJ) pathways, evidenced by RAD51 and 53BP-1 foci formation, respectively. These data demonstrate enhanced sensitivity to cytarabine in cells expressing mutant DNMT3A is due to increased susceptibility to DNA damage during replication, rather than defects in double-strand DNA break repair. In support of this, cells with mutant DNMT3Awere characterized by accentuated replication stress as evidenced by high levels of phospho-RPA, which persisted after drug wash-out (Figure 1F). Consistently, DNMT3A-mutant cells treated with cytarabine were characterized by a higher number and a larger area of PCNA foci. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine wash-out demonstrated that while the overall kinetics of replication restart remained unchanged, cells with DNMT3A(mut) showed higher rate of fork collapse and increased reliance on latent replication origins (Figure 1G). Gene expression profiling by RNA-seq identified dysregulation of pathways associated with cell cycle progression, specifically G1/S phase transition, DNA replication, DNA integrity checkpoint, and chromatin. Our studies show cells with DNMT3A mutations have a defect in recovery from replication fork arrest and subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during DNA replication and suggest that cytarabine-induced replication fork stalling may further synergize with other agents aimed at DNA damage and replication. Figure 1 Disclosures No relevant conflicts of interest to declare.

Nonmuscle myosin 2 (NM-2) powers cell motility and tissue morphogenesis by assembling into bipolar filaments that interact with actin. Although the enzymatic properties of purified NM-2 motor fragments have been determined, the emergent properties of filament ensembles are unknown. Using single myosin filament in vitro motility assays, we report fundamental differences in filaments formed of different NM-2 motors. Filaments consisting of NM2-B moved processively along actin, while under identical conditions, NM2-A filaments did not. By more closely mimicking the physiological milieu, either by increasing solution viscosity or by copolymerization with NM2-B, NM2-A containing filaments moved processively. Our data demonstrate that both the kinetic and mechanical properties of these two myosins, in addition to the stochiometry of NM-2 subunits, can tune filament mechanical output. We propose altering NM-2 filament composition is a general cellular strategy for tailoring force production of filaments to specific functions such as maintaining tension or remodeling actin.

Nature Communications 8: Article number: 14907 (2017); Published: 7 April 2017; Updated: 25 May 2018 The original version of this Article contained an error in the spelling of the author Aysegul Gezer, which was incorrectly given as Aysegul Geyser. This has now been corrected in both the PDF and HTML versions of the Article.

Myosin-6 is a molecular motor that translocates and generates force towards the pointed-end of the actin filament, and is important in a variety of cellular processes including the maintenance of the Golgi complex, exocytosis and cell migration. Myosin-6 has direct relevance to diseases such as cancer, cardiomyopathy, deafness, and neurodegenerative diseases and has thus been a focus of many investigations. Previous in vitro mechanical studies have shown that forced dimers of myosin-6 have a large step-size in comparison to its rather short lever arm, as well as a broad step-size distribution. Furthermore, a recent study has shown evidence that myosin-6 moves via both "hand-over-hand" and "inchworm-like" mechanisms. To elucidate this dualistic mechanism of myosin-6 motility we have generated a human myosin-6 (MYO6) heavy meromyosin-like construct (amino acids1-1021), with a N-terminal AviTag, a C-terminal GCN4 leucine zipper to force dimerization of two heavy-chains, a GFP moiety for fluorescence imaging and FLAG-tag for affinity purification. To examine acto-myosin-6-HMM interactions at both high spatial and temporal resolutions, we utilized negative-stained electron microscopy, fluorescence microscopy and interferometric scattering microscopy (iSCAT) assays. We present here direct evidence that the forced dimers of human myosin-6 dwell in an actin filament bound state where the two heads are directly next to each other and that the possible explanation of this mechanical state may be due to the inchworm-like mechanism. Further studies are underway to examine these dwell states, as well as the stepping mechanism, in greater detail. This work was supported by the Intramural Research Program of the NHLBI (to J.R.S.) and the NIDCD (to T.B.F.), and the ERC NanoScope (to P.K.).