Single-molecule fluorescence microscopy reveals regulatory mechanisms of MYO7A-driven cargo transport in stereocilia of live inner ear hair cells

Abstract

Stereocilia are F-actin-based cylindrical protrusions on the apical surface of inner ear hair cells that function as biological mechanosensors of sound and acceleration. During stereocilia development, specific unconventional myosins transport proteins and phospholipids as cargo and mediate elongation, differentiation and acquisition of the mechanoelectrical transduction (MET). How unconventional myosins localize themselves and cargo in stereocilia using energy from ATP hydrolysis is only partially understood. Here, we developed STELLA-SPIM microscopy to visualize movement of single myosin molecules in live hair cell stereocilia. STELLA-SPIM demonstrated that MYO7A, a component of MET machinery, shows processive movement toward stereocilia tips when chemically dimerized or constitutively activated by missense mutations disabling tail-mediated autoinhibition. Conversely, MYO7A shows step-wise but not processive movement in stereocilia when its tail is tethered to the plasma membrane or F-actin in the presence of MYO7A interacting partners. We posit that MYO7A dimerizes and moves processively in stereocilia when unleashed from autoinhibition.

Fig. 1. Development of single-molecule microscopy in live hair cells.

a Our workflow for single-molecule microscopy. b Labeling density optimization using vestibular hair cells expressing HaloTag-actin. At 0.3 nM JFX554-ligand, the entire cells are labeled, with dense signal at stereocilia tips (arrows) and in the cuticular plate. Fluorescent puncta appear in the cell body at 0.1 nM (arrowheads) and in stereocilia around 0.01 nM (open arrowheads). Maximum projections of volume scans. Exposure, 100 ms per plane at 0.2 kW/cm². Bars, 5 µm. c Classification of fluorescent puncta using the Gaussian Mixture model. The sum intensity is calculated by integrating the pixel values after background subtraction. Among the three populations (Pop1, Pop2 and Pop3), the peak intensity of Pop2 (985) is approximately twice that of Pop1 (408), indicating that Pop1 and Pop2 are emitted from one and two fluorophores, respectively. A total of 76 puncta were analyzed from six cells (including b, 0.01 nM), using average projections of 12 planes per volume scan. d Comparison between the average line intensity profile of fluorescent puncta (orange solid line) and the theoretical point spread function (PSF) of the objective lens calculated using the Born & Wolf 3D Optical Model^, (black dashed line). The similarity between these two intensity curves suggests that these puncta are emitted from a point source. Fluorescence intensity is an average of 10 puncta in b (0.01 nM, Pop1 only). SD, orange dotted lines. e, f Representative kymograms of non-fused HaloTag (e) and HaloTag-actin (f) labeled with 0.1 nM and 0.01 nM JFX554-ligands, respectively. Single-plane images are acquired every 1 s for comparison with MYO7A movement. Kymograms are generated from the line scans between arrowheads. Most non-fused HaloTag molecules disappear after one frame (e, arrows) due to diffusion, except for a few molecules (e, open arrows). Most HaloTag-actin molecules stay in the same place and disappear due to photobleaching or transition to the dark state (f, arrows). Imaging conditions are similar to (b). Bars, 5 µm (cell images); 2 µm and 20 s (kymograms). Source data are provided as a Source Data file.

Fig. 2. MYO7A-HMM dimers directionally moving in stereocilia.

a Domain structures of mouse MYO7A (NM_001256083.1) and its heavy-meromyosin-like fragment (MYO7A-HMM) used in this study. b Schematic diagrams showing the structure of HaloTag-MYO7A-HMM-FKBP and conditional dimerization under AP20187 treatment. Note that only a small portion of HaloTag is labeled in our single-molecule microscopy. c Confocal microscopy showing AP20187-dependent accumulation of HaloTag-MYO7A-HMM-FKBP at stereocilia tips. Vestibular hair cells (P2) expressing HaloTag-MYO7A-HMM-FKBP are incubated with or without 100 nM AP20187 for 2 h. Samples are fixed and stained with 200 nM JFX554-conjugated HaloTag ligands (yellow) and Alexa405-phalloidin (magenta). HaloTag-MYO7A-HMM-FKBP localizes to large protein blobs at stereocilia tips in AP20187-treated cells (arrows), and diffusely in the cuticular plate (arrowhead) and stereocilia (open arrow) in non-treated cells. Bars, 5 µm. d Directional and processive movement of MYO7A-HMM dimers in stereocilia under 200 nM AP20187 treatment. Time-lapse images (grayscale) are shown for the rectangular region in the pseudo-colored image (HaloTag in green and EGFP in magenta). A moving molecule is indicated by magenta circles in time-lapse images and as a continuous trajectory in the kymogram (arrows). JFX554, 0.3 nM. Single-plane time-lapse, every 1 s. Bars, 5 µm (time-lapse images); 2 µm and 20 s (kymogram). e, f Velocity and run lengths of MYO7A-HMM dimers directionally moving in stereocilia. The means ± SDs are 101 ± 53 nm/s and 2.3 ± 1.0 µm (n = 42). g Behavior of HaloTag-MYO7A-HMM-FKBP molecules in non-treated cells. Trajectories in the kymogram (arrows) are parallel to the time axis and consistent with molecules staying at the same position. Stepwise movement toward the stereocilia tips is occasionally detected (open arrows in the inset, magnifying the rectangle). Imaging conditions and scale bars are the same as in grey-scale images in (d). Source data are provided as a Source Data file.

