A mammal’s sense of hearing and balance are helped by so-called hair cells within the inner ear. These cells are named after their long, hair-like protrusions called stereocilia, and mutations in the genes involved in stereocilia development and maintenance can lead to hearing loss in humans. Damage to the stereocilia caused by excessive exposure to loud noises can also have the same effect.

Stereocilia are full of filaments made of a protein called actin. Other proteins called class III myosins and Espin are also both required for normal development of stereocilia. Mutations in the genes that encode these proteins can cause hereditary deafness in humans. However, it remains unclear exactly how these two proteins (myosins and Espin) interact with each other in stereocilia.

Using biochemical and structural studies, Liu, Li et al. have now discovered that the so-called ‘tail’ part of the myosins contains a pair of repeated sequences that can each interact with an Espin protein called Espin1. This interaction allows each myosin to cross-link two Espin1 proteins. Espins assemble actin filaments into bundles, and further experiments showed that this cross-linking interaction between myosins and Espins helped this process, which is linked to stereocilia development and maintenance.

Mammals actually have two related copies of class III myosins that play overlapping but slightly different roles. The next challenge will be to try to understand the differences between these related proteins, as well as to try to uncover the roles of other forms of Espin in stereocilia.

Class III myosins (Myo3), together with class IX myosins, are two special groups of the myosin superfamily as these two sub-families of actin motors contain enzymatically active domains and thus are regarded as motorized signaling molecules (Bähler, 2000). The first member of Myo3 was identified in Drosophila photoreceptors and named as NinaC (neither inactivation nor afterpotential C) (Montell and Rubin, 1988). There are two paralogs of Myo3 in vertebrate, Myo3a and Myo3b, both of which are known to express in vertebrate retina and cochlea (Dose and Burnside, 2000, 2002; Shin et al., 2013). It is believed that they may play partially redundant roles as transporters that are crucial for vertebrate photoreceptor and stereocilia ultrastructure maintenance (Manor et al., 2012; Mecklenburg et al., 2015; Merritt et al., 2012).

Myo3 across different species all contain an N-terminal S/T kinase domain before their motor head. The kinase domain has been reported to regulate the motor’s ATPase activity (Komaba et al., 2010; Quintero et al., 2010). The tail regions of Myo3 from different species are less conserved. Drosophila NinaC contains a PDZ binding motif at its very C-terminus capable of binding to a master scaffold protein called INAD (Inactivation no afterpotential D) (Wes et al., 1999). Vertebrate Myo3 tails share a conserved vertebrate specific domain referred to as tail homology I motif (THDI) (Figure 1A) (Dose et al., 2003). The THDI mediates binding of Myo3 to its cargo protein Espin1 (Ectoplasmic specialization protein 1) and allows Myo3 to transport Espin1 to the tips of actin bundle-based structures such as filopodia and stereocilia. Once tip localized, Espin1 WH2 domain promotes the elongation of actin protrusions (Merritt et al., 2012; Salles et al., 2009). However, the detailed molecular basis governing the Myo3 and Espin1 interaction is not clear.

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Espin1 was first identified in Sertoli cell-spermatid junctions (Bartles et al., 1996), encoded by the gene Espin. Later, shorter spliced isoforms of Espin gene products (Espin2B, Espin3A and Espin4) were shown to be expressed in other F-actin rich structures such as brush border microvilli and Purkinje cell dendritic spines (Bartles et al., 1998; Sekerkova et al., 2003). They share a common 14 kDa C-terminal actin binding domain (ABD; Figure 1A), which was reported to be necessary and sufficient for F-actin bundling activity (Bartles, 2000; Bartles et al., 1998). Besides the ABD, all Espin isoforms contain a WH2 motif which can bind to actin monomer and a proline rich (PR) region which can interact with profilins (Sekerkova et al., 2006). Espin2B contains one more PR region and an extra actin binding site (xAB) at the N-terminus (Chen et al., 1999). Espin1 is the longest isoform in the family and contains a stretch of ankyrin repeats (AR) in its N-terminus (Figure 1A). The AR of Espin1 is responsible for directly interacting with Myo3 tail THDI (Merritt et al., 2012; Salles et al., 2009). Recently, it was reported that Espin1 contains an AR binding sequence immediately C-terminal to xAB, and binding of AR to this sequence prevents xAB from binding to actin (Zheng et al., 2014). As such, the actin binding activity of the N-terminal part of Espin1 is auto-inhibited, and this AR-binding region is named as the auto-inhibitory region (AI, Figure 1A). The authors also proposed that Myo3 binding can release the auto-inhibition and increase the diameter of Espin1-promoted actin bundles (Zheng et al., 2014).

