Two proteins implicated in inherited deafness, myosin IIIa1, a plus end directed motor2, and espin3–5, an actin bundling protein containing the actin-monomer-binding motif WH26, have been shown to influence the length of mechanosensory stereocilia7, 8. Here we report that espin 1, an ankyrin repeat-containing isoform of espin6, colocalizes with myosin IIIa at stereocilia tips and interacts with a unique conserved domain of myosin IIIa. We show that overexpression of these proteins causes elongation of stereocilia greater that when myosin IIIa alone or espin 1 alone are overexpressed. When these two proteins are co-expressed in the fibroblast-like COS-7 cell line they induce a ten-fold elongation of filopodia. This extraordinary filopodia elongation results from the transport of espin 1 to the plus ends of F-actin by myosin IIIa and depends on espin 1 WH2 activity. This study provides the basis for understanding the role myosin IIIa and espin 1 play in regulating stereocilia length, and presents a physiological example where myosins can boost elongation of actin protrusions by transporting actin regulatory factors to the plus ends of actin filaments.
Stereocilia, the prominent actin protrusions on the apical surfaces sensory hair cells, emerge early during development and their lengths are maintained at fixed heights for the lifetime of the organism. The bundle of parallel actin filaments that make up the core of each stereocilium is continuously renewed, with the entire actin bundle constantly assembled at the tip, treadmilling downward, and disassembling at the base9–11. Given that stereocilia can be up to 100 µm in length, it is likely that some form of regulated transport is necessary to localize components of the actin polymerization machinery to the plus end of the actin filaments. Although several myosins have been shown to alter stereocilia lengths and shapes depending on their expression levels9, 12–16, the mechanisms by which these motors or their binding partners regulate actin dynamics and stereocilia lengths remain unclear.
Using antibodies specific to the ankyrin repeat domain (ARD) of espin 1 (Supplementary Information, Fig. S1), we show localization at stereocilia tips with a tip-to-base gradient distribution (Fig. 1) similar to what was previously described for myosin IIIa. The immunofluorescence of espin 1 is more intense in the longer stereocilia and the characteristic tip-to-base fluorescence intensity gradient has a longer decay length (Fig. 1g,h). In contrast to other espin isoforms, which are present inside the actin core and along the entire stereocilia length8, espin 1 is excluded from the actin cores and forms a thimble-like distribution at the tips of stereocilia (Fig. 1). Immunofluorescence in developing hair cells of the rat organ of Corti shows that espin 1 can be detected at the tips of stereocilia during their elongation and maturation phases (Fig. 1k–m). To confirm the localization of espin 1 at the tips of stereocilia, we overexpressed GFP-espin 1 in organotypic cultures of hair cells. Transfected hair cells show that GFP-espin 1 localizes at the tips of stereocilia, exhibiting a tip-to-base gradient of intensity (Fig. 2) comparable to the immunolocalization (Fig. 1). These localization patterns – tip-to-base gradients and thimble-like distributions7, as well as the temporal expression pattern17 – closely match those of myosin IIIa. We hypothesized that targeting espin 1 to stereocilia tips, which is the site of actin polymerization9, 11, influences actin polymerization and stereocilia elongation. An analysis of the heights of stereocilia of cochlear and vestibular hair cells transfected with GFP-espin 1 shows elongation of stereocilia upon overexpression of espin 1 (Fig. 2), consistent with our hypothesis.
The striking similarity of the tip-to-base gradient localization of both espin 1 and myosin IIIa prompted us to investigate whether myosin IIIa helps localize espin 1 to the tips of stereocilia and whether they have a combined role in the regulation of stereocilia length. We compared stereocilia length in hair cells transfected with espin alone, myosin IIIa alone and with a combination of both plasmids (Fig. 2). Hair cells transfected with myosin IIIa and espin 1 show an increase in stereocilia length, higher than the combined increase observed for myosin IIIa alone and espin 1 alone (Fig. 2). It is important to note that any analysis of lengthening due to overexpression of myosin IIIa and espin 1 in hair cells must take into account intrinsic limitations due to natural stereocilia length variations and, importantly, the fact that stereocilia are already quite elongated and express robust amounts of endogenous espin 1 and myosin IIIa.