Fig. 3. Directional movement of constitutively active MYO7A mutants.

a Structures of two constitutively active HaloTag-fused MYO7A mutants. HaloTag-MYO7A-RK/AA has two missense mutations, p.R2127A and p.K2130A, that we insert in the second MyTH4-FERM (M/F2) domain, referring to the study using human MYO7A. HaloTag-MYO7A-ΔSH3-ΔM/F2 has a truncated tail. These mutations are introduced to remove the tail-mediated autoinhibition of the motor domain. b Representative confocal images of HaloTag-fused full-length MYO7A and HaloTag-MYO7A-RK/AA expressed in vestibular hair cells (P2–5). Full-length MYO7A diffusely distributes in stereocilia (arrowhead) while HaloTag-MYO7A-RK/AA accumulates at stereocilia tips (arrows) of some cells, indicating directional movement toward stereocilia tips. Bars, 5 µm. c Confocal images of MYO7A-ΔSH3-ΔM/F2 expressed in vestibular hair cells (P2–5). This mutant accumulates at stereocilia tips in a small number of cells (arrows) and localizes diffusely in stereocilia in other cells (arrowhead). Bars, 5 µm. d Single-molecule microscopy of HaloTag-MYO7A-RK/AA. Time-lapse images (lower panels) show a molecule directionally moving in stereocilia (magenta circles). The kymogram (upper right panel) illustrates the processive movement of this molecule (arrows) and another molecule (open arrows). JFX554, 0.3 nM. Single-plane time-lapse, every 1 s. Bars, 5 µm (time-lapse images); 2 µm and 20 s (kymogram). e Single-molecule microscopy of HaloTag-MYO7A-ΔSH3-ΔM/F2. Time-lapse images and the kymogram show the processive and directional movement of a molecule (magenta circles and arrows). A stationary molecule is indicated for comparison (cyan circles and open arrows). Imaging conditions and scale bars are the same as (d).

Fig. 4. Movement of membrane-anchored MYO7A-HMM and MYO10-MD.

a Domain structures of HaloTag-MYO7A-HMM-FRB and HaloTag-MYO10-MD-FRB and a schematic diagram showing membrane anchoring by IL2Rα-EGFP-FKBP (human Interleukin 2 receptor alpha chain fused with EGFP and FKBP). Membrane anchoring is mediated by conditional heterodimerization between FRB and FKBP under AP21987 treatment. MYO10-MD is a control monomeric myosin motor head. b Confocal images showing AP21987-dependent membrane anchoring of HaloTag-MYO7A-HMM-FRB. Vestibular hair cells (P2) co-expressing HaloTag-MYO7A-HMM-FRB and IL2Rα-EGFP-FKBP are incubated with or without 500 nM AP21987 for 2 h. HaloTag-MYO7A-HMM-FRB distributes diffusely along stereocilia of untreated cells (arrowhead) but accumulates along the plasma membrane of stereocilia (arrow) and at the edge of the cuticular plate (open arrow) in AP21987-treated cells. Excess IL2Rα-EGFP-FKBP sometimes accumulates in vesicles without apparent damage to stereocilia (open arrowheads). Bars, 5 µm. c Confocal images showing AP21987-dependent membrane anchoring of MYO10-MD and its localization changes. HaloTag-MYO10-MD-FRB accumulates at stereocilia tips weakly in a few untreated cells (arrowhead), suggesting that a small number of MYO10-MD molecules can move directionally without a scaffold. Increased accumulation of HaloTag-MYO10-MD-FRB at stereocilia tips in AP21987-treated cells (arrows) indicates directional movement enhanced by membrane anchoring. This localization change in stereocilia is consistent with a previous study of MYO10-MD in filopodia. Bars, 5 µm. d Single-molecule microscopy of membrane-anchored MYO7A-HMM. Staircase-like trajectories in the kymogram indicate stepwise movement of MYO7A-HMM (arrows and open arrows). The molecule indicated by arrows is shown in time-lapse images (magenta circles). AP21987, 500 nM. Single-plane time-lapse, every 1 s. Bars, 5 µm (cell image); 20 s and 2 µm (kymogram). e Single-molecule microscopy of unanchored and membrane-anchored MYO10-MD. Before AP21987 treatment, a small number of MYO10-MD molecules show rapid directional and processive movement toward stereocilia tips (arrows and open arrows in the upper kymogram). After AP21987 treatment, MYO10-MD molecules show slower directional and processive movement (arrows and open arrows in the lower kymogram). Molecules indicated by arrows are shown in time-lapse images (magenta circles). AP21987, 500 nM. Single-plane images, every 100 ms. Bars, 5 µm (cell images); 2 s and 2 µm (kymograms).