Myo3a/Myo3b are known to co-localize with Espin1 at the tips of stereocilia of hair cells (Merritt et al., 2012; Salles et al., 2009; Schneider et al., 2006). Importantly, co-expression of Myo3a and Espin1 can further stimulate elongation of stereocilia in cultured organ of Corti hair cells (Salles et al., 2009). When expressed in heterologous cells, Myo3a gets enriched at the tip of filopodia (Les Erickson et al., 2003; Salles et al., 2009; Schneider et al., 2006). Mutations of human Myo3a gene is known to cause progressive non-syndromic hearing loss, DFNB30 (Walsh et al., 2002). Also, transgenic mice with DFNB30 mutation undergo age-dependent outer hair cell degeneration (Walsh et al., 2011). The jerker mouse carrying espin frame-shift mutation suffers from hair cells degeneration, deafness and vestibular dysfunction (Sekerkova et al., 2011; Zheng et al., 2000). Mutations in espin were also reported to be associated with non-syndromic autosomal recessive deafness DFNB36 and non-syndromic autosomal dominant deafness (Boulouiz et al., 2008; Donaudy et al., 2006; Naz et al., 2004). A prominent phenotype of the jerker mice is that their hair cell stereocilia are uniformly thinner and shorter, degenerate faster than those of the wild type littermates (Sekerkova et al., 2011; Zheng et al., 2000). These genetic findings convincingly point to critical roles of espin in stereocilia development and maintenance, likely by promoting the assembly and stabilization of parallel actin filament bundles in stereocilia (Bartles, 2000; Sekerkova et al., 2006).

Here, we discover that both Myo3a and Myo3b contain two highly similar repeat sequences in their THDI region, each capable of independently binding to Espin1-AR with high affinity. The high resolution crystal structures of each of the two binding sequences from Myo3b in complex with Espin1-AR not only reveal the molecular basis governing the specific interaction between Myo3 and Espin1, but also allow us to discern the Myo3-mediated release of the auto-inhibition mechanism of Espin1. Based on these structural findings, we predict that binding of Myo3 to Espin1 can cluster Espin1 and thus enable Espin1 to further cross-link actin filaments into higher order fibers. Consistent with this prediction, we demonstrate by electron and fluorescence microscopic studies that binding of Myo3 to Espin1 can further promote formation of Espin1-mediated thicker actin bundles.

The most important findings of this study are the structure-based discovery of two strong and independent Espin1 binding sites in the tail region of each class III myosins, and Myo3 binding-induced auto-inhibition release of Espin1. These discoveries, together with our cell-based filopodia formation and localization assay and microscopic-based actin bundling assay, provide compelling evidences showing that Myo3 and Espin1 work together to assemble and promote higher order parallel actin bundle formation in cellular processes such as stereocilia. We believe that our model reveals the probable underlying molecular mechanism of hearing loss development (stereocilia degeneration) in humans with mutations in Myo3a (DFNB30) (Walsh et al., 2002). It is believed that the delayed hearing loss phenotype in DFNB30 subjects could be due to Myo3b compensatory mechanism (Manor et al., 2012), a hypothesis supported by our results as well. With the help of the motor domain-mediated high affinity binding of Myo3 to actin filaments (Dose et al., 2007; Kambara et al., 2006), the full-length Myo3/Espin1 complex may have even higher actin fiber bundling activity than the Myo3-ABR12/Espin1 complex studied in this work. As such, our study reveals a previously unrecognized direct myosin binding-induced actin bundling activity regulation mechanism of an actin filament cross-linker protein. It also demonstrates an intimate synergistic action mechanism between two classes of actin binding proteins, namely actin filament-based myosin motors and actin filament cross-linking protein, in controlling actin fiber size and stability. It has been reported that another unconventional myosin in stereocilia, Myosin XVa, can form a complex with an actin capping protein Eps8 to regulate stereocilia elongations (Manor et al., 2011), suggesting that working together with actin binding proteins to regulate actin skeletal dynamics might be a new mode of function for unconventional myosins.