We tested whether myosin IIIa can effectively interact with and transport espin 1 in COS-7 cells, using filopodia as a model to study actin protrusions. Myosin IIIa and espin 1 are not naturally expressed at detectable levels in COS-7 cells 7. Myosin IIIa has been shown to induce filopodial actin protrusions and localize to their tips in cultured cells particularly well when its kinase domain has been removed (myosin IIIa ΔK)7,7, 18 suggesting that the kinase could serve to down-regulate the functional activity of myosin IIIa. We examined the distribution of co-expressed mCherry-ARD of espin 1 with GFP-tagged myosin IIIa ΔK (Fig. 3). We also co-expressed mCherry-ARD with GFP-tagged myosin X and GFP-tagged myosin XVa. Since all of these myosins accumulate at the tips of filopodia13, 16, 19, they provide a well-defined spatial compartment where any potential interaction can be clearly visualized. Co-transfections showed that mCherry-ARD is efficiently targeted to the tips of filopodia initiated by myosin IIIa ΔK, but not by myosins X or XVa (Fig. 3a), demonstrating a specific colocalization of ARD with myosin IIIa. Live imaging of COS-7 cells transfected with GFP-myosin IIIa ΔK and mCherry-ARD showed dynamic colocalization at the filopodia tips from the early steps of their initiation and elongation (Fig. 3b; Supplementary Information, Video S1). Live imaging also showed matching forward and rearward intra-filopodial movements of the GFP-myosin IIIa ΔK and mCherry-ARD fluorescence puncta while maintaining steady-state tip-to-base distributions (Supplementary Information, Video S2 and Video S3, and Fig. 3c), similar to the distributions of myosin IIIa and espin 1 observed in stereocilia (Fig. 1 and Fig 2). The intensity profiles for mCherry-ARD and GFP-myosin IIIa ΔK within each frame of the video (Fig. 3c) are highly correlated (cross-correlation = ~0.990), supporting the model that these two proteins are trafficking together in the filopodia. The interaction between espin 1 ARD and myosin IIIa ΔK was confirmed with a GST pull-down assay (Fig. 3e). The dynamic localization of espin 1 ARD at filopodia tips when co-transfected with myosin IIIa, but not with myosin X or with myosin XVa, along with our GST pull-down assay results, led us to hypothesize that myosin IIIa transports espin 1 to the tips of stereocilia.
We next asked which specific region of myosin IIIa is involved in the interaction with espin 1. Myosin IIIa has two conserved tail homology domains, designated as 3THDI and 3THDII20. We first co-transfected COS-7 cells with espin 1 and GFP-myosin IIIa and showed that the two proteins colocalize at actin bundles as well as at filopodia tips (Fig. 4a,b). This pattern of colocalization is abolished when we use a GFP-myosin IIIa construct that lacks the portion of the tail containing both 3THDI and 3THDII (GFP-myoIIIa Δ32; Supplementary Information, Table S1, Fig. S2; Fig. 4a,b). We next co-transfected COS-7 cells with espin 1 and with a GFP tagged tail portion lacking 3THDII (GFP-tailΔ3THDII; Fig. 4a,b) and narrowed down the region of interaction to the 3THDI and its immediate flanking regions. We observed colocalization of espin 1 with GFP-myosin IIIa tail that contained only the 3THDI domain (GFP-3THDI; Fig. 4a,b), but not with regions of only the myosin IIIa tail immediately amino-terminal (pre3THDI) or carboxyl-terminal (post3THDI) to the 3THDI domain (Fig. 4a,b). This suggests that the 3THDI domain is necessary for the myosin IIIa:espin 1 interaction. Together these data suggest that espin 1 and myosin IIIa specifically interact via their ARD and 3THDI domains, respectively. We verified this interaction in vitro using a GST pull-down assay and demonstrate that GST-ARD binds to GFP-3THDI, but not to the pre3THDI or post3THDI regions (Fig. 4c).