Fig. 5. Movement of MYO7A-HMM and MYO10-MD coupled with a harmonin b fragment.

a Major harmonin isoform classes and FRB-DFCR-EGFP, the F-actin anchor used in this study. FRB-DFCR-EGFP contains a harmonin b fragment lacking in dfcr mice (double asterisks; residues 296–728 of NM_1163733). Harmonin d was recently identified in the retina (asterisk). b Schematic of HaloTag-MYO7A-HMM-FKBP, HaloTag-MYO10-MD-FKBP and their AP21987-dependent coupling with FRB-DFCR-EGFP. The PST domain interacts with F-actin. Two coiled-coil (CC) domains may interact with other harmonin isoforms. c Confocal images showing AP21987-dependent coupling between MYO7A-HMM and FRB-DFCR-EGFP. See Fig. 4 for sample preparation. FRB-DFCR-EGFP localizes to stereocilia (white arrowhead) and at the cuticular plate edge (white arrow), with weak accumulation near stereocilia tips (orange open arrowheads). HaloTag-MYO7A-HMM-FKBP distributes diffusely in stereocilia of untreated cells (orange arrowhead) and co-localizes with FRB-DFCR-EGFP in treated cells (orange arrows). A subset of HaloTag-MYO7A-HMM-FKBP localizes to stereocilia tips without dense FRB-DFCR-EGFP puncta (yellow arrows), possibly trafficked there using FRB-DFCR-EGFP as a scaffold. Bars, 5 µm. d Confocal images showing relocalization of MYO10-MD via AP21987-dependent coupling with FRB-DFCR-EGFP. In untreated cells, FRB-DFCR-EGFP localizes similarly to (c). MYO10-MD distributes diffusely in stereocilia in untreated cells (orange arrowhead) and accumulates at stereocilia tips with FRB-DFCR-EGFP after AP21987 treatment (orange arrows and open arrows). The amounts of MYO10-MD and FRB-DFCR-EGFP at stereocilia tips increase compared to untreated cells, suggesting that MYO10-MD moves using FRB-DFCR-EGFP as a scaffold. Bars, 5 µm. e Single-molecule microscopy of MYO7A-HMM coupled with FRB-DFCR-EGFP. The kymogram shows stepwise, directional movement toward stereocilia tips (arrows). The molecule indicated by arrows is also shown in time-lapse images (magenta circles). AP21987, 500 nM. Single-plane time-lapse, every 1 s. Bars, 5 µm (cell image); 20 s and 2 µm (kymogram). f Single-molecule microscopy of MYO10-MD coupled with FRB-DFCR-EGFP. The kymogram shows directional movement toward stereocilia tips (arrows, open arrows and open arrowheads). The molecules indicated by arrows and open arrows in the kymogram are also shown in time-lapse images (magenta and cyan circles, respectively). AP21987, 500 nM. Single-plane time-lapse, every 100 ms. Bars, 5 µm (cell image); 2 s and 2 µm (kymogram).

Fig. 6. Possible scenarios of MYO7A-driven cargo transport in stereocilia.

a Schematic of stereocilia and MET machinery. MET channels are localized to the distal ends of stereocilia and physically connected to the upper tip-link density (UTLD) of adjacent longer stereocilia. b Major interacting partners of MYO7A in stereocilia. SANS and harmonin bridge interactions with other partners. SANS binds to the first MyTH4-FERM domain of MYO7A in an anti-parallel orientation. The PST domain of harmonin b can bind to F-actin. The single α-helix (SAH) of MYO7A has weak dimerization activity^,. SANS, PCDH15 and CDH23 can dimerize with each other^,, and may keep multiple MYO7A molecules in proximity. Oligomerization has been reported for harmonin. PCDH15 and CDH23 have one transmembrane motif (TM)^,. The SH3 domain of MYO7A can interact with the intracellular region (IC) of PCDH15 (CD2 isoform shown). c MYO7A activation in stereocilia predicted from the behaviors of MYO7A-RK/AA and MYO7A-ΔSH3-ΔM/F2. From its backfolded autoinhibitory conformation, MYO7A could be dimerized by SANS and harmonin as proposed by a previous X-ray crystallography study (blue panel). MYO7A may also move processively when a PCDH15 dimer brings two MYO7A molecules into proximity (green panel). Considering the low-frequency processive movement of MYO7A-ΔSH3-ΔM/F2, there may be transitional forms (red panel). d–f Possible MYO7A-driven tip-link assembly. The scaffold-first scenario (d) assumes that MYO7A (with SANS) settles at the future UTLD region via the PST domain of harmonin b and recruits tip-link components. However, MYO7A and SANS lack mechanisms to recognize the future UTLD location. The travel-and-connect scenario (e) assumes that either CDH23 or PCDH15 travels toward stereocilia tips. The other side may simply diffuse. Harmonin isoforms may switch from harmonin a or c (lacking the PST domain) to harmonin b (with the PST domain), given that MYO7A coupled with harmonin b fragments does not traffic efficiently (see Fig. 5). PCDH15 may form temporary links, as indicated by BAPTA-mediated remodeling experiments. The walking-links scenario (f) supported by a previous cryo-electron microscopy study assumes that pre-assembled tip links are transported toward stereocilia tips.