Fitting with this model, stereocilia in jerker mice are thinner in their diameters likely due to loss of Espin1-mediated higher order actin fiber assembly, easier to form tapered structures and easier to degenerate presumably due to stability decrease of less bundled actin fibers in stereocilia (Sekerkova et al., 2011; Zheng et al., 2000). As one of the three known actin cross-linkers in stereocilia, the abundance of Espin is relatively low compared to fascin and plastin (Shin et al., 2013). It has been proposed that Espin may play a regulating role in elongation and widening of actin bundle by concentrating at the tip of stereocilia instead of major structural cross-linking roles (Avenarius et al., 2014; Loomis et al., 2003). Interestingly, the abundance of Myo3 (both Myo3a and Myo3b together) is similar to that of Espin (a few hundred copies per stereocilium) (Shin et al., 2013), indicating that Myo3 and Espin1 may work together to regulate higher order actin bundle structure formation and stability in stereocilia. Both Myo3 and Espin1 are known to concentrate at the tips of stereocilia in hair cells and at the tips of filopodia when expressed in heterologous cells (Merritt et al., 2012; Salles et al., 2009; Schneider et al., 2006). It is possible that stabilization of the growing end of the actin filaments at the plus ends by both Myo3 and Espin1 is a critical driving force for actin filament elongations (Avenarius et al., 2014; Loomis et al., 2003; Shin et al., 2013).

The Myo3 binding-mediated cross-linking of Espin1 cargo is in sharp contrast to the cargo binding-mediated motor dimerization and processivity induction known in a number of other unconventional myosins (myosin V, VI and VII) (Lu et al., 2014; Sakai et al., 2011; Shi et al., 2014; Yu et al., 2009). Such large differences in their cargo bindings probably match with the distinct functional properties of these myosins. For a fast-moving cargo transporting motor such as myosin VI, it is important that the motor assumes as a dimer and gets activated for moving in one direction upon binding to a cognate cargo. In such a case, the binding of a cargo protein to the motor tail exerts large impact on the motor activity regulations. As a high duty ratio motor, processive and rapid movement along the actin filaments is not likely to be the main function of Myo3, although the motor can still move towards the plus end of actin filaments (Les Erickson et al., 2003; Merritt et al., 2012; Salles et al., 2009; Schneider et al., 2006). Instead, one of Myo3’s key functions appears to coordinate with its cargo protein Espin1 to regulate Espin1-mediated actin filaments assembly and stability. Whether such myosin binding-mediated cargo activity regulation is also adopted by other unconventional myosins, particularly for those not known to play transporting roles, is an interesting research topic in the future.

We provide detailed structural information regarding Myo3b/Espin1 interaction. We demonstrate that the Espin1 binding sites in Myo3a and Myo3b are essentially identical and therefore are predicted to be functionally interchangeable. These findings provide a molecular explanation for the partially redundant functions of Myo3a and Myo3b in hair cells. This analysis also predicts that Espin-like proteins can also bind to Myo3 as Espin1 does, suggesting that the functions of Espin1 and Espin-like protein may also be partially redundant in tissues like stereocilia (Shin et al., 2013). Such redundancies suggest that mutation of single myo3a or myo3b or defects in espin1 or espin-like may not always develop severe phenotypes in vertebrates. Moreover, we note that forked, the Drosophila ortholog of Espin, is expressed in bristle cells, a cell type that is enriched in bundled actin filaments (Petersen et al., 1994). Drosophila Forked protein is predicted to contain five ANK repeats, corresponding to Espin1 repeats 5–9. The structures solved here of mammalian Espin1-AR should help for identifying potential binding partners of Forked-AR.