The fact that stereocilia length can be influenced by either espin 13, 8 or myosin IIIa7, along with the observation that they both localize to the same compartment at stereocilia tips and interact biochemically, suggests a combined functional role for the myosin IIIa:espin 1 complex in the elongation of stereocilia F-actin. We discovered that COS-7 cells co-transfected with myosin IIIa ΔK and espin 1 (Fig. 5a–c) display filopodial actin protrusions that can be up to ten times longer (mean length = 14.3 ± 9.1 µm; number of cells, nc =18; number of filopodia, nf=56) than those transfected with myosin IIIa ΔK alone (1.7 ± 0.83 µm, nc=12, nf=49), or with espin 1 alone (1.3 ± 0.28 µm, nc=13, nf=104). Mean lengths of filopodia of COS-7 cells transfected with empty GFP vector was 1.26 ± 0.7 (nc = 10, nf = 59). The synergistic effect between myosin IIIa and espin 1 is specific for myosin IIIa, since we found no enhanced elongation when espin 1 was co-expressed with either myosin X (2.40 ± 1.50 µm, nc=16, nf=165) or myosin XVa (2.08 ± 1.63 µm, nc=15, nf=134).
We used myosin IIIa without the kinase domain to observe the behavior of the dephosphorylated and more functionally active myosin. To exclude the possibility that the deletion of the kinase domain produces aberrant behavior, we developed a kinase-dead construct, myosin IIIa K50R (Supplementary Information, Table. S1). This construct allowed us to examine the role of autophosphorylation in the regulation of motor function, which in turn enabled us to investigate the role of myosin IIIa motor function in espin 1 tip-localization activity. We have determined that inactivation of the myosin IIIa kinase in a myosin IIIa 2IQ construct reduces the KATPase yet it does not affect maximal ATPase activity (Supplementary Information, Table S2 and Figure S3). We next evaluated the role of the kinase activity in myosin IIIa tip-localization in COS-7 cells using GFP tagged constructs. Full-length myosin IIIa K50R localizes more efficiently to the tips of filopodia in COS-7 cells (39% at tips nc=137) than wild-type (5% at tips nc=200), although not as strikingly as myosin IIIa ΔK (93% at tips n=105). Furthermore, co-expression of myosin IIIa K50R and espin 1 (Fig. 5e) yielded longer filopodia (mean length = 5.93 ± 3.10 µm, nc=15, nf=89) than co-expression of wild-type myosin IIIa and espin 1 (3.7 ± 3.2 µm, nc=15, nf=63; Fig. 5d), although not as long as the myosin IIIa ΔK:espin 1 co-expression. This data shows that myosin IIIa motor ATPase activity parallels the ability of myosin IIIa to localize to filopodia tips and to elongate filopodia when co-expressed with espin 1.
Interestingly, espin 1 co-expressed with a myosin IIIa ΔK lacking the tail domain downstream of exon 32 (myosin IIIa ΔK,33,34; Supplementary Information, Table S1, Fig. S2) resulted in slightly shorter filopodia (10.0 ± 4.74 µm, n=64; Fig. 5f) than co-expression with myosin IIIa ΔK. Using COS-7 cell co-expression and GST pull-down assays, we confirmed that the upstream portion of 3THDI (3THDI Δ33, Supplementary Information, Fig. S4) binds to espin 1. The 3THDII of myosin IIIa has previously been shown to be an actin-binding site18. Previous studies reported that myosin IIIa lacking the 3THDII actin-binding domain does not localize to filopodia tips 7, 18, but here we show that when co-expressed with espin 1 myosin IIIa goes to the tip and promotes filopodia elongation (Fig. 5f). It appears that the association with espin 1, which does have actin-binding sites, compensates for the missing actin-binding site in the myosin IIIa without the 3THDII domain.
