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

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

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

Fig. 5

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 mice123 (double asterisks; residues 296–728 of NM_1163733). Harmonin d was recently identified in the retina124 (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 isoforms80. 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).

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