Although Myo3a and Myo3b are highly similar, there are clear differences in terms of filopodia tip localizations (Manor et al., 2012; Merritt et al., 2012). Myo3a localize to the very tip of filopodia, while Myo3b distributes in a wider tip-base gradient (Merritt et al., 2012). The different tip localization patterns of Myo3a and Myo3b does not originate from the unique ABM of Myo3a (Manor et al., 2012). Our study here suggests that their Espin1 binding THDI regions are not responsible for different localizations of Myo3a and Myo3b along stereocilia/filopodia either. It is possible that the kinetics and the F-actin binding affinities of their motor domains or other variable regions in their tails may contribute to such differences.

Most of the unconventional myosins use their globular cargo binding domains to recognize their cargoes (Lu et al., 2014). However, Myo3 use their unstructured, yet highly conserved tails to recognize globular domains from their cargoes. Myo3 and another unconventional myosins, Myosin XIX, are not predicted to contain folded globular domains in their tail regions (Lu et al., 2014). Based on the results in this study, we anticipate that these two myosins might use different unstructured fragments in their tails to specifically recognize various cargoes. A very recent report showed that another region in Myo3a’s tail, which is located immediately N-terminal to the Espin1 binding ARB12 region, can bind to MORN4 (Mecklenburg et al., 2015), the mammalian ortholog of Drosophila Retinophilin. It is noted that Retinophilin can also bind to Drosophila NinaC (Venkatachalam et al., 2010). Future studies are required to elucidate the molecular basis governing the Myo3/MORN4 and NinaC/Retinophilin interactions for better understanding of the interactions.

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Author details

1. Haiyang Liu

   Shenzhen Key Laboratory for Neuronal Structural Biology, Biomedical Research Institute, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, Shenzhen, China

   Contribution

   HL, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Jianchao Li

   Competing interests

   No competing interests declared.

2. Jianchao Li

   Division of Life Science, State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China

   Contribution

   JL, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Haiyang Liu

   Competing interests

   No competing interests declared.

3. Manmeet H Raval

   Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, United States

   Contribution

   MHR, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

4. Ningning Yao

   Shenzhen Key Laboratory for Neuronal Structural Biology, Biomedical Research Institute, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, Shenzhen, China

   Contribution

   NY, Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared.

5. Xiaoying Deng

   Shenzhen Key Laboratory for Neuronal Structural Biology, Biomedical Research Institute, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, Shenzhen, China

   Contribution

   XD, Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared.

6. Qing Lu

   Division of Life Science, State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China

   Contribution

   QL, Conception and design, Analysis and interpretation of data

   Competing interests

   No competing interests declared.

7. Si Nie

   National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

   Contribution

   SN, Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared.

8. Wei Feng

   National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

   Contribution

   WF, Conception and design, Analysis and interpretation of data

   Competing interests

   No competing interests declared.

9. Jun Wan

   1. Shenzhen Key Laboratory for Neuronal Structural Biology, Biomedical Research Institute, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, Shenzhen, China
   2. Division of Life Science, State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China

   Contribution

   JW, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

10. Christopher M Yengo

    Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, United States

    Contribution

    CMY, Analysis and interpretation of data, Drafting or revising the article

    Competing interests

    No competing interests declared.

11. Wei Liu

    1. Shenzhen Key Laboratory for Neuronal Structural Biology, Biomedical Research Institute, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, Shenzhen, China
    2. Division of Life Science, State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China

    Contribution

    WL, Conception and design, Analysis and interpretation of data, Drafting or revising the article

    For correspondence

    liuw@ust.hk

    Competing interests

    No competing interests declared.

12. Mingjie Zhang

    1. Shenzhen Key Laboratory for Neuronal Structural Biology, Biomedical Research Institute, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, Shenzhen, China
    2. Division of Life Science, State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China
    3. Center of Systems Biology and Human Health, School of Science and Institute for Advanced Study, Hong Kong University of Science and Technology, Hong Kong, China

    Contribution

    MZ, Conception and design, Analysis and interpretation of data, Drafting or revising the article

    For correspondence

    mzhang@ust.hk

    Competing interests

    MZ: Reviewing editor, eLife

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