Co-expression of espin 1 and myosin IIIa results in enhanced localization of espin 1 at filopodia tips (Supplementary Information, Fig. S5). When myosin IIIa ΔK is co-expressed with espin 1 lacking the ARD domain, we observed that both espin tip localization and filopodia elongation are abolished (Fig. 5g). These results demonstrate that the actin cross-linking activity of espin 1 is not solely responsible for the enhanced filopodia or stereocilia elongation observed in our experiments. We conclude that espin 1 promotes enhanced elongation of filopodia only when transported to the polymerization end of actin filaments by myosin IIIa. The fact that espin 1 elongates filopodia only when localized to the F-actin plus ends by myosin IIIa suggests that WH2-dependent polymerization activity is involved in elongation. We tested this hypothesis by substituting the first two of three highly conserved leucine residues of the espin 1 WH2 motif (L655A, L656A), which have been shown to be essential for its actin-monomer-binding activity21, 22. In COS-7 cells co-transfected with the WH2-mutated espin 1 construct (espin 1 mWH2) and myosin IIIa ΔK (Fig. 5h), the average length of filopodia (2.65 ±1.50 µm, nc=10, nf=75) remains comparable to the protrusions induced by myosin IIIa ΔK alone. The lack of enhanced elongation despite the colocalization of espin 1 mWH2 and myosin IIIa ΔK at the tips of filopodia (Fig. 5h) demonstrates that the WH2 motif is critical for mediating the role of espin 1 in elongation.
The steady-state distribution of myosin IIIa in a tip-to-base gradient is likely dynamically maintained. The length of the myosin IIIa distribution should be inversely proportional to the net velocity of the myosin towards the tip23, which will be slower for faster treadmilling actin cores (i.e. in longer stereocilia9 and filopodia24). This prediction is also consistent with our observation that wild-type myosin IIIa, which has relatively low activity, has decreased tip localization in the filopodia compared to the more active kinase mutant forms of myosin IIIa used in our experiments (Fig. 5). However, in stereocilia where the actin treadmilling is much slower, the wild-type myosin IIIa self-localizes effectively to the tip7 (Fig. 2). Similarly, the observed steady-state tip-to-base gradient distribution of espin 1 is not compatible with a model where espin 1 passively diffuses and binds to myosin IIIa resident at the tip, since this scenario would result in a homogenous distribution along the entire length of the stereocilia with no detectable concentration gradient at steady-state. The gradient distribution of espin 1 at steady-state is reminiscent of a myosin VI-driven gradient for the stereocilia membrane protein PTPRQ, and is best explained by a model that includes binding, directed transport, and diffusion of myosins and their cargo25. A more detailed consideration of this dynamic process that also accounts for actin treadmilling and plus-end directed motors predicts a similar distribution, which can be several microns long for longer stereocilia23. Thus, we favor a model where myosin IIIa:espin 1 complexes are dynamically associated with the treadmilling actin core. This model suggests that espin 1 is transported to the tips of stereocilia by myosin IIIa, whereupon it remains bound to the surface of the actin core for a period of time. Interestingly, abolishing or reducing myosin IIIa kinase activity enhances the affinity of the myosin IIIa for actin, providing further evidence that the kinase domain plays a role in regulating the myosin IIIa motor kinetics and actin-binding properties26, 27. While the myosin IIIa:espin 1 complex is tightly bound to actin, it travels back towards the base of the stereocilia along with the treadmilling actin core. In support of this model, live video imaging in transfected COS-7 cells shows fluorescent puncta of GFP-myosin IIIa ΔK and mCherry-ARD (Supplementary Information, Video S4) that move rearwards at rates matching the rates reported for actin treadmilling in filopodia (~0.5 µm/min)24. We suggest that these puncta are stably bound to the surface of the treadmilling actin filament bundle.
It is noteworthy that the stereocilia tips are also the site of mechanoelectrical transduction (MET)28, that the myosin IIIa developmental expression level is correlated with maturation of MET in stereocilia17, and that myosin IIIa has been shown to transport components of the photoreceptor transduction machinery in Drosophila29, 30. We cannot exclude the possibility that the localization and dynamics of the myosin IIIa:espin 1 complex are also affected by interactions with other proteins at the stereocilia tip. Furthermore, ankyrin repeats have been shown to be promiscuous binders of membrane proteins31. It is possible that the turnover and dynamic localization of the espin 1:myosin IIIa complex are influenced by interactions with components of the MET machinery, and vice-versa.